Michiko M. Nakano

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.

Nucleotide sequence of 5′ portion of srfA that contains the region required for competence establishment in Bacillus subtilus

The nucleotide sequence of the 20,535 base pairs of the 5 end of the srfA operon, containing the region required for competence development, was determined. This included the srfA promoter region, the first open reading frame, srfAA, encoding surfactin synthetase I and part of the second open reading frame, srfAB, encoding surfactin synthetase II. Three amino acid-activating domains characteristic of those found in peptide synthetases could be discerned in both srfAA (activating Glu, Leu and D-Leu) and srfAB (activating Val, Asp, and D-Leu). The presence of a conserved spacer motif in the amino-terminal end of srfAA suggests that the srfAA product may not initiate surfactin synthesis. The portion of srfA that contains the region required for competence is composed of srfAA and the first amino acid-activating domain of srfAB.

Interaction of ResD with regulatory regions of anaerobically induced genes in Bacillus subtilis.

The two‐component regulatory proteins ResD and ResE are required for anaerobic nitrate respiration in Bacillus subtilis. ResD, when it undergoes ResE‐dependent phosphorylation, is thought to activate transcriptionally anaerobically induced genes such as fnr, hmp and nasD. In this report, deletion analysis of the fnr, hmp and nasD promoter regions was carried out to identify cis‐acting sequences required for ResDE‐dependent transcription. The results suggest that the hmp and nasD promoters have multiple target sequences for ResDE‐dependent regulation and that fnr has a single target site. Gel mobility shift assays and DNase I footprinting analyses were performed to determine whether ResD interacts directly with the regulatory regions of the three genes. Our results indicate that ResD specifically binds to sequences residing upstream of the hmp and nasD promoters and that phosphorylation of ResD significantly stimulates this binding. In contrast, a higher concentration of ResD is required for binding to the fnr promoter region and no stimulation of the binding by ResD phosphorylation was observed. Taken together, these results suggest that ResD activates transcription of fnr, hmp and nasD by interacting with DNA upstream of these promoters. Our results suggest that phosphorylation of ResD stimulates binding to multiple ResD binding sites, but is much less stimulatory if only a single binding site exists.

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. ComSI13u2003→u2003A and ComSW43u2003→u2003A were selected for further analysis as representative mutants for both regions required for ComS activity. We observed that ComSI13u2003→u2003A showed significantly reduced affinity for MecA, whereas ComSW43u2003→u2003A 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.