mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis

Here, we present a novel method for the directed genetic manipulation of the Bacillus subtilis chromosome free of any selection marker. Our new approach employed the Escherichia coli toxin gene mazF as a counter-selectable marker. The mazF gene was placed under the control of an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression system and associated with a spectomycin-resistance gene to form the MazF cassette, which was flanked by two directly-repeated (DR) sequences. A double-crossover event between the linearized delivery vector and the chromosome integrated the MazF cassette into a target locus and yielded an IPTG-sensitive strain with spectomycin-resistance, in which the wild-type chromosome copy had been replaced by the modified copy at the targeted locus. Another single-crossover event between the two DR sequences led to the excision of the MazF cassette and generated a strain with IPTG resistance, thereby realizing the desired alteration to the chromosome without introducing any unwanted selection markers. We used this method repeatedly and successfully to inactivate a specific gene, to introduce a gene of interest and to realize the in-frame deletion of a target gene in the same strain. As there is no prerequisite strain for this method, it will be a powerful and universal tool

OLE RNA, an RNA motif that is highly conserved in several extremophilic bacteria, is a substrate for and can be regulated by RNase P RNA

OLE (ornate, large, and extremophilic) RNA is a noncoding RNA that is found in several extremophilic bacteria, including Bacillus halodurans. The function of OLE RNA has not been clarified. In this study, we found that RNase P cleaves OLE RNA and that the cleavage leads to a small reduction of expression of a downstream gene determined by analyses in vitro and in vivo. Under RNase P-deficient conditions, the amount of OLE RNA increased. Our results imply that RNase P could play a role in the regulation of gene expression in relation to conserved RNA motifs like OLE RNA as well as in riboswitches and operons

Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR–DNA complexes

The Bacillus subtilis GlnR repressor controls gene expression in response to nitrogen availability. Because all GlnR-regulated genes are expressed constitutively in mutants lacking glutamine synthetase (GS), GS is required for repression by GlnR. Feedback-inhibited GS (FBI-GS) was shown to activate GlnR DNA binding with an in vitro electophoretic mobility shift assay (EMSA). The activation of GlnR DNA binding by GS in these experiments depended on the feedback inhibitor glutamine and did not occur with mutant GS proteins defective in regulating GlnR activity in vivo. Although stable GS–GlnR–DNA ternary complexes were not observed in the EMSA experiments, cross-linking experiments showed that a protein–protein interaction occurs between GlnR and FBI-GS. This interaction was reduced in the absence of the feedback inhibitor glutamine and with mutant GS proteins. Because FBI-GS significantly reduced the dissociation rate of the GlnR–DNA complexes, the stability of these complexes is enhanced by FBI-GS. These results argue that FBI-GS acts as a chaperone that activates GlnR DNA binding through a transient protein–protein interaction that stabilizes GlnR–DNA complexes. GS was shown to control the activity of the B. subtilis nitrogen transcription factor TnrA by forming a stable complex between FBI-GS and TnrA that inhibits TnrA DNA binding. Thus, B. subtilis GS is an enzyme with dual catalytic and regulatory functions that uses distinct mechanisms to control the activity of two different transcription factors

Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor

Clostridium difficile, a causative agent of antibiotic-associated diarrhea and its potentially lethal form, pseudomembranous colitis, produces two large protein toxins that are responsible for the cellular damage associated with the disease. The level of toxin production appears to be critical for determining the severity of the disease, but the mechanism by which toxin synthesis is regulated is unknown. The product of a gene, txeR, that lies just upstream of the tox gene cluster was shown to be needed for tox gene expression in vivo and to activate promoter-specific transcription of the tox genes in vitro in conjunction with RNA polymerases from C. difficile, Bacillus subtilis, or Escherichia coli. TxeR was shown to function as an alternative sigma factor for RNA polymerase. Because homologs of TxeR regulate synthesis of toxins and a bacteriocin in other Clostridium species, TxeR appears to be a prototype for a novel mode of regulation of toxin genes

Structural and biological studies on bacterial nitric oxide synthase inhibitors

Nitric oxide (NO) produced by bacterial NOS functions as a cytoprotective agent against oxidative stress in Staphylococcus aureus, Bacillus anthracis, and Bacillus subtilis. The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds impede the growth of B. subtilis under oxidative stress, and crystal structures show that each compound exhibits a unique binding mode. Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria

Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response

Horizontal gene transfer contributes to the evolution of bacterial species. Mobile genetic elements play an important role in horizontal gene transfer, and characterization of the regulation of these elements should provide insight into conditions that influence bacterial evolution. We characterized a mobile genetic element, ICEBs1, in the Gram-positive bacterium Bacillus subtilis and found that it is a functional integrative and conjugative element (ICE) capable of transferring to Bacillus and Listeria species. We identified two conditions that promote ICEBs1 transfer: conditions that induce the global DNA damage response and crowding by potential recipients that lack ICEBs1. Transfer of ICEBs1 into cells that already contain the element is inhibited by an intercellular signaling peptide encoded by ICEBs1. The dual regulation of ICEBs1 allows for passive propagation in the host cell until either the potential mating partners lacking ICEBs1 are present or the host cell is in distress