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![Gene organization in wild-type and mutant strains of L. biflexa. (A) Genetic organization of bat genes and flanking genes on chromosome II of L. biflexa (not drawn to scale). The corresponding deleted regions in mutant strains are depicted with the respective bat genes replaced by the kanamycin-resistance cassette [13]. (B) Southern blot analysis of L. biflexa strains confirms the absence of the respective bat genes in mutant strains. Genomic DNA for the Southern blot was double-digested with restriction endonucleases NdeI and PstI. Three independently isolated transformants from each mutant were compared to wild-type and hybridized with either a labeled batA fragment or with a labeled fragment spanning batB to batD. The weak signal observed at ~3 kb in the batA mutant strains hybridized with the batA probe is likely due to cross-hybridization with batB. +, purified plasmid DNA from E. coli with a cloned region of L. biflexa DNA containing batABD.](profile/James-Carroll-12/publication/233907938/figure/fig2/AS:214218453327874@1428085067412/Gene-organization-in-wild-type-and-mutant-strains-of-L-biflexa-A-Genetic-organization.png)
Gene organization in wild-type and mutant strains of L. biflexa. (A) Genetic organization of bat genes and flanking genes on chromosome II of L. biflexa (not drawn to scale). The corresponding deleted regions in mutant strains are depicted with the respective bat genes replaced by the kanamycin-resistance cassette [13]. (B) Southern blot analysis of L. biflexa strains confirms the absence of the respective bat genes in mutant strains. Genomic DNA for the Southern blot was double-digested with restriction endonucleases NdeI and PstI. Three independently isolated transformants from each mutant were compared to wild-type and hybridized with either a labeled batA fragment or with a labeled fragment spanning batB to batD. The weak signal observed at ~3 kb in the batA mutant strains hybridized with the batA probe is likely due to cross-hybridization with batB. +, purified plasmid DNA from E. coli with a cloned region of L. biflexa DNA containing batABD.
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Background
Leptospires lack many of the homologs for oxidative defense present in other bacteria, but do encode homologs of the Bacteriodes aerotolerance (Bat) proteins, which have been proposed to fulfill this function. Bat homologs have been identified in all families of the phylum Spirochaetes, yet a specific function for these proteins has not...
Citations
... Prooxidants that include reactive oxygen species (ROS) are produced mainly produced during aerobic respiration (Khaleque et al., 2020;Farías et al., 2021). The ROS causes oxidative damage to cellular components by attacking nucleic acids, cell membranes, and proteins, which causes mutation, protein denaturation, enzyme inactivation, or lipid peroxidation and disrupts intracellular homeostasis (Stewart et al., 2012;Zhang et al., 2012;Ren et al., 2020). The three main ROSs are superoxide (O 2 − ), hydrogen peroxide (H 2 O 2 ), and, hydroxyl radical (OH) which are produced either during microbial metabolic processes or following exposure to physical and chemical agents such as ionizing radiation, desiccation, ultraviolet radiation, and mitomycin (Gao et al., 2020). ...
Release of dye-containing textile wastewater into the environment causes severe pollution with serious consequences on aquatic life. Bioremediation of dyes using thermophilic microorganisms has recently attracted attention over conventional treatment techniques. Thermophiles have the natural ability to survive under extreme environmental conditions, including high dye concentration, because they possess stress response adaptation and regulation mechanisms. Therefore, dye detoxification by thermophiles could offer enormous opportunities for bioremediation at elevated temperatures. In addition, the processes of degradation generate reactive oxygen species (ROS) and subject cells to oxidative stress. However, thermophiles exhibit better adaptation to resist the effects of oxidative stress. Some of the major adaptation mechanisms of thermophiles include macromolecule repair system; enzymes such as superoxide dismutase, catalase, and glutathione peroxidase; and non-enzymatic antioxidants like extracellular polymeric substance (EPSs), polyhydroxyalkanoates (PHAs), etc. In addition, different bacteria also possess enzymes that are directly involved in dye degradation such as azoreductase, laccase, and peroxidase. Therefore, through these processes, dyes are first degraded into smaller intermediate products finally releasing products that are non-toxic or of low toxicity. In this review, we discuss the sources of oxidative stress in thermophiles, the adaptive response of thermophiles to redox stress and their roles in dye removal, and the regulation and crosstalk between responses to oxidative stress.
... The pathogenic and saprophytic BatA and BatB share 50 and 38% similarity, respectively, yet both exhibit conserved VWF A domains and MIDAS motifs. Stewart and colleagues excluded the proposed oxidative stress protection function of the Bat proteins in L. biflexa by employing mutant strains [36]. Hence, the L. interrogans BatA/BatB proteins might likewise not participate in aerotolerance, although experimental validations are required. ...
Leptospirosis is a worldwide spread zoonosis, caused by pathogenic Leptospira. Evidences suggest that compromised hemostasis might be involved in the leptospirosis pathophysiology. In the genome of L. interrogans serovar Copenhageni, we found two genes coding for proteins which comprise von Willebrand factor (VWF) A domains (BatA and BatB). As VWF A domains exhibit multiple binding sites which contributes to human VWF hemostatic functions, we hypothesized that the L. interrogans BatA and BatB proteins could be involved in the hemostatic impairment during leptospirosis. We have cloned, expressed in Escherichia coli, and purified recombinant BatA and BatB. The influence of recombinant BatA and BatB on different in vitro hemostatic assays evaluating the enzymatic activity, platelet aggregation and fibrinogen integrity was investigated. We describe BatB as a new serine protease which is able to cleave thrombin chromogenic substrate, fibrin, fibrinogen, gelatin and casein; while BatA is active only towards fibrinogen. BatA and BatB interfere with the platelet aggregation induced by VWF/ristocetin and thrombin. Our results suggest an important role of the L. interrogans serovar Copenhageni Bat proteins in the hemostasis dysfunction observed during leptospirosis and contribute to the understanding of the leptospirosis pathophysiological mechanisms.
... RNA was purified using Nucleospin kits (Macherey-Nagel Co., Bethlehem, PA) per manufacturer's recommendations, or TRIzol reagent (Invitrogen, Carlsbad, CA). After DNase treatment, 500 ng of RNA was converted to cDNA using High-Capacity cDNA Reverse Transcription kits (Applied Biosystems, Foster City, CA) as previously described (Stewart et al., 2012). The cDNA samples were diluted 1:20 with water and 5 µl of each was used in the subsequent quantitative PCR reactions. ...
When the Lyme disease spirochete, Borrelia burgdorferi, transfers from a feeding tick into a human or other vertebrate host, the bacterium produces vertebrate‐specific proteins and represses factors needed for arthropod colonization. Previous studies determined that the B. burgdorferi BpuR protein binds to its own mRNA and autoregulates its translation, and also serves as co‐repressor of erp transcription. Here we demonstrate that B. burgdorferi controls transcription of bpuR, expressing high levels of bpuR during tick colonization but significantly less during mammalian infection. The master regulator of chromosomal replication, DnaA, was found to bind specifically to a DNA sequence that overlaps the bpuR promoter. Cultured B. burgdorferi that were genetically manipulated to produce elevated levels of BpuR exhibited altered levels of several proteins, although BpuR did not impact mRNA levels. Among these was the SodA superoxide dismutase, which is essential for mammalian infection. BpuR bound to sodA mRNA in live B. burgdorferi, and a specific BpuR‐binding site was mapped 5' of the sodA open reading frame. Recognition of post‐transcriptional regulation of protein levels by BpuR adds another layer to our understanding of the B. burgdorferi regulome, and provides further evidence that bacterial protein levels do not always correlate directly with mRNA levels. This article is protected by copyright. All rights reserved.
... RNA was isolated using the Nucleospin kit (Macherey-Nagel Co., Bethlehem, PA), according to the manufacturer's recommendations, or TRIzol reagent (Invitrogen, Carlsbad, CA). RNA was treated with DNase and converted to cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA), as described previously (44). Three biological replicates (independent pools of ticks), with triplicate technical replicates, were assessed for gene expression by qRT-PCR, as described previously (45). ...
The SpoVG protein of Borrelia burgdorferi , the Lyme disease spirochete, binds to specific sites of DNA and RNA. The bacterium regulates transcription of spoVG during the natural tick-mammal infectious cycle, and in response to some changes in culture conditions. Bacterial levels of spoVG mRNA and SpoVG protein did not necessarily correlate, suggesting that post-transcriptional mechanism(s) also control protein levels. Consistent with this, SpoVG binds to its own mRNA, adjacent to the ribosome-binding site. SpoVG also binds to two DNA sites in the glpFKD operon and to two RNA sites in glpFKD mRNA. That operon encodes genes necessary for glycerol catabolism, and is important for colonization in ticks. In addition, spirochetes engineered to dysregulate spoVG exhibited physiological alterations.
IMPORTANCEB. burgdorferi persists in nature by cycling between ticks and vertebrates. Little is known about how the bacterium senses and adapts to each niche of the cycle. The present studies indicate that B. burgdorferi controls production of SpoVG, and that this protein binds to specific sites of DNA and RNA in the genome and transcriptome, respectively. Altered expression spoVG exerts effects on bacterial replication and other aspects of the spirochete's physiology.
... The presence of the VWFA domain within Borrelial proteins is of note because of the known function of this domain in eukaryotes, playing roles in cell adhesion, particularly regarding interactions with extracellular matrix (ECM) components [22]. Additionally, bb0170 to bb0176 genes are found to have close similarity to a region of the genome of the aerotolerant anaerobe Bacteriodes fragilis, the BatI (Bacteriodes aerotolerance) complex [23]. This conserved genomic region has also been described in Rhizobium leguminosarum and Leptopsira interrogans, although no definite function has been determined [23,24]. ...
... Additionally, bb0170 to bb0176 genes are found to have close similarity to a region of the genome of the aerotolerant anaerobe Bacteriodes fragilis, the BatI (Bacteriodes aerotolerance) complex [23]. This conserved genomic region has also been described in Rhizobium leguminosarum and Leptopsira interrogans, although no definite function has been determined [23,24]. ...
... BatI-like genes have also been noted in other spirochetes, such as Leptospira interrogans and Treponema denticoloa [21,23]. In each of these cases as well as in B. burgdorferi, the VWFA domain containing proteins are found to be associated with a methanol dehydrogenase regulatory (MoxR) ATPase Associated with diverse cellular Activities (AAA). ...
Background
The bacterial spirochete Borrelia burgdorferi is the causative agent of the most commonly reported arthropod-borne illness in the United States, Lyme disease. A family of proteins containing von Willebrand Factor A (VWFA) domains adjacent to a MoxR AAA+ ATPase have been found to be highly conserved in the genus Borrelia. Previously, a VWFA domain containing protein of B. burgdorferi, BB0172, was determined to be an outer membrane protein capable of binding integrin α3β1. In this study, the characterization of a new VWFA domain containing membrane protein, BB0173, is evaluated in order to define the location and topology of this multi-spanning membrane protein. In addition, functional predictions are made.
Results
Our results show that BB0173, in contrast to BB0172, is an inner membrane protein, in which the VWFA domain is exposed to the periplasmic space. Further, BB0173 was predicted to have an aerotolerance regulator domain, and expression of BB0173 and the surrounding genes was evaluated under aerobic and microaerophilic conditions, revealing that these genes are downregulated under aerobic conditions. Since the VWFA domain containing proteins of B. burgdorferi are highly conserved, they are likely required for survival of the pathogen through sensing diverse environmental oxygen conditions.
Conclusions
Presently, the complex mechanisms that B. burgdorferi uses to detect and respond to environmental changes are not completely understood. However, studying the mechanisms that allow B. burgdorferi to survive in the highly disparate environments of the tick vector and mammalian host could allow for the development of novel methods of preventing acquisition, survival, or transmission of the spirochete. In this regard, a putative membrane protein, BB0173, was characterized. BB0173 was found to be highly conserved across pathogenic Borrelia, and additionally contains several truly transmembrane domains, and a Bacteroides aerotolerance-like domain. The presence of these functional domains and the highly conserved nature of this protein, strongly suggests a required function of BB0173 in the survival of B. burgdorferi.
... All proteomic work was repeated with at least 3 independent biological replicates. Soluble and membrane-associated proteins of L. biflexa were separated by mechanical disruption followed by ultracentrifugation, a method frequently used for spirochetes (27)(28)(29)(30). Cells were harvested by centrifugation at 10,000 ϫ g at 23°C for 10 min, and cell pellets were washed with distilled H 2 O and frozen at Ϫ80°C until analyzed. ...
... Four independent L. biflexa cultures were harvested in mid-exponential growth phase for RNA isolation. Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA), as previously described (30). RNA quality was assessed using the 2200 Tapestation (Agilent Technologies, Santa Clara, CA), and 500 ng of RNA was converted to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA), according to the manufacturer's recommendations. ...
... RNA quality was assessed using the 2200 Tapestation (Agilent Technologies, Santa Clara, CA), and 500 ng of RNA was converted to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA), according to the manufacturer's recommendations. The cDNA reactions were diluted 1:10, and 5 l of the dilution was used in each quantitative PCR using the TaqMan Universal PCR master mix kit (Applied Biosystems, Foster City, CA), as described previously (30,31). Primer/probe sets were obtained from Sigma-Aldrich (Table 1). ...
The saprophyte Leptospira biflexa is an excellent model for studying the physiology of this medically important genus, the pathogenic members of which are
more recalcitrant to genetic manipulation and have a significantly longer in vitro growth rate. However, relatively little
is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated
proteins of L. biflexa during exponential growth and in stationary phase. Using these data we have identified abundantly-produced proteins in each
cellular fraction and quantified the transcript levels from a subset of these genes using quantitative RT-PCR. These proteins
should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number
of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several post-translational modifications suggested by the protein patterns. Methylation and
acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation
was detected mainly among soluble proteins. These three post-translational modification systems appear to be conserved between
the free-living L. biflexa and the pathogenic L. interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species.
... The ribosome fraction was separated on a 4 to 20% SDS-polyacrylamide gel and stained with Coomassie brilliant blue. Visible bands were excised and sent to the Protein Chemistry Section of the NIAID Research Technologies Branch, NIH, for mass spectrometry identification using the protocol described previously (59,60). ...
Borrelia burgdorferi, a causative agent of Lyme borreliosis, is a zoonotic pathogen that survives in nutrient-limited environments within a tick,
prior to transmission to its mammalian host. Survival under these prolonged nutrient-limited conditions is thought to be similar
to survival during stationary phase, which is characterized by growth cessation and decreased protein production. Multiple
ribosome-associated proteins are implicated in stationary-phase survival of Escherichia coli. These proteins include hibernation-promoting factor (HPF), which dimerizes ribosomes and prevents translation of mRNA. Bioinformatic
analyses indicate that B. burgdorferi harbors an hpf homolog, the bb0449 gene. BB0449 protein secondary structure modeling also predicted HPF-like structure and function. However, BB0449 protein
was not localized in the ribosome-associated protein fraction of in vitro-grown B. burgdorferi. In wild-type B. burgdorferi, bb0449 transcript and BB0449 protein levels are low during various growth phases. These results are inconsistent with patterns of
synthesis of HPF-like proteins in other bacterial species. In addition, two independently derived bb0449 mutants successfully completed the mouse-tick infectious cycle, indicating that bb0449 is not required for prolonged survival in the nutrient-limited environment in the unfed tick or any other stage of infection
by B. burgdorferi. We suggest either that BB0449 is associated with ribosomes under specific conditions not yet identified or that BB0449 of
B. burgdorferi has a function other than ribosome conformation modulation.
... The supernatant and two washes [5% (vol/vol) formic acid in 50% (vol/vol) acetonitrile] of the gel digests were pooled and concentrated by speed vac (Labconco) to dryness in 200-μL polypropylene auto-sampler vials (Sun Sri). The recovered peptides were resuspended in 5 μL of solvent A [0.1% formic acid, 2% (vol/vol) acetonitrile, and 97.9% (vol/vol) water] and analyzed as previously described (41). ...
Yersinia pestis is the etiological agent of plague, one of the most devastating diseases in human history. Because of the identification of the plague bacillus, the understanding of its epidemiological cycle, and the advent of efficient antibiotic therapies, the death toll due to the plague has dramatically decreased over the last 100 y. However, this disease has never been eradicated, and because of its rodent reservoir, it will not be eradicated in the near future. On the contrary, human plague cases have been reported since the 1990s in countries where the disease was thought to be extinct for decades, and therefore, the plague is now categorized as a reemerging disease. Y. pestis has two characteristics that distinguish it from most other bacterial pathogens. One is its exceptional pathogenicity for humans, with a mortality rate of 40–70% in ∼1 wk for the bubonic form and close to 100% in around 3 d for pneumonic plague. The other characteristic of Y. pestis is its transmission from rodent to rodent and from rodent to human by an infectious fleabite. These two characteristics are probably linked (see below), and therefore deciphering the mechanisms that explain the peculiar mode of transmission of Y. pestis by fleas is a key step in the understanding of pathogen evolution and gain of virulence. In PNAS, Chouikha and Hinnebusch identify a fundamental event in the adaptation of the plague bacillus to its flea vector (1).
... The results were analyzed using Bio-Rad CFX Manager v3.1 software (Bio-Rad). The expression difference was determined using the ⌬⌬C T comparative quantification algorithm with the housekeeping gene flaB3 as a normalizer and the wild-type LEPBIa0072 gene as a calibrator (41). An unpaired t test with Welch's correction was used to determine significant differences in expression. ...
Leptospira spp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyte L. biflexa is a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this
study, we constructed a library of 4,996 random transposon mutants in L. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to
identify genetic determinants that reduce L. biflexa susceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29
genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional
level, these genes could be categorized by function as follows: regulation and signaling (n = 11), transport (n = 6), membrane structure (n = 5), stress response (n = 2), DNA damage repair (n = 1), and other processes (n = 3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component
systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors
to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical
for EtBr resistance. We identified genes required for the growth of L. biflexa in the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants.
This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic
resistance development in pathogenic L. interrogans.
... LB054 to LB057 share strong similarity with BatABCD from the batI operon in Bacteroides fragilis, involved in the aerotolerance of this obligate anaerobe (46). A recent study characterized the batI operon in L. biflexa but found no involvement in oxidative stress (47). Additionally, despite the operon structure surrounding htpG, this gene and lb059, located downstream of batD, are independently transcribed (47). ...
... A recent study characterized the batI operon in L. biflexa but found no involvement in oxidative stress (47). Additionally, despite the operon structure surrounding htpG, this gene and lb059, located downstream of batD, are independently transcribed (47). The last gene in the operon, lb059 encodes a hypothetical protein with only limited similarity to a hypothetical protein in Leptonema illini, Lepil_4068 (28% identity; 55% similarity). ...
Leptospira interrogans is a global zoonotic pathogen and is the causative agent of leptospirosis, an endemic disease of humans and animals worldwide.
There is limited understanding of leptospiral pathogenesis; therefore, further elucidation of the mechanisms involved would
aid in vaccine development and the prevention of infection. HtpG (high-temperature protein G) is the bacterial homolog to the highly conserved molecular chaperone Hsp90 and is important in the stress responses of many
bacteria. The specific role of HtpG, especially in bacterial pathogenesis, remains largely unknown. Through the use of an
L. interrogans htpG transposon insertion mutant, this study demonstrates that L. interrogans HtpG is essential for virulence in the hamster model of acute leptospirosis. Complementation of the htpG mutant completely restored virulence. Surprisingly, the htpG mutant did not appear to show sensitivity to heat or oxidative stress, phenotypes common in htpG mutants in other bacterial species. Furthermore, the mutant did not show increased sensitivity to serum complement, reduced
survival within macrophages, or altered protein or lipopolysaccharide expression. The underlying cause for attenuation thus
remains unknown, but HtpG is a novel leptospiral virulence factor and one of only a very small number identified to date.
