Identification of CodY targets in Bacillus anthracis by genome-wide in vitro binding analysis.

In Gram-positive bacteria, CodY is an important regulator of genes whose expression changes under conditions of nutrient limitation. Bacillus anthracis CodY represses or activates directly or indirectly approximately 500 genes. Affinity purification of CodY-DNA complexes was used to identify the direct targets of CodY. Of the 389 DNA binding sites that were copurified with CodY, 132 sites were in or near the regulatory regions governing the expression of 197 CodY-controlled genes, indicating that CodY controls many other genes indirectly. CodY-binding specificity was verified using electrophoretic mobility shift and DNase I footprinting assays for three CodY targets. Analysis of the bound sequences led to the identification of a B. anthracis CodY-binding consensus motif that was found in 366 of the 389 affinity-purified DNA regions. Regulation of the expression of the two genes directly controlled by CodY, sap and eag, encoding the two surface layer (S-layer) proteins, was analyzed further by monitoring the expression of transcriptional lacZ reporter fusions in parental and codY mutant strains. CodY proved to be a direct repressor of both sap and eag expression. Since the expression of the S-layer genes is under the control of both CodY and PagR (a regulator that responds to bicarbonate), their expression levels respond to both metabolic and environmental cues.

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

Bacillus anthracis cell envelope components.

Bacillus anthracis is a Gram-positive bacterium harboring a complex parietal architecture. The cytoplasmic membrane is surrounded by a thick peptidoglycan of the A1 gamma type. Only one associated polymer, a polysaccharide composed of galactose, N-acetylglucosamine, and N-acetylmannosamine, is covalently linked to the peptidoglycan. Outside the cell wall is an S-layer. Two proteins can each compose the S-layer. They are noncovalently anchored to the cell wall polysaccharide by their SLH N-terminal domain. The poly-gamma-D-glutamate capsule, which covers the S-layer, has an antiphagocytic role and its synthesis is dependent on environmental factors mimicking the mammalian host, such as bicarbonate and a temperature of 37 degrees C.

Highly virulent M1 Streptococcus pyogenes isolates resistant to clindamycin.

CONTEXT: Emm1-type group A Streptococcus (GAS), or Streptococcus pyogenes, is mostly responsible for invasive infections such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The recommended treatment of severe invasive GAS infections is a combination of clindamycin and penicillin. Until 2012, almost all emm1 isolates were susceptible to clindamycin. OBJECTIVES: We aimed to identify the phenotypic and genotypic characteristics of emm1 GAS clone resistant to clindamycin. METHODS: GAS strains were characterized by emm sequence typing, detection of genes encoding pyrogenic exotoxins or superantigens. Cluster analysis was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antibiotic susceptibility was assessed using disk diffusion and resistance genes were detected by PCR. RESULTS: A total of 1321 GAS invasive isolates were analyzed between January 2011 and December 2012. The overall number of invasive isolates resistant to clindamycin was 52 (3.9%); seven of them were emm1 isolates. All isolates had the same genomic markers: macrolide resistance due to the presence of the erm(B) gene, emm subtype 1.0, the same toxin or superantigen profile, PFGE pattern and sequence type. CONCLUSION: This is the first description of highly virulent GAS emm1 isolates resistant to clindamycin in France. This article strengthens the need for monitoring the epidemiology of invasive GAS strains as they could lead to changes in treatment guidelines.

Group B streptococcus neonatal invasive infections, France 2007-2012.

Streptococcus agalactiae (group B streptococcus (GBS)) is the leading cause of invasive infections among newborns in industrialized countries, with two described syndromes: early-onset disease (EOD) and late-onset disease (LOD). Since the introduction in many countries of intrapartum antibioprophylaxis (IAP), the incidence of EOD has dramatically decreased, whereas that of LOD remains unchanged. We describe the clinical and bacteriological characteristics of 438 GBS neonatal invasive infections notified to the French National Reference Centre for Streptococci in France from 2007 to 2012. Clinical data were retrieved from hospitalization reports or questionnaires. Capsular type, assignment to the hypervirulent clonal complex (CC)17 and antibiotic susceptibility profiles were determined. One hundred and seventy-four (39.7%) and 264 (60.3%) isolates were responsible for EOD, including death in utero, and LOD, respectively. EOD was associated with bacteraemia (n = 103, 61%) and LOD with meningitis (n = 145, 55%). EOD was mainly due to capsular polysaccharide (CPS) III isolates (n = 99, 57%) and CPS Ia isolates (n = 40, 23%), and CPS III isolates were responsible for 80% (n = 211) of LOD cases. CC17 accounted for 80% (n = 121) of CPS III isolates responsible for meningitis (n = 151; total cases of meningitis, 188). Bad outcome risk factors were low gestational age and low birthweight. LOD represents almost 60% of cases of neonatal GBS disease in France and other countries in which IAP has been implemented. This observation reinforces the need to develop new prevention strategies targeting CC17, which is predominant in GBS neonatal infections.

Nucleotide sequence of the sucrase gene of Bacillus subtilis.

The sucrase gene (sacA) and part of the sacP locus, which corresponds to a membrane component of the phosphotransferase system (PTS) of sucrose transport of Bacillus subtilis, were previously cloned on a 2.1-kb EcoRI DNA fragment. Genes sacA and sacP were localized on this DNA fragment and the nucleotide sequence of the 2.1-kb DNA fragment was determined. A 1440-bp open reading frame (480 codons) was identified coding for a deduced polypeptide of Mr54827, which corresponds to that of purified sucrase. The amino acid sequence shares homology with that of yeast invertase (SUC2 gene product). The sacA gene and the preceding sacP gene seem to belong to the same operon.

Molecular tools for the study of transcriptional regulation in Bacillus anthracis.

Bacillus anthracis produces two toxins composed of three proteins. Genetic tools were constructed to study the regulation of toxin synthesis. They included transcriptional fusions with various reporter genes, in replicative and integrative vectors. The reporter gene xylE, encoding catechol 2,3-dioxygenase, may be valuable for screening of strong promoters, as expression of the gene can be visualized directly and the studies of regulation in B. anthracis. Therefore, transcriptional fusions between a lacZ reporter gene and the toxin genes were constructed. Experiments with a multicopy plasmid in trans suggested that the transcriptional activator(s) of the toxin genes were not titrated. B. anthracis strains, which contain pXO1 carrying multiple copies of fusions, were analysed. Expression of the reporter gene was proportional to the fusion copy number. Indeed, single integration of a suicide plasmid can be distinguished from multiple integration according to the level of resistance to an appropriate antibiotic. Finally, recombination in B. anthracis was found to be very efficient (approximately 10(-2) recombinants per transconjugant cell.

AtxA activates the transcription of genes harbored by both Bacillus anthracis virulence plasmids.

Fully virulent Bacillus anthracis bacilli are encapsulated and toxinogenic. These bacteria carry two plasmids, pXO1, and pXO2, encoding toxins and capsule synthetic-enzymes (capB, C, A, dep), respectively. The PXO1 plasmid strongly enhances capsule formation. This influence was studied by analysing the expression of a capB-lacZ fusion in various backgrounds. The beta-galactosidase activities were similar in a delta atxA strain and a pXO1 cured strain. Moreover, the capB-lacZ expression level could be restored, in a pXO1 cured strain, by addition of atxA in trans. Thus, we conclude that the pX01 influence on capsule synthesis is mediated by AtxA, the pXO1-encoded trans-activator of the toxin gene expression.

Regulation of pag gene expression in Bacillus anthracis: use of a pag-lacZ transcriptional fusion.

The production of protective antigen (PA), the common component of the two anthrax toxins, is influenced by the environment. In order to examine factors involved in its regulation, a transcriptional fusion between the promoter region of the PA gene (pag) and the lacZ gene was constructed and introduced into Bacillus anthracis Sterne. Activity of the pag promoter was followed by measuring beta-galactosidase activities under various growth and medium conditions. Expression from the pag promoter was observed throughout exponential-phase and was maximal in early stationary phase. The activity of the pag promoter was stimulated by the addition of glucose in the medium.

Co-existence of clpB and clpC in the Bacillaceae.

The gene encoding ClpC in Bacillus anthracis was amplified from the chromosome by polymerase chain reaction using degenerate oligonucleotide primers. These primers also amplified a second DNA fragment identified as a clpB homolog. Both genes were suggested to be functional. Contrary to Bacillus subtilis which possesses clpC but not clpB, many Bacillus species were found to harbor both clpC and clpB. We also found that Clostridium strains could possess clpB, clpC, or both. All the Gram-negative strains tested had clpB only.

[Epidemiology of Streptococcus pyogenes invasive diseases in France (2007-2011)]. / Épidémiologie des infections invasives à Streptococcus pyogenes (France 2007-2011).

Group A Streptococcus (GAS) is a human pathogen responsible for a wide range of clinical manifestations. An increase of GAS invasive infections has been described since the mid 1980s. To study the French epidemiology of invasive infections (i) we characterized all GAS invasive strains received at the French National Reference Center for streptococci (CNR-Strep) between 2007 and 2011; (ii) we analyzed the epidemiological data on the corresponding strains. For each strain, emm genotype, superantigen genes and antibiotics susceptibility were determined. Among the 2 603 non redundant invasive GAS strains, 65.1 % (n=1 695) were isolated from blood culture. A streptococcal toxic shock syndrome (STSS) was described in 16.4 % (n=428) of cases, mostly associated with necrotizing fasciitis (NF), pleuropulmonary or osteoarticular infections (p ≤0.001). The case fatality rate was 10.6 %. A total of 102 different emm genotypes were identified. Three emm genotypes predominated, reaching nearly 60 % of the strains: emm 1 (26.7 %), emm 28 (16.4 %), and emm 89 (12.8 %). The proportion of each emm genotype varied according to the year and the age of patients. Among those < 15 years old, the three main genotypes were emm 1 (36.8 %), emm 12 (12.9 %) and emm 4 (9.5 %). The distribution of superantigen genes (SpeA, SpeC and Ssa) was restricted to several emm genotypes. Between 2007 and 2011, the rate of macrolides resistant GAS strains decreased from 7.8 to 5.5 %. emm 1 strains are still the most common especially in most severe clinical manifestations including STSS and NF.

Differential influence of the two Bacillus anthracis plasmids on regulation of virulence gene expression.

Fully virulent Bacillus anthracis bacilli are encapsulated and toxinogenic. These bacteria contain two plasmids, pXO1 and pXO2, carrying genes coding for toxins (pag, lef, and cya) and for capsule synthetic enzymes (capB, capC, capA, and dep), respectively. A transcriptional fusion between the capB regulatory region and the lacZ reporter gene was constructed to study the regulation of capsule synthesis. A single copy of this fusion was inserted into the cap region of pXO2. The influence of environmental factors on the capB-lacZ fusion expression was initially analyzed in a pXO1-negative background: bicarbonate but not temperature induced the transcription from the capB promoter. A strain carrying the recombinant pXO2 and (delta)pag pXO1 was constructed for transregulatory studies. The pXO1 plasmid strongly enhanced capsule formation without modifying the bicarbonate-dependent induction level. A (delta)cap pXO2 was transduced into a strain containing pXO1 harboring a pag-lacZ transcriptional fusion (19). pXO2 showed no influence on the toxin gene transcription.

Anthrax.

Bacillus anthracis was shown to be the etiological agent of anthrax by R. Koch and L. Pasteur at the end of the nineteenth century. The concepts on which medical microbiology are based arose from their work on this bacterium. The link between plasmids and major virulence factors of B. anthracis was not discovered until the 1980s. The three toxin components are organized in two A-B type toxins, and the bacilli are covered by an antiphagocytic polyglutamic capsule. Structure-function analysis of the toxins indicated that the common B-domain binds to a ubiquitous cell receptor and forms a heptamer after proteolytic activation. One enzyme moiety is an adenylate cyclase and the other is a Zn(2+) metalloprotease, which is able to cleave MAPKKs. The capsule covers an S-layer sequentially composed of two distinct proteins. Knowledge of the toxins facilitates the design of safer veterinary vaccines. Spore-structure analysis could contribute to the improvement of human nonliving vaccines. The phylogeny of B. anthracis within the Bacillus cereus group is also reviewed.

Characterization of the operon encoding the alternative sigma(B) factor from Bacillus anthracis and its role in virulence.

The operon encoding the general stress transcription factor sigma(B) and two proteins of its regulatory network, RsbV and RsbW, was cloned from the gram-positive bacterium Bacillus anthracis by PCR amplification of chromosomal DNA with degenerate primers, by inverse PCR, and by direct cloning. The gene cluster was very similar to the Bacillus subtilis sigB operon both in the primary sequences of the gene products and in the order of its three genes. However, the deduced products of sequences upstream and downstream from this operon showed no similarity to other proteins encoded by the B. subtilis sigB operon. Therefore, the B. anthracis sigB operon contains three genes rather than eight as in B. subtilis. The B. anthracis operon is preceded by a sigma(B)-like promoter sequence, the expression of which depends on an intact sigma(B) transcription factor in B. subtilis. It is followed by another open reading frame that is also preceded by a promoter sequence similarly dependent on B. subtilis sigma(B). We found that in B. anthracis, both these promoters were induced during the stationary phase and induction required an intact sigB gene. The sigB operon was induced by heat shock. Mutants from which sigB was deleted were constructed in a toxinogenic and a plasmidless strain. These mutants differed from the parental strains in terms of morphology. The toxinogenic sigB mutant strain was also less virulent than the parental strain in the mouse model. B. anthracis sigma(B) may therefore be a minor virulence factor.

Cloning and expression in Escherichia coli of the sucrase gene from Bacillus subtilis.

A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank of E. coli harboring recombinant cosmids representative of the B. subtilis genome. It was shown that the sacA gene is located in a 2kb EcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for in B. subtilis and E. coli. Complementation of a sacA mutation was observed in Rec+ and REc- strains of B. subtilis. Expression of sucrase was also demonstrated in E. coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic in E. coli since the presence of the recombinant plasmids did not allow the transport of [U14C] sucrose and the growth of the cells. It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport of B. subtilis.

A target for carbon source-dependent negative regulation of the citB promoter of Bacillus subtilis.

Expression of the aconitase (citB) gene of Bacillus subtilis is subject to catabolite repression in cells grown in minimal media. In nutrient broth medium, citB expression is low in growing cells but is induced when cells enter sporulation. A 600-base-pair DNA fragment that extends from positions -400 through +200, relative to the transcription start site, was shown to include all of the cis-acting sequences necessary for catabolite repression and sporulation-associated regulation. This was demonstrated by fusing this DNA fragment to the Escherichia coli lacZ gene, integrating the fusion in the amyE locus of the B. subtilis chromosome, and measuring the regulation of expression of beta-galactosidase. By creating a series of deletions from either end of the 600-base-pair fragment, it was possible to define a target for catabolite repression; at least part of this target lies within the sequence between positions -84 and -68. DNA fragments that included positions -84 through +36, when carried on high-copy plasmids, caused derepression of aconitase synthesis, as if a negative regulator were being titrated. The same plasmids caused derepression of citrate synthase activity as well. Deletion of the sequence between positions -84 and -67 abolished this titration effect for both enzymes. Mutations that altered the target for catabolite repression also affected the inducibility of citB at the onset of sporulation, at least when sporulation was induced by the addition of decoyinine, an inhibitor of guanine nucleotide synthesis. When sporulation was induced by exhaustion of nutrient broth, there was no detectable difference in expression of citB-lacZ fusions whether or not they had the citB sequence from positions -84 to -67, suggesting that the mechanisms of regulation of citB in minimal medium and nutrient broth are different.

Characterization of the precursor form of the exocellular levansucrase from Bacillus subtilis.

Expression of the cloned levansucrase gene (sacB) was demonstrated in E. coli minicells by assay of the enzyme in crude extracts, SDS-polyacrylamide gel electrophoresis and immunoblotting. The existence of a precursor form of the enzyme of MW 53000 was also demonstrated and confirmed by the DNA sequence corresponding to the NH2 terminal region of the protein.

Bacillus subtilis sucrose-specific enzyme II of the phosphotransferase system: expression in Escherichia coli and homology to enzymes II from enteric bacteria.

Sucrose is transported into Bacillus subtilis cells by way of a phosphotransferase system, which consists of a specific enzyme II, a nonspecific enzyme I, and a histidine-containing phosphocarrier protein. Mutations in the sacP locus abolish the specific transport of sucrose. The B. subtilis sacP gene was cloned and expressed in Escherichia coli, and transformed cells could transport and phosphorylate sucrose. This indicates that the sacP gene product is enzyme II of the sucrose phosphotransferase system of B. subtilis. The nucleotide sequence of the sacP gene was determined and was found to overlap with the sacA gene at the tetranucleotide ATGA, which may allow a translational coupling between sacP and sacA. The two genes are therefore probably organized in an operon structure with the promoter located 5' to sacP gene. The deduced amino acid sequence gave a Mr of 48,945 for the sucrose-specific enzyme II polypeptide. The amino acid sequence was compared to that of three other known enteric bacterial enzymes II (beta-glucoside-specific enzyme II, mannitol-specific enzyme II, and glucose-specific enzyme II). Homology was found with beta-glucoside enzyme II, and well conserved regions were identified through the comparison of the proteins.

The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature.

The two Bacillus anthracis toxins are composed of three proteins, protective antigen, lethal factor, and edema factor. The structural genes for these three components are located on the virulence plasmid pXO1. We constructed transcriptional fusions between the regulatory region of each of these genes and lacZ. Each construct was then inserted as a single copy at the corresponding toxin gene locus on pXO1, resulting in three isogenic strains. Two environmental factors, bicarbonate and temperature, were found to induce beta-galactosidase synthesis in each recombinant strain. Furthermore, the transcription of the three toxin genes appears to be coordinately regulated.