Detection and extraction of anti-Listerial compounds from Calligonum comosum, a medicinal plant from arid regions of Tunisia.

Calligonum comosum, a Tunisian plant from arid regions, is traditionally used in folk medicine to treat rural population microbial infections. The plant was investigated in vitro for its ability to inhibit the growth of Listeria ivanovii. Various aqueous and organic extracts were prepared from different plant tissues. Results indicated that ethanolic, methanolic and acetonic extracts from whole plant tissues except seeds, exhibited significant antibacterial activity with growth inhibition zones (9 - 18mm) as shown by the agar-well diffusion method. Minimum Inhibitory Concentration (MIC) of 0.65mg/ml was obtained in acetonic extract generated from C. comosum roots. Preliminary phytochemical analysis based on heat and protease treatments showed that bioactive extracts were stable up to 10m in heating at 100°C and that they resist protease digestion. Based on these latter results, the activity of organic extracts may be related to the presence of sterols, terpenoids, and/or phenolics. Overall, these results indicate that C. comosum organic extracts are probably useful in the control of food contamination by listerial species.

Identification of Bacillus cereus genes specifically expressed during growth at low temperatures.

The mechanisms involved in the ability of Bacillus cereus to multiply at low temperatures were investigated. It was assumed that many genes involved in cold acclimation would be upregulated at low temperatures. Recombinase-based in vivo expression technology (IVET) was adapted to the detection of the transient activation of B. cereus promoters during growth at 10 degrees C. Four independent screenings of a promoter library from type strain ATCC 14579 were performed, and 17 clones were isolated. They corresponded to 17 promoter regions that displayed reproducibly elevated expression at 10 degrees C relative to expression at 30 degrees C. This analysis revealed several genes that may be important for B. cereus to grow successfully under the restrictive conditions of cold habitats. Among them, a locus corresponding to open reading frames BC5402 to BC5398, harboring a lipase-encoding gene and a putative transcriptional regulator, was identified three times. While a mutation in the putative regulator-encoding gene did not cause any particular phenotype, a mutant deficient in the lipase-encoding gene showed reduced growth abilities at low temperatures compared with the parental strain. The mutant did not change its fatty acid profiles in the same way as the wild type when grown at 12 degrees C instead of 37 degrees C. This study demonstrates the feasibility of a promoter trap strategy for identifying cold-induced genes. It outlines a first picture of the different processes involved in B. cereus cold acclimation.

Identification of Bacillus cereus internalin and other candidate virulence genes specifically induced during oral infection in insects.

Bacillus cereus is an opportunistic bacterium frequently associated with food-borne infections causing gastroenteritis. We developed an in vivo expression technology (IVET), with an insect host, for identification of the B. cereus genes specifically expressed during infection. This IVET-based approach uses site-specific recombinase TnpI to identify transient promoter activation. We constructed a genomic library of B. cereus ATCC14579 by cloning DNA fragments upstream from tnpI. The library was screened in vivo by oral infection of the insect Galleria mellonella. We selected 100 clones from dead larvae. Sequencing of the inserts followed by a second screen for specific in vivo induction led to the identification of 20 in vivo-induced genes (ivi genes). They belonged to several different functional classes: regulation, metabolism, DNA repair and replication, cell division, transport, virulence and adaptation. A strongly induced gene, ivi29, was further analysed. It encodes an internalin-like protein with four distinct domains: an N-terminal signal peptide for export, a NEAT domain thought to be involved in iron transport, a leucine-rich repeat domain that may interact with host cells, and a C-terminal SLH domain presumably binding the protein to the peptidoglycan. As suggested by a Fur box in the promoter, transcriptional analysis showed ivi29 expression to be repressed by iron, suggesting that expression was induced in vivo due to iron deprivation in the host. This iron-regulated, leucine-rich surface protein was designated IlsA. Disruption of ilsA reduced the virulence of the bacteria to the insect larvae indicating its role in the overall pathogenesis of B. cereus.

The enhancin-like metalloprotease from the Bacillus cereus group is regulated by the pleiotropic transcriptional activator PlcR but is not essential for larvicidal activity.

Bacillus cereus group bacteria produce virulence factors. Many of these are regulated by the pleiotropic transcriptional activator PlcR, which is implicated in insect virulence. In silico analysis of the B. cereus strain ATCC14579 genome showed an enhancin-like gene preceded by a typical PlcR binding sequence. The gene is predicted to encode a polypeptide showing 23-25% identity with enhancins from several baculoviruses and 31% with that of Yersinia pestis. Viral enhancin acts after oral infection and degrades the peritrophic matrix of various Lepidopteran larvae. To rule out a possible implication of Bacillus enhancin in insect virulence, we sequenced the enhancin gene from the Bacillus thuringiensis 407-crystal minus strain and investigated its gene regulation and larvicidal activity. A typical metalloprotease zinc-binding domain (HEIAH) was detected and the gene was named mpbE (metalloprotease bacillus enhancin). An mpbE'-lacZ transcriptional fusion demonstrated that mpbE belongs to the PlcR regulon. The mpbE mutant was fed to Galleria mellonella larvae, and no significant reduction in virulence was observed. However, this may not exclude MpbE from a role in pathogenesis.

Characterization of two Bacillus thuringiensis genes identified by in vivo screening of virulence factors.

Bacillus thuringiensis vegetative cells are known to be highly pathogenic when injected into the hemocoel of susceptible insect larvae. This pathogenicity is due to the capacity of B. thuringiensis to cause septicemia in the host. We screened a B. thuringiensis mini-Tn10 insertion library for loss of virulence against Bombyx mori larvae on injection into the hemocoel. Three clones with attenuated virulence were isolated, corresponding to two different mini-Tn10 insertions mapping to the yqgB/yqfZ locus. Single disruptions of the yqgB and yqfZ genes did not affect virulence against B. mori. In contrast, the inactivation of both genes simultaneously reproduced the effect of the mini-Tn10 insertion and resulted in a significant delay to infection. The double DeltayqgB DeltayqfZ mutant was also nonmotile, and its growth was affected at 25 degrees C. We analyzed lacZ transcriptional fusions and detected promoter activity upstream from yqgB at 25 and 37 degrees C. Overall, our findings suggest that the yqgB and yqfZ 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.

The Bacillus thuringiensis PlcR-regulated gene inhA2 is necessary, but not sufficient, for virulence.

We previously reported that Bacillus thuringiensis strain 407 Cry 32(-) secretes a zinc-requiring metalloprotease, InhA2, that is essential for virulence in orally infected insects. Analysis of the inhA2-lacZ transcriptional fusion showed that inhA2 expression is repressed in a PlcR(-) background. Using DNase I footprinting experiments, we demonstrated that PlcR activates inhA2 transcription directly by binding to a DNA sequence showing a one-residue mismatch with the previously reported PlcR box. It was previously reported that PlcR is essential for B. thuringiensis virulence in oral infection by contributing to the synergistic properties of the spores on the insecticidal activity of the Cry1C protein. We used complementation experiments to investigate whether the PlcR(-) phenotype was due to the absence of InhA2. The results indicated that overexpression of inhA2 in the (Delta)plcR strain did not restore the wild-type phenotype. However, virulence was fully restored in the (Delta)inhA2 complemented mutant. Thus, inhA2 is the first example of a PlcR-regulated gene found to be directly involved in virulence. However, it is not sufficient for pathogenicity when the other members of the PlcR regulon are lacking. This suggests that InhA2 may act in concert with other PlcR-regulated gene products to provide virulence.

Distinct clpP genes control specific adaptive responses in Bacillus thuringiensis.

ClpP and ClpC are subunits of the Clp ATP-dependent protease, which is ubiquitous among prokaryotic and eukaryotic organisms. The role of these proteins in stress tolerance, stationary-phase adaptive responses, and virulence in many bacterial species has been demonstrated. Based on the amino acid sequences of the Bacillus subtilis clpC and clpP genes, we identified one clpC gene and two clpP genes (designated clpP1 and clpP2) in Bacillus thuringiensis. Predicted proteins ClpP1 and ClpP2 have approximately 88 and 67% amino acid sequence identity with ClpP of B. subtilis, respectively. Inactivation of clpC in B. thuringiensis impaired sporulation efficiency. The clpP1 and clpP2 mutants were both slightly susceptible to salt stress, whereas disruption of clpP2 negatively affected sporulation and abolished motility. Virulence of the clp mutants was assessed by injecting bacteria into the hemocoel of Bombyx mori larvae. The clpP1 mutant displayed attenuated virulence, which appeared to be related to its inability to grow at low temperature (25 degrees C), suggesting an essential role for ClpP1 in tolerance of low temperature. Microscopic examination of clpP1 mutant cells grown at 25 degrees C showed altered bacterial division, with cells remaining attached after septum formation. Analysis of lacZ transcriptional fusions showed that clpP1 was expressed at 25 and 37 degrees C during the entire growth cycle. In contrast, clpP2 was expressed at 37 degrees C but not at 25 degrees C, suggesting that ClpP2 cannot compensate for the absence of ClpP1 in the clpP1 mutant cells at low temperature. Our study demonstrates that ClpP1 and ClpP2 control distinct cellular regulatory pathways in B. thuringiensis.

The InhA2 metalloprotease of Bacillus thuringiensis strain 407 is required for pathogenicity in insects infected via the oral route.

The entomopathogenic bacterium Bacillus thuringiensis is known to secrete a zinc metalloprotease (InhA) that specifically cleaves antibacterial peptides produced by insect hosts. We identified a second copy of the inhA gene, named inhA2, in B. thuringiensis strain 407 Cry(-). The inhA2 gene encodes a putative polypeptide showing 66.2% overall identity with the InhA protein and harboring the zinc-binding domain (HEXXH), which is characteristic of the zinc-requiring metalloproteases. We used a transcriptional inhA2'-lacZ fusion to show that inhA2 expression is induced at the onset of the stationary phase and is overexpressed in a Spo0A minus background. The presence of a reverse Spo0A box in the promoter region of inhA2 suggests that Spo0A directly regulates the transcription of inhA2. To determine the role of the InhA and InhA2 metalloproteases in pathogenesis, we used allelic exchange to isolate single and double mutant strains for the two genes. Spores and vegetative cells of the mutant strains were as virulent as those of the parental strain in immunized Bombyx mori larvae infected by the intrahemocoelic route. Exponential phase cells of all the strains displayed the same in vitro potential for colonizing the vaccinated hemocoel. We investigated the synergistic effect of the mutant strain spores on the toxicity of Cry1C proteins against Galleria mellonella larvae infected via the oral pathway. The spores of DeltainhA2 mutant strain were ineffective in providing synergism whereas those of the DeltainhA mutant strain were not. These results indicate that the B. thuringiensis InhA2 zinc metalloprotease has a vital role in virulence when the host is infected via the oral route.