Peter Zuber

Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis.

SummaryThe sfp gene is required for cells of Bacillus subtilis to become producers of the lipopeptide antibiotic surfactin. sfp was isolated and its nucleotide sequence was determined. sfp was expressed in Escherichia coli and its putative product was purified for use in antibody production and in amino acid sequence analysis. The gene was plasmid-amplified in B. subtilis, where it conferred a Srf+ phenotype on sfp0 (surfactin non-producing) cells. Overproduction of Sfp in B. subtilis did not cause production of an increased amount of surfactin and resulted in the repression of a lacZ transcriptional fusion of the srfA operon, which encodes enzymes that catalyze surfactin synthesis. We propose that sfp represents an essential component of peptide synthesis systems and also plays a role, either directly or indirectly, in the regulation of surfactin biosynthesis gene expression.

Role of Lon and ClpX in the post‐translational regulation of a sigma subunit of RNA polymerase required for cellular differentiation in Bacillus subtilis

The RNA polymerase sigma subunit, σH (Spo0H) of Bacillus subtilis, is essential for the transcription of genes that function in sporulation and genetic competence. Although spo0H is transcriptionally regulated by the key regulatory device that controls sporulation initiation, the Spo0 phosphorelay, there is considerable evidence implicating a mechanism of post‐translational control that governs the activity and concentration of σH. Post‐translational control of spo0H is responsible for the reduced expression of genes requiring σH under conditions of low environmental pH. It is also responsible for heightened σH activity upon relief of acid stress and during nutritional depletion. In this study, the ATP‐dependent proteases LonA and B and the regulatory ATPase ClpX were found to function in the post‐translational control of σH. Mutations in lonA and lonB result in elevated σH protein concentrations in low‐pH cultures. However, this is not sufficient to increase σH‐dependent transcription. Activation of σH‐dependent transcription upon raising medium pH and in cells undergoing sporulation requires clpX, as shown by measuring the expression of lacZ fusions that require σH for transcription and by complementation of a clpX null mutation. A hypothesis is presented that low environmental pH results in the Lon‐dependent degradation of σH, but the activity of σH in sporulating cells and in cultures at neutral pH is stimulated by a ClpX‐dependent mechanism in response to nutritional stress.

Interaction of AbrB, a transcriptional regulator from Bacillus subtilis with the promoters of the transition state-activated genes tycA and spoVG

SummaryIn Bacillus subtilis the abrB gene product negatively affects the transcription of some genes activated during the transition from vegetative to stationary phase of growth. Interaction of AbrB with the promoters of two such genes, spoVG, a sporulation gene, and tycA, an antibiotic biosynthesis gene, was studied by DNase I and hydroxyl radical footprinting. Two binding areas within the leader and promoter regions of tycA were identified. In spoVG the binding site is located at the A+T-rich region upstream of the promoter. Hydroxyl radical footprinting revealed that the AbrB-protected regions, in both the tycA and spoVG promoters, are short A+T-rich regions that are separated by one helical turn, indicating that AbrB binds to one face of the helix. To examine the role of spoOA in the expression of abrB-controlled genes, the levels of AbrB protein in Spo+ and in spoOA cells were determined by Western blot analysis. In wild-type cells AbrB was detected only during vegetative growth, whereas in spoOA cells a high level of AbrB was detected during both the vegetative and stationary phases of growth. These findings support a model in which (i) spoOA negatively affects abrB expression, and (ii) the repression of the transition state-activated genes tycA and spoVG in spoOA cells is due to constitutive expression of AbrB, which acts as a repressor.

Mutational analysis of ComS: evidence for the interaction of ComS and MecA in the regulation of competence development in Bacillus subtilis

The development of Bacillus subtilis genetic competence is a highly regulated adaptive response to stationary‐phase stress. A key step in competence development is the activation of the transcriptional regulator ComK, which is required for the expression of genes encoding the products that function in DNA uptake. In log‐phase cultures, ComK is trapped in a complex composed of MecA and ClpC, in which it is rendered inactive. The comS gene, contained within the srf operon, is induced in response to high culture cell density and nutritional stress. Its product functions to release active ComK from the complex, allowing ComK to stimulate the transcription initiation of its own gene as well as that of the late competence operons. Western analysis showed that ComS accumulates to maximal levels between T3 and T4, mirroring the pattern of competence cell development and late competence gene expression. Experiments to examine the target of ComS activity in vitro showed that ComS binds to MecA. This is further supported by coimmunoprecipitation using anti‐MecA antiserum. To clarify the role of ComS in competence regulation, a system for evaluating the effect of comS and mutant derivatives on the expression of comG, one of the late competence operons, was constructed. comS mutations, created by alanine‐scanning mutagenesis, that significantly reduced comG–lacZ expression were clustered within two regions, one at the N‐terminus and the other at the C‐terminus of ComS. ComSI13 → A and ComSW43 → A were selected for further analysis as representative mutants for both regions required for ComS activity. We observed that ComSI13 → A showed significantly reduced affinity for MecA, whereas ComSW43 → A showed near normal binding affinity for MecA. The results show that binding to MecA is critical for ComS function, but do not rule out the possibility that ComS possesses other activities.

Analysis of a mutant amino acid-activating domain of surfactin synthetase bearing a serine-to-alanine substitution at the site of carboxylthioester formation

The reactive serine of the TGGHSL thioester binding motif of the first amino acid‐activating domain of surfactin synthetase was replaced by alanine using site‐directed mutagenesis. The multienzyme from cells of the resulting mutant lost its ability for thioester formation with l‐Glu and was therefore inactive in surfactin production. The thiolation reactions catalyzed by the other amino acid‐activating domains of surfactin synthetase were not affected by the mutation. The results show that l‐Glu is acativated at the first domain of surfactin synthetase, and give further evidence that a serine residue is essential for substrate amino acid activation at the reaction centers of peptide synthetases.

The modular organization of multifunctional peptide synthetases.

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme–inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4′-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the “Multiple Carrier Model” of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.