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Bacillus thuringiensis is the leading biopesticide used to control insect pests worldwide. Although they have a long record of safe use, under certain conditions commercial strains of B. thuringiensis have the ability to produce numerous putative enterotoxins that have been associated with food poisoning attributed to Bacillus cereus. Therefore, we designed a strategy to delete the genes encoding these toxins. B. thuringiensis strain VBTS 2477 contained genes encoding NHE, CytK-2 and three homologues of haemolysin BL (HBL, [HBL.sub.a1] and [HBL.sub.a2]). This is the first report, to our knowledge, of a strain of B. cereus or B. thuringiensis containing three sets of hbl operons. The genes encoding [HBL.sub.a1] and [HBL.sub.a2] were 96-97% identical to each other and 76-84% identical to those encoding HBL. The [hbl.sub.a2] operon was detected by PCR amplification only after hbla1 was deleted. We used sequential gene replacement to replace the wild-type copies of the NHE and three HBL operons with copies that contained internal deletions that span the three genes in each operon. The insecticidal activity of the quadruple-enterotoxin-deficient mutant was similar to that of the wild-type strain against larvae of Trichoplusia ni, Spodoptera exigua and Plutella xylostella. This demonstrates that the genes for enterotoxins can be deleted, eliminating the possibility of enterotoxin production without compromising the insecticidal efficacy of a strain of B. thuringiensis. DOI 10.1099/mic.0.039925-0

The frequency at which Cytophaga-Flavobacterium (CF) bacteria coisolated with Bacillus cereus strains from soybean rhizosphere and the mechanism by which Bacillus cereus stimulates growth of CF rhizosphere strains in root exudate media is determined. The results suggest that Bacillus cereus and CF rhizospheria have a commensal relationship in which peptidoglycan produced by Bacillus cereus stimulates the growth of CF bacteria.

Research identifies genes involved in zwittermicin A biosynthesis, which seem to be related to those involved in peptide and polyketide antibiotics production. Analyses of 4,800 transposon mutants of Bacillus cereus showed that all except nine accumulate zwittermicin A with one producing eightfold more than the wild-type.

In a fluorescence induction system consisting of a promoter trap in which the genes whose promoter activity is influenced by the host seed exudate, from a recombinant inbred line supporting growth of Bacillus cereus or Pseudomonas aureofaciens, clones carrying genes regulated by biotic environment can be identified. Results show that lipA is one such plant-regulated gene in Bacillus cereus.

Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system ( T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5′ untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion. [ABSTRACT FROM AUTHOR]

We have explored the microbial community in a nonpermafrost, cold Alaskan soil using both culture-based and culture-independent approaches. In the present study, we cultured >1000 bacterial isolates from this soil and characterized the collection of isolates phylogenetically and functionally. A screen for antibiosis identified an atypical, red-pigmented strain of Janthinobacterium lividum (strain BR01) that produced prodigiosin when grown at cool temperatures as well as strains (e.g., strain BP01) that are more typical of J. lividium, which produce a purple pigment, violacein. Both purple- and red-pigmented strains exhibited high levels of resistance to β-lactam antibiotics. The prodigiosin pathway cloned from J. lividium BR01 was expressed in the heterologous host, Escherichia coli, and the responsible gene cluster differs from that of a well-studied prodigiosin producer, Serratia sp. J. lividum BR01 is the first example of a prodigiosin-producer among the β-Proteobacteria. The results show that characterization of cultured organisms from previously unexplored environments can expand the current portrait of the microbial world. [ABSTRACT FROM AUTHOR]

Infection with Neisseria gonorrhoeae leads to inflammation that is responsible for the disease symptoms of gonococcal urethritis, cervicitis, and pelvic inflammatory disease. During growth, these bacteria release significant amounts of peptidoglycan (PG) fragments which elicit inflammatory responses in the human host. To better understand the mechanisms involved in PG synthesis and breakdown in N. gonorrhoeae, we characterized the effects of loss of function mutations in mltG. MltG has been identified in other bacterial species as a terminase that stops PG strand growth by cleaving the growing glycan. Mutation of mltG in N. gonorrhoeae did not affect bacterial growth rate but resulted in increased PG turnover, more cells of large size, decreased autolysis under non-growth conditions, and increased sensitivity to antibiotics that affect PG crosslinking. An mltG mutant released greatly increased amounts of PG monomers, PG dimers, and larger oligomers. In the mltG background, mutation of either ltgA or ltgD, encoding the lytic transglycosylases responsible for PG monomer liberation, resulted in wild-type levels of PG monomer release. Bacterial two-hybrid assays identified positive interactions of MltG with synthetic penicillin-binding proteins PBP1 and PBP2 and the PG-degrading endopeptidase PBP4 (PbpG). These data are consistent with MltG acting as a terminase in N. gonorrhoeae and suggest that the absence of MltG activity results in excessive PG growth and extra PG in the sacculus that must be degraded by lytic transglycosylases including LtgA and LtgD. Furthermore, the absence of MltG causes a cell wall defect that is manifested as large cell size and antibiotic sensitivity. [ABSTRACT FROM AUTHOR]