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The authors discuss molecular mechanism to control bacterial motility. Results indicate that crp and hns mutants are nonmotile because of lack of flagellin accumulation in Escherichia coli.

Abstract Background H-NS regulates the acid stress resistance. The present study aimed to characterize the H-NS-dependent cascade governing the acid stress resistance pathways and to define the interplay between the different regulators. Results We combined mutational, phenotypic and gene expression analyses, to unravel the regulatory hierarchy in acid resistance involving H-NS, RcsB-P/GadE complex, HdfR, CadC, AdiY regulators, and DNA-binding assays to separate direct effects from indirect ones. RcsB-P/GadE regulatory complex, the general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways plays a central role in the regulatory cascade. However, H-NS also directly controls specific regulators of these pathways (e.g. cadC) and genes involved in general stress resistance (hdeAB, hdeD, dps, adiY). Finally, we found that in addition to H-NS and RcsB, a third regulator, HdfR, inversely controls glutamate-dependent acid resistance pathway and motility. Conclusions H-NS lies near the top of the hierarchy orchestrating acid response centred on RcsB-P/GadE regulatory complex, the general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways.

Importance: The RavA-ViaA complex was previously found to sensitize Escherichia coli to aminoglycosides (AGs) in anaerobic conditions, but the mechanism is unknown. AGs are antibiotics known for their high efficiency against Gram-negative bacteria. In order to elucidate how the expression of the ravA-viaA genes increases bacterial susceptibility to aminoglycosides, we aimed at identifying partner functions necessary for increased tolerance in the absence of RavA-ViaA, in Vibrio cholerae . We show that membrane stress response systems Cpx and Zra2 are required in the absence of RavA-ViaA, for the tolerance to AGs and for outer membrane integrity. In the absence of these systems, the ∆ravvia strain's membrane becomes permeable to external agents such as the antibiotic vancomycin., Competing Interests: The authors declare no conflict of interest.

Vibrio cholerae N16961 genome encodes 18 type II Toxin/Antitoxin (TA) systems, all but one located inside gene cassettes of its chromosomal superintegron (SI). This study aims to investigate additional TA systems in this genome. We screened for all two-genes operons of uncharacterized function by analyzing previous RNAseq data. Assays on nine candidates, revealed one additional functional type II TA encoded by the VCA0497-0498 operon, carried inside a SI cassette. We showed that VCA0498 antitoxin alone and in complex with VCA0497 represses its own operon promoter. VCA0497-0498 is the second element of the recently identified dhiT/dhiA superfamily uncharacterized type II TA system. RNAseq analysis revealed that another SI cassette encodes a novel type I TA system: VCA0495 gene and its two associated antisense non-coding RNAs, ncRNA495 and ncRNA496. Silencing of both antisense ncRNAs lead to cell death, demonstrating the type I TA function. Both VCA0497 and VCA0495 toxins do not show any homology to functionally characterized toxins, however our preliminary data suggest that their activity may end up in mRNA degradation, directly or indirectly. Our findings increase the TA systems number carried in this SI to 19, preferentially located in its distal end, confirming their importance in this large cassette array., Competing Interests: Declaration of competing interest None., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Antimicrobial resistance develops as a major problem in infectious diseases treatment. While antibiotic resistance mechanisms are usually studied using lethal antibiotic doses, lower doses allowing bacterial growth are now considered as factors influencing the development and selection of resistance. Starting with a high-density Tn insertion library in Vibrio cholerae and following its evolution by TN-seq in the presence of subinhibitory concentrations of antibiotics, we discovered that RNA modification genes can have opposite fates, being selected or counter-selected. We, thus have undertaken the phenotypic characterization of 23 transfer RNA (tRNA) and ribosomal RNA (rRNA) modifications deletion mutants, for which growth is globally not affected in the absence of stress. We uncover a specific involvement of different RNA modification genes in the response to aminoglycosides (tobramycin and gentamicin), fluoroquinolones (ciprofloxacin), β-lactams (carbenicillin), chloramphenicol, and trimethoprim. Our results identify t/rRNA modification genes, not previously associated to any antibiotic resistance phenotype, as important factors affecting the bacterial response to low doses of antibiotics from different families. This suggests differential translation and codon decoding as critical factors involved in the bacterial response to stress., Competing Interests: None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Antibiotics are well known drugs which, when present above certain concentrations, are able to inhibit the growth of certain bacteria. However, a growing body of evidence shows that even when present at lower doses (subMIC, for sub-minimal inhibitory concentration), unable to inhibit or affect microbial growth, antibiotics work as signaling molecules, affect gene expression and trigger important bacterial stress responses. However, how subMIC antibiotic signaling interplays with other well-known signaling networks in bacteria (and the consequences of such interplay) is not well understood. In this work, through transcriptomic and genetic approaches, we have explored how quorum-sensing (QS) proficiency of V. cholerae affects this pathogen's response to subMIC doses of the aminoglycoside tobramycin (TOB). We show that the transcriptomic signature of V. cholerae in response to subMIC TOB depends highly on the presence of QS master regulator HapR. In parallel, we show that subMIC doses of TOB are able to negatively interfere with the AI-2/LuxS QS network of V. cholerae , which seems critical for survival to aminoglycoside treatment and TOB-mediated induction of SOS response in this species. This interplay between QS and aminoglycosides suggests that targeting QS signaling may be a strategy to enhance aminoglycoside efficacy in V. cholerae .

Indole is a molecule proposed to be involved in bacterial signaling. We find that indole secretion is induced by sublethal tobramycin concentrations and increases persistence to aminoglycosides in V. cholerae . Indole transcriptomics showed increased expression of raiA , a ribosome associated factor. Deletion of raiA abolishes the appearance of indole dependent persisters to aminoglycosides, although its overexpression leads to 100-fold increase of persisters, and a reduction in lag phase, evocative of increased active 70S ribosome content, confirmed by sucrose gradient analysis. We propose that, under stress conditions, RaiA-bound inactive 70S ribosomes are stored as "sleeping ribosomes", and are rapidly reactivated upon stress relief. Our results point to an active process of persister formation through ribosome protection during translational stress (e.g., aminoglycoside treatment) and reactivation upon antibiotic removal. Translation is a universal process, and these results could help elucidate a mechanism of persistence formation in a controlled, thus inducible way., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Background: In fast-growing bacteria, the genomic location of ribosomal protein (RP) genes is biased towards the replication origin (oriC). This trait allows optimizing their expression during exponential phase since oriC neighboring regions are in higher dose due to multifork replication. Relocation of s10-spc-α locus (S10), which codes for most of the RP, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction on its dosage, its expression, and bacterial growth rate. However, a mechanism linking S10 dosage to cell physiology has still not been determined., Results: We hypothesized that S10 dosage perturbations impact protein synthesis capacity. Strikingly, we observed that in Vibrio cholerae, protein production capacity was independent of S10 position. Deep sequencing revealed that S10 relocation altered chromosomal replication dynamics and genome-wide transcription. Such changes increased as a function of oriC-S10 distance. Since RP constitutes a large proportion of cell mass, lower S10 dosage could lead to changes in macromolecular crowding, impacting cell physiology. Accordingly, cytoplasm fluidity was higher in mutants where S10 is most distant from oriC. In hyperosmotic conditions, when crowding differences are minimized, the growth rate and replication dynamics were highly alleviated in these strains., Conclusions: The genomic location of RP genes ensures its optimal dosage. However, besides of its essential function in translation, their genomic position sustains an optimal macromolecular crowding essential for maximizing growth. Hence, this could be another mechanism coordinating DNA replication to bacterial growth.

We have previously identified Vibrio cholerae mutants in which the stress response to subinhibitory concentrations of aminoglycoside is altered. One gene identified, VC1636, encodes a putative DNA/RNA helicase, recently named RadD in Escherichia coli Here we combined extensive genetic characterization and high-throughput approaches in order to identify partners and molecular mechanisms involving RadD. We show that double-strand DNA breaks (DSBs) are formed upon subinhibitory tobramycin treatment in the absence of radD and recBCD and that formation of these DSBs can be overcome by RNase H1 overexpression. Loss of RNase H1, or of the transcription-translation coupling factor EF-P, is lethal in the radD deletion mutant. We propose that R-loops are formed upon sublethal aminoglycoside treatment, leading to the formation of DSBs that can be repaired by the RecBCD homologous recombination pathway, and that RadD counteracts such R-loop accumulation. We discuss how R-loops that can occur upon translation-transcription uncoupling could be the link between tobramycin treatment and DNA break formation. IMPORTANCE Bacteria frequently encounter low concentrations of antibiotics. Active antibiotics are commonly detected in soil and water at concentrations much below lethal concentration. Although sub-MICs of antibiotics do not kill bacteria, they can have a major impact on bacterial populations by contributing to the development of antibiotic resistance through mutations in originally sensitive bacteria or acquisition of DNA from resistant bacteria. It was shown that concentrations as low as 100-fold below the MIC can actually lead to the selection of antibiotic-resistant cells. We seek to understand how bacterial cells react to such antibiotic concentrations using E. coli , the Gram-negative bacterial paradigm, and V. cholerae , the causative agent of cholera. Our findings shed light on the processes triggered at the DNA level by antibiotics targeting translation, how damage occurs, and what the bacterial strategies are to respond to such DNA damage., (Copyright © 2019 Negro et al.)

Background: The SOS response is an almost ubiquitous response of cells to genotoxic stresses. The full complement of genes in the SOS regulon for Vibrio species has only been addressed through bioinformatic analyses predicting LexA binding box consensus and in vitro validation. Here, we perform whole transcriptome sequencing from Vibrio cholerae treated with mitomycin C as an SOS inducer to characterize the SOS regulon and other pathways affected by this treatment., Results: Comprehensive transcriptional profiling allowed us to define the full landscape of promoters and transcripts active in V. cholerae. We performed extensive transcription start site (TSS) mapping as well as detection/quantification of the coding and non-coding RNA (ncRNA) repertoire in strain N16961. To improve TSS detection, we developed a new technique to treat RNA extracted from cells grown in various conditions. This allowed for identification of 3078 TSSs with an average 5'UTR of 116 nucleotides, and peak distribution between 16 and 64 nucleotides; as well as 629 ncRNAs. Mitomycin C treatment induced transcription of 737 genes and 28 ncRNAs at least 2 fold, while it repressed 231 genes and 17 ncRNAs. Data analysis revealed that in addition to the core genes known to integrate the SOS regulon, several metabolic pathways were induced. This study allowed for expansion of the Vibrio SOS regulon, as twelve genes (ubiEJB, tatABC, smpA, cep, VC0091, VC1190, VC1369-1370) were found to be co-induced with their adjacent canonical SOS regulon gene(s), through transcriptional read-through. Characterization of UV and mitomycin C susceptibility for mutants of these newly identified SOS regulon genes and other highly induced genes and ncRNAs confirmed their role in DNA damage rescue and protection., Conclusions: We show that genotoxic stress induces a pervasive transcriptional response, affecting almost 20% of the V. cholerae genes. We also demonstrate that the SOS regulon is larger than previously known, and its syntenic organization is conserved among Vibrio species. Furthermore, this specific co-localization is found in other γ-proteobacteria for genes recN-smpA and rmuC-tatABC, suggesting SOS regulon conservation in this phylum. Finally, we comment on the limitations of widespread NGS approaches for identification of all RNA species in bacteria.

Unlabelled: The role of chromosomal toxin-antitoxin (TA) systems, which are ubiquitous within the genomes of free-living bacteria, is still debated. We have scanned the Vibrio cholerae N16961 genome for class 2 TA genes and identified 18 gene pair candidates. Interestingly, all but one are located in the chromosome 2 superintegron (SI). The single TA found outside the SI is located on chromosome 1 and is related to the well-characterized HipAB family, which is known to play a role in antibiotic persistence. We investigated this clustering within the SI and its possible biological consequences by performing a comprehensive functional analysis on all of the putative TA systems. We demonstrate that the 18 TAs identified encode functional toxins and that their cognate antitoxins are able to neutralize their deleterious effects when expressed in Escherichia coli. In addition, we reveal that the 17 predicted TA systems of the SI are transcribed and expressed in their native context from their own promoters, a situation rarely found in integron cassettes. We tested the possibility of interactions between noncognate pairs of all toxins and antitoxins and found no cross-interaction between any of the different TAs. Although these observations do not exclude other roles, they clearly strengthen the role of TA systems in stabilizing the massive SI cassette array of V. cholerae., Importance: The chromosomal toxin-antitoxin systems have been shown to play various, sometimes contradictory roles, ranging from genomic stabilization to bacterial survival via persistence. Determining the interactions between TA systems hosted within the same bacteria is essential to understand the hierarchy between these different roles. We identify here the full set of class 2 TAs carried in the Vibrio cholerae N16961 genome and found they are all, with a single exception, located in the chromosome 2 superintegron. Their characterization, in terms of functionality, expression, and possible cross-interactions, supports their main role as being the stabilization of the 176-cassette-long array of the superintegron but does not exclude dual roles, such as stress response elements, persistence, and bacteriophage defense through abortive infection mechanisms., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Chromosome 2 of Vibrio cholerae carries a chromosomal superintegron, composed of an integrase, a cassette integration site (attI) and an array of mostly promoterless gene cassettes. We determined the precise location of the promoter, Pc, which drives the transcription of the first cassettes of the V. cholerae superintegron. We found that cassette mRNA starts 65 bp upstream of the attI site, so that the inversely oriented promoters Pc and Pint (integrase promoter) partly overlap, allowing for their potential co-regulation. Pint was previously shown to be induced during the SOS response and is further controlled by the catabolite repression cAMP-CRP complex. We found that cassette expression from Pc was also controlled by the cAMP-CRP complex, but is not part of the SOS regulon. Pint and Pc promoters were both found to be induced in rich medium, at high temperature, high salinity and at the end of exponential growth phase, although at very different levels and independently of sigma factor RpoS. All these results show that expression from the integrase and cassette promoters can take place at the same time, thus leading to coordinated excisions and integrations within the superintegron and potentially coupling cassette shuffling to immediate selective advantage.

Vibrio nigripulchritudo is an emerging pathogen of farmed shrimp in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have suggested that pathogenicity is linked to particular lineages. Here, we performed high-throughput sequencing-based comparative genome analysis of 16 V. nigripulchritudo strains to explore the genomic diversity and evolutionary history of pathogen-containing lineages and to identify pathogen-specific genetic elements. Our phylogenetic analysis revealed three pathogen-containing V. nigripulchritudo clades, including two clades previously identified from New Caledonia and one novel clade comprising putatively pathogenic isolates from septicemic shrimp in Madagascar. The similar genetic distance between the three clades indicates that they have diverged from an ancestral population roughly at the same time and recombination analysis indicates that these genomes have, in the past, shared a common gene pool and exchanged genes. As each contemporary lineage is comprised of nearly identical strains, comparative genomics allowed differentiation of genetic elements specific to shrimp pathogenesis of varying severity. Notably, only a large plasmid present in all highly pathogenic (HP) strains encodes a toxin. Although less/non-pathogenic strains contain related plasmids, these are differentiated by a putative toxin locus. Expression of this gene by a non-pathogenic V. nigripulchritudo strain resulted in production of toxic culture supernatant, normally an exclusive feature of HP strains. Thus, this protein, here termed 'nigritoxin', is implicated to an extent that remains to be precisely determined in the toxicity of V. nigripulchritudo.

Bacteria encounter sub-inhibitory concentrations of antibiotics in various niches, where these low doses play a key role for antibiotic resistance selection. However, the physiological effects of these sub-lethal concentrations and their observed connection to the cellular mechanisms generating genetic diversification are still poorly understood. It is known that, unlike for the model bacterium Escherichia coli, sub-minimal inhibitory concentrations (sub-MIC) of aminoglycosides (AGs) induce the SOS response in Vibrio cholerae. SOS is induced upon DNA damage, and since AGs do not directly target DNA, we addressed two issues in this study: how sub-MIC AGs induce SOS in V. cholerae and why they do not do so in E. coli. We found that when bacteria are grown with tobramycin at a concentration 100-fold below the MIC, intracellular reactive oxygen species strongly increase in V. cholerae but not in E. coli. Using flow cytometry and gfp fusions with the SOS regulated promoter of intIA, we followed AG-dependent SOS induction. Testing the different mutation repair pathways, we found that over-expression of the base excision repair (BER) pathway protein MutY relieved this SOS induction in V. cholerae, suggesting a role for oxidized guanine in AG-mediated indirect DNA damage. As a corollary, we established that a BER pathway deficient E. coli strain induces SOS in response to sub-MIC AGs. We finally demonstrate that the RpoS general stress regulator prevents oxidative stress-mediated DNA damage formation in E. coli. We further show that AG-mediated SOS induction is conserved among the distantly related Gram negative pathogens Klebsiella pneumoniae and Photorhabdus luminescens, suggesting that E. coli is more of an exception than a paradigm for the physiological response to antibiotics sub-MIC., Competing Interests: The authors have declared that no competing interests exist.

The human pathogen Vibrio cholerae carries a chromosomal superintegron (SI). The SI contains an array of hundreds of gene cassettes organized in tandem which are stable under conditions when no particular stress is applied to bacteria (such as during laboratory growth). Rearrangements of these cassettes are catalyzed by the activity of the associated integron integrase. Understanding the regulation of integrase expression is pivotal to fully comprehending the role played by this genetic reservoir for bacterial adaptation and its connection with the development of antibiotic resistance. Our previous work established that the integrase is regulated by the bacterial SOS response and that it is induced during bacterial conjugation. Here, we show that transformation, another horizontal gene transfer (HGT) mechanism, also triggers integrase expression through SOS induction, underlining the importance of HGT in genome plasticity. Moreover, we report a new cyclic AMP (cAMP)-cAMP receptor protein (CRP)-dependent regulation mechanism of the integrase, highlighting the influence of the extracellular environment on chromosomal gene content. Altogether, our data suggest an interplay between different stress responses and regulatory pathways for the modulation of the recombinase expression, thus showing how the SI remodeling mechanism is merged into bacterial physiology.

Background: H-NS regulates the acid stress resistance. The present study aimed to characterize the H-NS-dependent cascade governing the acid stress resistance pathways and to define the interplay between the different regulators., Results: We combined mutational, phenotypic and gene expression analyses, to unravel the regulatory hierarchy in acid resistance involving H-NS, RcsB-P/GadE complex, HdfR, CadC, AdiY regulators, and DNA-binding assays to separate direct effects from indirect ones. RcsB-P/GadE regulatory complex, the general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways plays a central role in the regulatory cascade. However, H-NS also directly controls specific regulators of these pathways (e.g. cadC) and genes involved in general stress resistance (hdeAB, hdeD, dps, adiY). Finally, we found that in addition to H-NS and RcsB, a third regulator, HdfR, inversely controls glutamate-dependent acid resistance pathway and motility., Conclusions: H-NS lies near the top of the hierarchy orchestrating acid response centred on RcsB-P/GadE regulatory complex, the general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways.

In Escherichia coli, hns mutants lack flagellar motility and display an increase in acid stress resistance. Spontaneous phenotypic revertants showed reversion of both H-NS-controlled phenotypes. In the present study, suppressor mutations were identified in the rcsB gene. In addition to RcsA, our experiments establish that H-NS indirectly controlled the RcsB regulator via repression of RcsD. We also show that RcsB(D56E), mimicking phosphorylated RcsB, interacts with GadE to form a RcsB-P/GadE complex, a general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways. In addition, we showed that H-NS positively affects motility via the flhDC master operon repression by RcsB. This substantiates the central role of RcsB in H-NS-mediated control of motility and acid stress resistance., (2010 Elsevier Masson SAS. All rights reserved.)

Bacteria of the Thiomonas genus are ubiquitous in extreme environments, such as arsenic-rich acid mine drainage (AMD). The genome of one of these strains, Thiomonas sp. 3As, was sequenced, annotated, and examined, revealing specific adaptations allowing this bacterium to survive and grow in its highly toxic environment. In order to explore genomic diversity as well as genetic evolution in Thiomonas spp., a comparative genomic hybridization (CGH) approach was used on eight different strains of the Thiomonas genus, including five strains of the same species. Our results suggest that the Thiomonas genome has evolved through the gain or loss of genomic islands and that this evolution is influenced by the specific environmental conditions in which the strains live., Competing Interests: The authors have declared that no competing interests exist.

We report global expression profiling of a uvrY-deficient mutant of Photorhabdus luminescens. We found that the regulator moiety of the two-component regulatory system BarA/UvrY regulated more than 500 target genes coding for functions involved in the synthesis of major compartments and metabolic pathways of the cell. This regulation appeared to be in part indirect as UvrY affected the expression of several regulators. Indeed, the flagellum biosynthesis transcription activator FlhC and the flagella regulon were induced in the absence of UvrY, leading to a hyperflagellated phenotype and an increase in motility and biofilm formation. Two major regulatory systems were also altered: the type 2 quorum-sensing inducer AI-2 was activated by UvrY, and the CsrA regulator function appeared to be repressed by the increase of the small-untranslated RNA csrB, the CsrA activity inhibitor TldD and the chaperonin GroESL. Both through and independently of these systems, UvrY regulated oxidative stress resistance; bioluminescence; iron, sugar and peptide transport; proteases; polyketide synthesis enzymes and nucleobases recycling, related to insect degradation and assimilation by bacteria. As a consequence, the uvrY-deficient strain exhibited a decreased killing of insect cells and a reduced growth on insect cells culture, suggesting a UvrY role in the adaptation of P. luminescens inside the insect.

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments-including ground and surface waters-from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the beta-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Bacterial virulence is an integrative process that may involve quorum sensing. In this work, we compared by global expression profiling the wild-type entomopathogenic Photorhabdus luminescens subsp. laumondii TT01 to a luxS-deficient mutant unable to synthesize the type 2 quorum-sensing inducer AI-2. AI-2 was shown to regulate more than 300 targets involved in most compartments and metabolic pathways of the cell. AI-2 is located high in the hierarchy, as it controls the expression of several transcriptional regulators. The regulatory effect of AI-2 appeared to be dose dependent. The luxS-deficient strain exhibited decreased biofilm formation and increased type IV/V pilus-dependent twitching motility. AI-2 activated its own synthesis and transport. It also modulated bioluminescence by regulating the synthesis of spermidine. AI-2 was further shown to increase oxidative stress resistance, which is necessary to overcome part of the innate immune response of the host insect involving reactive oxygen species. Finally, we showed that the luxS-deficient strain had attenuated virulence against the lepidopteran Spodoptera littoralis. We concluded that AI-2 is involved mainly in early steps of insect invasion in P. luminescens.

Cell-free supernatants from growing Bacillus cereus strain ATCC 10987 induced luminescence in a Photorhabdus luminescens DeltaluxS mutant, indicating the production of functional autoinducer 2 (AI-2). The exogenous addition of in vitro synthesized AI-2 had an inhibitory effect on biofilm formation by B. cereus and promoted release of the cells from a preformed biofilm.

A considerable fraction of life develops in the sea at temperatures lower than 15 degrees C. Little is known about the adaptive features selected under those conditions. We present the analysis of the genome sequence of the fast growing Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. We find that it copes with the increased solubility of oxygen at low temperature by multiplying dioxygen scavenging while deleting whole pathways producing reactive oxygen species. Dioxygen-consuming lipid desaturases achieve both protection against oxygen and synthesis of lipids making the membrane fluid. A remarkable strategy for avoidance of reactive oxygen species generation is developed by P. haloplanktis, with elimination of the ubiquitous molybdopterin-dependent metabolism. The P. haloplanktis proteome reveals a concerted amino acid usage bias specific to psychrophiles, consistently appearing apt to accommodate asparagine, a residue prone to make proteins age. Adding to its originality, P. haloplanktis further differs from its marine counterparts with recruitment of a plasmid origin of replication for its second chromosome.

The nucleoid-associated protein H-NS is thought to play an essential role in the organization of bacterial chromatin in Escherichia coli. Homologues, often with very low sequence identity, are found in most gram-negative bacteria. Microscopic analysis reveals that, despite limited sequence identity, their structural organization results in similar DNA binding properties.

In several Gram-positive and Gram-negative bacteria glutamate decarboxylases play an important role in the maintenance of cellular homeostasis in acid environments. Here, new insight is brought to the regulation of the acid response in Escherichia coli. Overexpression of yhiE, similarly to overexpression of gadX, a known regulator of glutamate decarboxylase expression, leads to increased resistance of E. coli strains under high acid conditions, suggesting that YhiE is a regulator of gene expression in the acid response. Target genes of both YhiE (renamed GadE) and GadX were identified by a transcriptomic approach. In vitro experiments with GadE purified protein provided evidence that this regulator binds to the promoter region of these target genes. Several of them are clustered together on the chromosome and this chromosomal organization is conserved in many E. coli strains. Detailed structural (in silico) analysis of this chromosomal region suggests that the promoters of the corresponding genes are preferentially denatured. These results, along with the G+C signature of the chromosomal region, support the existence of a fitness island for acid adaptation on the E. coli chromosome.

We describe here new members of the H-NS protein family identified in a psychrotrophic Acinetobacter spp. bacterium collected in Siberia and in a psychrophilic Psychrobacter spp. bacterium collected in Antarctica. Both are phylogenetically closely related to the HvrA and SPB Rhodobacter transcriptional regulators. Their amino acid sequence shares 40% identity, and their predicted secondary structure displays a structural and functional organization in two modules similar to that of H-NS in Escherichia coli. Remarkably, the Acinetobacter protein fully restores to the wild-type H-NS-dependent phenotypes, whereas the Psychrobacter protein is no longer able to reverse the effects of H-NS deficiency in an E. coli mutant strain above 30 degrees C. Moreover, in vitro experiments demonstrate that the ability of the Psychrobacter H-NS protein to bind curved DNA and to form dimers is altered at 37 degrees C. The construction of hybrid proteins containing the N- or the C-terminal part of E. coli H-NS fused to the C- or N-terminal part of the Psychrobacter protein demonstrates the role of the N-terminal domain in this process. Finally, circular dichroism analysis of purified H-NS proteins suggests that, as compared with the E. coli and Acinetobacter proteins, the alpha-helical domain displays weaker intermolecular interactions in the Psychrobacter protein, which may account for the low thermal stability observed at 37 degrees C.

The cAMP-catabolite activator protein (CAP) complex is a pleiotropic regulator that regulates a vast number of Escherichia coli genes, including those involved in carbon metabolism. We identified two new targets of this complex: argG, which encodes the arginosuccinate synthase involved in the arginine biosynthetic pathway, and metY, which encodes one of the two methionine tRNA initiators, tRNAf2Met. The cAMP-CAP complex activates argG transcription and inhibits metY transcription from the same DNA position. We also show that ArgR, the specific repressor of the arginine biosynthetic pathway, together with its arginine cofactor, acts on the regulation of metY mediated by CAP. The regulation of the two divergent promoters is thus simultaneously controlled not only by the cAMP-CAP complex, a global regulator, but also by a specific regulator of arginine metabolism, suggesting a previously unsuspected link between carbon metabolism and translation initiation.

In this paper, we initiated the first two-dimensional electrophoresis map of Vibrio cholerae, the aetiological agent of cholera disease. In this pathogen the efficient adaptation to detrimental conditions plays an important role in its survival in both the aquatic reservoir and human intestine. By proteome analysis we investigated the effect of mild acid treatment on the physiology of V. cholerae. More than 50 proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database searching. Amongst them, pH regulated proteins belong to various functional classes such as intermediary metabolism and bacterial envelope. Several proteins whose accumulation level was decreased in response to acidic pH are known to be involved in the organization and the functioning of membranes, including lipopolysaccharide. Consistent with this, we observed an increased susceptibility to hydrophobic drugs, a loss of motility and a reduction in the ability to form a biofilm in cells grown at pH 6. Our results suggest that V. cholerae is able to sense a moderate decrease in pH and to modify accordingly its structure and physiology.

The effect of detrimental conditions on bacterial motility in Escherichia coli was investigated. Expression profiling of mutant E. coli strains by DNA arrays and analysis of phenotypic traits demonstrated that motility and low-pH resistance are coordinately regulated. Analysis of transcriptional fusions suggests that bacterial motility in response to an acidic environment is mediated via the control by H-NS of flhDC expression. Moreover, the results suggested that the presence of an extended mRNA 5' end and DNA topology are required in this process. Finally, the presence of a similar regulatory region in several Gram-negative bacteria implies that this mechanism is largely conserved.

During the last few years, several genes, such as pap, bgl and flhDC, have been shown to be coregulated by the histone-like nucleoid-structuring (H-NS) protein and the cyclic AMP-catabolite activator protein (cAMP/CAP) complex, suggesting an interaction between both systems in the control of some cellular functions. In this study, the possible effect of H-NS on the cAMP level was investigated. In a CAP-deficient strain, the presence of an hns mutation results in a strong reduction in the amount of cAMP, due to a decrease in adenylate cyclase activity. This is caused by the reduced expression of crr, which encodes the Enzyme IIA(Glc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), from its specific P2 promoter. This leads to a twofold reduction in the global amount of Enzyme IIA(Glc), the adenylate cyclase activator, responsible for the decrease in adenylate cyclase activity observed in the hns crp strain.

We describe a rapid method for determining nucleotide sequences directly from total genomic DNA. This technique was used to determine genomic DNA sequences in various prokaryotic and eukaryotic microorganisms with a G+C content between 40 and 50%, e.g. Escherichia coli, Vibrio cholerae, Bacillus subtilis and Saccharomyces cerevisiae. Furthermore, the method was applied to accurately sequence up to 300 DNA base pairs in Photorhabdus luminescens, whose genome sequencing is currently under way. Taken together, these results provide evidence that our technique can be widely used to easily and efficiently determine genomic DNA sequences.

Despite many years of intense work investigating the function of nucleoid-associated proteins in prokaryotes, their role in bacterial physiology remains largely unknown. The two-dimensional protein patterns were compared and expression profiling was carried out on H-NS-deficient and wild-type strains of Escherichia coli K-12. The expression of approximately 5% of the genes and/or the accumulation of their protein was directly or indirectly altered in the hns mutant strain. About one-fifth of these genes encode proteins that are involved in transcription or translation and one-third are known to or were in silico predicted to encode cell envelope components or proteins that are usually involved in bacterial adaptation to changes in environmental conditions. The increased expression of several genes in the mutant resulted in a better ability of this strain to survive at low pH and high osmolarity than the wild-type strain. In particular, the putative regulator, YhiX, plays a central role in the H-NS control of genes required in the glutamate-dependent acid stress response. These results suggest that there is a strong relationship between the H-NS regulon and the maintenance of intracellular homeostasis.

During the last decade, the hns gene and its product, the H-NS protein, have been extensively studied in Escherichia coli. H-NS-like proteins seem to be widespread in gram-negative bacteria. However, unlike in E. coli and in Salmonella enterica serovar Typhimurium, little is known about their role in the physiology of those organisms. In this report, we describe the isolation of vicH, an hns-like gene in Vibrio cholerae, the etiological agent of cholera. This gene was isolated from a V. cholerae genomic library by complementation of different phenotypes associated with an hns mutation in E. coli. It encodes a 135-amino-acid protein showing approximately 50% identity with both H-NS and StpA in E. coli. Despite a low amino acid conservation in the N-terminal part, VicH is able to cross-react with anti-H-NS antibodies and to form oligomers in vitro. The vicH gene is expressed as a single gene from two promoters in tandem and is induced by cold shock. A V. cholerae wild-type strain expressing a vicHDelta92 gene lacking its 3' end shows pleiotropic alterations with regard to mucoidy and salicin metabolism. Moreover, this strain is unable to swarm on semisolid medium. Similarly, overexpression of the vicH wild-type gene results in an alteration of swarming behavior. This suggests that VicH could be involved in the virulence process in V. cholerae, in particular by affecting flagellum biosynthesis.

The structural gene of the H-NS protein, a global regulator of bacterial metabolism, has been identified in the group of enterobacteria as well as in closely related bacteria, such as Erwinia chrysanthemi and Haemophilus influenzae. Isolated outside these groups, the BpH3 protein of Bordetella pertussis exhibits a low amino acid conservation with H-NS, particularly in the N-terminal domain. To obtain information on the structure, function and/or evolution of H-NS, we searched for other H-NS-related proteins in the latest databases. We found that HvrA, a trans-activator protein in Rhodobacter capsulatus, has a low but significant similarity with H-NS and H-NS-like proteins. This Gram-negative bacterium is phylogenetically distant from Escherichia coli. Using theoretical analysis (e.g. secondary structure prediction and DNA binding domain modelling) of the amino acid sequence of H-NS, StpA (an H-NS-like protein in E. coli), BpH3 and HvrA and by in vivo and in vitro experiments (e.g. complementation of various H-NS-related phenotypes and competitive gel shift assay), we present evidence that these proteins belong to the same class of DNA binding proteins. In silico analysis suggests that this family also includes SPB in R. sphaeroides, XrvA in Xanthomonas oryzae and VicH in Vibrio cholerae. These results demonstrate that proteins structurally and functionally related to H-NS are widespread in Gram-negative bacteria.

The pyrH gene, encoding UMP-kinase from Escherichia coli, was cloned using as a genetic probe the property of the carAB operon to be controlled for its expression by the concentration of cytoplasmic UTP. The open reading frame of the pyrH gene of 723 bp was found to be identical to that of the smbA gene [Yamanaka, K., et al. (1992) J. Bacteriol. 174, 7517-7526], previously described as being involved in chromosome partitioning in E. coli. The bacterial UMP-kinase did not display significant sequence similarity to known nucleoside monophosphate kinases. On the contrary, it exhibited similarity with three families of enzymes including aspartokinases, glutamate kinases, and Pseudomonas aeruginosa carbamate kinase. UMP-kinase overproduced in E. coli was purified to homogeneity and analyzed for its structural and catalytic properties. The protein consists of six identical subunits, each of 240 amino acid residues (the N-terminal methionine residue is missing in the expressed protein). Upon excitation at 295 nm, the bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 332 nm which indicates that the single tryptophan residue of the protein (Trp119) is located in a hydrophobic environment. Like other enzymes involved in the de novo synthesis of pyrimidine nucleotides, UMP-kinase of E. coli is subject to regulation by nucleotides: GTP is an allosteric activator, whereas UTP serves as an allosteric inhibitor. UTP and UDP, but none of the other nucleotides tested such as GTP, ATP, and UMP, enhanced the fluorescence of the protein. The sigmoidal shape of the dose-response curve indicated cooperativity in binding of UTP and UDP.(ABSTRACT TRUNCATED AT 250 WORDS)

A DNA fragment of Streptomyces coelicolor encoding the carboxy-terminal catalytic domain of adenylate cyclase was cloned, sequenced and expressed in an Escherichia coli cya-defective strain where it produced nanomole levels of cAMP. The amino acid sequence of the enzyme displays similarities with the Brevibacterium liquefaciens pyruvate regulated adenylate cyclase.

The catalytic domain of Bordetella pertussis adenylate cyclase, a calmodulin-activated enzyme with toxic properties, is a modular construct cleaved by trypsin into two subdomains of 224 (T25) and 175 (T18) amino acids. The calmodulin-binding locus of the bacterial enzyme consists of approximately 70 amino acids and overlaps the C-terminus of T25 and the N-terminus of T18. This region, exposed to the solvent or proteases, also exhibits an unusual high flexibility and allows, as demonstrated in this study, reconstitution in the presence of calmodulin of active species of adenylate cyclase from overlapping inactive fragments of the enzyme. Moreover, several combinations of inactive variants of the bacterial enzyme obtained by site-directed mutagenesis can yield active species. Heterodimers, resulting from a few selected combinations of inactive species of adenylate cyclase, exhibit specific activity similar to that of the native enzyme. Productive complementation from inactive fragments is a unique phenomenon among calmodulin-activated enzymes and represents a new and helpful tool in the understanding of the molecular mechanism of activation of B. pertussis adenylate cyclase upon binding of calmodulin.

Alignments between amino acid sequences of eukaryotic adenylate (ACase) and guanylate (GCase) cyclases and the prokaryotic Rhizobium meliloti ACase revealed four conserved regions. Two were the target of site-directed mutagenesis. A positive charge at position 44 converted the enzyme to GCase, a negative charge at this position had no effect. A second modification indicated that residues 107 and 124 contribute to the nucleoside triphosphate binding pocket's conformation. This latter region was used to scan protein sequences data banks. A similar region was detected in the family of E1-E2 type ATPases. Topographical resemblance between these ATPases, eukaryotic ACases and several transporters suggest that they evolved from a common ancestor.

A truncated Bordetella pertussis cya gene product was expressed in Escherichia coli and purified by affinity chromatography on calmodulin-agarose. Trypsin cleavage of the 432-residue recombinant protein (Mr = 46,659) generated two fragments of 28 kDa and 19 kDa. These fragments, each containing a single Trp residue, were purified and analyzed for their catalytic and calmodulin-binding properties. The 28-kDa peptide, corresponding to the N-terminal domain of the recombinant adenylate cyclase, exhibited very low catalytic activity, and was still able to bind calmodulin weakly, as evidenced by using a fluorescent derivative of the activator protein. The 19-kDa peptide, corresponding to the C-terminal domain of the recombinant adenylate cyclase, interacted only with calmodulin as indicated by a shift in its intrinsic fluorescence emission spectrum or by the enhancement of fluorescence of dansyl-calmodulin. T28 and T19 fragments exhibited an increased sensitivity to denaturation by urea as compared to uncleaved adenylate cyclase, suggesting that interactive contacts between ordered portions of T28 and T19 in the intact protein participate both in their own stabilization and in stabilization of the whole tertiary structure. The two fragments reassociated into a highly active calmodulin-dependent species. Reassociation was enhanced by calmodulin itself, which 'trapped' the two complementary peptides into a stable, native-like, ternary complex, which shows similar catalytic properties to intact adenylate cyclase.