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Subverting the host immune system is a major task for any given pathogen to assure its survival and proliferation. For the opportunistic human pathogen Bacillus cereus (Bc), immune evasion enables the establishment of potent infections. In various species of the Bc group, the pleiotropic regulator PlcR and its cognate cell--cell signaling peptide PapR7 regulate virulence gene expression in response to fluctuations in population density, i.e., a quorum-sensing (QS) system. However, how QS exerts its effects during infections and whether PlcR confers the immune evading ability remain unclear. Herein, we report how interception of the QS communication in Bc obliterates the ability to affect the host immune system. Here, we designed a peptide-based QS inhibitor that suppresses PlcR- dependent virulence factor expression and attenuates Bc infectivity in mouse models. We demonstrate that the QS peptidic inhibitor blocks host immune system--mediated eradication by reducing the expression of PlcR-regulated major toxins similarly to the profile that was observed for isogenic strains. Our findings provide evidence that Bc infectivity is regulated by QS circuit-mediated destruction of host immunity, thus reveal a interesting strategy to limit Bc virulence and enhance host defense. This peptidic quorum-quenching agent constitutes a readily accessible chemical tool for studying how other pathogen QS systems modulate host immunity and forms a basis for development of anti-infective therapeutics. [ABSTRACT FROM AUTHOR]

Bacillus cereus EntFM displays an NlpC/P60 domain, characteristic of cell wall peptidases. The protein is involved in bacterial shape, motility, adhesion to epithelial cells, biofilm formation, vacuolization of macrophages, and virulence. These data provide new information on this, so far, poorly studied toxin and suggest that this protein is a cell wall peptidase, which we propose to rename CwpFM. doi: 10.1128/JB.01315-09

The virulence of Bacillus cereus requires that bacteria have the capacity to colonize their host, degrade specific tissues, and circumvent the host immune system. To study this aspect of pathogenesis, we focused on three metalioproteases, InhA1, InhA2, and InhA3, which share more than 66% identity. The expression of these metalloprotease genes was assessed by transcriptional fusions with a lacZ reporter gene. The expression profiles suggest a complementary time course of InhA production. Indeed, the genes are simultaneously expressed but are oppositely controlled during stationary phase. We constructed single and multiple inhA mutants and assessed the bacterial locations of the proteins as well as their individual or additive roles in macrophage escape and toxicity, antibacterial-peptide cleavage, and virulence. InhA1, a major component of the spore exosporium, is the only InhA metalloprotease involved in bacterial escape from macrophages. A mutant lacking inhA1, inhA2, and inhA3 shows a strong decrease in the level of virulence for insects. Taken together, these results show that the InhA metalloproteases of B. cereus are important virulence factors that may allow the bacteria to counteract the host immune system. doi: 10.1128/JB.00264-09

The novel protein MclX (mother cell lysis X) in Bacillus thuringiensis subsp. kurstaki strain HD73 (B. thuringiensis HD73) was characterized in this work. MclX has no known domain and its gene deletion in HD73 resulted in Cry1Ac encapsulation in the mother cell and did not influence Cry1Ac protein production or insecticidal activity. In vitro cell wall hydrolysis experiments showed that MclX cannot hydrolyze the cell wall. In mclX deletion mutants, the expression of cwlC (which encodes a key cell wall hydrolase) was significantly decreased, as shown by the β-galactosidase activity assay. MclX cannot directly bind to the cwlC promoter, based on the electrophoretic mobility shift assay (EMSA). The cwlC was reported to be regulated by σK and GerE. However, the transcriptional activities of sigK and gerE showed no difference between HD73 and the mclX deletion mutant. It is indicated that MclX influenced cwlC expression independently of σK or GerE, through a new pathway to regulate cwlC expression. mclX deletion could be a new approach for insecticidal protein encapsulation in Bacillus thuringiensis. [ABSTRACT FROM AUTHOR]

Several approaches are used to characterize Bacillus thuringiensis FlhA mutant and elucidate an eventual relationship between motility, secretion of virulence factors and pathogenesis. The results have indicated that the transcription of many PlcR-regulated virulence factors is coordinated with the flagellar apparatus and hence, FlhA appears to be an essential virulence factor with a pleiotropic role.

The expression of extracellular virulence factors in various species of the Bacillus cereus group is controlled by the plcR and papR genes, which encode a transcriptional regulator and a cell-cell signaling peptide, respectively. A processed form of PapR, presumably a pentapeptide, specifically interacts with plcR to facilitate its binding to its DNA targets. This activating mechanism is strain specific, with this specificity being determined by the first residue of the pentapeptide. We carried out in vivo complementation assays and compared the PlcR-PapR sequences of 29 strains from the B. cereus group. Our findings suggested that the fifth amino acid of the pentapeptide is also involved in the specificity of activation. We identified four classes of PlcR-PapR pairs, defining four distinct pherotypes in the B. cereus group. We used these findings to look at the evolution of the PlcR-PapR quorum-sensing system with regard to the phylogeny of the species forming the B. cereus group.

The cytotoxin CytK produced by Bacillus cereus is believed to be involved in food-borne diseases. The transcriptional activity of the cytK promoter region in a food-poisoning strain was studied using a reporter gene and compared with that in the reference B. cereus strain ATCC 14579. In the food-poisoning strain, cytK is more strongly transcribed, possibly explaining the pathogenicity. The global regulator PlcR in B. cereus controls several putative virulence factors. It was found that PlcR regulates cytK in this clinical strain despite a mismatch in the PlcR recognition site, as currently defined. This suggests that the PlcR box consensus should be reconsidered and that the PlcR regulon might be larger than suspected. It is also shown that the high level of cytK transcription is not caused by a modification in the PlcR recognition site.

The identification and characterization of two genes, yqgB and yqfZ, of unknown function are reported. The findings suggest that the yqgB and ygfZ genes encode adaptive factors that may act in synergy, enabling the bacteria to cope with the physical environment in vivo, facilitating colonization of the host.

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.sup.-] [Lec.sup.-] 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.

[beta]-Exotoxin I is an insecticidal nucleotide analogue secreted by various Bacillus thuringiensis strains. In this report, we describe the characterization and transcriptional analysis of a gene cluster, designated sigW-ecfX-ecfY, that is essential for [beta]-exotoxin I production in B. thuringiensis subsp, thuringiensis strain 407-1. In this strain, the disruption of the sigW cluster resulted in nontoxic culture supernatants. sigW encodes a protein of 177 residues that is 97 and 94% identical to two putative RNA polymerase extracytoplasmic-function-type sigma factors from Bacillus anthracis strain Ames and Bacillus cereus strain ATCC 14579, respectively. It is also 50, 30, and 26% identical to SigW from Clostridium perfringens and SigW and SigX from Bacillus subtilis, respectively. EcfX, encoded by the gene following sigW, significantly repressed the expression of sigW when both genes were overtranscribed, suggesting that it could be the anti-sigma factor of SigW. Following the loss of its curable cry plasmid, strain 407 became unable to synthesize crystal toxins, in contrast to the mutant strain 407-1([Cry.sup.-])([Pig.sup.+]), which overproduced this molecule in the absence of this plasmid. Transcriptional analysis of sigW indicated that this gene was expressed during the stationary phase and only in the 407-1 ([Cry.sup.-])([Pig.sup.+]) mutant. This suggests that in the wild type-407([Cry.sup.+]) strain, [beta]-exotoxin I was produced from determinants located on a cry gene-bearing plasmid and that sigW is able to induce [beta]-exotoxin I production in B. thuringiensis in the absence of cry gene-bearing plasmids. Although the signal responsible for this activation is unknown, these results indicate that [beta]-exotoxin I production in B. thuringiensis can be restored or induced via an alternative pathway that requires sigW expression.

Research has been conducted on Bacillus thuringiensis mutant. The characterization of this mutant which is defective in flagellum gilament assembly, in swimming and swarming motility and in produvtion of hemolysin BL has been carried out and is described.

Research has been conducted on Bacillus thuringiensis strains. Bacillus thuringiensis acrystalliferrous mutant strain which produces brown soluble pigment identified as beta-exotoxin I has been investigated, and the results demonstrate that the genes involed in beta-exotoxin I production have been identified via the use of the random mutant library and the mini-transposon delivery system.

Gene mutation studies demonstrate that the clpP1 and clpP2 genes encoded proteins control distinct and separate regulatory pathways in Bacillus thuringiensis. Bacteria carrying mutations in both the genes show susceptibility to salt stress and differences in virulence, whereas mutation only in clpP2 affects sporulation and cell motility.

Results show that the Bacillus thuringiensis InhA2 zinc metalloprotease plays a significant part in promoting virulence when the host is infected orally as indicated by the ineffectivity of the spores of the inhA2 deletion mutant strains and effectivity of the inhA deletion mutants in providing synergism to infection.

Research has been conducted on Bacillus cereus and Bacillus thuringiensis sympatric strains. The isolation of these strains from different geographic sites has been carried out in investigating genetic structure of B. cereus and B. thuringiensis populations and in exploring the intra- and interspecific genetic exchange and the results are discussed.

Genes with a role in the activation of the Bacillus thuringiensis immune inhibitor A (inhA) metalloprotease gene at the onset of sporulation have been identified. Study has shown that the major regulator of inhA expression is AbrB. It acts as a repressor of inhA transcription.

Results show that pIcR gene involved in encoding pleiotropic regulator of extracellular factors, controls pathogenicity of Bacillus thuringiensis and B. cereus as shown in an insect strain and mice. Data further indicate that bacterial spores also contribute to the overall entomopathogenicity.

Research was conducted to characterize the PlcR-regulated three virulence gene loci employing cloning and sequencing techniques. The data from analyzing a 37.1 kb DNA sequence surrounding hbl, plcA and plcR genes indicated they were not involved in the virulence process.

The development of, and field trials conducted on, a novel recombinant Bacillus thuringiensis strain that produces large amounts of two different crystal proteins are reported. It was demonstrated that encapsulation of the toxins within the cell protects them from deactivation by UV radiation. The strain was found to be a sporulation-deficient mutant that cannot survive in the environment, thus, minimizing any environmental effects that arise from the dissemination of large numbers of viable spores.

The plasmid pAW63, the minor plasmid of the two large plasmids of Bacillus thuringiensis subspecies kurstaki strain HD73 was shown to be self-transmissible. Further, it can be transmitted to the other species of the genus Bacillus. The plasmid was tagged with the tetracycline resistance gene and was observed to be transmitted to all recipients within two hours. Bacillus thuringiensis subspecies kurstaki strain HD73 colonizes the larvae of lepidopterans.

A TnpI-mediated site-specific recombinant system removes antibiotic resistance gene markers from genetically modified Bacillus thuringiensis strains. The system introduces a cryIC gene, that is active against Spodoptera littoralis, into Bacillus thuringiensis Kto containing cryIA(c) gene, that is active against Ostrinia nubilalis. This broadens the activity spectrum of the parent strain. The method can facilitate the development of B. thuringiensis into a commercial biopesticide.

CryIIIA gene expression in Bacillus thuringiensis is independent of the key sporulation-specific sigma factors. This was gleaned from a genetic analysis of cryIIIA expression in Bt. It was also found that a different mechanism than that of most of the other cry genes regulates the cryIIIA gene. Unlike the other classes of cry genes, its expression starts during late-exponential growth, and not during sporulation. CryIIIA expression is also increased in a spoOA genetic framework.

A Bacillus thuringiensis trans activator gene, plcR, auto regulates its own expression and the transcription of the phosphatidylinositol-specific phospholipase C (plcA) gene. The gene is active at the beginning of the stationary phase in B. thuringiensis and B. subtilis and has a putative coding sequence of 855-bp which encodes a protein, PlcR of molecular weight 33762. A highly conserved DNA sequence has been located, which is the likely recognition target for activation of PlcR.

The different ways by which Bacillus thuringiensis accumulates large amounts of insecticidal protein crystals, used as biopesticides, are examined. B. thuringiensis creates protease-resistant inclusion bodies which are made easily digestible in the insect stomach, by several factors, among which are the secondary structure of the Cry proteins, the energy of the disulfide bonds, and the presence of additional chemicals.

Use of primer extension analysis method and lacZ fusions to study Bacillus thuringiensis cryIIIA toxin gene of B. subtilis reveals that the toxin gene expression is independent of sporulation-specific sigma factors sigma(super E) and sigma(super H), and that B. subtilis strains lacking the two factors exhibit normal toxin genes expression. The cryIIIA expression is high in the spoOA mutants that also display strong cryIIIA-mediated beta-galactosidase activities. The gene expression in spoOA mutants develops into a rectangular crystal structure resembling Coleoptera-specific B. thuringiensis strain structure.