Search

Genetic tools derived from the Cas9 RNA-guided nuclease are providing essential capabilities to study and engineer bacteria. While the importance of off-target effects was noted early in Cas9’s application to mammalian cells, off-target cleavage by Cas9 in bacterial genomes is easily avoided due to their smaller size. Despite this, several studies have reported experimental setups in which Cas9 expression was toxic, even when using the catalytic dead variant of Cas9 (dCas9). Specifically, dCas9 was shown to be toxic when in complex with guide RNAs sharing specific PAM (protospacer adjacent motif)-proximal sequence motifs. Here, we demonstrate that this toxicity is caused by off-target binding of Cas9 to the promoter of essential genes, with silencing of off-target genes occurring with as little as 4 nt of identity in the PAM-proximal sequence. Screens performed in various strains of Escherichia coli and other enterobacteria show that the nature of toxic guide RNAs changes together with the evolution of sequences at off-target positions. These results highlight the potential for Cas9 to bind to hundreds of off-target positions in bacterial genomes, leading to undesired effects. This phenomenon must be considered in the design and interpretation of CRISPR–Cas experiments in bacteria.

International audience; Abundance and diversity of bacteria and their viral predators, bacteriophages (phages), in the digestive tract are associated with human health. Particularly intriguing is the long-term coexistence of these two antagonistic populations. We performed genome-wide RNA sequencing on a human enteroaggregative Escherichia coli isolate to identify genes differentially expressed between in vitro conditions and in murine intestines. We experimentally demonstrated that four of these differentially expressed genes modified the interactions between E. coli and three virulent phages by either increasing or decreasing its susceptibility/resistance pattern and also by interfering with biofilm formation. Therefore, the regulation of bacterial genes expression during the colonization of the digestive tract influences the coexistence of phages and bacteria, highlighting the intricacy of tripartite relationships between phages, bacteria, and the animal host in intestinal homeostasis.

A beige-pigmented, oxidase-positive bacterial isolate, Wesi-4T, isolated from charcoal in 2012, was examined in detail by applying a polyphasic taxonomic approach. Cells of the isolates were rod shaped and Gram-stain negative. Examination of the 16S rRNA gene sequence of the isolate revealed highest sequence similarities to the type strains of Pseudomonas matsuisoli and Pseudomonas nosocomialis (both 97.3 %). Phylogenetic analyses on the basis of the 16S rRNA gene sequences indicated a separate position of Wesi-4T, which was confirmed by multilocus sequence analyses (MLSA) based on the three loci gyrB, rpoB and rpoD and a core genome-based phylogenetic tree. Genome sequence based comparison of Wesi-4T and the type strains of P. matsuisoli and P. nosocomialis yielded average nucleotide identity values in silico DNA-DNA hybridization values 16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and summed feature 8 (C18 : 1ω7c and/or C18 : 1 ω6c). In addition, physiological and biochemical tests revealed a clear phenotypic difference from P. matsuisoli . These cumulative data indicate that the isolate represents a novel species of the genus Pseudomonas for which the name Pseudomonas carbonaria sp. nov. is proposed with Wesi-4T (=DSM 110367T=CIP 111764T=CCM 9017T) as the type strain.

Background Klebsiella pneumoniae is a leading cause of intractable hospital-acquired multidrug-resistant infections and carbapenemase-producing K. pneumoniae (CPKp) are particularly feared. Most of the clinical isolates produce capsule as a major virulence factor. Recombination events at the capsule locus are frequent and responsible for capsule diversity within Klebsiella spp. Capsule diversity may also occur within clonal bacterial populations generating differences in colony aspect. However, little is known about this phenomenon of phenotypic variation in CPKp and its consequences. Results Here, we explored the genetic causes of in vitro switching from capsulated, mucoid to non-mucoid, non-capsulated phenotype in eight clinical CPKp isolates. We compared capsulated, mucoid colony variants with one of their non-capsulated, non-mucoid isogenic variant. The two colony variants were distinguished by their appearance on solid medium. Whole genome comparison was used to infer mutations causing phenotypic differences. The frequency of phenotypic switch was strain-dependent and increased along with colony development on plate. We observed, for 72 non-capsulated variants that the loss of the mucoid phenotype correlates with capsule deficiency and diverse genetic events, including transposition of insertion sequences or point mutations, affecting genes belonging to the capsule operon. Reduced or loss of capsular production was associated with various in vitro phenotypic changes, affecting susceptibility to carbapenem but not to colistin, in vitro biofilm formation and autoaggregation. Conclusions The different impact of the phenotypic variation among the eight isolates in terms of capsule content, biofilm production and carbapenem susceptibility suggested heterogeneous selective advantage for capsular loss according to the strain and the mutation. Based on our results, we believe that attention should be paid in the phenotypic characterization of CPKp clinical isolates, particularly of traits related to virulence and carbapenem resistance.

Background Limited epidemiological and antimicrobial resistance data are available on Salmonella enterica from sub-Saharan Africa. We determine the prevalence of resistance to antibiotics in isolates in the Central African Republic (CAR) between 2004 and 2013 and the genetic basis for resistance to third-generation cephalosporin (C3G). Methodology/Principal findings A total of 582 non-duplicate human clinical isolates were collected. The most common serotype was Typhimurium (n = 180, 31% of the isolates). A randomly selected subset of S. Typhimurium isolates were subtyped by clustered regularly interspaced short palindromic repeat polymorphism (CRISPOL) typing. All but one invasive isolate tested (66/68, 96%) were associated with sequence type 313. Overall, the rates of resistance were high to traditional first-line drugs (18–40%) but low to many other antimicrobials, including fluoroquinolones (one resistant isolate) and C3G (only one ESBL-producing isolate). The extended-spectrum beta-lactamase (ESBL)-producing isolate and three additional ESBL isolates from West Africa were studied by whole genome sequencing. The blaCTX-M-15 gene and the majority of antimicrobial resistance genes found in the ESBL isolate were present in a large conjugative IncHI2 plasmid highly similar (> 99% nucleotide identity) to ESBL-carrying plasmids found in Kenya (S. Typhimurium ST313) and also in West Africa (serotypes Grumpensis, Havana, Telelkebir and Typhimurium). Conclusions/Significance Although the prevalence of ESBL-producing Salmonella isolates was low in CAR, we found that a single IncHI2 plasmid-carrying blaCTX-M-15 was widespread among Salmonella serotypes from sub-Saharan Africa, which is of concern., Author summary Salmonella enterica infections are common causes of bloodstream infection in sub-Saharan Africa and associated with a high mortality rate. Levels of multidrug resistance have become alarmingly high. Then, third-generation cephalosporin (C3G) and fluoroquinolones have become standard for first-line empirical treatment. Recently, C3G-resistant Salmonella populations have emerged and spread over all continents. This resistance is mainly mediated by acquired extended-spectrum beta-lactamase (ESBL) genes carried by mobile genetic elements such as plasmids. We report here the prevalence of resistance to antibiotics in isolates in the Central African Republic (CAR) between 2004 and 2013 and the genetic basis for resistance to C3G. Overall, resistance rates to antimicrobials were low during the study period, for all classes other than conventional antimicrobials, confirming recommendations for first-line treatment based on C3G and fluoroquinolones. Only one ESBL-producing isolate was recovered. The ESBL gene and the majority of antimicrobial resistance genes found were present in a large plasmid highly similar to ESBL-carrying plasmids found in East and West Africa, highlighting its significant role in the spread of ESBL genes in Salmonella isolates in sub-Saharan Africa. These finding have implications for treatment of salmonellosis and support the growing necessity for increased microbiological surveillance based on networks of clinical laboratories in order to control dissemination of antibiotic resistance among Salmonella isolates.

International audience; In 1986, Bouvet and Grimont delineated two related taxa of the genus Acinetobacter termed genospecies (GS) 8 and 9. They proposed the name Acinetobacter lwoffii for GS8, which included the supposed type strain (CIP 64.10). As the authenticity of CIP 64.10 was later questioned, this study aimed at reassessing the taxonomy of these genospecies. We investigated 52 strains of GS8 or GS9, including CIP 64.10 and the genuine type strain of A. lwoffii (NCTC 5866T). All strains were subjected to the genus-wide comparative analyses of MALDI-TOF whole-cell mass spectra, rpoB gene sequences and metabolic traits while whole-genome sequences were analysed for 16 strains. The strains were classified into two distinct groups corresponding to GS8 (n=15) and GS9 (n=37). CIP 64.10 fell within GS8 whereas NCTC 5866T belonged to GS9. Intraspecies ANIb values for the genomes of GS8 (n=6) and GS9 (n=10) were ≥96.1% and ≥95.4%, respectively, whereas the ANIb values between them were 86.8-88.6%. Based on core genome phylogeny, GS8 and GS9 formed a distinct clade within the genus, with two respective, strongly supported subclades. GS8 and GS9 were similar in physiological and catabolic properties but were separable by MALDI-TOF MS. We conclude that the name A. lwoffii pertains to GS9 and not to GS8 as originally assumed and that these groups represent two species. We propose the name Acinetobacter pseudolwoffii sp. nov. for GS8, with ANC 5044T (=CCM 8638T=CCUG 67963T=CIP 111642T) as the type strain, and provide the emended description of A. lwoffii.

International audience; Antimicrobial resistance, genotype, and virulence gene content of Escherichia coli isolated from bovine clinical mastitis in Tehran Province, Iran, were studied. Two hundred seven (207) milk samples from individual cows presenting mastitis symptoms collected from different dairy farms were used to determine the presence of specific genes of E. coli responsible for this pathology. Multiplex PCR was used to differentiate E. coli isolates into different phylogenetic groups/subgroups and to detect their virulence and involved resistance genes. All the isolated strains were tested for the susceptibility to 21 antimicrobial agents. The results showed that E. coli was detected in 42 (20.3%) samples and 69% of them belonged to the phylogenetic groups A and B1. The phylogenetic subgroup A1 (31%) and subgroup B1 (28.6%) demonstrated the highest prevalence of virulence genes (f17c-A, and eae (n = 6), f17b-A, and f17d-A (n = 5), afaD-8, afaE-8, aucD, and bfpA (n = 4), clpG and VT (n = 2), and LT and ST genes (n = 1)). The highest antimicrobial resistance was observed for tetracycline (45.2%) followed by streptomycin (26.2%). The antimicrobial resistance genes tetB (31%), tetA (28.6%), and aadA (26.2%) were the most prevalent. Moreover, integron class 1 and 2 were found in 24 (57.1%) and 8 (19%) of the E. coli isolates.

International audience; Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which RNA polymerase "kicks out" dCas9 from the target and completes transcription. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. Alternatively, tuning repression by changing dCas9 concentration is noisy and promoter-strength dependent. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto-repressor. Second, we study the impact of amount variations of cell-wall synthesizing enzymes on cell morphology. Finally, we multiplex the system to obtain any combination of fractional repression of two genes.

Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment., Author summary Nucleotide-based second messengers play central functions in bacterial physiology and host-pathogen interactions. Among these signalling nucleotides, cyclic-di-AMP (c-di-AMP) synthesis was originally assumed to be essential for bacterial growth. In this study, we confirmed that the only di-adenylate cyclase enzyme in the opportunistic pathogen Streptococcus agalactiae is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable by accumulating spontaneous mutations in genes involved in osmotic regulation. We identified that c-di-AMP binds directly to four proteins necessary to maintain osmotic homeostasis, including three osmolyte transporters and the BusR transcriptional factor. We demonstrated that BusR negatively controls the expression of the busAB operon and that it is the main component leading to growth inhibition in the absence of c-di-AMP synthesis if osmoprotectants are present in the environment. Overall, c-di-AMP is essential to maintain osmotic homeostasis by coordinating osmolyte uptake and thus bacteria have developed specific mechanisms to keep c-di-AMP as the central regulator of osmotic homeostasis.

International audience; Genome dynamics is crucial for bacterial genome evolution and adaptation. Here we reveal the unexpectedly fast genome diversification occurring within a highly genetically cohesive population of soil bacteria Streptomyces. We applied a reverse ecology sampling strategy in order to isolate sympatric Streptomyces that have diverged within a short evolutionary time. For this purpose, we isolated bacteria from rhizospheric soil aggregates in the range of mm3 size and distant at the centimeter scale. We selected isolates that shared 100% of identity in 16S rRNA sequences and presented a high degree of similarity in MLST and ANI genome comparisons. We concluded that the isolated strains were all highly related at the intra-specific level and according to the sampling scheme should derive from a common ancestor over a very short evolutionary time. By comparing the genome sequences of 18 isolates (genome size ca. 11.7 Mb), we identified at least 25 genomic islands (>10 kb) scattered along the chromosome, but with a higher frequency in the terminal regions of the linear chromosome, including in some cases the chromosomal end. Some of them exhibit clear signatures of integrated and conjugative elements, while some other include secondary metabolite biosynthetic genes. In addition, some isolates also diverge by the presence of large plasmids (100 to 400 kb). Different patterns of inhibitory capacities were distinguished within the population. Hence, while only some Streptomyces isolates exhibited inhibitory activities against other bacterial coinhabitants (e.g. Bacillus), all the Streptomyces strains could develop together. This suggests that inhibitory activities provided by some isolates may constitute ‘public goods’ to the Streptomyces community, and this by the mean of differentiation of the secondary metabolism gene repertoire. As a proof of concept that rapid genome dynamics impacts population ecology, we inactivated a biosynthetic gene cluster gene (i.e. NRPS) specific of one inhibitory strain and observed the abolition of the inhibitory activity. Together our data support the hypothesis that the high level of evolvability of the Streptomyces genome impacts structuring and adaptation of natural bacterial communities.

International audience; The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) "hypervirulent" ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus. Deletion of this 43-bp sequence in strain BM110 resulted in a 3-to 5-fold increased transcription of PI-2b. Characterization of PI-2b promoter region in A909 and BM110 strains was carried out by RNAseq, primer extension, qRT-PCR and transcriptional fusions with gfpas reporter gene. Our results indicate the presence of a single promoter (Ppi2b) with a transcriptional start site (TSS) mapped 37 bases upstream of the start codon of the first PI-2b gene. The large operon of 16 genes located upstream of PI-2b codes for the group B carbohydrate (also known as antigen B), a major constituent of the bacterial cell wall. We showed that the hairpin sequence located between antigen B and PI-2b operons is a transcriptional terminator. In A909, increased expression of PI-2b probably results from read-through transcription from antigen B operon. In addition, we showed that an extended 5′ promoter region is required for maximal transcription of gfpas a reporter gene in S. agalactiae from Ppi2b promoter. Gene reporter assays performed in Lactococcus lactis strain NZ9000, a related non-pathogenic Gram-positive species, revealed that GBS-specific regulatory factors are required to drive PI-2b transcription. PI-2b expression is up-regulated in the BM110AcovR mutant as compared to the parental BM110 strain, but this effect is probably indirect. Collectively, our results indicate that PI-2b expression is regulated in GBS ST17 strains, which may confer a selective advantage in the human host either by reducing host immune responses and/or increasing their dissemination potential.

Summary Background A novel multicomponent vaccine against meningococcal capsular group B (MenB) disease contains four major components: factor-H-binding protein, neisserial heparin binding antigen, neisserial adhesin A, and outer-membrane vesicles derived from the strain NZ98/254. Because the public health effect of the vaccine, 4CMenB (Novartis Vaccines and Diagnostics, Siena, Italy), is unclear, we assessed the predicted strain coverage in Europe. Methods We assessed invasive MenB strains isolated mainly in the most recent full epidemiological year in England and Wales, France, Germany, Italy, and Norway. Meningococcal antigen typing system (MATS) results were linked to multilocus sequence typing and antigen sequence data. To investigate whether generalisation of coverage applied to the rest of Europe, we also assessed isolates from the Czech Republic and Spain. Findings 1052 strains collected from July, 2007, to June, 2008, were assessed from England and Wales, France, Germany, Italy, and Norway. All MenB strains contained at least one gene encoding a major antigen in the vaccine. MATS predicted that 78% of all MenB strains would be killed by postvaccination sera (95% CI 63–90, range of point estimates 73–87% in individual country panels). Half of all strains and 64% of covered strains could be targeted by bactericidal antibodies against more than one vaccine antigen. Results for the 108 isolates from the Czech Republic and 300 from Spain were consistent with those for the other countries. Interpretation MATS analysis showed that a multicomponent vaccine could protect against a substantial proportion of invasive MenB strains isolated in Europe. Monitoring of antigen expression, however, will be needed in the future. Funding Novartis Vaccines and Diagnostics.

International audience; Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens whose survival and virulence in the human digestive tract remain unclear owing to paucity of relevant models. EHEC interact with the follicle-associated epithelium of Peyer's patches of the distal ileum and translocate across the intestinal epithelium via M-cells, but the underlying molecular mechanisms are still unknown. Here, we investigated the involvement of Long polar fimbriae (Lpf) in EHEC pathogenesis. Of the 236 strains tested, a significant association was observed between the presence of lpf operons and pathogenicity. In sophisticated in vitro models of the human gastro-intestinal tract, lpf expression was induced during transit through the simulated stomach and small intestine, but not in the colonic compartment. To investigate the involvement of Lpf in EHEC pathogenesis, lpf isogenic mutants and their relative trans-complemented strains were generated. Translocation across M-cells, interactions with murine ileal biopsies containing Peyer's patches and the number of hemorrhagic lesions were significantly reduced with the lpf mutants compared to the wild-type strain. Complementation of lpf mutants fully restored the wild-type phenotypes. Our results indicate that (i) EHEC might colonize the terminal ileum at the early stages of infection, (ii) Lpf are an important player in the interactions with Peyer's patches and M-cells, and could contribute to intestinal colonization.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited - © 2006 Salaün and Saunders; licensee BioMed Central Ltd. Background: Population structures are normally determined using genes under minimal functional selection. In this study we have assessed genes that are not always essential, show differences in alleles between strains, and are involved in the directly host-selectable phenotype of LPS biosynthesis. Results: Eight complete LPS biosynthesis genes, seven of which are associated with phase variation in some or all strains of Helicobacter pylori, have been sequenced and their divergence analyzed. The differences observed indicate that recombination within these genes largely reflects exchange between strains within the population lineages previously determined on the basis of MLST using housekeeping genes. This indicates that the differences that are used for MLST are likely to broadly associate with genes under functional selection, and differences in strain behaviour. However, instances of exchange between the subpopulations were identified, including the hpAfrica2 subpopulation. Further, there were other differences in gene complements and the chromosomal location of genes indicative of greater diversity within the population than is revealed by the available genome sequences and comparative genome hybridization studies. Conclusion: These results indicate that the described population structure based upon MLST is broadly a good basis for studying the biology of H. pylori, but that individual alleles may not follow these associations. As a consequence, when working in unsequenced strains, it is necessary to carefully check the presence, sequence, and distribution of any individual gene of interest. This work is supported by a Wellcome Trust Advanced Fellowship.

International audience; OBJECTIVES:Reduced susceptibility to penicillin G in Neisseria meningitidis is mainly due to alterations in PBP2 encoded by the penA gene. However, this phenotype was not associated with reduced susceptibility to third-generation cephalosporins (C3Gs). We aimed to study the emergence of meningococci with reduced susceptibility to C3Gs (MIC >0.06 mg/L) in France since 2012.METHODS:Invasive meningococcal isolates were typed by MLST and penA sequencing and their antibiotic susceptibility was tested.RESULTS:Isolates with reduced susceptibility to C3Gs represented 2% of all invasive isolates from 2012-15, but were absent before. They harboured a new penA allele, penA327, that was also detected in isolates from urethritis cases and in gonococci.CONCLUSIONS:Surveillance of these isolates should be enhanced as they may jeopardize the use of C3Gs in the management of invasive meningococcal disease.

International audience; The presence of extended-spectrum beta-lactamase and plasmid-mediated AmpC beta-lactamase producing Enterobacteriaceae (ESBL-Eb and pAmpC-Eb, respectively) was analyzed in 57 wastewater and 57 surface-water samples in Tunisia. Twenty-four of the 57 wastewater samples (42.1%) and one of the 57 surface-water samples (1.7%, a river that received effluents of a wastewater-treatment-plant) contained ESBL-Eb or pAmpC-Eb; one ESBL/pAmpC-Eb per positive sample was further characterized. Beta-lactamase genes detected were as follows: blaCTX-M-1 (10 Escherichia coli),blaCTX-M-15 (eight E. coli, one Klebsiella pneumoniae, one Citrobacter freundii), blaCTX-M-14 (one E. coli) and blaCMY-2 (four E. coli). The blaTEM-1, blaOXA-1 or blaSHV-1 genes were also found in 72% of these isolates. The ISEcp1, orf477 or IS903 sequences were found upstream or downstream of blaCTX-M genes. Class 1 integrons were present in 16 of the 25 ESBL-Eb/pAmpC-Eb strains (64%), and contained five different gene-cassette arrays. Most of the strains (76%) showed a multiresistant phenotype and qnr genes were identified in four strains. Molecular typing of ESBL/CMY-2-producing E. coli isolates showed 23 different PFGE-patterns and 15 different sequence-types (ST10, ST46, ST48, ST58, ST69, ST101, ST117, ST131, ST141, ST288, ST359, ST399, ST405, ST617, and the new ST4530); these strains were ascribed to phylogroups A (11 isolates), B1 (3 isolates), D (6 isolates) and B2 (3 isolates). From one to five plasmids were detected in each strain (size from 30 kb to > 240 kb) and ESBL or pAmpC genes were transferred by conjugation in 69.5% of the E. coli strains. In conclusion, ESBL-Eb and pAmpC-Eb strains are frequently detected in wastewater samples and they might be a source for dissemination in other environments with repercussion in public health.

International audience; The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cis-tronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcel-lular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ.

The aim of this study was to assess and characterize Mycobacterium tuberculosis complex (MTBC) genotypic diversity in Tanzania, as well as in neighbouring East and other several African countries. We used spoligotyping to identify a total of 293 M. tuberculosis clinical isolates (one isolate per patient) collected in the Bunda, Dar es Salaam, Ngorongoro and Serengeti areas in Tanzania. The results were compared with results in the SITVIT2 international database of the Pasteur Institute of Guadeloupe. Genotyping and phylogeographical analyses highlighted the predominance of the CAS, T, EAI, and LAM MTBC lineages in Tanzania. The three most frequent Spoligotype International Types (SITs) were: SIT21/CAS1-Kili (n = 76; 25.94%), SIT59/LAM11-ZWE (n = 22; 7.51%), and SIT126/EAI5 tentatively reclassified as EAI3-TZA (n = 18; 6.14%). Furthermore, three SITs were newly created in this study (SIT4056/EAI5 n = 2, SIT4057/T1 n = 1, and SIT4058/EAI5 n = 1). We noted that the East-African-Indian (EAI) lineage was more predominant in Bunda, the Manu lineage was more common among strains isolated in Ngorongoro, and the Central-Asian (CAS) lineage was more predominant in Dar es Salaam (p-value

International audience; in the coding sequence. These mutations have previously been shown to result in increased azithromycin resistance due to induced expression of the MtrCDE efflux pump. 8 The genes erm(A), erm(B), erm(C) and erm(F), encoding rRNA methylases, and mef(A), encoding an efflux pump, were not present in these isolates. Molecular epidemiology analysis of the isolates was performed using the N. gonorrhoeae multiantigen sequence typing (NG-MAST) method, 9 which assigns an ST based on a combination of the highly variable por and tbpB alleles. The isolates were assigned ST3102 and ST1866, which are almost identical and differ only by one SNP in por. Thus far, the high-level azithromycin-resistant N. gonorrhoeae strains that have been isolated in various countries belong to 16 different STs, of which ST649 isolates appear to be most widespread. 5 – 7 The por and tbpB alleles of these STs differ by a total of 33–92 SNPs from the por and tbpB alleles of the strains described in this study, except for the Australian ST10133 isolate, 7 which contains an identical tbpB allele (33) and a por allele (2573) that only differs by 16 and 17 SNPs from the por alleles of ST3102 and ST1866, respectively. Interestingly, it was reported that this isolate was most likely contracted in China or Hong Kong, which might explain its closer genetic relationship. How widespread high-level azithromycin-resistant strains are in China remains to be determined. Several years earlier, in 2009, two ST1866 strains were isolated from patients in the city of Nanjing, 10 which is located in a neighbouring province. These strains were reported to have an azithromycin MIC of .64 mg/L. It is not known whether these strains actually are directly related to the ST1866 isolate described in this study, but if they are related it would indicate that this high-level azithromycin-resistant ST might be spreading in China. Azithromycin susceptibility testing is currently not the standard in China. However, the identification of high-level azithromycin-resistant N. gonorrhoeae isolates stresses the need for a more thorough overview of azithromycin susceptibility in China, particularly when ceftriaxone and azithromycin dual antimicrobial therapy is considered as a future first-line therapy. These high-level azithromycin-resistant isolates might have a major impact on the efficacy of this dual antimicrobial therapy.

OBJECTIVES In group B Streptococcus (GBS), cross-resistance to lincosamides, streptogramin A and pleuromutilins (LSAP) is mediated by the acquisition of lsa genes. Here, we characterized the diversity, mobility and ecology of lsa genes in this species. METHODS lsa variants were systematically identified by BLAST searches in the genomes of 531 GBS strains from different hosts and geographical origins. The associated phenotypes were determined by a microdilution MIC method. Acquisition of resistance genes was deduced from comparative genomics and phylogeny. Their mobility was tested by conjugation experiments. RESULTS lsa(E) and three variants of lsa(C) were identified in GBS strains. Two lsa(C) variants had not been previously reported. All four variants conferred LSAP phenotypes. lsa(E) was located in a multiresistance gene cluster of a single human strain. This gene was transferred by a high-frequency recombination-type mechanism between GBS strains. Two lsa(C) variants are carried in six unrelated human strains by two similar elements specifically integrated in the oriT site of four different classes of integrative and conjugative elements (ICEs). Strikingly, the acquisition of the resistance gene always occurred by the integration of the element into a resident ICE. The third lsa(C) variant was located at the same site in the core genome of 11 genetically distant bovine strains and was likely propagated by horizontal transfer of the corresponding chromosomal region. CONCLUSIONS lsa genes in GBS show distinct host specificities and modes of transfer. In general, their dissemination is mediated by recombination rather than by the transfer of conjugative elements.

We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii , a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1 , RD Rio , and fbpC , the insertion of IS 6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates ( n = 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA , rpoB , rpoC , katG , and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG , and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) in inhA . Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB . No putative compensatory mutation was found in either rpoA or rpoC . This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers.

International audience; Insertion sequence (IS) elements are present in almost all bacterial genomes and are mobile enough to provide genomic tools to differentiate closely related isolates. They can be used to estimate genetic diversity and identify fitness-enhancing mutations during evolution experiments. Here, we determined the genomic distribution of eight IS elements in 120 genomes sampled from Escherichia coli populations that evolved in glucose- and phosphate-limited chemostats by comparison to the ancestral pattern. No significant differential transposition of the various IS types was detected across the environments. The phylogenies revealed substantial diversity amongst clones sampled from each chemostat, consistent with the phenotypic diversity within populations. Two IS-related changes were common to independent chemostats, suggesting parallel evolution. One of them corresponded to insertions of IS1 elements within rpoS encoding the master regulator of stress conditions. The other parallel event was an IS5-dependent deletion including mutY involved in DNA repair, thereby providing the molecular mechanism of generation of mutator clones in these evolving populations. These deletions occurred in different co-existing genotypes within single populations and were of various sizes. Moreover, differential locations of IS elements combined with their transpositional activity provided evolved clones with different phenotypic landscapes. Therefore, IS elements strongly influenced the evolutionary processes in continuous E. coli cultures by providing a way to modify both the global regulatory network and the mutation rates of evolving cells.

The objective of this study was to implement a semimechanistic pharmacokinetic-pharmacodynamic (PK-PD) model to describe the effects of ciprofloxacin against Pseudomonas aeruginosa in vitro. Time-kill curves were generated with an initial inoculum close to 5 × 10 6 CFU/ml of P. aeruginosa PAO1 and constant ciprofloxacin concentrations between 0.12 and 4.0 μg/ml (corresponding to 0.5× and 16× MIC). To support the model, phenotypic experiments were conducted with the PAO7H mutant strain, which overexpresses the MexEF OprN efflux pump and phenyl arginine β-naphthylamide (PAβN), a known efflux inhibitor of main Mex multidrug efflux systems. A population approach was used for parameter estimation. At subinhibitory ciprofloxacin concentrations (0.12 and 0.25 μg/ml), an initial CFU decay followed by regrowth was observed, attesting to rapid emergence of bacteria with increased but moderate resistance (8-fold increase of MIC). This phenomenon was mainly due to an overexpression of the Mex protein efflux pumps, as shown by a 16-fold diminution of the MIC in the presence of PAβN in these strains with low-level resistance. A PK-PD model with adaptation development was successfully used to describe these data. However, additional experiments are required to validate the robustness of this model after longer exposure periods and multiple dosing regimens, as well as in vivo.

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida . The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C 10 OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila . While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila , including strong biocontrol abilities.

The aminocoumarin antibiotic novobiocin is a gyrase inhibitor formed by a Streptomyces strain. The biosynthetic gene cluster of novobiocin spans 23.4 kb and contains 20 coding sequences, among them the two regulatory genes novE and novG. We investigated the location of transcriptional promoters within this cluster by insertion of transcriptional terminator cassettes and RT-PCR analysis of the resulting mutants. The cluster was found to contain eight DNA regions with promoter activity. The regulatory protein NovG binds to a previously identified binding site within the promoter region located upstream of novH, but apparently not to any of the other seven promoters. Quantitative real-time PCR was used to compare the number of transcripts in a strain carrying an intact novobiocin cluster with strains carrying mutated clusters. Both in-frame deletion of the regulatory gene novG and insertion of a terminator cassette into the biosynthetic gene novH led to a strong reduction of the number of transcripts of the genes located between novH and novW. This suggested that these 16 biosynthetic genes form a single operon. Three internal promoters are located within this operon but appear to be of minor importance, if any, under our experimental conditions. Transcription of novG was found to depend on the presence of NovE, suggesting that the two regulatory genes, novE and novG, act in a cascade-like mechanism. The resistance gene gyrBR , encoding an aminocoumarin-resistant gyrase B subunit, may initially be co-transcribed with the genes from novH to novW. However, when the gyrase inhibitor novobiocin accumulates in the cultures, gyrBR is transcribed from its own promoter. Previous work has suggested that this promoter is controlled by the superhelical density of chromosomal DNA.

International audience; The nucleoid of radioresistant bacteria, including D. radiodurans, adopts a highly condensed structure that remains unaltered after exposure to high doses of irradiation. This structure may contribute to radioresistance by preventing the dispersion of DNA fragments generated by irradiation. In this report, we focused our study on the role of HU protein, a nucleoid-associated protein referred to as a histone-like protein, in the nucleoid compaction of D. radiodurans. We demonstrate, using a new system allowing conditional gene expression, that HU is essential for viability in D. radiodurans. Using a tagged HU protein and immunofluorescence microscopy, we show that HU protein localizes all over the nucleoid and that when HU is expressed from a thermosensitive plasmid, its progressive depletion at the non-permissive temperature generates decondensation of DNA before fractionation of the nucleoid into several entities and subsequent cell lysis. We also tested the effect of the absence of Dps, a protein also involved in nucleoid structure. In contrast to the drastic effect of HU depletion, no change in nucleoid morphology and cell viability was observed in dps mutants compared with the wild-type, reinforcing the major role of HU in nucleoid organization and DNA compaction in D. radiodurans.

International audience; Comparative genomics with Staphylococcus aureus suggested the existence of a regulatory system governing beta-lactamase (BlaC) production in Mycobacterium tuberculosis. The crystal structure of Rv1846c, a winged helix regulator of previously unknown function, was solved thus revealing strong similarity to the BlaI and MecI repressors of S. aureus, which both respond to beta-lactam treatment. Using chromatin immunoprecipitation and hybridization to microarrays (ChIP-on-chip), the Rv1846c regulon was shown to comprise five separate genomic loci. Two of these mediate responses and resistance to beta-lactam antibiotics (rv1845c, rv1846c-rv1847; blaC-sigC); two encode membrane proteins of unknown function (rv1456c, rv3921c) while the last codes for ATP synthase (rv1303-atpBEFHAGDC-rv1312). The ChIP-on-chip findings were confirmed independently using electrophoretic mobility shift assays, DNAse footprinting and transcript analysis leading to Rv1846c being renamed BlaI. When cells were treated with beta-lactams, BlaI was released from its operator sites causing derepression of the regulon and upregulation of ATP synthase transcription. The existence of a potential regulatory loop between cell wall integrity and ATP production was previously unknown.

The Beijing genotype of Mycobacterium tuberculosis has been shown in many settings to be hypervirulent and associated with multi-drug resistance. Its presently global and rapid dissemination makes it an important issue of public health. Here, I present a significantly enlarged update of the MIRU-VNTR global database of the M. tuberculosis Beijing genotype (11 loci). I further attempted to link the observed mycobacterial diversity with relevant events of the known human history. Large water masses have been the most efficient and drastic generators of the genetic divergence between human populations. The same situation appears true also for M. tuberculosis, which general diversity pattern amazingly resembles that of its human host. At the same time, less expected affinities observed between distant populations of M. tuberculosis may reflect hidden patterns of human migrations or yet unknown epidemiological links between distant regions.

During the last few years, the use of type III secretion system-based bacterial vectors for immunotherapy purposes has been assessed in various applications. We showed that a type III secretion-based Pseudomonas aeruginosa vector delivering the ovalbumin (OVA) antigen induced an efficient specific CD8 + T-lymphocyte immune response against OVA-expressing cells. Because of the intrinsic toxicity of the vector, further virulence attenuation was needed. Therefore, we explored the effects of the deletion of quorum-sensing genes and the aroA gene toward toxicity and efficiency of the vector strain. The aroA mutation in our strain (making the strain auxotrophic for aromatic amino acids) conferred a strikingly reduced toxicity, with the bacterial lethal dose being more than 100 times higher than that of the parental strain. The quorum-sensing gene mutation alone was associated with a slightly reduced toxicity. In a prophylactic OVA-expressing melanoma mouse model, an OVA-delivering aroA -deficient mutant was the most efficient at a low dose (10 5 ), but dose enhancement was not associated with a greater immune response. The quorum-sensing-deficient strain was the most efficient at a mild dose (10 6 ), but this dose was close to the toxic dose. Combination of both mutations conferred the highest efficiency at an elevated dose (10 7 ), in agreement with the known negative effects of quorum-sensing molecules upon T-cell activation. In conclusion, we have obtained a promising immunotherapy vector regarding toxicity and efficiency for further developments in both antitumor and anti-infectious strategies.

CggR belongs to the SorC family of bacterial transcriptional regulators which control the expression of genes and operons involved in carbohydrate catabolism. CggR was first identified in Bacillus subtilis where it represses the gapA operon encoding the five enzymes that catalyze the central part of glycolysis. Here we present a structure/function study demonstrating that the C-terminal region of CggR regulates the DNA binding activity of this repressor in response to binding of a phosphorylated sugar. Molecular modeling of CggR revealed a winged-helix DNA-binding motif followed by a C-terminal domain presenting weak but significant homology with glucosamine-6-phosphate deaminases from the NagB family. In silico ligand screening suggested that the CggR C-terminal domain would bind preferentially bi-phosphorylated compounds, in agreement with previous studies that proposed fructuose-1,6-biphosphate (FBP) as the inducer metabolite. In vitro, FBP was the only sugar compound capable of interfering with CggR cooperative binding to DNA. FBP was also found to protect CggR against trypsin degradation at two arginine residues predicted to reside in a mobile loop forming the active site lid of the NagB enzymes. Replacement of residues predicted to interact with FBP led to mutant CggR with altered repressor activity in vivo but retaining their structural integrity and DNA binding activity in vitro. Interestingly, some of the mutant repressors responded with different specificity towards mono- and di-phospho-fructosides. Based on these results, we propose that the activity of the CggR-like repressors is controlled by a phospho-sugar binding (PSB) domain presenting structural and functional homology with NagB enzymes.

International audience; The qnr genes are transferable genes that confer low-level quinolone resistance by protection of topoisomerase. The occurrence of mutations in DNA gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) genes in strains harbouring qnr was investigated in 28 qnrA-positive clinical isolates, among which 7 strains also harboured qnrS. Topoisomerase mutations were found in 25 (89%) of the 28 strains, with at least two mutations (gyrA and parC) in 13 strains and one mutation in 12 strains. Isolates of the Enterobacter cloacae complex were compared with reference strains of the new Enterobacter species. gyrA mutations were found at position 83 (Ser or Thr for Ile, Tyr, Leu or Phe depending on the species), and new gyrB mutations were described (S463A, S464F). qnrA had an additive effect of a 10-fold increase in the minimum inhibitory concentration (MIC) whatever the number of topoisomerase mutations, and qnrS was additive to qnrA with a further 2- to 10-fold increase in the MIC. Comparison of MICs with susceptibility breakpoints showed that strains combining qnrA and topoisomerase mutations were resistant to fluoroquinolones, but the three strains lacking a topoisomerase mutation were susceptible using ciprofloxacin and levofloxacin but not using nalidixic acid or moxifloxacin testing.

Using signature-tagged transposon mutagenesis, we isolated 23 Mycobacterium tuberculosis mutants, corresponding to 21 genes or genetic regions, attenuated in their ability to parasitize human macrophages. Mutants disrupted in the ABC transporter-encoding genes Rv0986 and Rv0987 were further characterized as being impaired in their ability to bind to host cells.

A study was undertaken to determine the prevalence and diversity of species of the genus Arcobacter in pig and dairy cattle manure, which led to the identification of strains AF1440T, AF1430 and AF1581. Initially identified as Arcobacter butzleri based on colony morphology and initial PCR-confirmation tests, analyses of 16S rRNA gene sequences of these strains confirmed that they belonged to the genus Arcobacter and were different from all known species of the genus. The isolates formed a distinct group within the genus Arcobacter based on their 16S rRNA, gyrB, rpoB, cpn60, gyrA and atpA gene sequences and fatty acid profiles. Their unique species status was further supported by physiological properties and DNA–DNA hybridization that allowed phenotypic and genotypic differentiation of the strains from other species of the genus Arcobacter. The isolates were found to be oxidase, catalase and esterase positive and urease negative; they grew well at 30 °C under microaerophilic conditions and produced nitrite and acetoin. Based on their common origin and various physiological properties, it is proposed that the isolates are classified as members of a novel species with the name Arcobacter lanthieri sp. nov. The type strain is AF1440T ( = LMG 28516T = CCUG 66485T); strains AF1430 ( = LMG 28515 = CCUG 66486) and AF1581 ( = LMG 28517 = CCUG 66487) are reference strains.

International audience; Advances in high-throughput DNA sequencing allow for a comprehensive analysis of bacterial genes that contribute to virulence in a specific infectious setting. Such information can yield new insights that affect decisions on how to best manage major public health issues such as the threat posed by increasing antimicrobial drug resistance. Much of the focus has been on the consequences of the selective advantage conferred on drug-resistant strains during antibiotic therapy. It is thought that the genetic and phenotypic changes that confer resistance also result in concomitant reductions in in vivo fitness, virulence, and transmission. However, experimental validation of this accepted paradigm is modest. Using a saturated transposon library of Pseudomonas aeruginosa, we identified genes across many functional categories and operons that contributed to maximal in vivo fitness during lung infections in animal models. Genes that bestowed both intrinsic and acquired antibiotic resistance provided a positive in vivo fitness advantage to P. aeruginosa during infection. We confirmed these findings in the pathogenic bacteria Acinetobacter baumannii and Vibrio cholerae using murine and rabbit infection models, respectively. Our results show that efforts to confront the worldwide increase in antibiotic resistance might be exacerbated by fitness advantages that enhance virulence in drug-resistant microbes.

Objectives The objective was to study a new vanG-type locus in Clostridium argentinense vanGCar and to determine its impact on glycopeptide susceptibility of the host. Methods The whole genome of C. argentinense NCIB 10714 was sequenced using Illumina single-reads sequencing technology. The presence of vanGCar in seven C. argentinense strains was tested by PCR and its expression was tested by quantitative RT–PCR (qRT–PCR). Glycopeptide susceptibility was determined by the Etest procedure. Results The vanGCar locus contained four genes encoding a carboxypeptidase, a d-alanine:d-serine ligase, a serine transporter and a serine racemase, and was present in the seven C. argentinense studied. An AraC-type transcriptional regulator was found upstream from the genes. C. argentinense NCIB 10714 was susceptible to vancomycin and to teicoplanin. qRT–PCR experiments revealed that vanGCar was not expressed without or with induction by a subinhibitory concentration of vancomycin. Conclusions The new vanGCar locus was cryptic in C. argentinense and intrinsic to this species. Emergence of vancomycin resistance in C. argentinense due to decryptification of the vanGCar gene cluster could occur.

N.R. and I.M. are Editorial Board members of the International Journal of Mycobacteriology.; International audience; Tuberculosis (TB) control remains an important public health priority for Bulgaria. The population structure of Mycobacterium tuberculosis is clonal and certain genetic families of this species (e.g., Latin-American-Mediterranean [LAM]) have attracted more attention due to their global dissemination and/or particular pathogenic properties, e.g., association with multidrug resistance (MDR). The aim of this study was to evaluate the prevalence of the M. tuberculosis LAM family in Bulgaria based on the use of different molecular markers. A total of 101 previously spoligotyped M. tuberculosis strains were studied by LAM-specific PCR assay to detect an insertion of IS6110 in the specific genome region. On the whole, clear-cut results were obtained for most strains; spoligotype-based family was reassigned in some of them. At the same time, double bands were amplified in some cases and warrant further validation studies of this method. The higher MDR rate among LAM versus other genotype isolates was observed (P = 0.04). In conclusion, these results suggest a low (

The Bacillus subtilis genome contains several sets of paralogs. An extreme case is the four putative malic enzyme genes maeA , malS , ytsJ , and mleA. maeA was demonstrated to encode malic enzyme activity, to be inducible by malate, but also to be dispensable for growth on malate. We report systematic experiments to test whether these four genes ensure backup or cover different functions. Analysis of single- and multiple-mutant strains demonstrated that ytsJ has a major physiological role in malate utilization for which none of the other three genes could compensate. In contrast, maeA , malS , and mleA had distinct roles in malate utilization for which they could compensate one another. The four proteins exhibited malic enzyme activity; MalS, MleA, and MaeA exhibited 4- to 90-fold higher activities with NAD + than with NADP + . YtsJ activity, in contrast, was 70-fold higher with NADP + than with NAD + , with K m values of 0.055 and 2.8 mM, respectively. lacZ fusions revealed strong transcription of ytsJ , twofold higher in malate than in glucose medium, but weak transcription of malS and mleA . In contrast, mleA was strongly transcribed in complex medium. Metabolic flux analysis confirmed the major role of YtsJ in malate-to-pyruvate interconversion. While overexpression of the NADP-dependent Escherichia coli malic enzyme MaeB did not suppress the growth defect of a ytsJ mutant on malate, overexpression of the transhydrogenase UdhA from E. coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion.

To understand the mechanisms of ectoine-induced osmoprotection in Sinorhizobium meliloti , a proteomic examination of S. meliloti cells grown in minimal medium supplemented with ectoine was undertaken. This revealed the induction of 10 proteins. The protein products of eight genes were identified by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Five of these genes, with four other genes whose products were not detected on two-dimensional gels, belong to the same gene cluster, which is localized on the pSymB megaplasmid. Four of the nine genes encode the characteristic components of an ATP-binding cassette transporter that was named ehu , for ectoine/hydroxyectoine uptake. This transporter was encoded by four genes ( ehuA , ehuB , ehuC , and ehuD ) that formed an operon with another gene cluster that contains five genes, named eutABCDE for ectoine utilization. On the basis of sequence homologies, eutABCDE encode enzymes with putative and hypothetical functions in ectoine catabolism. Analysis of the properties of ehuA and eutA mutants suggests that S. meliloti possesses at least one additional ectoine catabolic pathway as well as a lower-affinity transport system for ectoine and hydroxyectoine. The expression of ehuB , as determined by measurements of UidA activity, was shown to be induced by ectoine and hydroxyectoine but not by glycine betaine or by high osmolality.

A DNA macroarray containing 465 intragenic amplicons was designed to identify Staphylococcus aureus at the species level and to type S. aureus isolates. The genes selected included those encoding (i) S. aureus -specific proteins, (ii) staphylococcal and enterococcal proteins mediating antibiotic resistance and factors involved in their expression, (iii) putative virulence proteins and factors controlling their expression, and (iv) proteins produced by mobile elements. The macroarray was hybridized with the cellular DNAs of 80 S. aureus clinical isolates that were previously typed by analyses of their antibiograms and SmaI patterns. The set selected contained unrelated, endemic, and outbreak-related isolates belonging to 45 SmaI genotypes. In a gene content dendrogram, the 80 isolates were distributed into 52 clusters. The outbreak-related isolates were linked in the same or a closely related cluster(s). Clustering based on gene content provided a better discrimination than SmaI pattern analysis for the tested mecA + isolates that were endemic to Europe. All of the antibiotic resistance genes detected could be correlated with their corresponding phenotypes, except for one isolate which carried a mecA gene without being resistant. The 16 isolates responsible for bone infections were distinguishable from the 12 isolates from uninfected nasal carriers by a significantly higher prevalence of the sdrD gene coding for a putative SD (serine-aspartate) adhesin (in 15 and 7 isolates, respectively). In conclusion, the macroarray designed for this study offers an attractive and rapid typing method which has the advantage of providing additional information concerning the gene content of the isolate of interest.

International audience; In the field of tuberculosis, the challenge is to interpret genome information in the context of the host–pathogen interaction, and then translate the resulting knowledge into a new generation of drugs and vaccines with which to arm ourselves in the fight against this ancient disease.

International audience; Prochlorococcus, the most abundant genus of photosynthetic organisms, owes its remarkably large depth distribution in the oceans to the occurrence of distinct genotypes adapted to either low- or high-light niches. The pcb genes, encoding the major chlorophyll-binding, light-harvesting antenna proteins in this genus, are present in multiple copies in low-light strains but as a single copy in high-light strains. The basis of this differentiation, however, has remained obscure. Here we show that the moderate low-light-adapted strain Prochlorococcus sp. MIT 9313 has one iron-stress-induced pcb gene encoding an antenna protein serving photosystem I (PSI)--comparable to isiA genes from cyanobacteria--and a constitutively expressed pcb gene encoding a photosystem II (PSII) antenna protein. By comparison, the very low-light-adapted strain SS120 has seven pcb genes encoding constitutive PSI and PSII antennae, plus one PSI iron-regulated pcb gene, whereas the high-light-adapted strain MED4 has only a constitutive PSII antenna. Thus, it seems that the adaptation of Prochlorococcus to low light environments has triggered a multiplication and specialization of Pcb proteins comparable to that found for Cab proteins in plants and green algae.

Size determination represents a fundamental requirement for multicomponent biological structures. Some pathogenic bacteria possess a weapon derived from the flagellum. Like the flagellum, this type-III secretion apparatus, called the injectisome, has a transmembrane basal body, but the external component is a needle-like structure instead of a hook and a filament. Here, we provide evidence that the length of this needle is determined by the size of a protein, YscP, acting as a molecular ruler.

International audience; Phospholipases C play a role in the pathogenesis of several bacteria. Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses four genes encoding putative phospholipases C, plcA, plcB, plcC and plcD. However, the contribution of these genes to virulence is unknown. We constructed four single mutants of M. tuberculosis each inactivated in one of the plc genes, a triple plcABC mutant and a quadruple plcABCD mutant. The mutants all exhibited a lower phospholipase C activity than the wild-type parent strain, demonstrating that the four plc genes encode a functional phospholipase C in M. tuberculosis. Functional complementation of the Delta plcABC triple mutant with the individual plcA, plcB and plcC genes restored in each case about 20% of the total Plc activity detected in the parental strain, suggesting that the three enzymes contribute equally to the overall Plc activity of M. tuberculosis. RT-PCR analysis of the plc genes transcripts showed that the expression of these genes is strongly upregulated during the first 24 h of macrophage infection. Moreover, the growth kinetics of the triple and quadruple mutants in a mouse model of infection revealed that both mutants are attenuated in the late phase of the infection emphasizing the importance of phospholipases C in the virulence of the tubercle bacillus.

International audience; Members of a family of ATP-dependent proteases related to Lon from Escherichia coli are present in most prokaryotes and eukaryotes. These proteases are generally reported to be heat induced, and various regulatory systems have been described. The authors cloned and disrupted the lon gene and studied the regulation of its expression in Streptomyces lividans. lon is negatively regulated by the HspR/HAIR repressor/operator system, suggesting that Lon is produced concomitantly with the other members of this regulon, DnaK and ClpB. The lon mutant grew more slowly than the wild-type and spore germination was impaired at high temperature. Nevertheless its cell cycle was not greatly affected and it sporulated normally.

Pseudomonas sp. strain KIE171 was able to grow with isopropylamine or l -alaninol [ S -(+)-2-amino-1-propanol] as the sole carbon source, but not with d -alaninol. To investigate the hypothesis that l -alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn 5 mutants unable to utilize both isopropylamine and l -alaninol were isolated. Whereas mutant KIE171-BI transformed isopropylamine to l -alaninol, mutant KIE171-BII failed to do so. The two genes containing a transposon insertion were cloned, and the DNA regions flanking the insertions were sequenced. Two clusters, one comprising eight ipu (isopropylamine utilization) genes ( ipuABCDEFGH ) and the other encompassing two genes ( ipuI and orf259 ), were identified. Comparisons of sequences of the deduced Ipu proteins and those in the database suggested that isopropylamine is transported into the cytoplasm by a putative permease, IpuG. The next step, the formation of γ-glutamyl-isopropylamide from isopropylamine, ATP, and l -glutamate, was shown to be catalyzed by IpuC, a γ-glutamylamide synthetase. γ-Glutamyl-isopropylamide is then subjected to stereospecific monooxygenation by the hypothetical four-component system IpuABDE, thereby yielding γ-glutamyl- l -alaninol [γ( l -glutamyl)- l -hydroxy-isopropylamide]. Enzymatic hydrolysis by a hydrolase, IpuF, was shown to finally liberate l -alaninol and to regenerate l -glutamate. No gene(s) encoding an enzyme for the next step in the degradation of isopropylamine was found in the ipu clusters. Presumably, l -alaninol is oxidized by an alcohol dehydrogenase to yield l -2-aminopropionaldehyde or it is deaminated by an ammonia lyase to propionaldehyde. Genetic evidence indicated that the aldehyde formed is then further oxidized by the hypothetical aldehyde dehydrogenases IpuI and IpuH to either l -alanine or propionic acid, compounds which can be processed by reactions of the intermediary metabolism.

Clostridium perfringensis a ubiquitous gram-positive pathogen that is present in the air, soil, animals, and humans. AlthoughC. perfringensis strictly anaerobic, vegetative and stationary cells can survive in a growth-arrested stage in the presence of oxygen and/or low concentrations of superoxide and hydroxyl radicals. Indeed, it possesses an adaptive response to oxidative stress, which can be activated in both aerobic and anaerobic conditions. To identify the genes involved in this oxidative stress response,C. perfringensstrain 13 mutants were generated by Tn916insertional mutagenesis and screened for resistance or sensitivity to various oxidative stresses. Three of the 12 sensitive mutants examined harbored an independently inserted single copy of the transposon in the same operon as two genes orthologous to theydaDandycdFgenes ofBacillus subtilis, which encode a putative NADPH dehydrogenase. Complementation experiments and knockout experiments demonstrated that these genes are both required for efficient resistance to oxidative stress inC. perfringensand are probably responsible for the production of NADPH, which is required for maintenance of the intracellular redox balance in growth-arrested cells. Other Tn916disrupted genes were also shown to play important roles in the oxidative stress response. This is the first time that some of these genes (e.g., a gene encoding an ATP-dependent RNA helicase, the β-glucuronidase gene, and the gene encoding the atypical iron sulfur prismane protein) have been shown to be involved in the oxidative response.

Sub-inhibitory concentrations (sub-MIC) of antibiotics play a very important role in selection and development of resistances. Unlike Escherichia coli, Vibrio cholerae induces its SOS response in presence of sub-MIC aminoglycosides. A role for oxidized guanine residues was observed, but the mechanisms of this induction remained unclear. To select for V. cholerae mutants that do not induce low aminoglycoside-mediated SOS induction, we developed a genetic screen that renders induction of SOS lethal. We identified genes involved in this pathway using two strategies, inactivation by transposition and gene overexpression. Interestingly, we obtained mutants inactivated for the expression of proteins known to destabilize the RNA polymerase complex. Reconstruction of the corresponding mutants confirmed their specific involvement in induction of SOS by low aminoglycoside concentrations. We propose that DNA lesions formed on aminoglycoside treatment are repaired through the formation of single-stranded DNA intermediates, inducing SOS. Inactivation of functions that dislodge RNA polymerase leads to prolonged stalling on these lesions, which hampers SOS induction and repair and reduces viability under antibiotic stress. The importance of these mechanisms is illustrated by a reduction of aminoglycoside sub-MIC. Our results point to a central role for transcription blocking at DNA lesions in SOS induction, so far underestimated.

Cerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health. We have previously described a Bacillus subtilis cerulenin-resistant strain, expressing a point-mutated condensing enzyme FabF (FabF[I108F]) (i.e. FabF with isoleucine 108 substituted by phenylalanine). We now report the crystal structures of wild-type FabF from B. subtilis, both alone and in complex with cerulenin, as well as of the FabF[I108F] mutant protein. The three-dimensional structure of FabF[I108F] constitutes the first atomic model of a condensing enzyme that remains active in the presence of the inhibitor. Soaking the mycotoxin into preformed wild-type FabF crystals allowed for noncovalent binding into its specific pocket within the FabF core. Interestingly, only co-crystallization experiments allowed us to trap the covalent complex. Our structure shows that the covalent bond between Cys163 and cerulenin, in contrast to that previously proposed, implicates carbon C3 of the inhibitor. The similarities between Escherichia coli and B. subtilis FabF structures did not explain the reported inability of ecFabF[I108F] (i.e. FabF from Escherichia coli with isoleucine 108 substituted by phenylalanine) to elongate medium and long-chain acyl-ACPs. We now demonstrate that the E. coli modified enzyme efficiently catalyzes the synthesis of medium and long-chain ketoacyl-ACPs. We also characterized another cerulenin-insensitive form of FabF, conferring a different phenotype in B. subtilis. The structural, biochemical and physiological data presented, shed light on the mechanisms of FabF catalysis and resistance to cerulenin. Database Crystallographic data (including atomic coordinates and structure factors) have been deposited in the Protein Data Bank under accession codes 4LS5, 4LS6, 4LS7 and 4LS8.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the “ntrPR operon,” which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.