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Cytoplasmic free-Ca level rises with repellents and falls with attractants in Escherichia coli chemotaxis
Abstract
Cytoplasmic free-Ca2+ levels in Escherichia coli were measured by use of the fluorescent Ca(2+)-indicator dye fura-2. Chemotactically wild-type E. coli regulated cytoplasmic free Ca2+ at approximately 100 nM when no stimuli were encountered, but changes in bacterial behavior correlated with changes in cytoplasmic free-Ca2+ concentration. For chemotactically wild-type E. coli, addition of a repellent resulted in cells tumbling and a transient increase in cytoplasmic free-Ca2+ levels. Conversely, addition of an attractant to wild-type cells caused running and produced a transient decrease in cytoplasmic free-Ca2+ levels. Studies with mutant strains showed that the chemoreceptors were required for the observed changes in cytoplasmic free-Ca2+ levels in response to chemical stimuli.
... The role of calcium ion participation in bacterial behaviour and signal transduction has been established by several lines of evidence [2][3][4][5][6][7] . Previous studies demonstrate the following observations: (1) Changing cytoplasmic free Ca 2+ levels in cells of E. coli alters their behavior 2 . ...
... Tumbling ensues following the release of free Ca 2+ that is produced from caged Ca 2+ compounds such as nitr-5 upon irradiation. (2) Changes in bacterial behaviour are correlated with changes in cytoplasmic free Ca 2+ level 4 . Fluorescent Ca 2+ indicator dye fura-2 was used to show that repellents caused a temporary rise in cytoplasmic free Ca 2+ levels, while attractants caused a temporary fall, and the receptor proteins were required for these effects. ...
... Four genes (calA, calC, calD, and chaA) have been identified in E. coli that are involved in calcium homeostasis. All three of these loci, A, C, and D, are associated with Ca 2+ /PO 4 2symporter activity 15 . The chaA gene is involved in Ca 2+ /H + antiporter activity 16 . ...
While the role of calcium ions as secondary messengers has been well described in eukaryotic cells, little is known about calcium homeostasis in bacteria at the physiological and molecular levels. Genetic and genomic approaches were used to address calcium regulation and to identify genes (cal) involved in calcium homeostasis. Transposon mutagenesis of Escherichia coli generated several calcium-sensitive mutants that fell into three categories: (i) Ca2+-sensitive chemotaxis mutants, (ii) Ca2+-sensitive cell division mutants, and (iii) Ca2+-sensitive mutants that showed no defects in cell division or chemotaxis. The physiological properties of these Ca2+-sensitive mutants were determined. Besides calcium-sensitivity to 75 mM calcium, all of the mutants exhibited increased sensitivities to several divalent cations including Ni2+, Mg2+, Mn2+, Co2+, Zn2+, Cu2+, and Cd2+. To identify the cal gene sequence in the Ca2+-sensitive mutants, the region of the genes fused to the reporter gene (phoA) on the transposon TnphoA was amplified by PCR and sequenced. The sites of the gene fusion for three cal mutants were at the fdoG, gpt and pqi5 genes. The pleiotropic nature for the cal mutations suggested that many genes may be globally regulated by calcium. We then investigated global gene expression patterns of wild-type E. coli under calcium-depleted (addition of 10 mM EGTA) and calcium-elevated (addition of 75 mM Ca2+) conditions as compared to cultures grown under unstressed conditions. A comprehensive transcriptome analysis using macroarrys exhibited a global regulation of diverse genes within the E. coli genome during calcium homeostasis.Bangladesh J Microbiol, Volume 31, Number 1-2,June-Dec 2014, pp 1-8
... While the role of Ca 2+ in eukaryotes has been extensively studied, the role of Ca 2+ in prokaryotes still remains elusive. Indirect evidence suggests that Ca 2+ may play a role in various bacterial physiological processes such as spore formation, chemotaxis, heterocyst differentiation, transport and virulence [9][10][11][12]. Several reports have shown that bacteria are capable of maintaining intracellular Ca 2+ homeostasis, and Ca 2+ transients are produced in response to adaptation to nitrogen starvation, environmental stress [13][14][15][16], and metabolites of carbohydrate metabolism [17,18]. ...
... The role of Ca 2+ in chemotaxis has been documented in several reports [11,22]. The switch from tumbling to swimming, after the addition of repellants and attractants, is modulated by Ca 2+ [11,50]. ...
... The role of Ca 2+ in chemotaxis has been documented in several reports [11,22]. The switch from tumbling to swimming, after the addition of repellants and attractants, is modulated by Ca 2+ [11,50]. However, the molecular events for Ca 2+ regulation in bacterial chemotaxis are still unknown. ...
While the role of calcium binding proteins (CaBPs) in cell signaling pathways and homeostasis is well established in eukaryotic cells, the physiological function of CaBPs in prokaryotes is unknown. Although several CaBPs have been identified and sequences predicted in a variety of prokaryotic genomes, biochemical and functional characterization is lacking. We hypothesize that CaBPs play an important role in Ca2+ homeostasis and that Ca2+ ions regulate several processes in bacterial cells. The purpose of this work was to study the effects of Ca2+ in the B. subtilis proteome, to identify CaBPs altered (increased or decreased) by the addition of Ca2+ -chelators (EGTA, BAPTA) or CaCl2, and to examine Ca2+ homeostasis in B. subtilis cells utilizing various analytical techniques. 45Ca-autoradiography and antibody-crossreactivity were used to detect CaBPs. These proteins were identified by LC-MS/MS. Intracellular calcium levels [Ca2+]i were measured using the photoprotein aequorin. Our results show that remarkable global changes in protein abundance occurred in the B. subtilis proteome as a result of CaCl2 or chelator 58 treatments compared to control cells. Six proteins appeared to be modulated by high levels of extracellular Ca2+. These proteins were increased after Ca2+ -chelator treatments and reduced upon Ca2+ addition. Moreover, these proteins bound radioactive 45Ca2+, and showed a shift in molecular weight in the presence of Ca2+ /EGTA. B. subtilis cells thightly regulate cytosolic Ca2+ levels. Taken together, these results suggest an important role of Ca2+ ions in B. subtilis.
... However, due to their small cell size, the selective permeability of their cell walls and cell membrane and the toxicity of many chelators used in these Ca 2+ studies, it remains complex to monitor Ca 2+ concentrations inside bacterial cells, which is nevertheless indispensable to increase our understanding of the connection between Ca 2+ influx and other cellular processes. The use of the Ca 2+ reporters aequorin (Watkins et al., 1995) and Fura 2 (1-[2-(5-carboxyoxazol-2-yl)-6-amino-benzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy) ethan-N,N,N′,N′-tetraacetic acid) (Gangola and Rosen, 1987;Tisa and Adler, 1995) revealed that variations in cytosolic Ca 2+ levels also regulate many important bacterial cellular processes. For example, Ca 2+ acts in bacteria, including plant pathogenic bacteria, as a versatile intracellular messenger involved in the maintenance of cell structure (Domínguez et al., 2015), motility (Cruz et al., 2012;Fishman et al., 2018;Gode-Potratz et al., 2010;Guragain et al., 2013;Parker et al., 2015;Tisa and Adler, 1995), cell division (Domínguez et al., 2015), gene expression (Domínguez et al., 2015), type III secretion (Dasgupta et al., 2006;DeBord et al., 2003;Fishman et al., 2018;Gode-Potratz et al., 2010), exopolysaccharide production (Kierek and Watnick, 2003;Kim et al., 1999;Patrauchan et al., 2007), iron scavenging (Domínguez et al., 2015;Patrauchan et al., 2007), quorum sensing (Werthén and Lundgren, 2001), biofilm formation (Cruz et al., 2012;Das et al., 2014;Parker Jennifer et al., 2016;Patrauchan et al., 2005;Rinaudi et al., 2006;Sarkisova et al., 2005;Zhou et al., 2013) or biofilm suppression (Bilecen and Yildiz, 2009;Shukla and Rao, 2013). ...
... The use of the Ca 2+ reporters aequorin (Watkins et al., 1995) and Fura 2 (1-[2-(5-carboxyoxazol-2-yl)-6-amino-benzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy) ethan-N,N,N′,N′-tetraacetic acid) (Gangola and Rosen, 1987;Tisa and Adler, 1995) revealed that variations in cytosolic Ca 2+ levels also regulate many important bacterial cellular processes. For example, Ca 2+ acts in bacteria, including plant pathogenic bacteria, as a versatile intracellular messenger involved in the maintenance of cell structure (Domínguez et al., 2015), motility (Cruz et al., 2012;Fishman et al., 2018;Gode-Potratz et al., 2010;Guragain et al., 2013;Parker et al., 2015;Tisa and Adler, 1995), cell division (Domínguez et al., 2015), gene expression (Domínguez et al., 2015), type III secretion (Dasgupta et al., 2006;DeBord et al., 2003;Fishman et al., 2018;Gode-Potratz et al., 2010), exopolysaccharide production (Kierek and Watnick, 2003;Kim et al., 1999;Patrauchan et al., 2007), iron scavenging (Domínguez et al., 2015;Patrauchan et al., 2007), quorum sensing (Werthén and Lundgren, 2001), biofilm formation (Cruz et al., 2012;Das et al., 2014;Parker Jennifer et al., 2016;Patrauchan et al., 2005;Rinaudi et al., 2006;Sarkisova et al., 2005;Zhou et al., 2013) or biofilm suppression (Bilecen and Yildiz, 2009;Shukla and Rao, 2013). Furthermore, Ca 2+ appears to determine the virulence of the facultative human pathogen Pseudomonas aeruginosa (Guragain et al., 2016;Patrauchan et al., 2007;Sarkisova et al., 2014) and of all species of Yersinia (Mekalanos, 1992). ...
In a number of compatible plant-bacterium interactions, a rise in apoplastic Ca2+ levels is observed, suggesting that Ca2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na+/Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2-AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or ATP. Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+/Ca2+ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na+/Ca2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav-cneA mutant. Complementation of the Psav-cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a ‘host signal’ that allows and promotes Psav pathogenicity on olive plants.
... However, due to their small cell size, the selective permeability of their cell walls and cell membrane and the toxicity of many chelators used in these Ca 2+ studies, it remains complex to monitor Ca 2+ concentrations inside bacterial cells, which is nevertheless indispensable to increase our understanding of the connection between Ca 2+ influx and other cellular processes. The use of the Ca 2+ reporters aequorin (Watkins et al., 1995) and Fura 2 (1-[2-(5-carboxyoxazol-2-yl)-6-amino-benzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy) ethan-N,N,N′,N′-tetraacetic acid) (Gangola and Rosen, 1987;Tisa and Adler, 1995) revealed that variations in cytosolic Ca 2+ levels also regulate many important bacterial cellular processes. For example, Ca 2+ acts in bacteria, including plant pathogenic bacteria, as a versatile intracellular messenger involved in the maintenance of cell structure (Domínguez et al., 2015), motility (Cruz et al., 2012;Fishman et al., 2018;Gode-Potratz et al., 2010;Guragain et al., 2013;Parker et al., 2015;Tisa and Adler, 1995), cell division (Domínguez et al., 2015), gene expression (Domínguez et al., 2015), type III secretion (Dasgupta et al., 2006;DeBord et al., 2003;Fishman et al., 2018;Gode-Potratz et al., 2010), exopolysaccharide production (Kierek and Watnick, 2003;Kim et al., 1999;Patrauchan et al., 2007), iron scavenging (Domínguez et al., 2015;Patrauchan et al., 2007), quorum sensing (Werthén and Lundgren, 2001), biofilm formation (Cruz et al., 2012;Das et al., 2014;Parker Jennifer et al., 2016;Patrauchan et al., 2005;Rinaudi et al., 2006;Sarkisova et al., 2005;Zhou et al., 2013) or biofilm suppression (Bilecen and Yildiz, 2009;Shukla and Rao, 2013). ...
... The use of the Ca 2+ reporters aequorin (Watkins et al., 1995) and Fura 2 (1-[2-(5-carboxyoxazol-2-yl)-6-amino-benzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy) ethan-N,N,N′,N′-tetraacetic acid) (Gangola and Rosen, 1987;Tisa and Adler, 1995) revealed that variations in cytosolic Ca 2+ levels also regulate many important bacterial cellular processes. For example, Ca 2+ acts in bacteria, including plant pathogenic bacteria, as a versatile intracellular messenger involved in the maintenance of cell structure (Domínguez et al., 2015), motility (Cruz et al., 2012;Fishman et al., 2018;Gode-Potratz et al., 2010;Guragain et al., 2013;Parker et al., 2015;Tisa and Adler, 1995), cell division (Domínguez et al., 2015), gene expression (Domínguez et al., 2015), type III secretion (Dasgupta et al., 2006;DeBord et al., 2003;Fishman et al., 2018;Gode-Potratz et al., 2010), exopolysaccharide production (Kierek and Watnick, 2003;Kim et al., 1999;Patrauchan et al., 2007), iron scavenging (Domínguez et al., 2015;Patrauchan et al., 2007), quorum sensing (Werthén and Lundgren, 2001), biofilm formation (Cruz et al., 2012;Das et al., 2014;Parker Jennifer et al., 2016;Patrauchan et al., 2005;Rinaudi et al., 2006;Sarkisova et al., 2005;Zhou et al., 2013) or biofilm suppression (Bilecen and Yildiz, 2009;Shukla and Rao, 2013). Furthermore, Ca 2+ appears to determine the virulence of the facultative human pathogen Pseudomonas aeruginosa (Guragain et al., 2016;Patrauchan et al., 2007;Sarkisova et al., 2014) and of all species of Yersinia (Mekalanos, 1992). ...
In a number of compatible plant-bacterium interactions, a rise in apoplastic Ca2+ levels is observed, suggesting that Ca2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na+/Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2-AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or ATP. Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+/Ca2+ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na+/Ca2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav-cneA mutant. Complementation of the Psav-cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a ‘host signal’ that allows and promotes Psav pathogenicity on olive plants.
... Free Ca 2 , produced from caged Ca 2 compounds such as nitr-5 upon irradiation, results in tumbling [3]. Changes in bacterial behavior were correlated with changes in cytoplasmic free Ca 2 levels that were measured by the use of the £uorescent Ca 2 indicator dye fura-2 [4] or by the use of the Ca 2 -sensitive photoprotein aequorin [5]. Repellents cause a temporary rise in cytoplasmic free Ca 2 levels, while attractants cause a temporary fall. ...
... Bacteria maintain and regulate cytosolic calcium levels at about 90 nM, similar to eukaryotic cells [4,8]. There are two calcium e¥ux systems in E. coli: a Ca 2 /PO 23 4 symporter and a Ca 2 /H antiporter [8,9]. ...
omega-Conotoxin, a calcium channel blocker, inhibits chemotaxis by Escherichia coli. To test whether omega-conotoxin acts at the cytoplasmic membrane, the kinetics of I-125-omega-conotoxin binding was investigated. I-125-omega-Conotoxin bound to Tris-EDTA-permeabilized cells or right-side-out membrane vesicles with saturation kinetics. Binding of I-125-omega-conotoxin to membrane vesicles was inhibited by Ca2+ ions, but not by Mg2+ ions. The calA mutant, defective in calcium transport, was more resistant to omega-conotoxin inhibition of chemotaxis than the parental wild-type. I-125-omega-Conotoxin binding to membrane vesicles indicated that both the wild-type and the calA mutant had similar K(D)s for omega-conotoxin binding. However, the saturation level was higher with the calA mutant, indicating that there are more binding sites in the calA mutant. Thus, calA does not directly affect the affinity of the omega-conotoxin binding site. Chemical cross-linking experiments identified two proteins as potential omega-conotoxin receptors. (C) 2000 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
... acidocalcisomes; Dominguez, 2004;Docampo and Moreno 2011;Dominguez et al., 2015). As a result, these prokaryotic cells, similar to eukaryotic cells, possess a low internal concentration of calcium, in the vicinity of 0.1 to 0.3 mM (Tisa and Adler, 1995;Watkins et al., 1995;Dominguez, 2004;Dominguez et al., 2015). Some of the calcium pumps in prokaryotes are similar to the phosphorylation or P-type ATPases of eukaryotic cells. ...
... With regard to regulating the cytoskeleton, calcium seems far less involved in prokaryotes compared with eukaryotes. Nevertheless, calcium has been implicated as a potential general regulator in chemotaxis and motility in E. coli and Bacillus subtilis cells (Tisa and Adler, 1995;Tisa et al., 2000). Thus, agents that repel bacteria cause a spike in calcium, which then causes the cell to tumble and change direction. ...
... While the role of Ca 2+ in eukaryotes has been extensively studied, the role of Ca 2+ in prokaryotes still remains elusive. Indirect evidence suggests that Ca 2+ affects various bacterial physiological processes such as spore formation, chemotaxis, heterocyst differentiation, transport and virulence [9][10][11][12][13][14]. Several reports have shown that bacteria are capable of maintaining intracellular Ca 2+ homeostasis [15][16][17][18]. ...
... For example, it was shown that ChaA from E.coli and ApCAX and SynCAX from cyanobacteria provide tolerance to high Ca 2+ and enhance salt tolerance [85,89]. Indirect evidence using inhibitors suggested that Ca 2+ channels may be involved in chemotaxis in E. coli [10] and B. subtilis [97]. Ca 2+ ATPases have been shown to play role in heat-resistance and germination of spores in B. subtilis, B. anthracis and C. perfringes [79]. ...
... The role of Ca as a regulator and secondary messenger is well established in eukaryotes (51-53) but poorly understood in prokaryotes (53,54). Multiple aspects of physiology are assumed to be controlled by Ca in prokaryotes, including cell cycle progression, virulence, and competence (55)(56)(57)(58). Ca modulates the phosphorylation state of the Che proteins, which are the basis for chemotaxis (54). ...
Lanthanides (Ln) are the most recently described life metals and are central to methylotrophy (type of metabolism in which organic substrates without carbon-carbon bonds serve as carbon and energy source) in diverse taxa. We recently characterized a novel, Ln-dependent, and Ln-accumulating methylotroph, Beijerinckiaceae bacterium RH AL1, which requires lighter Ln (La, Ce, Nd) for methanol oxidation. Starting from two sets of incubations, one with different La concentrations (50 nM and 1 µM) and one with different Ln elements [La, Nd, or an Ln cocktail (containing Ce, Nd, Dy, Ho, Er, Yb)], we could show that La concentration and different Ln elements strongly affect gene expression and intracellular Ln accumulation. Differential gene expression analysis revealed that up to 41% of the encoded genes were differentially expressed. The effects of La concentration and Ln elements were not limited to Ln-dependent methanol oxidation but reached into many aspects of metabolism. We observed that Ln influence the flagellar and chemotactic machinery and that they affect polyhydroxyalkanoate biosynthesis. The most differentially expressed genes included lanM , coding for the well-characterized lanthanide-binding protein lanmodulin, and a glucose dehydrogenase gene linked to the conversion of β-D-glucose to D-glucono-1,5-lactone, a known potential metal chelator. Electron microscopy, together with RNAseq, suggested that Beijerinckiaceae bacterium RH AL1 can discriminate between Ln elements and that they are differently taken up and accumulated. The discrimination of Ln and links between Ln and various aspects of metabolism underline a broader physiological role for Ln in Beijerinckiaceae bacterium RH AL1.
IMPORTANCE
Since its discovery, Ln-dependent metabolism in bacteria attracted a lot of attention due to its bio-metallurgical application potential regarding Ln recycling and circular economy. The physiological role of Ln is mostly studied dependent on presence and absence. Comparisons of how different (utilizable) Ln affect metabolism have rarely been done. We noticed unexpectedly pronounced changes in gene expression caused by different Ln supplementation. Our research suggests that strain RH AL1 distinguishes different Ln elements and that the effect of Ln reaches into many aspects of metabolism, for instance, chemotaxis, motility, and polyhydroxyalkanoate metabolism. Our findings regarding Ln accumulation suggest a distinction between individual Ln elements and provide insights relating to intracellular Ln homeostasis. Understanding comprehensively how microbes distinguish and handle different Ln elements is key for turning knowledge into application regarding Ln-centered biometallurgy.
... The role of calcium as a regulator and secondary messenger is well-established in eukaryotes (49)(50)(51), but poorly understood in prokaryotes (51, 52). Multiple aspects of physiology are assumed to be controlled by calcium in prokaryotes, including cell cycle progression, virulence, and competence (53)(54)(55)(56). Calcium modulates the phosphorylation state of the Che proteins, which are the basis for chemotaxis (52). ...
Lanthanides are the most recently described life metals and are central to methylotrophy in diverse taxa. We recently characterized a novel, lanthanide-dependent, and lanthanide-accumulating methylotroph, Beijerinckiaceae bacterium RH AL1, that utilizes lighter lanthanides (La, Ce, Nd) for methanol oxidation. We show that lanthanum concentration and different lanthanide (Ln) elements strongly affect gene expression and intracellular lanthanide accumulation. Differential gene expression analysis based on incubations with either La (50 nM or 1 µM), Nd (1 µM), or a lanthanide cocktail ([La, Ce, Nd, Dy, Ho, Er, Yb], equimolarly pooled, 1 µM), revealed that up to 41% of the encoded genes were differentially expressed. The effects of lanthanum concentration and Ln elements were not limited to lanthanide-dependent methanol oxidation but reached into many aspects of metabolism. We observed that lanthanides control the flagellar and chemotactic machinery and that they affect polyhydroxyalkanoate (PHA) biosynthesis. Secretion and various uptake systems, and carbohydrate metabolism were highly responsive. The most differentially expressed genes encode various unknown or hypothetical proteins, but also lanM , coding for the well-characterized lanthanide-binding protein lanmodulin, and a glucose dehydrogenase gene linked to the conversion of β-D-glucose to gluconolactone, a known metal chelator. Electron microscopy, together with RNAseq, suggested different and potentially selective mechanisms for the uptake and accumulation of individual Ln elements. Mechanisms for discriminating lanthanides and links between lanthanides and various aspects of metabolism underline a broader functional role for lanthanides, possibly by functioning as calcium complements or antagonists.
Importance
Since its discovery, lanthanide-dependent metabolism in bacteria attracted a lot of attention due to its bio-metallurgical application potential regarding lanthanide recycling and circular economy. The physiological role of lanthanides is mostly studied dependent on presence and absence. Comparisons of how different (utilizable) lanthanides affect metabolism have rarely been done. Our research shows that strain RH AL1 distinguishes different lanthanide elements and that the effect of lanthanides reaches into many aspects of physiology, for instance, motility and polyhydroxyalkanoate metabolism. Numerous differentially expressed genes coding for unknown or hypothetical proteins might hide so far unknown lanthanide-binding proteins. Our findings regarding lanthanide accumulation suggest different mechanisms for dealing with individual lanthanide elements and provide insights relating to intracellular lanthanide homeostasis. Understanding comprehensively how microbes distinguish and handle different lanthanide elements is key for turning knowledge into application regarding lanthanide-centered biometallurgy.
... Calcium is required for the bacterial cytochrome c oxidase [33][34][35], which is in agreement with our finding that genes for this enzyme were up-regulated when more calcium was supplied. It is reported that calcium ions may play an essential role in chemotaxis, cell cycle, and competence by using calcium ions from an external source and calcium antagonists in prokaryotes [36][37][38]. However, direct evidence of the role of intercellular calcium ions in prokaryotes is still poorly understood and remains to be elucidated [39,40]. ...
The bacterium Gemmatimonas phototrophica AP64 isolated from a freshwater lake in the western Gobi Desert represents the first phototrophic member of the bacterial phylum Gemmatimonadota. This strain was originally cultured on agar plates because it did not grow in liquid medium. In contrast, the closely related species G. groenlandica TET16 grows both on solid and in liquid media. Here, we show that the growth of G. phototrophica in liquid medium can be induced by supplementing the medium with 20 mg CaCl2 L−1. When grown at a lower concentration of calcium (2 mg CaCl2 L−1) in the liquid medium, the growth was significantly delayed, cells were elongated and lacked flagella. The elevated requirement for calcium is relatively specific as it can be partially substituted by strontium, but not by magnesium. The transcriptome analysis documented that several groups of genes involved in flagella biosynthesis and transport of transition metals were co-activated after amendment of 20 mg CaCl2 L−1 to the medium. The presented results document that G. phototrophica requires a higher concentration of calcium for its metabolism and growth compared to other Gemmatimonas species.
... Other studies also found lanthanides such as La 3+ and Gd 3+ significantly constrain chemotaxis (Kinoshita et al., 2017;Wheeler, 2017). It was noted that by blocking Ca 2+ channels in the plasma membrane and membranes of intracellular Ca 2+ stores, the lanthanides limit the availability of free cytosolic Ca 2+ , the movement of which is required to phosphorylate ATP and drive motility and chemotaxis (Tisa and Adler, 1995). Conversely, the calmodulin proteins that bind Ca 2+ and translate cytosolic Ca 2+ transients associated with environmental stimuli also play crucial role in chemotactic response (Bothwell et al., 2006). ...
Ca ²⁺ signaling regulates physiological processes including chemotaxis in eukaryotes and prokaryotes. Its inhibition has formed the basis for control of human disease but remains largely unexplored for plant disease. This study investigated the role of Ca ²⁺ signaling on motility and chemotaxis of Spongospora subterranea zoospores, responsible for root infections leading to potato root and tuber disease. Cytosolic Ca ²⁺ flux inhibition with Ca ²⁺ antagonists were found to alter zoospore swimming patterns and constrain zoospore chemotaxis, root attachment and zoosporangia infection. LaCl 3 and GdCl 3 , both Ca ²⁺ channel blockers, at concentrations ≥ 50 μM showed complete inhibition of zoospore chemotaxis, root attachment and zoosporangia root infection. The Ca ²⁺ chelator EGTA, showed efficient chemotaxis inhibition but had relatively less effect on root attachment. Conversely the calmodulin antagonist trifluoperazine had lesser effect on zoospore chemotaxis but showed strong inhibition of zoospore root attachment. Amiloride hydrochloride had a significant inhibitory effect on chemotaxis, root attachment, and zoosporangia root infection with dose rates ≥ 150 μM. As expected, zoospore attachment was directly associated with root infection and zoosporangia development. These results highlight the fundamental role of Ca ²⁺ signaling in zoospore chemotaxis and disease establishment. Their efficient interruption may provide durable and practical control of Phytomyxea soilborne diseases in the field.
... [K] in is 2 to 28 fold greater than [K] out (E. coli: [30,41,[45][46][47], marine and nonmarine archaea: [48], marine bacteria: [39], nonmarine bacteria: [40], review of bacteria: [33]). [Ca] in is always lower than [Ca] out (E. coli: [49][50][51][52], reviews of bacteria: [53][54][55]). Further evidence of the importance of regulating crossmembrane ion flow in prokaryotes is provided by the SecY protein transport system, which has a gasket-like seal that prevents flow of small molecules, including ions, across the cell membrane during protein transport [56]. ...
Background: Internalization-based hypotheses of eukaryotic origin require close physical association of host and symbiont. Prior hypotheses of how these associations arose include chance, specific metabolic couplings between partners, and prey-predator/parasite interactions. Since these hypotheses were proposed, it has become apparent that mixed-species, close-association assemblages (biofilms) are widespread and predominant components of prokaryotic ecology. Which forces drove prokaryotes to evolve the ability to form these assemblages are uncertain. Bacteria and archaea have also been found to form membrane-lined interconnections (nanotubes) through which proteins and RNA pass. These observations, combined with the structure of the nuclear envelope and an energetic benefit of close association (see below), lead us to propose a novel hypothesis of the driving force underlying prokaryotic close association and the origin of eukaryotes.
Results: Respiratory proton transport does not alter external pH when external volume is effectively infinite. Close physical association decreases external volume. For small external volumes, proton transport decreases external pH, resulting in each transported proton increasing proton motor force to a greater extent. We calculate here that in biofilms this effect could substantially decrease how many protons need to be transported to achieve a given proton motor force. Based as it is solely on geometry, this energetic benefit would occur for all prokaryotes using proton-based respiration.
Conclusions: This benefit may be a driving force in biofilm formation. Under this hypothesis a very wide range of prokaryotic species combinations could serve as eukaryotic progenitors. We use this observation and the discovery of prokaryotic nanotubes to propose that eukaryotes arose from physically distinct, functionally specialized (energy factory, protein factory, DNA repository/RNA factory), obligatorily symbiotic prokaryotes in which the protein factory and DNA repository/RNA factory cells were coupled by nanotubes and the protein factory ultimately internalized the other two. This hypothesis naturally explains many aspects of eukaryotic physiology, including the nuclear envelope being a folded single membrane repeatedly pierced by membrane-bound tubules (the nuclear pores), suggests that species analogous or homologous to eukaryotic progenitors are likely unculturable as monocultures, and makes a large number of testable predictions.
... Prokaryotes also have channels and transporters involved in various aspects of signalling and cell physiology such as chemotaxis [204][205][206][207][208]. These channels can be homologous to the eukaryotic channels, for example the TRIC channels are conserved across bacteria, archaea and eukaryotes [209]. ...
All living cells interact dynamically with a constantly changing world. Eukaryotes, in particular, evolved radically new ways to sense and react to their environment. These advances enabled new and more complex forms of cellular behaviour in eukaryotes, including directional movement, active feeding, mating, and responses to predation. But what are the key events and innovations during eukaryogenesis that made all of this possible? Here we describe the ancestral repertoire of eukaryotic excitability and discuss five major cellular innovations that enabled its evolutionary origin. The innovations include a vastly expanded repertoire of ion channels, the emergence of cilia and pseudopodia, endomembranes as intracellular capacitors, a flexible plasma membrane and the relocation of chemiosmotic ATP synthesis to mitochondria, which liberated the plasma membrane for more complex electrical signalling involved in sensing and reacting. We conjecture that together with an increase in cell size, these new forms of excitability greatly amplified the degrees of freedom associated with cellular responses, allowing eukaryotes to vastly outperform prokaryotes in terms of both speed and accuracy. This comprehensive new perspective on the evolution of excitability enriches our view of eukaryogenesis and emphasizes behaviour and sensing as major contributors to the success of eukaryotes.
This article is part of the theme issue ‘Basal cognition: conceptual tools and the view from the single cell’.
... Prokaryotes also have channels and transporters involved in various aspects of signalling and cell physiology such as chemotaxis [191][192][193][194][195]. These channels can be homologous to the eukaryotic channels, for example the trimeric intracellular cation-specific (TRIC) channels are conserved across bacteria, archaea and eukaryotes [196]. ...
All living cells interact dynamically with a constantly changing world. Eukaryotes in particular, evolved radically new ways to sense and react to their environment. These advances enabled new and more complex forms of cellular behavior in eukaryotes, including directional movement, active feeding, mating, or responses to predation. But what are the key events and innovations during eukaryogenesis that made all of this possible? Here we describe the ancestral repertoire of eukaryotic excitability and discuss five major cellular innovations that enabled its evolutionary origin. The innovations include a vastly expanded repertoire of ion channels, endomembranes as intracellular capacitors, a flexible plasma membrane, the emergence of cilia and pseudopodia, and the relocation of chemiosmotic ATP synthesis to mitochondria that liberated the plasma membrane for more complex electrical signaling involved in sensing and reacting. We conjecture that together with an increase in cell size, these new forms of excitability greatly amplified the degrees of freedom associated with cellular responses, allowing eukaryotes to vastly outperform prokaryotes in terms of both speed and accuracy. This comprehensive new perspective on the evolution of excitability enriches our view of eukaryogenesis and emphasizes behaviour and sensing as major contributors to the success of eukaryotes.
... The strong ability of CavMr to exclude Na + and K + along with Ca 2+ permeation suggests that its primary physiological role is Ca 2+ intake in response to a voltage change (Figure 3e and f). In some bacteria, the direction of flagellar rotation and chemotaxis changes depending on the internal Ca 2+ concentration (Ordal, 1977;Tisa et al., 1993;Tisa and Adler, 1995). M. ruber was isolated from hot springs, and therefore a sufficient amount of Ca 2+ is likely to exist in its native environment (Loginova et al., 1984). ...
Voltage-dependent Ca²⁺ channels (Cavs) are indispensable for coupling action potentials with Ca²⁺ signaling in living organisms. The structure of Cavs is similar to that of voltage-dependent Na⁺ channels (Navs). It is known that prokaryotic Navs can obtain Ca²⁺ selectivity by negative charge mutations of the selectivity filter, but native prokaryotic Cavs had not yet been identified. We report the first identification of a native prokaryotic Cav, CavMr, whose selectivity filter contains a smaller number of negatively charged residues than that of artificial prokaryotic Cavs. A relative mutant whose selectivity filter was replaced with that of CavMr exhibits high Ca²⁺ selectivity. Mutational analyses revealed that the glycine residue of the CavMr selectivity filter is a determinant for Ca²⁺ selectivity. This glycine residue is well conserved among subdomains I and III of eukaryotic Cavs. These findings provide new insight into the Ca²⁺ selectivity mechanism that is conserved from prokaryotes to eukaryotes.
... The increase in cytosolic Ca 2+ (Ca i 2+ ) levels is often used as a signal for cells to avoid repellents or dangers. Transient increases in Ca i 2+ levels have been reported in various species when cells change their polarity [13][14][15][16][17][18] . In addition, a Ca i 2+ gradient has been indicated to participate in cell migration [19][20][21][22][23] . ...
Living organisms employ various mechanisms to escape harm. At the cellular level, mobile cells employ movement to avoid harmful chemicals or repellents. The present study is the first to report that cells move away from the site of injury in response to local wounding. When a migrating Dictyostelium cell was locally wounded at its anterior region by laserporation, the cell retracted its anterior pseudopods, extended a new pseudopod at the posterior region, and migrated in the opposite direction with increasing velocity. When wounded in the posterior region, the cell did not change its polarity and moved away from the site of wounding. Since the cells repair wounds within a short period, we successfully manipulated cell migration by applying multiple wounds. Herein, we discussed the signals that contributed to the wound-induced escape behavior of Dictyostelium cells. Our findings provide important insights into the mechanisms by which cells establish their polarity.
... Since 1977, Ordal reported that cytosolic Ca 2+ controlled the rotation of the flagella in B. subtilis cells. Later work corroborated that cytosolic Ca 2+ transients affect bacterial motility in E. coli, possibly through the phosphorylation of the Che proteins [78][79][80]. The involvement of Ca 2+ as a signal transducer in a variety of environmental conditions, where cytosolic free Ca 2+ is elevated as a result of the stimulus, has been shown in various organisms including: oxidative stress in B. subtilis [81], heat/cold shock, and salt and osmotic stress in Anabaena strain PCC7120 [14,82], carbohydrate fermentation products in E. coli [19], organic solvents, pharmaceuticals and antibiotics in cyanobacteria [16,17]. ...
... The EDS mapping (Fig. 6c) identified Si, O, C, S, N and Ca element in area around the damaged cell, and among these elements Si, O, C and S came from silicone rubber substrate and coupling MPTS, N and Ca came only from cell itself. Nitrogen is critical for bacteria growth and cytoplasmic free-Ca 2+ plays a role in bacteria behavior [36]. So it was believed that cytoplasm of E. coli was leaked after incubation on GO coatings surface and resulted in the bacteria cell disruption. ...
Graphene oxide (GO) was thought to be a promising antibacterial material. In this work, graphene oxide coatings on polymer substrate were prepared and the antibacterial activity against E. coli and S. aureus was investigated. It was demonstrated that the coatings exhibited stronger antibacterial activity against E. coli with thin membrane than S. aureus with thick membrane. Take into consideration the fact that the coatings presented smooth, sharp edges-free morphology and bonded parallelly to substrate, which was in mark contrast with their precursor GO nanosheets, oxidative stress mechanism was considered the main factor of antibacterial activity. The coatings, which are easy to recycle and have no inhalation risk, provide an alternative for application in antibacterial medical instruments.
... Though the specific mechanisms are not understood, Ca modulates a variety of bacterial phenotypes including motility (Tisa and Adler, 1995;Gode-Potratz et al., 2010;Cruz et al., 2012;Guragain et al., 2013;Parker et al., 2015), type III secretion system activity (DeBord et al., 2003;Dasgupta et al., 2006;Gode-Potratz et al., 2010), spore germination (Wang et al., 2008), quorum sensing (Werthen and Lundgren, 2001), and production of extracellular proteases , pectinolytic enzymes (Flego et al., 1997), and exopolysaccharides (EPS) (Kim et al., 1999;Patrauchan et al., 2007). Of particular interest is the formation of bacterial biofilm, an important virulence characteristic in many bacterial pathogens. ...
The functions of calcium (Ca) in bacteria are less characterized than in eukaryotes, where its role has been studied extensively. The plant-pathogenic bacterium Xylella fastidiosa has several virulence features that are enhanced by increased Ca concentrations, including biofilm formation. However, the specific mechanisms driving modulation of this feature are unclear. Characterization of biofilm formation over time showed that 4mM Ca supplementation produced denser biofilms that were still developing at 96 hrs, while biofilm in non-supplemented media had reached the dispersal stage by 72 hrs. To identify changes in global gene expression in X. fastidiosa grown in supplemental Ca, RNA-Seq of batch culture biofilm cells was conducted at three 24-hr time intervals. Results indicate that a variety of genes are differentially expressed in response to Ca, including genes related to attachment, motility, exopolysaccharide synthesis, biofilm formation, peptidoglycan synthesis, regulatory functions, iron homeostasis, and phages. Collectively, results demonstrate that Ca supplementation induces a transcriptional response that promotes continued biofilm development, while biofilm cells in non-supplemented media are driven towards dispersion of cells from the biofilm structure. These results have important implications for disease progression in planta, where xylem sap is the source of Ca and other nutrients for X. fastidiosa. This article is protected by copyright. All rights reserved.
... [K] in is 2 to 28 fold greater than [K] out (E. coli: [30,41,[45][46][47], marine and nonmarine archaea: [48], marine bacteria: [39], nonmarine bacteria: [40], review of bacteria: [33]). [Ca] in is always lower than [Ca] out (E. coli: [49][50][51][52], reviews of bacteria: [53][54][55]). Further evidence of the importance of regulating crossmembrane ion flow in prokaryotes is provided by the SecY protein transport system, which has a gasket-like seal that prevents flow of small molecules, including ions, across the cell membrane during protein transport [56]. ...
Following the publication of this article [1] it was noticed that, due to an error on the part of the publisher, the 2nd round of comments submitted by Reviewer 1, Dr. López-García, were unintentionally omitted during the peer review process. As a consequence of this error, the authors were unable to reply to Dr. López-García’s comments and subsequently revise their manuscript accordingly (where appropriate).
In fairness to both the authors and reviewer, Dr. López-García’s (Reviewer 1) 2nd round of comments are now included below and Scott L Hooper and Helaine J Burstein (author) were given the opportunity to reply. Any consequent amendments to the research article [1] are outlined in the author’s replies.
... Bacteria can sense calcium and respond in diverse ways (reviewed in (Norris et al., 1996, Dominguez, 2004. Fluxes in the intracellular calcium level influence chemotactic behavior in B. subtilis and E. coli (Ordal, 1977, Tisa & Adler, 1995. The concentrations of MgCl 2 and CaCl 2 that are found in seawater enhance the swimming motility of Vibrio fischeri (O'Shea et al., 2005). ...
... Biochemical and physiological data confirm that calcium ions are involved in a variety of bacterial cellular processes, including cell cycles and division~Yu & Margolin, 1997!, competence~Trombe et al., 1994!, pathogenesis~Straley et al., 1993!, motility, and chemotaxis~Onek & Smith, 1992Tisa & Adler, 1995;Norris et al., 1996;Michiels et al., 2002!. As in eukaryotic cells, mechanisms of calcium signaling in bacterial cells are based on local changes of free calcium concentration in the cytoplasm~Shemarova & Nesterov, 2005!. ...
Many different bacterial species precipitate carbonate in alkaline environments rich in Ca2+ ions. Numerous authors have described the capability of bacteria to precipitate carbonate in both natural habitats and laboratory culture.
Bacterially induced carbonate mineralization has recently been proposed as an environmentally friendly method to protect decayed ornamental carbonate stone. This new conservation method mimics what nature has been doing for eons, since many carbonate rocks have been cemented by microbe-induced calcium carbonate precipitation.
Biocalcification by bacteria is an emerging restoration technique that is still being developed, and requires further research. However, the production of acid substances, the development of colored spots by microbial metabolism or bacterial survival within the carbonate crystal could have serious implications in restoration techniques. It has already been demonstrated that uncontrolled bacterial growth can damage stone. To over-come these problems, development of stone treatment without viable cells seems a better biotechnological tool.
This study highlights the role of specific outer bacterial structures, such as glycocalix, in calcium carbonate crystallization in vitro. We described the formation of calcite crystals by extracellular polymeric materials (EPM), such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) isolated from Bacillus firmus and Nocardia calcarea. EPM were isolated from calcifying bacteria grown on synthetic medium - in the presence or in the absence of calcium ions - and their effect on calcite precipitation was assessed. SEM observations and EDS analysis showed that CPS and EPS fractions were involved in calcium carbonate precipitation, not only serving as nucleation sites but also through a direct role in crystals formation. The utilization of different synthetic media, with and without addition of calcium ions, influenced the EPS/CPS, biofilm production and protein profile of EPM. Proteins of CPS fractions isolated from Nocardia calcarea with a molecular mass between 25 and 70 kDa were overexpressed when calcium ions were present in the medium. SEM observations showed that some CPS overexpressed proteins were involved in calcium carbonate precipitation. The discovery that outer structures such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) and /or proteins isolated from bacterial strains are able to mediate CaCO3 precipitation in vitro, shows new perspectives in their biothecnological applications such as stone treatment without viable cells.
... Biochemical and physiological studies have previously shown that Ca 2? is involved in a variety of bacterial cellular processes, including the cell cycle and cell division [32], competence [29], pathogenesis [25], motility, and chemotaxis [27,28]. Evidence for the role of Ca 2? at the molecular level has also been demonstrated. ...
Legionella pneumophila is the causative agent of 90 % of Legionnaires' disease cases. This bacterium lives naturally in fresh water and can colonize biofilms, which play an important role in the protection of Legionella against environmental stress factors. Relationship between the presence of minerals in water and Legionella adherence to surfaces is not well-known. In this study, we studied influence of minerals on bacterial adherence. For the first time, to our knowledge, this report shows that calcium and magnesium in a less extent, enhances the adherence of Legionella to surfaces compared to the bacteria behavior in distilled water. Treatment with proteinase K of live cells showed that surface proteins do not seem to play a crucial role in bacteria adherence to surfaces. Our results represent a first step in understanding effect of ions on Legionella adherence to surfaces. Such field of research could be helpful to better understand biofilm colonization by this bacterium to improve Legionella risk management in water networks.
... Similar to eukaryotic cells, eubacterial cells are equipped with transporters to efflux Ca 2ϩ and maintain Ca 2ϩ homeostasis (17). The cytosolic Ca 2ϩ concentration is usually significantly lower than in the extracellular environment, roughly between 0.1 and 1 M in E. coli (18,19). Early studies by Rosen and McClees (20) and Harold and co-workers (21) first identified a Ca 2ϩ /H ϩ antiporter in E. coli and an ATP-dependent P-type Ca 2ϩ efflux pump in Streptococcus faecalis, and these types of systems were later shown to also exist in other bacteria (22,23) including L. lactis (24,25). ...
LmrP is a major facilitator superfamily multidrug transporter from Lactococcus lactis that mediates the efflux of cationic amphiphilic substrates from the cell in a proton-motive force-dependent fashion. Interestingly,
motif searches and docking studies suggested the presence of a putative Ca2+-binding site close to the interface between the two halves of inward facing LmrP. Binding experiments with radioactive 45Ca2+ demonstrated the presence of a high affinity Ca2+-binding site in purified LmrP, with an apparent Kd of 7.2 μm, which is selective for Ca2+ and Ba2+ but not for Mn2+, Mg2+, or Co2+. Consistent with our structure model and analogous to crystal structures of EF hand Ca2+-binding proteins, two carboxylates (Asp-235 and Glu-327) were found to be critical for 45Ca2+ binding. Using 45Ca2+ and a fluorescent Ca2+-selective probe, calcium transport measurements in intact cells, inside-out membrane vesicles, and proteoliposomes containing
functionally reconstituted purified protein provided strong evidence for active efflux of Ca2+ by LmrP with an apparent Kt of 8.6 μm via electrogenic exchange with three or more protons. These observations demonstrate for the first time that LmrP mediates
selective calcium/proton antiport and raise interesting questions about the functional and physiological links between this
reaction and that of multidrug transport.
... Biochemical and physiological data confirm that calcium ions are involved in a variety of bacterial cellular processes, including cell cycles and division~Yu & Margolin, 1997!, competence~Trombe et al., 1994!, pathogenesis~Straley et al., 1993!, motility, and chemotaxis~Onek & Smith, 1992Tisa & Adler, 1995;Norris et al., 1996;Michiels et al., 2002!. As in eukaryotic cells, mechanisms of calcium signaling in bacterial cells are based on local changes of free calcium concentration in the cytoplasm~Shemarova & Nesterov, 2005!. ...
This study highlights the role of specific outer bacterial structures, such as the glycocalix, in calcium carbonate crystallization in vitro. We describe the formation of calcite crystals by extracellular polymeric materials, such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) isolated from Bacillus firmus and Nocardia calcarea. Organic matrices were isolated from calcifying bacteria grown on synthetic medium--in the presence or absence of calcium ions--and their effect on calcite precipitation was assessed. Scanning electron microscopy observations and energy dispersive X-ray spectrometry analysis showed that CPS and EPS fractions were involved in calcium carbonate precipitation, not only serving as nucleation sites but also through a direct role in crystal formation. The utilization of different synthetic media, with and without addition of calcium ions, influenced the biofilm production and protein profile of extracellular polymeric materials. Proteins of CPS fractions with a molecular mass between 25 and 70 kDa were overexpressed when calcium ions were present in the medium. This higher level of protein synthesis could be related to the active process of bioprecipitation.
... Bacteria can sense calcium and respond in diverse ways (reviewed in references 10 and 39). Fluxes in the intracellular calcium level influence chemotactic behavior in Bacillus subtilis and Escherichia coli (42,52). The concentrations of MgCl 2 and CaCl 2 that are found in seawater enhance the swimming motility of Vibrio fischeri (43). ...
Here, we probe the response to calcium during growth on a surface and show that calcium influences the transcriptome and stimulates
motility and virulence of Vibrio parahaemolyticus. Swarming (but not swimming) gene expression and motility were enhanced by calcium. Calcium also elevated transcription of
one of the organism's two type III secretion systems (T3SS1 but not T3SS2) and heightened cytotoxicity toward host cells in
coculture. Calcium stimulation of T3SS gene expression has not been reported before, although low calcium is an inducing signal
for the T3SS of many organisms. EGTA was also found to increase T3SS1 gene expression and virulence; however, this was demonstrated
to be the consequence of iron rather than calcium chelation. Ectopic expression of exsA, encoding the T3SS1 AraC-type regulator, was used to define the extent of the T3SS1 regulon and verify its coincident induction
by calcium and EGTA. To begin to understand the regulatory mechanisms modulating the calcium response, a calcium-repressed,
LysR-type transcription factor named CalR was identified and shown to repress swarming and T3SS1 gene expression. Swarming
and T3SS1 gene expression were also demonstrated to be linked by LafK, a σ54-dependent regulator of swarming, and additionally connected by a negative-feedback loop on the swarming regulon propagated
by ExsA. Thus, calcium and iron, two ions pertinent for a marine organism and pathogen, play a signaling role with global
consequences on the regulation of gene sets that are relevant for surface colonization and infection.
ABSTRACT
Calcium ions (Ca2+) play an important role in eukaryote cell signaling and regulation of
physiological functions. Although evidence of a similar role for Ca2+ in prokaryotes has been
difficult to demonstrate, there is mounting evidence that Ca2+ acts as a cell regulator in bacteria.
The purpose of this study was to investigate Ca2+ signaling and the effect of Ca2+ on gene
expression of various multidrug resistant (MDR) efflux pumps and their regulator MgrA in
Staphylococcus aureus and clinically isolated MRSA. We hypothesized that the presence of Ca2+
increased gene expression of multidrug resistance pumps, LmrS, NorA, and the regulator, MgrA.
This hypothesis was based on our previous evidence of unique intracellular Ca2+concentration
signatures in response to various antibiotics and the enhancement of ethidium bromide efflux by
Ca2+ in S. aureus. The effect of Ca2+ on efflux gene expression was evaluated by the addition of
increasing concentrations of CaCl2, and the Ca2+ chelator, EGTA. Gene expression of the efflux
genes and the regulator, MgrA were measured and compared to housekeeping genes, GMK and
GyrA under the same conditions. Our results indicate that the presence of Ca2+ significantly
enhanced gene expression of MDR efflux genes as compared to untreated and EGTA treated cells.
The results indicate that the presence of EGTA significantly decreased efflux gene activity as
compared to untreated cells and Ca2+ treated cells. We conclude that Ca2+ may be a signaling
molecule for the regulation of gene expression of efflux in S. aureus. Transcriptomic studies are
needed to determine the proteins involved in potential calcium transduction pathways for genetic
regulation of efflux in S. aureus
Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca2+]i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca2+ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca2+]i in the range of 0.1-39.8 µM in small cells like those of prokaryotes.
The ubiquitous divalent cations magnesium and calcium are important nutrients required by bacteria for growth and cell maintenance. Multi-faceted roles are shown both in bacterial initial attachment and biofilm maturation. The effects of calcium and magnesium can be highlighted in physio-chemical interactions, gene regulation and bio-macromolecular structural modification, which lead to either promotion or inhibition of biofilms. This review outlines recent research addressing phenotypic changes and mechanisms undertaken by calcium and magnesium in affecting bacterial biofilm formation.
Despite a wealth of knowledge regarding calcium’s role as secondary messenger in eukaryotic cells, relatively little is known about calcium homeostasis in bacteria. Our study employing molecular genetics and transcriptome analysis of calcium homeostasis previously exhibited a calcium regulation of gene expression in elevated or depleted calcium throughout E. coli genome. Here we report the results of our subsequent investigation into quantitative analysis using quantitative reverse transcriptase PCR (qRT-PCR) of gene expression in subsets of genes from wild-type and mutant strains with addition of calcium and chelation of calcium. Data from qRT-PCR reveal very high upregulation of genes yjeE and secA indicating their possible role as overactive calcium efflux systems in E. coli. Calcium is highly likely to be involved in stress response as evidenced by upregulation of marB in transposon mutants and by 10-fold induction of rpoS in elevated calcium condition in mutants defective in calcium transport.
This chapter discusses chemiluminescence and its advantages and disadvantages over fluorescence, as well as bioluminescence. Chemiluminescence is the emission of light because of a chemical reaction. In chemiluminescence, the energy for exciting the electron comes from the enthalpy of the chemical reaction. Bioluminescence is visible light-emission from luminous organisms. The term “bioluminescence” is also used to describe reactions extracted, or DNA cloned and engineered, from luminous organisms. The chapter describes the analytical potential of chemiluminescent compounds along with applications of chemi- and bioluminescence to living cells and the various advantages of bioluminescence. New bioluminescent systems are likely to become available by cloning proteins from deep-sea organisms. Three-dimensional structural information will also establish whether the evolution of bioluminescence and its puzzling phylogenetic distribution are simply dependent on the generation of the appropriate solvent cage. Thus, bioluminescent proteins may provide a model for the first threshold in the evolution of an enzyme. Bioluminescence and the probes developed from its components offer great potential for investigating one of the key problems in biology: whether a process is digital or analog.
The role of Ca2+ ions in the regulation of motility, cell cycle, and division of prokaryotes is discussed, as well as their involvement in the pathogenesis of some infectious diseases. The structural and functional organization of the prokaryotic Ca2+ signaling system and the mechanisms of Ca2+ membrane transport and homeostasis are described. Special attention is paid to the role of Ca2+ cation channels, Ca2+ transporters, and Ca2+-binding proteins in the regulation of the intercellular Ca2+ concentration.
Previous studies from our laboratory have indicated that audible sound field stimulation can significantly affect E.coli growth and metabolic action, which can enhance or inhibit the growth of Escherichia coli (E.coli), and the effects of sound field on E.coli growth depended greatly on the intensity and frequency of sound field. However, very little efforts have been put forth in studying the potential mechanism of bacterial cells responses to audible sound stimulation. In this paper, the potential role of calcium signaling in mediating the response of E.coli to audible sound stimulation was firstly reported. We found that audible sound wave stimulation could evidently enhance total intracellular Ca2+ content. And the lack of calcium ion in medium significantly alleviated audible sound wave biological effects. Moreover, by adding appropriate level of calcium chloride to the LB medium contained 1.2 mM EDTA, the promotion effect of audible sound wave to E.coli growth was gradually resumed. On the basis of these findings, we speculate that calcium signaling may play an important role in mediating the response of E.coli to audible sound stimulation.
Several theories consider the brain to be a network of neurons that process perception with simple activation functions. Real neurons, however, are far more intricate.Through reviews of literature and results from original experiments, Neural Cell Behavior and Fuzzy Logic offers a comprehensive look at these complex systems, supplying trustworthy evidence that neurons can predict the consequences of input signals and transiently change their own excitability to suit. The book also examines how fuzzy logic, the computing of perceptions, can be used to provide a theoretical description of real neuron behavior, and as a model for the "logic" the brain uses to describe environments and make decisions. This book includes sections for general and advanced readers, and will be particularly useful to neuroscience students, academics and researchers as well as to mathematicians and theoretical physicists. © 2008 Springer Science+Business Media, LLC. All rights reserved.
Cover crops can reduce the dependence of farmers on agrochemicals while enhancing overall agrosystem’s performance. However,
the inherent complexity of cover-crop-based systems hampers their adoption by conventional farmers. Therefore, special management
skills and alternative research and technology transfer approaches may be required to facilitate their adoptive use by conventional
farmers. We propose that development and adoption of suitable cover-crop-based production systems may require the use of an
“innovation framework” that includes (1) identification of system constraints, (2) analysis of system behavior, (3) exploration
of alternative systems, and (4) system design and selection. We describe case studies from four regions of the Americas (Florida,
USA; Paraná and Santa Catarina, Brazil; and Canelones, Uruguay) that illustrate the relationships between this innovation
framework and the development and adoption of cover-crop-based production systems. Where successful, development and adoption
of such systems appear to relate to a number of attributes including (1) active involvement by farmers in research and dissemination
programs; (2) integration of cover crops into production systems without net loss of land or labor resources; (3) informing
farmers of the (direct) benefits of cover crop use; (4) provision of multiple benefits by cover crops, (5) sufficient access
to information, inputs, and technologies required for cover crop use; and (6) provision of skills and experience necessary
to manage cover crops effectively. Where these attributes are absent and failure to innovate has prevented development and
adoption of cover-crop-based systems, policy initiatives to reward farmers for ecological services provided by cover crops
may be required.
KeywordsCover crops-green technologies-system analysis-innovation-adoption-sustainability-Americas-• green manure-• living mulch
Consumers are demanding more organic products, in part because of concerns over environmental issues in conventional agriculture.
Modern, high-input agriculture can cause groundwater contamination, soil erosion, and eutrophication of surface waters. It
may be possible to enhance natural nutrient cycling and reduce our dependence on inorganic fertilizers in cropping systems.
To do so, we have to manage our cropping systems to encourage diverse soil microbial communities and arbuscular mycorrhizal
fungi. This chapter reviews the impacts of cropping management practices on soil microbial diversity and arbuscular mycorrhizal
communities. Systems that have reduced tillage, diverse crop rotations or intercrops, low applications of inorganic fertilizers
and pesticides, and some organic fertility inputs tend to encourage a large and diverse microbial community with mycorrhizal
fungi. Organic systems should strive for minimum tillage and the avoidance of bare soil fallow in rotation. Well-managed conventional
systems with minimum tillage and inorganic crop inputs can be as effective as organic systems in encouraging soil biological
fertility. Both organic and conventional cropping systems should incorporate intercrops into their systems to encourage diversity
within the soil system.
KeywordsDiversity-arbuscular mycorrhizal fungi-organic management-conventional management-tillage-crop rotation-fertilizers and pesticides-organic farming-soil biodiversity-tillage-no till-crop rotation
Plants require inorganic nutrients in addition to carbon dioxide and water for growth and production. Nutrients are present
in soil, but get depleted unless supplied through fertilization. Soil feeding is the normal practice, but has limitations
with respect to its availability to the plants. The elements such as phosphorus, potassium, and most of the micronutrients
are fixed in the soil complex, while the more soluble nutrients such as nitrogen are easily leached down the soil. What is
lost through leaching reaches the aquifer and pollutes the groundwater. For instance nitrates and phosphates can be harmful
to humans. With increasing costs of fossil fuel, which provides the raw materials for fertilizer manufacture, there is a need
to find innovations in fertilizer usage techniques. Foliar application is one such technique. Here I review the extensive
work that has been carried out on the effectiveness of foliar-applied nutrients, the mechanisms of foliar absorption, and
transport. The leaf components such as the cuticular membranes, the trichomes, the cuticular pores, ectoteichodes, their properties,
and their role in the nutrient transport into the plant leaf are reviewed. Cuticles are permeable to nutrient ions present
in aqueous forms and have distinct structures like pores. But it is not known if these pores facilitate easy entry into the
leaf cells. The trichomes increase the amount transported into the leaf by providing more area for absorption. The cuticles
have two types of lipophilic substances, the cutin and the cuticular wax, which influence the permeability of nutrient ions
to varying degrees. It is clear that nutrients reach the leaf cells, after penetrating the cuticle, and are further transported
to other parts through plasmadesmata. Some micronutrients are not as freely mobile as the major nutrient elements such as
N, P, or K. The age of the leaf and the pH of the spray liquid are important for foliar absorption. The absence of plasmadesmatic
connections between the guard cells and the epidermal cells is also important. One element Cl has been found to be transported
from the applied leaf to other parts rapidly, showing it is freely mobile. This should be true for many anions. The concept
of limiting factors and the law of the maximum proposed by Wallace group are useful in raising the yield plateau, and when
soil supply poses the “limiting” factor, foliar feeding will help increase the crop yield. Modern technique of sprinkler irrigation
system can be exploited to supply the nutrient elements in the irrigation water, which will be economical in foliar fertilization.
Foliar nutrition is very practical to correct micronutrient deficiencies, which are very important for maximizing the yield.
Crop breeders could also help evolve cultivars, which give good response to foliar feeding.
KeywordsCritical growth stage-cuticle-eutrophication-inorganic nutrients-leaf uptake-sprinkler irrigation
The urgency of the global food crisis, coupled with the environmental impact of global warming and fuel shortages, indicate that transgenic methods may be required to enhance food production and quality. Widely used chemical insecticides, such as phosphine and methyl bromide, are losing their utility either due to insect resistance or to the environmental damage they cause. It is most unlikely that traditional plant-breeding methods for generating insect resistance will deliver the crop improvements required in the available time frame. In this review, we discuss the application of transgenic avidin, a protein naturally occurring in egg-white, for the protection of rice, maize, potato and apple leaf from insect pests. Avidin binds the vitamin biotin with extraordinary affinity (10−15 M). Biotin is a water-soluble vitamin required for normal cellular metabolism and growth. The presence of avidin in the diet of insect pests is lethal since biotin is unavailable to them. The use of streptavidin, a bacterial homologue of avidin, is also described. We discuss the sub-cellular targeting of avidin expression in plants to avoid toxicity to the plant host and we describe the qualities of avidin which make it suitable for crop protection during cultivation and storage. Avidin is stable under normal conditions of crop storage but biodegradable and destroyed by cooking. These combined qualities make it an excellent choice for the protection of crops from insects. Finally, we discuss the modification of the avidin gene to allow expression in plants, the methods for transfection of the gene into plants, and the approaches used to quantify gene expression and avidin function in plant tissues. These methods include: polymerase chain reaction; enzyme-linked immmunosorbent assay; polyacrylamide gel-electrophoresis; fluorescence polarisation (FP); capillary electrophoresis; tissue-printing; square-wave voltammetry (SWV) and the measurement of larvae morbidity and mortality.
Soilborne pathogens are difficult to manage, especially since the use of methyl bromide has been phased out in most countries.
Resistance against many soilborne pathogens is hardly available and fungicides are effective only to a limited extent. In
organic agriculture, many problems related to soilborne pathogens are avoided by applying wide rotations, but still some polyphagous
soilborne pathogens can be highly problematic, especially since most chemical crop protectants are not allowed. In addition,
wide rotations are often economically unprofitable. Therefore, alternative practices to manage soilborne pathogens are needed.
In this review, the occurrence of soilborne pathogens in three types of cropping systems are evaluated: (i) continuous cultivation
of single crops in monoculture, (ii) crop rotation, and (iii) mixed cropping, i.e., cultivation of multiple crops in the same
field at the same time. Both continuous cropping and crop rotation have been investigated extensively. Therefore, in this
chapter we focus on mixed-cropping systems in relation to soilborne pathogens, their potential to suppress soilborne diseases,
and the mechanisms underlying disease suppression. In general, mixed cropping is practiced to optimize nutrient uptake, control
soil erosion, suppress the epidemic spread of airborne pathogens, and improve crop yields per unit of area. While mixed cropping
has received attention for its effects on airborne pests and pathogens, the effects on soilborne pathogens are poorly known.
In 30 out of 36 publications, mixed cropping showed a significant reduction in soilborne disease and in six, no or a positive
effect on disease incidence or severity was found. Diseases caused by splash-dispersed pathogens were less severe in mixed-cropping
systems in ten out of 15 studies. The magnitude of disease reduction in mixed compared to single crops varied, from a 63%
reduction to a 100% increase in disease. Host dilution appeared to be the most important mechanism of disease suppression
for both soilborne and splash-dispersed pathogens (12 and five cases, respectively). Although the use of mixed cropping for
soilborne disease suppression is still in its infancy, the wide range of biological effects and interactions observed holds
promise for further optimization and management of soilborne diseases, for example, by selecting plant species and cultivars
that provide an optimal combination of root architectures.
KeywordsMixed cropping-intercropping-soilborne pathogens-crop rotation-microclimate-monocropping-multiple cropping-disease management-allelopathy-ISR (induced systemic resistance)-SAR (systemic acquired resistance)-microbial antagonism
Organic farming is a sustainable agricultural system that respects and relies on natural ecological systems. Its principles
exclude the use of synthetic pesticides and fertilizers. Instead it is based on management practices that sustain soil quality
and health. Composting of organic residues and the use of compost in agriculture bring back plant nutrients and organic matter
to the soil that otherwise would be lost. Nevertheless, there are some potential risks associated with compost use, such as
the accumulation of heavy metals or organic pollutants, which must not be neglected. Some types of organic farms, such as
stockless farms or vegetable farms, have difficulties sustaining soil humus using only organic farming sources. For such farms,
using biowaste compost from separately collected organic household waste might be a solution, which in addition helps to close
nutrient and organic matter loops of the whole society. Here we compile information on beneficial effects and potential risks
associated with compost use and on crop yields and quality, with compost under an organic farming perspective. The most important
benefit of using compost is the increase in soil organic matter (SOM). Under temperate climate conditions, 6–7 t ha−1 year−1 (dry wt.) compost is sufficient to maintain the soil humus level of medium-textured soils; higher rates increase the soil
humus content. Regular compost addition enhances soil fauna and soil microbial biomass and stimulates enzyme activity, leading
to increased mineralization of organic matter and improved resistance against pests and diseases, both features essential
for organic farming. Through the significant increase in the soil’s content of organic carbon, compost fertilization may make
agricultural soil a carbon sink and thus contribute to the mitigation of the greenhouse effect. Phosphorus and potassium in
compost become nearly completely plant-available within a few years after compost application. The nitrogen-fertilizer value
of compost is lower. In the first years of compost application, N mineralization may vary from −15% to +15%. Nitrogen recovery
in the following years depends on the site- and cultivation-specific mineralization characteristics and will roughly be the
same as that of soil organic matter (SOM). Soil cation exchange capacity (CEC) increases with compost use, improving nutrient
availability. Moderate rates of compost of 6–7 t ha−1 year−1 dry wt. are sufficient to substitute regular soil liming. In the available micronutrient status of the soil, only minor changes
are to be expected with high-quality composts. Increasing soil organic matter exerts a substantial influence on soil structure,
improving soil physical characteristics such as aggregate stability, bulk density, porosity, available water capacity, and
infiltration. Increased available water capacity may protect crops against drought stress. Plant-disease suppression through
compost is well established in container systems. In field systems, the same processes involving the suppression of pathogens
by a highly active microflora supported by the supply of appropriate organic matter are likely at work. When using high-quality
composts, such as specified by the EU regulation 2092/91, the risk of heavy metal accumulation in the soil is very low. Nitrogen
mineralization from compost takes place relatively slowly and there are virtually no reports of uncontrollable N-leaching.
Concentrations of persistent organic pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls
(PCBs), or polychlorinated dibenzodioxins and dibenzofurans (PCDD/F) in high-quality composts usually approach the usual soil
background values. Also the overall hygiene and hygiene concerning plant diseases and weeds are not a problem if quality composts
produced in a monitored system are used. Most studies found positive yield effects of biowaste compost. However, the effect
of biowaste compost applied at moderate rates usually takes some years to develop. It depends on the factors determining nutrient
mineralization from soil and compost and also on crop-related factors such as the nutrient requirements and uptake dynamics
of the respective crop rotation. Crops with longer growth periods can make better use of compost. Many vegetable crops respond
favorably to compost fertilization, often immediately after the first application. Crop quality is usually not affected by
compost fertilization in cereals and slightly positively influenced in vegetable crops.
KeywordsSoil humus-nitrogen-phosphorus-potassium-soil structure-heavy metals-organic pollutants-yield-crop quality-compost-organic farming-Cd-Zn-Ni-Pb-Hg-Cu-Cr-PAH-dioxin-CEC-soil pH-soil N-nitrate, P, K-micronutrients-soil aggregate-soil water-plant disease-maize-wheat-barley-potato-tomato-broccoli-cabbage-cauliflower-cantaloupe-legume-onion
The sustainable use of nutrients in agricultural food production represents a major emphasis for international research, and
evidence that clearly demonstrates the imbalance between nutrient inputs and outputs exists. Nutrient surpluses exist and
are most commonly associated with intensive livestock production and present a particular range of environmentally related
issues. Nutrient deficiency can also develop, and organically managed systems highlight the difficulties that are involved
in maintaining agronomically acceptable concentrations of soil phosphorus (P). A restricted range of P-containing sources,
often having poor solubility, exacerbate these difficulties, and obvious benefits would arise if the availability could be
“naturally” enhanced. Slow rates of phosphate rock (PR) solubilization under prevailing soil conditions reduce the general
agronomic usefulness and potential benefits that any direct applications might provide. Being able to improve rates of dissolution
through some control of the solubilization process would offer widespread potential advantages, particularly with respect
to better matching patterns of P supply with crop demand. A variety of pre and postapplication opportunities exist to improve
the solubility of rock phosphate. Some of these have particular relevance to organic agriculture where phosphate rock represents
an important and acceptable “external” source of P. A range of post-application, farm management practices that include green
manures and rotations using crops with favorable traits that improve P utilization have been successfully employed. Here,
we emphazise pre-application techniques, especially the co-composting of phosphate rock with various organic by-product materials
that include livestock manures and residual vegetable matter. A range of laboratory incubations have demonstrated the underlying
mechanisms involved with solubilization. The significance of microbially induced production of organic acids and acidity during
composting is particularly important in this respect. While co-composting with phosphate rock offers a great potential that
could be developed for use at the individual farm scale, the key controlling factors and underlying mechanisms are far from
being fully understood. A possible time sequence of reactions that might be envisaged include an initial production of protons
and organic acids leading to the mineralogical dissolution and release of Ca and P, followed finally by an extended period
during aging of the compost where secondary reactions appear to influence the form of P. The consequences of composting conditions
and individual processes on immediate and longer-term bioavailability of P once field applied are still poorly defined.
KeywordsPhosphate rock-composting-sustainability-nutrient use efficiency
There was a desperate need for food to recover the economy of the 1950s and 1960s. Farmers all over the world were advised to rely on intensive production methods and synthetic pesticide inputs to increase the productivity. No doubt, these chemical-based agricultural practices substantially increased crop yield. However, indiscriminate use of agrochemicals have contributed significantly to the environmental pollution and adversely affected human and animal health. In addition, the increasing cost of these agrochemicals has continued to lower the farmer’s net cash return. The global use of synthetic pesticides at the start of this millennium exceeded 2.5 million tons per year. A growing worldwide concern for these problems has motivated researchers, administrators, and farmers to seek alternatives to chemical-based, conventional agriculture. One such product is effective microorganisms (EM) developed by Japanese scientists. Effective microorganisms are a mixed culture of beneficial and naturally occurring microorganisms, such as species of photosynthetic bacteria (Rhodopseudomonas palustris and Rhodobacter sphaeroides), lactobacilli (Lactobacillus plantarum, L. casei, and Streptococcus lactis), yeasts (Saccharomyces spp.), and Actinomycetes (Streptomyces spp.). These beneficial microorganisms improve crop growth and yield by increasing photosynthesis, producing bioactive substances such as hormones and enzymes, controlling soil diseases, and accelerating decomposition of lignin materials in the soil. Experiments conducted on various agricultural crops in different parts of the world have shown good prospects for the practical application of these beneficial microorganisms in improving crop yield and soil fertility. Application of beneficial microorganisms generally improves soil physical and chemical properties and favors the growth and efficiency of symbiotic microorganisms such as nitrogen fixing rhizobia and arbuscular mycorrhizal (AM) fungi. Nonetheless experiences of some researchers revealed that the effect of these microorganisms on crop growth and yield was usually not evident or even negative in the first test crop. However, this adverse effect can be overcome through repeated applications of these microorganisms. Research on these microorganisms has shown that crop yields tend to increase gradually as subsequent crops are grown. Foliar application of beneficial microorganisms avoids many of the biotic and abiotic factors and constraints of the soil environment, and thus increases the crop growth and yield significantly. Application of beneficial microorganisms also reduces seed bank of weeds in agricultural soils by enhancing the rate of weed seeds germination. There are reports of management of various fungal and bacterial pathogens as well as insect pests due to application of beneficial microorganisms. These microorganisms have shown a great promise in dairy wastewater treatment. They can reduce NH3 concentration in poultry manure up to 70% possibly by transforming NH
4+ to NO
3−. Research conducted so far concludes that benefits of beneficial microorganisms can be best exploited through their repeated applications for few years in combination with organic amendments and applying them as foliar spray. Integrated use of organic matter plus beneficial microorganisms with half mineral NPK can yield equivalent to that of full recommended NPK fertilizers dose. Beneficial microorganisms can also be used for wastewater treatment, pest and disease management, and to reduce the abiotic stresses on crop growth and yield.
Within the last 2 decades the transition in livestock production technology and intensity has resulted in an increase in annual
livestock production and a drastic decrease in the number of livestock operations. Consequently, the susceptibility of current
livestock operations to experience manure spills is far greater relative to livestock farms 20 years ago, due to increased
herd size per farm. Therefore, manure spills in agricultural communities have become a pervasive issue and have led to the
catastrophic contributions of nutrients and pathogens to surface and groundwaters, human health issues, and large fish kills.
Furthermore, the current remediation methods for manure spills that reach surface waters focus on mitigating contaminants
in the water column and give no attention to the manure-exposed ditch sediments that remain in the fluvial system and continue
to impair the water column. Therefore, this chapter addresses the causes, environmental impacts, and current and alternative
remediation methods for manure spills in agricultural streams. Geographic data suggest that the location of animal-feeding
operations and the occurrence of manure spills were highly correlated with the location of tile-drained agriculture fields.
In addition, at least 14% of reported manure spills were separately attributed to the failure in waste storage equipment and
over-application of manure in the states of Iowa and Ontario, Canada. Evaluations of the downstream impacts of manure spills
have reported ammonia, total phosphorus, and total N concentrations that were at least 28 times the average upstream concentrations
before the spill occurred. Studies have also determined that the current manure spill remediation method results in soluble
phosphorus and nitrogen concentrations significantly greater than the Environmental Protection Agency total phosphorus nutrient
critical limit, 24 h after the plume of the spill has passed. However, supplemental treatment of manure exposed sediments
resulted in at least a 50% decrease in the soluble phosphorus concentrations which was in compliance with the phosphorus nutrient
criteria.
KeywordsManure spills-manure spill remediation methods-alum-ammonium-phosphorus-sediments
Rapid depletion of global fertilizer and fossil fuel reserves, combined with concerns about global warming, have resulted in increased interest in alternative strategies for sustaining agricultural production. Moreover, many farmers are being caught in a vicious spiral of unsustainability related to depletion and degradation of land and water resources, increasing labor and input costs, and decreasing profit margins. To reduce their dependence on external inputs and to enhance inherent soil fertility, farmers, thus, may opt to employ farm-generated renewable resources, including the use of cover crops. However, perceived risks and complexity of cover-crop-based systems may prevent their initial adoption and long-term use. In this review article, we provide a historic perspective on cover-crop use, discuss their current revival in the context of promotion of green technologies, and outline key selection and management considerations for their effective use.
The relatively low cost of fertiliser and the increasing demand and competition for cheap food have encouraged the over-fertilisation
of field vegetables over the past few decades. However, more recent scientific and public concern over eutrophication of water
and the accumulation of nitrates in vegetables for human consumption requires a more effective use of nitrogen fertilisers
in a more sustainable manner, which minimises the potential risk of negative effects on the environment and human health.
In this review, we present the current state of the art in knowledge of N dynamic in vegetable crops and the latest advances
in nutrient management, which could be used to mitigate nitrate losses from vegetables fields to the wider environment. Findings
are based on published data and personal communications with researchers and consultants across Europe. Areas of research
where further work is required are identified and described. A conclusive chapter reports on the economic and environmental
impact of technology transfer of improved nitrogen management in three south European states and in the Netherlands.
KeywordsVegetable crops-nitrate leaching-nutrient management-soil and water pollution-Decision Support System-Integrated Crop Management-soil N-chlorophyll meter-fertigation-slow release fertiliser-nitrification inhibitor-intercropping-mulch-cover crop
We have previously proposed that fluctuations in Ca2+ levels should play an important role in bacteria as in eukaryotes in regulating cell cycle events (Norris et al., J. Theor. Biol. 134 (1998) 341–350). This proposal implied the presence of Ca2+ uptake systems in bacteria, cell cycle mutants simultaneously defective in Ca2+-homeostasis, and perturbation of cell cycle processes when cellular Ca2+ levels are depleted. We review the properties of new cell cycle mutants in E. coli and B. subtilis resistant to inhibitors of calmodulin, PKC or Ca2+-channels; the evidence for Ca2+-binding proteins including Acp and FtsZ; and Ca2+-transporters. In addition, the effects of EGTA and verapamil (a Ca2+ channel inhibitor) on growth, protein synthesis and cell cycle events in E. coli are described. We also describe new measurements of free Ca2+-levels, using aequorin, in E. coli. Several new cell cycle mutants were obtained using this approach, affecting either initiation of DNA replication or in particular cell division at non-permissive temperature. Several of the mutants were also hypersensitive to EGTA and or Ca2+. However, none of the mutants apparently involved direct alteration of a drug target and surprisingly in some cases involved specific tRNAs or a tRNA synthetase. The results also indicate that the expression of several genes in E. coli may be regulated by Ca2+. Cell division in particular appears very sensitive to the level of cell Ca2+, with the frequency of division clearly reduced by EGTA and by verapamil. However, whilst the effect of EGTA was clearly correlated with depletion of cellular Ca2+ including free Ca2+, this was not the case with verapamil which appears to change membrane fluidity and the consequent activity of membrane proteins. Measurement of free Ca2+ in living cells indicated levels of 200–300 nM, tightly regulated in wild type cells in exponential phase, somewhat less so in stationary phase, with apparently La2+-sensitive PHB-polyphosphate complexes involved in Ca2+ influx. The evidence reviewed increasingly supports a role for Ca2+ in cellular processes in bacteria, however, any direct link to the control of cell cycle events remains to be established.
Escherichia coli regulates cytosolic free Ca2+ in the micromolar range through influx and efflux. Herein, we show for the first time that ATP is essential for Ca2+ efflux and that ATP levels also affect generation time. A transcriptome analysis identified 110 genes whose expression responded to an increase in cytosolic Ca2+ (41 elevated, 69 depressed). Of these, 3 transport proteins and 4 membrane proteins were identified as potential Ca2+ transport pathways. Expression of a further 943 genes was modified after 1 h in growth medium containing Ca2+ relative to time zero. Based on the microarray results and other predicted possible Ca2+ transporters, the level of cytosolic free Ca2+ was measured in selected mutants from the Keio knockout collection using intracellular aequorin. In this way, we identified a knockout of atpD, coding for a component of the FoF1 ATPase, as defective in Ca2+ efflux. Seven other putative Ca2+ transport proteins exhibited normal Ca2+ handling. The defect in the ΔatpD knockout cells could be explained by a 70% reduction in ATP. One millimolar glucose or 1 mM methylglyoxal raised ATP in the ΔatpD knockout cells to that of the wild type and restored Ca2+ efflux. One millimolar 2,4-dinitrophenol lowered the ATP in wild type to that in the ΔatpD cells. Under these conditions, a similar defect in Ca2+ efflux in wild type was observed in ΔatpD cells. Ten millimolar concentration of Ca2+ resulted in a 30% elevation in ATP in wild type and was accompanied by a 10% reduction in generation time under these conditions. Knockouts of pitB, a potential Ca2+ transporter, atoA, the β subunit of acetate CoA-transferase likely to be involved in polyhydroxybutyrate synthesis, and ppk, encoding polyphosphate kinase, all indicated no defect in Ca2+ efflux. We therefore propose that ATP is most likely to regulate Ca2+ efflux in E. coli through an ATPase.
The AtoSC two-component system in Escherichia coli is a key regulator of many physiological processes. We report here the contribution of AtoSC in E. coli motility and chemotaxis. AtoSC locus deletion in ΔatoSC cells renders cells not motile or responsive against any chemoattractant or repellent independently of the AtoSC inducer’s presence. AtoSC expression through plasmid complemented the ΔatoSC phenotype. Cells expressing either AtoS or AtoC demonstrated analogous motility and chemotactic phenotypes as ΔatoSC cells, independently of AtoSC inducer’s presence. Mutations of AtoC phosphate-acceptor sites diminished or abrogated E. coli chemotaxis. trAtoC, the AtoC constitutive active form which lacks its receiver domain, up-regulated E. coli motility. AtoSC enhanced the transcription of the flhDC and fliAZY operons and to a lesser extent of the flgBCDEFGHIJKL operon. The AtoSC-mediated regulation of motility and chemotactic response required also the expression of the CheAY system. The AtoSC inducers enhanced the AtoSC-mediated motility and chemotaxis. Acetoacetate or spermidine further promoted the responses of only AtoSC-expressing cells, while Ca2+ demonstrated its effects independently of AtoSC. Histamine regulated bacterial chemotaxis only in atoSC
+ cells in a concentration-dependent manner while reversed the AtoSC-mediated effects when added at high concentrations. The trAtoC-controlled motility effects were enhanced by acetoacetate or spermidine, but not by histamine. These data reveal that AtoSC system regulates the motility and chemotaxis of E. coli, participating in the transcriptional induction of the main promoters of the chemotactic regulon and modifying the motility and chemotactic phenotypes in an induction-dependent mechanism.
S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and exposes a hydrophobic protein-binding surface. Recently S100B was identified as a binding partner of the dopamine D2 receptor in a bacterial two-hybrid screen involving the third intracellular loop (IC3). The low in vivo calcium concentration in bacteria (100-300 nM) suggests this interaction may occur in the absence of calcium. In this work the calcium-sensitive ability for S100B to recruit the IC3 of the dopamine D2 receptor was examined, and regions in both proteins required for complex formation were identified. Peptide array experiments identified the C-terminal 58 residues of the IC3 (IC3-C58) as the major interacting site for S100B. These experiments along with pull-down assays showed the IC3 interacts with S100B in the absence and presence of calcium. (1)H-(15)N HSQC experiments were used to identify residues, primarily in helices III and IV, utilized in the IC3-C58 interaction. NMR titration data indicated that although an interaction between apo-S100B and IC3-C58 occurs without calcium, the binding was enhanced more than 100-fold upon calcium binding. Further, it was established that shorter regions within IC3-C58 comprising its N- and C-terminal halves had diminished binding to Ca(2+)-S100B and did not display any observable affinity in the absence of calcium. This indicates that residue or structural components within both regions are required for optimal interaction with Ca(2+)-S100B. This work represents the first example of an S100B target that interacts with both the apo- and calcium-saturated forms of S100B.
Escherichia coli regulates intracellular free Ca2+ at about 90 nM [Gangola, P. & Rosen, B. P. (1987) J. Biol. Chem. 262, 12570-12574]. To increase intracellular free Ca2+, nitr-5/Ca2+, a "caged" Ca2+ compound, was electroporated into cells and then its affinity for Ca2+ was reduced by exposure to 370-nm light. Upon release of the Ca2+ ions, the cells tumbled. Studies on mutant strains showed that the receptor proteins (methyl-accepting chemotaxis proteins, MCPs) were not required for the Ca(2+)-induced tumbling but that CheA, CheW, and CheY proteins were required. Similar results were obtained with DM-nitrophen/Ca2+, another caged calcium compound that releases Ca2+ upon illumination at 340 nm. Diazo-2, a caged Ca2+ chelator that takes up Ca2+ upon illumination at 340 nm, was used to decrease intracellular free Ca2+, and this caused smooth swimming.
Recently a series of fluorescent calcium indicator dyes have been developed for measurement of free intracellular calcium in eukaryotic cells. Here we report the use of one such dye, fura-2, for the study of intracellular calcium levels in the prokaryote Escherichia coli. Cells of E. coli were loaded with the membrane-permeable acetoxymethyl ester of fura-2, which was cleaved intracellularly to give the free pentaacid. The concentration of free [Ca2+]i in unstarved cells was maintained at 90 +/- 10 nM, irrespective of the Ca2+ concentration in the extracellular medium. Cells of a strain lacking the H+-translocating ATPase were depleted of endogenous energy reserves and loaded with calcium. In this strain oxidative phosphorylation is uncoupled, so ATP is not produced by respiration. In starved cells [Ca2+]i varied from 0.2 to 0.7 microM when the loading Ca2+ concentration varied from 10 microM to 10 mM. Addition of glucose lowered the Ca2+ levels to 90 nM. Addition of respiratory substrates as energy donors produced cyanide-sensitive efflux. Total cell Ca2+ increased in parallel to the extracellular calcium, but the pool of free calcium did not equilibrate with the total cellular pool. These results demonstrate that 1) the pool of total Ca2+ in the bacterial cell is large and responds to extracellular calcium, 2) the free [Ca2+]i is independent of extracellular calcium, and 3) energy in the form of a proton motive force is required for maintenance of the free intracellular pool of calcium.
The ability to glide on a solid surface was inducible by calcium ion in Stigmatella aurantiaca. The induction of motility but not motility itself was prevented by chloramphenicol and erythromycin. Calcium ion was also required for cells to glide, even when they were previously induced. The ability of Myxococcus xanthus to glide in groups using the S motility system but not as single cells (A system) was prevented by chloramphenicol and erythromycin.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
A new, fluorescent, highly selective Ca2+ indicator , "quin2", has been trapped inside intact mouse and pig lymphocytes, to measure and manipulate cytoplasmic free Ca2+ concentrations, [Ca2+]i. Quin2 is a tetracarboxylic acid which binds Ca2+ with 1:1 stoichiometry and an effective dissociation constant of 115 nM in a cationic background mimicking cytoplasm. Its fluorescence signal (excitation 339 nm, emission 492 nm) increases about fivefold going from Ca-free to CA-saturated forms. Cells are loaded with quin2 by incubation with its acetoxymethyl ester, which readily permeates the membrane and is hydrolyzed in the cytoplasm, thus trapping the impermeant quin2 there. The intracellular quin2 appears to be free in cytoplasm, not bound to membranes and not sequestered inside organelles. The fluorescence signal from resting cells indicates a [Ca2+]i of near 120 nM. The millimolar loadings of quin2 needed for accurately calibrated signals do not seem to perturb steady-state [Ca2+]i, but do somewhat slow or blunt [Ca2+]i transients. Loadings of up to 2mM are without serious toxic effects, though above this level some lowering of cellular ATP is observed. [Ca2+]i was well stabilized in the face of large changes in external Ca2+. Alterations of Na+ gradients, membrane potential, or intracellular pH had little effect. Mitochondrial poisons produced a small increase in [Ca2+]i, probably due mostly to the effects of severe ATP depletion on the plasma membrane. Thus intracellulary trapped chelators like quin2 offer a method to measure or buffer [Ca2+]i in hitherto intractable cell types.
Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.
Escherichia coli chemotaxis was inhibited by omega-conotoxin, a calcium ion channel blocker. With Tris-EDTA-permeabilized cells, nanomolar levels of omega-conotoxin inhibited chemotaxis without loss of motility. Cells treated with omega-conotoxin swam with a smooth bias, i.e., tumbling was inhibited.
The products of three chemotaxis-specific genes in Escherichia coli, cheM, cheD, and cheZ, are methylated. The cheZ gene codes for the synthesis of a 24,000 molecular weight polypeptide that appears in the cytoplasm. cheM codes for the synthesis of a membrane-bound polypeptide with a molecular weight of 61,000. cheD codes for another membrane-bound polypeptide with an apparent molecular weight of 64,000. CheM(-) mutants show chemotaxis toward some attractants (Tar(-) phenotype), while CheD(-) mutants respond to other attractants (Tsr(-) phenotype). The double mutant (CheD(-), CheM(-)) does not respond to any attractant or repellent tested. Therefore, these polypeptides play a central role in chemotaxis. They collect information from two subsets of chemoreceptors and act as the last step in the chemoreceptor pathway and the first step in the general processing of signals for transmission to the flagellar rotor. It is suggested that they may be involved in both an initial process that reflects the instantaneous state of the chemoreceptors and in an integrative, adaptive process. Two other genes, cheX and cheW, are required for the methylation of the cheD and cheM gene products.
The properties of two classes of behavioral mutants of Escherichia coli (called tsr and tar) are described. The mutations in these strains define two complementary pathways of information flow in bacterial chemotaxis: behavioral responses to one set of stimuli are defective in tsr mutants, while responses to a complementary set of stimuli are defective in tar mutants. A double mutant containing both genetic lesions is defective in responses to all stimuli tested. The behavioral defects are shown to correlate with alterations in the properties of a methylation reaction involved in chemotaxis. Two independent sets of methyl-accepting proteins are demonstrated in the wild type, each set functioning in one of the two pathways mentioned above. Methylation of one set of proteins is defective in tsr mutants, while methylation of the complementary set is defective in tar mutants. The double mutant shows no methylation of either set. The relationship between the genetic loci (tsr and tar) and the methyl-accepting proteins is discussed.
Mutants that at one time were thought to be specifically defective in taxis toward aspartate and related amino acids (tar mutants) or specifically defective in taxis toward serine and related amino acids (tar mutants) are now shown to be pleiotropic in their defects. The tar mutants also lack taxis toward maltose and away from Co2+ and Ni2+. The tsr mutants are altered in their response to a variety of repellents. Double mutants (tar tsr) fail in nearly all chemotactic responses. The tar and tsr mutants provide evidence for two complementary, converging pathways of information flow: certain chemoreceptors feed information into the tar pathway and others into the tsr pathway. The tar and tsr products have been shown to be two different sets of methylated proteins.
Mutants sensitive to growth inhibition by CaCl2 were found to have alterations in calcium uptake in everted membrane vesicles. These mutations map at different loci on the Escherichia coli chromosomes. A mutation at the calA locus results in vesicles which have two- to threefold higher levels of uptake activity than vesicles from wild-type cells. The calA mutation is phenotypically expressed as increased sensitivity to CaCl2 in a strain also harboring a mutation in the corA locus, which is involved in Mg2+ transport. The calA locus maps very close to purA and cycA at about min 97. The calB mutation results both in sensitivity to CaCl2 at pH 5.6 and in vesicles with diminished calcium transport capability. The CalB phenotype is also expressed only in a corA genetic background; the calB locus appears to map very near, yet separately from, the calA locus. When the cor+ allele is present, calA and calB mutations still result in a defect in calcium transport in vesicles. In addition, both calC and calD mutations result in vesicles with impaired calcium transport activity. calC is cotransducible with kdp and nagA, whereas calD is cotransducible with proC.
CHEMOTAXIS is the process by which bacteria travel to higher concentrations of attractant or lower concentrations of repellant. Its study has attracted wide interest as an example of the stimulus-response network since bacteria are the most primitive of living creatures. Behaviour in peritrichous bacteria is indeed simple: bacteria alternately swim smoothly and tumble, which results in random reorientation for the next swim1. Chemotaxis occurs by the increasing tendency of bacteria to tumble when headed in the `unfavourable' direction and by increasing inclination to swim when the bacteria are headed in the `favourable' direction (such as towards higher attractant concentrations)1,2. Tumbling is caused by clockwise rotation of flagella and swimming by counter-clockwise rotation3. Addition of repellent to bacteria causes tumbling; addition of attractant causes swimming4-6. Understanding how the switch that controls direction of flagellar rotation is controlled and how the controlling parameter is regulated by changes in attractant or repellent concentration is necessary to understand chemotaxis. Here I present evidence that for Bacillus subtilis the free concentration of internal Ca2+ ion controls direction of flagellar rotation (high concentrations bringing about tumbling; low concentrations, swimming) and that repellents, which cause tumbling when added to bacteria, work by increasing the flow of Ca2+ ion across the plasma membrane.
Signal transduction in bacterial chemotaxis involves transfer of a phosphoryl group between the cytoplasmic proteins CheA and CheY. In addition to the established metal ion requirement for autophosphorylation of CheA, divalent magnesium ions are necessary for the transfer of phosphate from CheA to CheY. The work described here demonstrates via fluorescence studies that CheY contains a magnesium ion binding site. This site is a strong candidate for the metal ion site required to facilitate phosphotransfer from phospho-CheA to CheY. The diminished magnesium ion interaction with CheY mutant D13N and the lack of metal ion binding to D57N along with significant reduction in phosphotransfer to these two mutants are in direct contrast to the behavior of wild-type CheY. This supports the hypothesis that the acidic pocket formed by Asp13 and Asp57 is essential to metal binding and phosphotransfer activity. Metal ion is also required for the dephosphorylation reaction, raising the possibility that the phosphotransfer and hydrolysis reactions occur by a common metal-phosphoprotein transition-state intermediate. The highly conserved nature of the proposed metal ion binding site and site of phosphorylation within the large family of phosphorylated regulatory proteins that are homologous to CheY supports the hypothesis that all these proteins function by a similar catalytic mechanism.
As research into the flagellar gene systems of Escherichia
coli and Salmonella typhimurium has progressed, their complexity
has proved to be far greater than originally imagined.
Currently, close to 40 genes are known to be involved in the
flagellar gene systems of both species (see, e.g., reference 2),
making them second only to the ribosomal gene system in
size.
The Trg transducer mediates chemotactic response to galactose and ribose by interacting, respectively, with sugar-occupied galactose- and ribose-binding proteins. Adaptation is linked to methylation of specific glutamyl residues of the Trg protein. This study characterized two trg mutations that affect interaction with binding protein ligands but do not affect methylation or adaptation. The mutant phenotypes indicated that the steady-state activity of methyl-accepting sites is independent of ligand-binding activity. The mutation trg-8 changed arginine 85 to histidine, and trg-19 changed glycine 151 to aspartate. The locations of the mutational changes provided direct evidence for functioning of the amino-terminal domain of Trg in ligand recognition. Cross-inhibition of tactic sensitivity by the two Trg-linked attractants implies competition for a common site on Trg. However, the single amino acid substitution caused by trg-19 greatly reduced the response to galactose but left unperturbed the response to ribose. Thus Trg must recognize the two sugar-binding proteins at nonidentical sites, and the complementary sites on the respective binding proteins should differ. trg-8 mutants were substantially defective in the response to both galactose and ribose. An increase in cellular content of Trg-8 protein improved the response to galactose but not to ribose. It appears that Trg-8 protein is defective in the generation of the putative conformational change induced by ligand interaction. The asymmetry of the mutational defect implies that functional separation of interaction sites could persist beyond the initial stage of ligand binding.
The effect of several Ca2+ channel blockers, which inhibit the voltage-dependent Ca2+ uptake in Bacillus subtilis, on chemotactic behaviour of the bacterium was studied. Nitrendipine, verapamil, LaCl3 and omega-conotoxin were tested and these blockers inhibited chemotactic behaviour in the bacterium toward L-alanine. Among these blockers, omega-conotoxin was the most effective inhibitor of chemotaxis. EGTA was also as effective as omega-conotoxin. In contrast, these blockers, did not inhibit the motility and the growth of the bacterium. These results suggest that internal Ca2+ plays an important role in the sensory system of bacterial chemotaxis.
Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.
Several methods for detecting or measuring negative chemotaxis are described. Using these, we have surveyed a number of chemicals for their ability to repel Escherichia coli. Although most of the repellents are harmful compounds, harmfulness is neither necessary nor sufficient to make a compound a repellent. The repellents can be grouped into at least nine classes according to (i) competition experiments, (ii) mutants lacking certain of the negative taxes, and (iii) their chemical structure. The specificity of each class was studied. It is suggested that each class corresponds to a distinct chemoreceptor. Generally, non-chemotactic mutants lack both positive and negative chemotaxis, and l-methionine is required for both kinds of taxis. Repellents at very low concentrations are not attractants, and attractants at very high concentrations are not repellents.
We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged.
Six Escherichia coli che loci (cheA,-B,-R,-W,-Y, and Z) are located in two adjacent operons that map at minute 42 on the chromosome. Point mutants defective in any of these six functions have aberrant swimming patterns and are generally nonchemotactic. Deletions within the two major che gene operons were isolated in order to examine epistatic interactions among these genes. We first constructed a specialized transducing phage (lambda che22), which carries both of the che operons and their associated promoters. Deleted lambda che22 derivatives were selected by chelating agent inactivation, and these derivatives were characterized by mapping them against a series of host strains with point mutations. Representative nonpolar deletions were then transferred into the E. coli chromosome by homologous recombination. Although the phenotype of cheR mutants (smooth swimming) was expected to be epistatic to that of cheB mutants (tumbly swimming), we found that deletion mutants lacking both of these functions exhibited frequent directional changes or tumbling episodes as they swam. An examination of larger deletions indicated that both the cheA-cheW and cheY-cheZ functions were required for the anomalous tumbling behavior observed in these mutants. Loss of the cheB function was also correlated with an inverted behavioral response to sodium acetate, a strong repellent of wild-type cells. These findings indicate that an important component of the signal transducing machinery may be altered in cheB mutants.
Intracellular Ca2+ has been previously implicated in the chemotactic response of Escherichia coli. However, no correlative measurements of intracellular free Ca2+ have been made during bacterial chemotaxis, essential if this is to be established. In order to monitor internal free Ca2+ in E. coli during challenge with chemotactic agents, the Ca(2+)-activated photoprotein aequorin was expressed in a chemotactic strain (AB1157) and a non-chemotactic strain [BL21(DE3)] of E. coli. Repellents were found to cause an increase (50-150 nM) in intracellular free Ca2+, whereas attractants caused a small but consistent decrease in intracellular free Ca2+. These data are in agreement with the proposed model that an increase in intracellular free Ca2+ causes tumbling. The effect of increasing external Ca2+ on the regulation of intracellular free Ca2+ in both strains was monitored by using aequorin. The resting level of free Ca2+ in E. coli (AB1157) was found to be 100 nM, which agrees with previous data [Gangola and Rosen (1987) J. Biol. Chem. 262, 12570-12574]. As these results also show differences in the regulation of intracellular free Ca2+ between the two strains in the presence of high external Ca2+ concentrations, this may have implications for the effect of high-Ca2+ environments on E. coli.
A new method is described for measuring intracellular free calcium concentrations, [(Ca2+)i], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)i] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells at all stages of development exhibit a sharp transient increase in [(Ca2+)i] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca2+)i] and show three times as large a response to cAMP stimulation as prespore cells.
Activation of surface folate receptors or cyclic AMP (cAMP) receptor (cAR) 1 in Dictyostelium triggers within 5-10 s an influx of extracellular Ca2+ that continues for 20 s. To further characterize the receptor-mediated Ca2+ entry, we analyzed 45Ca2+ uptake in amoebas overexpressing cAR2 or cAR3, cARs present during multicellular development. Both receptors induced a cAMP-dependent Ca2+ uptake that had comparable kinetics, ion selectivity, and inhibitor profiles as folate- and cAR1-mediated Ca2+ uptake. Analysis of mutants indicated that receptor-induced Ca2+ entry does not require G protein alpha subunits G alpha 1, G alpha 2, G alpha 3, G alpha 4, G alpha 7, or G alpha 8. Overexpression of cAR1 or cAR3 in g alpha 2- cells did not restore certain G alpha 2-dependent events, such as aggregation, or cAMP-mediated activation of adenylate and guanylate cyclases, but these strains displayed a cAMP-mediated Ca2+ influx with kinetics comparable to wild-type aggregation-competent cells. These results suggest that a plasma membrane-associated Ca(2+)-influx system may be activated by at least four distinct chemoreceptors during Dictyostelium development and that the response may be independent of G proteins.
Environmental and hormonal signals control diverse physiological processes in plants. The mechanisms by which plant cells perceive and transduce these signals are poorly understood. Understanding biochemical and molecular events involved in signal transduction pathways has become one of the most active areas of plant research. Research during the last 15 years has established that Ca2+ acts as a messenger in transducing external signals. The evidence in support of Ca2+ as a messenger is unequivocal and fulfills all the requirements of a messenger. The role of Ca2+ becomes even more important because it is the only messenger known so far in plants. Since our last review on the Ca2+ messenger system in 1987, there has been tremendous progress in elucidating various aspects of Ca(2+) -signaling pathways in plants. These include demonstration of signal-induced changes in cytosolic Ca2+, calmodulin and calmodulin-like proteins, identification of different Ca2+ channels, characterization of Ca(2+) -dependent protein kinases (CDPKs) both at the biochemical and molecular levels, evidence for the presence of calmodulin-dependent protein kinases, and increased evidence in support of the role of inositol phospholipids in the Ca(2+) -signaling system. Despite the progress in Ca2+ research in plants, it is still in its infancy and much more needs to be done to understand the precise mechanisms by which Ca2+ regulates a wide variety of physiological processes. The purpose of this review is to summarize some of these recent developments in Ca2+ research as it relates to signal transduction in plants.
Ca(2+) plays a key role in the regulation of ciliary and flagellar movement. This article focuses on the initial steps of this regulation: how and where Ca(2+) enters cilia and flagella to trigger specific changes in axonemal motility. This knowledge is fundamental for understanding the sites, molecular targets and mechanisms of action of Ca(2+) within the cilium of flagellum.
Chlamydomonas has long been a favourite organism for genetic and biochemical studies of flagellar motility and assembly, photosynthesis, and organelle genomes. With the recent development of procedures for the efficient transformation of its nuclear genome, Chlamydomonas has become accessible to a wide range of molecular genetic approaches, including gene tagging by insertional mutagenesis and cloning by complementation. The availability of these powerful techniques is stimulating interest in Chlamydomonas as a model system for research in areas where it previously has not been widely exploited. One such area that holds particular promise is phototransduction and the behavioural response to light.
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