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Regulation of Luminescence in Marine Bacteria
... The light producing reaction in bacteria is controlled by the enzyme luciferase. This reaction involves the oxidation of reduced flavin mononucleotide (FMNH2) and a long chain aldehyde (Figure 1.3.2.1) (Silverman et al., 1989). In V. fischeri (and most other naturally luminescent bacteria), bioluminescence is a density dependent reaction. ...
... Another theory is that the bioluminescent reaction provides an alternative pathway for the transfer of electrons at low oxygen tension (Silverman et al., 1989). ...
... Regulation Aldehyde Luciferase Aldehyde (Silverman et al., 1989). luxA and luxB code for the luciferase a and p subunits, respectively. ...
Every year, a substantial quantity of pesticides is introduced into the environment. The continued use of pesticides is necessary in order to meet the demands of the growing human population. However, only a small fraction of the applied pesticide reaches the target organisms, and there is a large potential for adverse effects on non-target organisms. Organophosphorus (OP) insecticides have largely replaced organochlorine compounds and are now one of the main groups of pesticides used in agriculture worldwide. The work presented in this thesis aimed to assess the impact of OP insecticides on the soil microbial community. The soil microbial community has many important functions in the soil, including carbon and nitrogen
transformations, maintenance of soil structural stability and the degradation of organic pollutants. Any adverse effects of OP insecticides on the soil microbial community could lead to a decrease in soil quality and a loss of agricultural sustainability.
The toxicity of OP insecticides was initially assessed using the lux-marked biosensors E. coli HB101 pUCD607 and P. fluorescens 10586r pl)CD607. The production of light is linked to metabolic activity in these genetically modified bacteria. Therefore, a toxic effect is observed as a decrease in light output. The biosensors were used to test OP insecticide toxicity in aqueous solution and in soil pore water. The effect of OP insecticides on soil microbial processes (respiration and nitrification) and the soil microbial community (viable counts and fatty acid analysis of whole soil extract) was investigated. Three soils (Insch, Countesswells and Boyndie) were used to determine the influence of soil type on the response of the microbial community to OP insecticides. These soils had contrasting soil texture, organic matter content and pH values, and were representative of many agricultural soils across the UK. The relative impact of a formulated insecticide (biomalathion)
and the corresponding active ingredient (malathion) was also investigated.
The lux-biosensors successfully reported on the toxicity of OP insecticides in aqueous bioassays. P. fluorescens was the most sensitive biosensor and biomalathion was the most toxic of the OP insecticides tested. When spiked soil pore water was used, the EC50 value both increased and decreased relative to the
aqueous bioassay, depending on soil type.
OP insecticides generally had stimulatory effects on soil microbial processes. The greatest increase in soil respiration rate was observed with biomalathion application. The effect of OP insecticides on potential nitrification rate was more complex, with stimulation and inhibition being reported for Insch and Countesswelis soil, respectively. The lux-biosensors detected toxicity in the OP spiked perfusate where no OP was detectable by GC-FID analysis. However, it did not prove possible to predict effects on soil microbial processes on the basis of lux-biosensor response. This is partly due to the difficulties in interpreting the results of the microbial process tests. For example, it is not clear whether a stimulatory effect should be classed as a toxic impact.
Plate counts of OP spiked soil showed an increase in the number of both total heterotrophic bacteria and Pseudomonas. However, fatty acid analysis of whole soil extracts failed to detect any effect of OP insecticide treatment on the soil microbial community. This may be because the OP insecticides are having a selective effect on the culturable fraction of the soil microbial community.
In this study, traditional methods for investigating pesticide effects on non-target soil microorganisms (plate counts, soil respiration, potential nitrification rate) were used in conjunction with more modern techniques (fatty acid analysis and lux-biosensors). These methods are complementary as they examine the impact of OP insecticide treatment on different levels of organisation of the soil microbial community (single species, whole community, process level). At present, it is not possible to adequately assess whether changes in the soil microbial community will have an effect on soil quality, as baseline values for a ‘healthy’ soil have not been
established. In this study, OP insecticides were shown to have a toxic effect towards bacteria. OP insecticide treatment also resulted in changes in soil processes and the soil bacterial populations, as determined by plate counts. Therefore, despite the lack of effect of OP insecticides on the soil fatty acid profile, there is evidence that OP insecticides can alter the soil microbial community.
The work presented in this thesis attempted to assess the impact of an agrochemical on the soil microbial community using an array of techniques. Both the traditional and modern techniques provided useful information about the effect of OP insecticides on the soil microbial community. Changes due to insecticide treatment were evident. However, further research is needed to assess whether these changes will have a long term impact on soil quality and agricultural sustainability.
... Several reviews have been published since 1985 on bacterial bioluminescence covering various aspects of the biochemistry, physiology, and molecular biology of bacterial bioluminescence (20,60,62,80,81,86,134). There have also been some short reviews describing applications of the lax genes (77,87,124,138). ...
... The possibility that the lux autoinducers are part of a larger class of signaling molecules (allomones, pheromones, or hormones) used to sense the local nutritional or chemical environment has been suggested (39,40,134). This proposal is supported by the close similarity in chemical structure of the autoinducers to a regulatory molecule (A factor), isocapryl-8-butyryl lactone which causes autoinduction of sporulation and antibiotic synthesis in certain Streptomyces species. ...
The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.
... If our observations in V. fischeri hold true for other Vibrio species, it may simply be that rounds of growth into stationary phase during genetic manipulations can give rise to luxO* mutants. In this regard, if we consider the reports by Keynan and Hastings (64) and Silverman et al. (65) of "luminescence variation" in V. harveyi resulting in genetically stable dim and dark mutants in old, statically grown cultures, it is tempting to speculate that at least some of these may have been luxO* mutants. One such dark mutant was used in the first description of the luxO locus (66), raising the possibility that a spontaneous luxO* mutant contributed to the discovery of luxO almost 30 years ago (65). ...
... In this regard, if we consider the reports by Keynan and Hastings (64) and Silverman et al. (65) of "luminescence variation" in V. harveyi resulting in genetically stable dim and dark mutants in old, statically grown cultures, it is tempting to speculate that at least some of these may have been luxO* mutants. One such dark mutant was used in the first description of the luxO locus (66), raising the possibility that a spontaneous luxO* mutant contributed to the discovery of luxO almost 30 years ago (65). ...
Importance:
Our results provide novel insight into the function of LuxO, which is a key component of pheromone signaling (PS) cascades in several members of the Vibrionaceae. Our results also contribute to an increasingly appreciated aspect of bacterial behavior and evolution whereby mutants that do not respond to a signal from like cells have a selective advantage. In this case, although "antisocial" mutants locked in the PS signal-off mode can outcompete parents, their survival advantage does not require wild-type cells to exploit. Finally, this work strikes a note of caution for those conducting or interpreting experiments in V. fischeri, as it illustrates how pleiotropic mutants could easily and inadvertently be enriched in this bacterium during prolonged culturing.
... It has been repeatedly postulated that the lux autoinducers are part of a larger class of signalling molecules; furthermore, far from being restricted to a very limited group of bacterial species, they may represent a hitherto barely recognised generic signalling mechanism in bacteria (Eberhard, 1972;Eberhard et al., 1981;Silverman et al., 1989;Meighen, 1991). To date, however, attempts to identify a wider spectrum of bacterial genera capable of synthesising and responding to the lux autoinducer family have not proved successful (Greenberg et al., 1979). ...
... Organisation and function of lux genes cloned from V. fischeri. Arrows denote operons containing lux genes; the leftward arrow marks operon L (containing luxR) and the rightward arrow marks operon R. The molecular sizes (in kDa) of the lux gene products and their functions are shown below the gene designations (adapted fromSilverman et al., 1989). ...
Micro-organisms have evolved complex and diverse mechanisms to sense environmental changes. Activation of a sensory mechanism typically leads to alterations in gene expression facilitating an adaptive response. This may take several forms, but many are mediated by response-regulator proteins. The luxR-encoded protein (LuxR) has previously been characterised as a member of the response-regulator superfamily and is known to respond to the small diffusible autoinducer signal molecule N-(beta-ketocaproyl) homoserine lactone (KHL). Observed previously in only a few marine bacteria, we now report that KHL is in fact produced by a diverse group of terrestrial bacteria. In one of these (Erwinia carotovora), we show that it acts as a molecular control signal for the expression of genes controlling carbapenem antibiotic biosynthesis. This represents the first substantive evidence to support the previous postulate that the lux autoinducer, KHL, is widely involved in bacterial signalling.
... As with the ES114 variant and original form, a particularly distinct dimorphism is seen with P. luminescens, which is symbiotic with entomopathogenic nematodes (Bleakley and Nealson 1988; Boemare et al. 1993). However, the conditions and mechanisms controlling variant formation in culture and the pleiotropic conversion of cells in ecological transitions between the symbiotic and free-living states at present are not understood (Doudoroff 1938; Giese 1943; 200 Nealson and Hastings 1979; Bleakley and Nealson 1988; Silverman et al. 1989). The pleiotropic nature of the differences between the ES114 variant and original form, including the expression of several membrane-associated and soluble proteins, indicates the involvement and differential expression of several genes. ...
Vibrio fischeri strains isolated from light organs of the sepiolid squidEuprymna scolpes are non-visibly luminous and fast growing in laboratory culture, whereas in the symbiosis they are visibly luminous and slow
growing. A spontaneous, visibly luminous, slowgrowing variant was isolated from a laboratory culture of the squid-symbioticV. fischeri strain ES114. Taxonomic and DNA-homology analyses demonstrated that the variant wasV. fischeri and was very similar to the original form. However, the variant grew at one-fourth the rate of the original form, produced
30,000-fold more luminescence, induced luminescence at a lower cell density, and produced a higher level ofV. fischeri luminescence autoinducer. Regulation of luminescence, nonetheless, was similar in the two forms and typical ofV. fischeri with respect to responses to autoinducer, glucose, the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid), and 3′:5′-cyclic AMP. Compared to the original form, cells of the variant were smaller, exhibited
from zero to two polar, sheathed flagella instead of a tuft of three to eight flagella, produced a deeper yellow-orange pigment,
did not acidify media containing glycerol, and produced a more distinct pellicle. The two forms also differed in the levels
of several outer membrane and soluble proteins. These results establish a distinctive physiological, morphological, and biochemical
dimorphism inV. fischeri ES114 in which the variant exhibits several traits similar toV. fischeri cells in the symbiotic state. The variant and its conversion from the original form in laboratory culture may provide insight
into the properties ofV. fischeri cells in the symbiosis and may serve as a model for elucidating the mechanism for their pleiotropic conversion upon colonization
of the squid.
... Relevant to the question of which species and strains of bacteria produce light is the observation that luminescence often is not phenotypically stable. Strains luminous on primary isolation often become dim or dark in laboratory culture (Nealson and Hastings, 1979b;Akhurst, 1980;Silverman et al., 1989;Nealson and Hastings, 1992). Therefore, it is reasonable to assume that luminescence has been overlooked in many species, especially those represented primarily by laboratory strains or those studied under clinical settings at temperatures where luminescence may not be produced. ...
“The smallest lamps in the world, luminous bacteria, are no different from ordinary bacteria except in their ability to luminesce.”
—E. N. Harvey, 1940
... While the functions of luminescence are quite clear for higher organisms, the ecological significance of bacterial and fungal luminescence is less straightforward. Bacterial bioluminescence predominates in marine ecosystems, particularly among fish (49). Studies of marine bioluminescence have provided great understanding on symbiotic relationships particularly from the Euprymna scolopes-V. ...
Bioluminescent bacteria are widespread in natural environments. Over the years, many researchers have been studying the physiology, biochemistry and genetic control of bacterial bioluminescence. These discoveries have revolutionized the area of Environmental Microbiology through the use of luminescent genes as biosensors for environmental studies. This paper will review the chronology of scientific discoveries on bacterial bioluminescence and the current applications of bioluminescence in environmental studies, with special emphasis on the Microtox toxicity bioassay. Also, the general ecological significance of bioluminescence will be addressed.
... In addition to 1uxA and luxB, the lux operon typically contains three further structural genes luxC, luxD and luxE that encode the subunits of a fatty acid reductase. There appears to be a universal conservation of the order of these genes in lux operons, as defined in Fig. 2. The remaining lux genes, luxR and luxl, are involved in the regulation of lux expression, an aspect which has been the subject of extensive review (Meighen, 1988(Meighen, , 1991Silverman et al., 1989). A number of additional lux genes (luxF, luxG, luxH and luxY) of unknown function have recently been identified and are reviewed by Meighen (1 99 1). ...
... Particular attention is given to the organization of the lux genes from the different luminescent bacteria, the regulation of expression of luminescence, and the application of the lux genes as reporters of gene expression. Several reviews have been published since 1985 on bacterial bioluminescence covering various aspects of the biochemistry , physiology, and molecular biology of bacterial bioluminescence (20, 60, 62, 80, 81, 86, 134). There have also been some short reviews describing applications of the lax genes (77, 87, 124, 138). ...
The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.
... We have no way of knowing whether these additional mutations occurred before or after the IS10 insertion in luxA. Dark variants have long been known in the bacterial bioluminescence field (15,22,33). Such variants occur most often in old cultures, especially in broth. ...
Bacterial bioluminescence can display a wide range of intensities among strains, from very bright to undetectable, and it
has been shown previously that there are nonluminous vibrios that possess lux genes. In this paper, we report the isolation and characterization of completely dark natural mutants in the genus Vibrio. Screening of over 600 Vibrio isolates with a luxA gene probe revealed that approximately 5% carried the luxA gene. Bioluminescence assays of the luxA-positive isolates, followed by repetitive extragenic palindromic-PCR fingerprinting, showed three unique genotypes that are
completely dark. The dark mutants show a variety of lesions, including an insertion sequence, point mutations, and deletions.
Strain BCB451 has an IS10 insertion sequence in luxA, a mutated luxE stop codon, and a truncated luxH. Strain BCB494 has a 396-bp deletion in luxC, and strain BCB440 has a frameshift in luxC. This paper represents the first molecular characterization of natural dark mutants and the first demonstration of incomplete
lux operons in natural isolates.
Current proposals for the deliberate release of genetically-engineered microorganisms (GEMs) into the open environment have prompted an increased need to understand processes that might lead to transfer, survival, expression and rearrangement of recombinant DNA molecules in microbial communities. Assessment of the impact of releasing large quantities of a specific microorganism is complicated since relatively little information is available concerning the genetic structure of natural populations and gene flux within natural microbial communities. Most information about gene transfer in bacteria comes from medical studies which address a rather specific and possibly atypical subset of the bacterial population. Nevertheless studies on resistance and pathogenicity of plasmids in clinically important bacteria have provided baselines for what might be expected with respect to both the nature and frequency of particular gene transfer events. Much emphasis is currently placed on measuring gene transfer frequencies using a variety of test systems and habitats. The evidence, both direct and indirect, suggests that the vast majority of plasmid transfer events are probably non- creative in an evolutionary sense. However, the penetration of the gene pool of a new species by a particular plasmid is nevertheless likely to be significant if particularly advantageous combinations of genes arise as a consequence.
Many bacteria grow rapidly by binary fission, with cell populations that are usually presumed to be virtually identical to each other except in cell cyclerelated processes or very rare events such as transposition or other DNA rearrangements. Generally, they do not have intracellular compartments. These qualities, along with their simple nutrition and exceptional amenability to a wide range of types of genetic analysis, make them wonderful objects for the investigation of many basic biological questions, for which they may act as models for higher organisms. On the other hand, many important aspects of higher organisms are a consequence of multicellularity and structural complexity. Some bacteria do indeed also show cellular differentiation, the formation of endospores by Bacillus subtilis discussed in the chapter by Errington being the best-known example; and a few are truly multicellular organisms — perhaps most notably the Gramnegative myxobacteria, in which unicellular motile cells cooperate to produce sporulating fruiting bodies, and the Gram-positive actinomycetes, many of which form mycelial colonies on which specialized spore-bearing organs develop. How is such multicellular organisation controlled and coordinated? How great are the genetic changes needed to account for the evolution of multicellular differentiation? Can students of eukaryotic developmental biology learn anything from prokaryotic developmental systems and vice versa? This chapter discusses current knowledge of the developmental biology of the most intensively studied actinomycetes, members of the genus Streptomyces. Much of this work has focused on S. coelicolor A3 (2), which is genetically the most studied species, though some important discoveries have also been made in other species, especially S. griseus and S. antibioticus, which sporulate particularly abundantly.
Bacteria occupy an extremely diverse range of habitats: from deep-sea volcanic vents to arctic soils and from animal organs to air-conditioning systems in modern buildings (Krieg and Holt, 1984). Given the diversity of genotypes within the eubacteria and archaebacteria this ubiquitous distribution is not surprising, but even at the genus level the ability to occupy disparate environments is often encountered. Moreover, this ability extends even to the species level, where, for example, Pseudomonas aeruginosa may be isolated from soil, water, and the lungs of humans compromised by cystic fibrosis (Palleroni, 1984). Evidently, the potential for adaptation within bacterial populations is prodigious. Even among genetically related bacteria, the ability to survive rapid and extensive changes in the environment is striking, reflecting in part the capacity for phenotypic variation or plasticity. Phenotypic plasticity has been defined as the ability of a single genotype to produce more than one alternative form of morphology, physiological state, and/or behavior in response to environmental conditions (West-Eberhard, 1989). Ultimately, however, phenotypic plasticity reflects altered gene expression and it is the essence and subtleties of these genetically determined adaptations which are addressed in this chapter.
Luminous bacteria are those bacteria that carry the lux genes, genes that code for proteins involved in light production. Many luminous bacteria emit light at high, easily visible levels in laboratory culture and in nature, and the phenomenon of light emission has generated interest in these bacteria for over 125 years. Luminous bacteria are especially common in ocean environments where they colonize a variety of habitats, but some species are found in brackish, freshwater, and terrestrial environments. This chapter, which begins with an historical perspective, summarizes current understanding of the biochemistry and genetics of bacterial light emission, the taxonomy and phylogenetics of light-emitting bacteria, the evolutionary origins and hypothesized physiological and ecological functions of bacterial luminescence, the distributions and activities of these bacteria in nature, their symbiotic interactions with animals and especially with marine fishes, and the quorum sensing regulatory circuitry controlling light production at the operon level. This chapter concludes with information on the isolation, cultivation, storage, and identification of luminous bacteria.
Bacterial light production involves enzymes-luciferase, fatty acid reductase, and flavin reductase-and substrates-reduced flavin mononucleotide and long-chain fatty aldehyde-that are specific to bioluminescence in bacteria. The bacterial genes coding for these enzymes, luxA and luxB for the subunits of luciferase; luxC, luxD, and luxE for the components of the fatty acid reductase; and luxG for flavin reductase, are found as an operon in light-emitting bacteria, with the gene order, luxCDABEG. Over 30 species of marine and terrestrial bacteria, which cluster phylogenetically in Aliivibrio, Photobacterium, and Vibrio (Vibrionaceae), Shewanella (Shewanellaceae), and Photorhabdus (Enterobacteriaceae), carry lux operon genes. The luminescence operons of some of these bacteria also contain genes involved in the synthesis of riboflavin, ribEBHA, and in some species, regulatory genes luxI and luxR are associated with the lux operon. In well-studied cases, lux genes are coordinately expressed in a population density-responsive, self-inducing manner called quorum sensing. The evolutionary origins and physiological function of bioluminescence in bacteria are not well understood but are thought to relate to utilization of oxygen as a substrate in the luminescence reaction.
A fusion gene usingluxA andluxB genes ofVibrio species has been designed to express light autonomously in plants.LuxA:luxB was introduced into plants by a high-efficiency transformation system consisting of a high-copy virulence helper plasmid pUCD2614 and T-vector pUCD2715 containingluxA:luxB. The expression ofluxA:luxB fusion gene was optimized by adjusting the spacing between the genes and by placing the translational efficiency of its mRNA under the control of the Ω-3 translational enhancer. The resulting transgenic plants synthesized luciferase at levels greater than 1% of the total leaf protein. These plants produced light autonomously and light intensity was enhanced by the addition of aldehyde. That theluxA:luxB fusion has been optimized enables its use as a reporter for gene activity in plants during development and under various stress-inducing conditions. These results show that a specific protein from an introduced foreign gene can be produced with high efficiency in cultivated plants and such a system is therefore amenable for production of desired proteins through conventional farming methods.
Mutagenesis with transposon mini-Mulac was used to identify loci containing genes for bioluminescence (lux) in the marine bacterium Vibrio harveyi. Transposon insertions which resulted in a Lux- phenotype were mapped to two unlinked regions of the genome. Region I contained the luxCDABE operon which was previously shown to encode the enzymes luciferase and fatty acid reductase, which are required for light production. The other locus, region II, which was identified for the first time in this study, appeared to have a regulatory function. In Northern blot analysis of mRNA from mutants with defects in this region, no transcription from the luxCDABE operon could be detected. Strains with transposon-generated lux::lacZ gene fusions were used to analyze control of the transcription of these regions. Expression of luminescence in the wild type was strongly influenced by the density of the culture, and in strains with the lacZ indicator gene coupled to the luxCDABE operon, beta-galactosidase synthesis was density dependent. So, transcription of this operon is responsive to a density-sensing mechanism. However, beta-galactosidase synthesis in strains with lacZ fused to the region II transcriptional unit did not respond to cell density. The organization and regulation of the lux genes of V. harveyi are discussed, particularly with regard to the contrasts observed with the lux system of the fish light-organ symbiont Vibrio fischeri.
Two reporter systems, lacZY and luxAB, were stably integrated into the chromosome of Pseudomonas aeruginosa UG2, a biosurfactant-producing strain. Growth and rhamnolipid production of the UG2 wild-type and reporter gene-bearing UG2L strains were similar in liquid culture. A spontaneous rifampin-resistant detecting UG2Lr, allowed antibiotic selection. Phenotypic characteristics were compared for usefulness in detecting UG2Lr colonies: morphology, fluorescent pigment production, light emission (lux), X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) cleavage (lac), and rifampin resistance. Survival patterns of UG2, UG2L, and UG2Lr strains were similar in sandy loam soil microcosms over 12 12 weeks. The lac marker was not suitable for monitoring P. aeruginosa UG2Lr in soil since 20 to 42% of cultured, aerobic, heterotrophic soil microorganisms formed blue, lactose-positive colonies. The lux genes provided a stable and unequivocal reporter system, as effective as conventional antibiotic plating, for tracking microorganisms nonselectively at 10(3) CFU/g of soil. Three months after inoculation into oil-contaminated and uncontaminated soil microcosms, UG2Lr cells were recovered at 10(7) and 10(4) cells per g (dry weight) of soil, respectively. Detection by PCR amplification of part of the luxA gene confirmed a decrease in UG2Lr cell numbers in uncontaminated soil. In combination, antibiotic resistance, bioluminescence, and PCR analyses provided sensitive detection and quantitative enumeration of P. aeruginosa UG2Lr in soil.
Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displayed a range of alterations in supernatant protein profiles. CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 microM copper. Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Copper-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype. A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP was isolated. The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) of V. alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes.
Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of an extracellular signal molecule (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode functions necessary for the synthesis of, and response to, a signal molecule. Sequence analysis of the region encoding these functions revealed three open reading frames, two (luxL and luxM) that are required for production of an autoinducer substance and a third (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of two-component, signal transduction proteins. The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal-response system controlling density-dependent expression of luminescence remains to be identified.
Although the study of microbe-host interactions has been traditionally dominated by an interest in pathogenic associations, there is an increasing awareness of the importance of cooperative symbiotic interactions in the biology of many bacteria and their animal and plant hosts. This review examines a model system for the study of such symbioses, the light organ association between the bobtail squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri. Specifically, the initiation, establishment, and persistence of the benign bacterial infection of the juvenile host light organ are described, as are efforts to understand the mechanisms underlying this specific colonization program. Using molecular genetic techniques, mutant strains of V. fischeri have been constructed that are defective at specific stages of the development of the association. Some of the lessons that these mutants have begun to teach us about the complex and long-term nature of this cooperative venture are summarized.
Bioluminescence measured with a luminometer and charge-coupled device was an effective marker in most-probable-number assays for luxAB-marked Pseudomonas aeruginosa UG2Lr in soil. Most-probable-number assays with microtiter plate wells and luminometer tubes gave estimates for UG2Lr that were similar to viable colony counts. Both methods detected five cells per g of soil.
Diversity among 130 strains of Bacillus polymyxa was studied; the bacteria were isolated by immunotrapping from nonrhizosphere soil (32 strains), rhizosphere soil (38 strains), and the rhizoplane (60 strains) of wheat plantlets growing in a growth chamber. The strains were characterized phenotypically by 63 auxanographic (API 50 CHB and API 20B strips) and morphological features, serologically by an enzyme-linked immunosorbent assay, and genetically by restriction fragment length polymorphism (RFLP) profiles of total DNA in combination with hybridization patterns obtained with an rRNA gene probe. Cluster analysis of phenotypic characters by the unweighted pair group method with averages indicated four groups at a similarity level of 93%. Clustering of B. polymyxa strains from the various fractions showed that the strains isolated from nonrhizosphere soil fell into two groups (I and II), while the third group (III) mainly comprised strains isolated from rhizosphere soil. The last group (IV) included strains isolated exclusively from the rhizoplane. Strains belonging to a particular group exhibited a similarity level of 96%. Serological properties revealed a higher variability among strains isolated from nonrhizosphere and rhizosphere soil than among rhizoplane strains. RFLP patterns also revealed a greater genetic diversity among strains isolated from nonrhizosphere and rhizosphere soil and therefore could not be clearly grouped. The RFLP patterns of sorbitol-positive strains isolated from the rhizoplane were identical. These results indicate that diversity within populations of B. polymyxa isolated from nonrhizosphere and rhizosphere soil is higher than that of B. polymyxa isolated from the rhizoplane. It therefore appears that wheat roots may select a specific subpopulation from the soil B. polymyxa population.
The synthesis of the luminous system of the marine luminous bacterium Photobacterium fischeri is subject to a complex, self-regulated control system called autoinduction. The bacteria produce an autoinducer which accumulates in the medium at a constant rate (as a function of cell growth). When autoinducer reaches a critical concentration it stimulates, at the level of transcription, the synthesis of the luminous system. Autoinduction is thus viewed as an environmental sensing mechanism, which curtails the synthesis of the luminous system under dilute conditions. For several isolates of P. fischeri it was found that variations in luminescence intensity could be accounted for by correlated variations in autoinducer production.
It has been previously demonstrated that luciferase synthesis in the luminous marine bacteria, Beneckea harveyi and Photobacterium fischeri is induced only when sufficient concentrations of metabolic products (autoinducers) of these bacteria accumulate in growth media. Thus, when cells are cultured in liquid medium there is a lag in luciferase synthesis. A quantitative bioassay for B. harveyi autoinducer was developed and it was shown that many marine bacteria produce a substance that mimics its action, but in different amounts, (20–130% of the activity produced by B. harveyi) depending on the species and strain. This is referred to as alloinduction. None of the bacteria tested produced detectable quantities of inducer for P. fischeri luciferase synthesis. These findings may have significance with respect to the ecology of B. harveyi and P. fischeri.