Agnès Fouet

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—One Species on the Basis of Genetic Evidence

ABSTRACT Bacillus anthracis, Bacillus cereus, andBacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes thatB. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.

Bacterial SLH domain proteins are non‐covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation

Several bacterial proteins are non‐covalently anchored to the cell surface via an S‐layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a non‐covalent interaction between the SLH domain and peptidoglycan‐associated polymers. Here we report the characterization of a two‐gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH‐containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan‐associated polysaccharide fraction, and that this modification was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLH‐containing proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.

Poly‐gamma‐glutamate in bacteria

Poly‐γ‐glutamate (PGA), a natural polymer, is synthesized by several bacteria (all Gram‐positive), one archaea and one eukaryote. PGA has diverse biochemical properties, enabling it to play different roles, depending on the organism and its environment. Indeed, PGA allows bacteria to survive at high salt concentrations and may also be involved in virulence. The minimal gene sets required for PGA synthesis were recently defined. There are currently two nomenclatures depending on the PGA final status: cap, for ‘capsule’, when PGA is surface associated or pgs, for ‘polyglutamate synthase’, when PGA is released. The minimal gene sets contain four genes termed cap or pgs B, C, A and E. The PGA synthesis complex is membrane‐anchored and uses glutamate and ATP as substrates. Schematically, the reaction may be divided into two steps, PGA synthesis and PGA transport through the membrane. PGA synthesis depends primarily on CapB‐CapC (or PgsB‐PgsC), whereas PGA transport requires the presence, or the addition, of CapA‐CapE (or PgsAA‐PgsE). The synthesis complex is probably responsible for the stereochemical specificity of PGA composition. Finally, PGA may be anchored to the bacterial surface or released. An additional enzyme is involved in this reaction: either CapD, a γ‐glutamyl‐transpeptidase that catalyses anchorage of the PGA, or PgsS, a hydrolase that facilitates release. The anchoring of PGA to the bacterial surface is important for virulence. All cap genes are therefore potential targets for inhibitors specifically blocking PGA synthesis or anchorage.

Bacillus anthracis CapD, belonging to the γ-glutamyltranspeptidase family, is required for the covalent anchoring of capsule to peptidoglycan

Several examples of bacterial surface‐structure anchoring have been described, but they do not include polyglutamate capsule. Bacillus anthracis capsule, which is composed only of poly‐γ‐ d‐glutamate, is one of the two major virulence factors of the bacterium. We analysed its anchoring. We report that the polyglutamate is anchored directly to the peptidoglycan and that the bond is covalent. We constructed a capD mutant strain, capD being the fourth gene of the capsule biosynthetic operon. The mutant bacilli are surrounded by polyglutamate material that is not covalently anchored. Thus, CapD is required for the covalent anchoring of polyglutamate to the peptidoglycan. Sequence similarities suggest that CapD is a γ‐glutamyltranspeptidase. Furthermore, CapD is cleaved at the γ‐glutamyltranspeptidase consensus cleavage site, and the two subunits remain associated, as necessary for γ‐glutamyltranspeptidase activity. Other Gram‐positive γ‐glutamyltranspeptidases are secreted, but CapD is located at the Bacillus surface, associated both with the membrane and the peptidoglycan. Polyglutamate is hydrolysed by CapD indicating that it is a CapD substrate. We suggest that CapD catalyses the capsule anchoring reaction. Interestingly, the CapD– strain is far less virulent than the parental strain.

The incompatibility between the PlcR- and AtxA-controlled regulons may have selected a nonsense mutation in Bacillus anthracis

Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are members of the Bacillus cereus group. These bacteria express virulence in diverse ways in mammals and insects. The pathogenic properties of B. cereus and B. thuringiensis in mammals results largely from the secretion of non‐specific toxins, including haemolysins, the production of which depends upon a pleiotropic activator PlcR. In B. anthracis, PlcR is inactive because of a nonsense mutation in the plcR gene. This suggests that the phenotypic differences between B. anthracis on the one hand and B. thuringiensis and B. cereus on the other could result at least partly from loss of the PlcR regulon. We expressed a functional PlcR in B. anthracis. This resulted in the transcriptional activation of genes weakly expressed in the absence of PlcR. The transcriptional activation correlated with the induction of enzymatic activities and toxins including haemolysins. The toxicity of a B. anthracis PlcR+ strain was assayed in the mouse subcutaneous and nasal models of infection. It was no greater than that of the parental strain, suggesting that the PlcR regulon has no influence on B. anthracis virulence. The PlcR regulon had dramatic effects on the sporulation of a B. anthracis strain containing the virulence plasmid pXO1. This resulted from incompatible interactions with the major AtxA‐controlled virulence regulon. We propose that the PlcR‐controlled regulon in B. anthracis has been counterselected on account of its disadvantageous effects.

Production and cell surface anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S‐layer proteins and the Bacillus subtilis levansucrase

Many surface proteins of Gram‐positive bacteria contain motifs, about 50 amino acids long, called S‐layer homology (SLH) motifs. Bacillus anthracis, the causal agent of anthrax, synthesizes two S‐layer proteins, each with three SLH motifs towards the amino‐terminus. We used biochemical and genetic approaches to investigate the involvement of these motifs in cell surface anchoring. Proteinase K digestion produced polypeptides lacking these motifs, and stable three‐motif polypeptides were produced in Escherichia coli that were able to bind the B. anthracis cell walls in vitrodemonstrating that the three SLH motifs were organized into a cell surface anchoring domain. We also determined the function of these SLH domains by constructing chimeric genes encoding the SLH domains fused to the normally secreted levansucrase of Bacillus subtilis. Cell fractionation and electron microscopy studies showed that each three‐motif domain was sufficient for the efficient anchoring of levansucrase onto the cell surface. Proteins consisting of truncated SLH domains fused to levansucrase were unstable and associated poorly with the cell surface. Surface‐exposed levansucrase retained its enzymatic and antigenic properties.

CapE, a 47-Amino-Acid Peptide, Is Necessary for Bacillus anthracis Polyglutamate Capsule Synthesis

Polyglutamate is found in various bacteria, but displays different functions depending on the species and their environment. Here, we describe a minimal polyglutamate synthesis system in Bacillus anthracis. In addition to the three genes previously described as sufficient for polyglutamate synthesis, this system includes a small open reading frame, capE, belonging to the cap operon. The polyglutamate systems requirement for the five cap genes, for capsulation and anchoring, was assayed in nonpolar mutants. The capA, capB, capC, and capE genes are all necessary and are sufficient for polyglutamate synthesis by B. anthracis. capD is required for polyglutamate anchoring to the peptidoglycan. The 47-amino-acid peptide encoded by capE is localized in the B. anthracis membrane. It is not a regulator and it is required for polyglutamate synthesis, suggesting that it has a structural role in polyglutamate synthesis. CapE appears to interact with CapA. Bacillus subtilis ywtC is similar to capE and we named it pgsE. Genes similar to capE or pgsE were found in B. subtilis natto, Bacillus licheniformis, and Staphylococcus epidermidis, species that produce polyglutamate. All the bacterial polyglutamate synthesis systems analyzed show a similar genetic organization and, we suggest, the same protein requirements.

A plasmid-encoded regulator couples the synthesis of toxins and surface structures in Bacillus anthracis

Transcription of the major Bacillus anthracis virulence genes is triggered by CO2, a signal believed to reflect the host environment. A 180 kb plasmid, pXO1, carries the anthrax toxin genes and the genes responsible for their regulation, pagR and atxA; the latter encodes a major trans‐activator. It has long been known that pXO1 genes have major effects on the physiology of B. anthracis, probably through regulatory cross‐talk between plasmid and chromosomal genes. Accordingly, we found that the chromosomal S‐layer genes, sap and eag, are regulated by pXO1 genes so that only eag is significantly expressed in the presence of CO2. This effect results from the product of pagR acting as the most downstream element of a signalling cascade initiated by AtxA. In vitro evidence showed that PagR is a transcription factor that controls the S‐layer genes by direct binding on their promoter regions. This work provides evidence that AtxA is a master regulator that co‐ordinates the response to host signals by orchestrating positive and negative controls over genes located on all genetic elements.

Developmental switch of S-layer protein synthesis in Bacillus anthracis

Adjustment of the synthesis of abundant protein to the requirements of the cell involves processes critical to the minimization of energy expenditure. The regulation of S‐layer genes might be a good model for such processes because expression must be controlled, such that the encoded proteins exactly cover the surface of the bacterium. Bacillus anthracis has two S‐layer genes, sap and eag, encoding the S‐layer proteins Sap and EA1 respectively. We report that the production and surface localization of Sap and EA1 are under developmental control, suggesting that an exponential phase ‘Sap layer’ is subsequently replaced by a stationary phase ‘EA1 layer’. This switch is controlled at the transcriptional level: sap is most certainly transcribed by RNA polymerase containing σA, whereas eag expression depends on σH. More importantly, Sap is required for the temporal control of eag, and EA1 is involved in strict feedback regulation of eag. This control may be direct because both S‐layer proteins bind, in vitro, the eag promoter, specifically suggesting that they might act as transcriptional repressors.

Distinct mutations in PlcR explain why some strains of the Bacillus cereus group are nonhemolytic.

Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis are closely related species belonging to the Bacillus cereus group. B. thuringiensis and B. cereus generally produce extracellular proteins, including phospholipases and hemolysins. Transcription of the genes encoding these factors is controlled by the pleiotropic regulator PlcR. Disruption of plcR in B. cereus and B. thuringiensis drastically reduces the hemolytic, lecithinase, and cytotoxic properties of these organisms. B. anthracis does not produce these proteins due to a nonsense mutation in the plcR gene. We screened 400 B. thuringiensis and B. cereus strains for their hemolytic and lecithinase properties. Eight Hly- Lec- strains were selected and analyzed to determine whether this unusual phenotype was due to a mutation similar to that found in B. anthracis. Sequence analysis of the DNA region including the plcR and papR genes of these strains and genetic complementation of the strains with functional copies of plcR and papR indicated that different types of mutations were responsible for these phenotypes. We also found that the plcR genes of three B. anthracis strains belonging to different phylogenetic groups contained the same nonsense mutation, suggesting that this mutation is a distinctive trait of this species.