The bolA gene, which is involved in the morphogenetic pathways of Escherichia coli, was sequenced and two potential promoters were identified. Expression from promoter P1, proximal to the bolA structural gene is specifically induced during the transition to the stationary phase of growth. This promoter contains an unusual‐‐10 region (CGGCTAGTA), which defines a new class of E. coli promoters necessary for the dramatic increase in the rate of synthesis of a large set of proteins during the cessation of logarithmic growth. This conclusion was confirmed by identifying two additional E. coli promoters and one plasmid promoter, which also were induced during the transition to the stationary phase of growth. Analysis of proteins produced during the exponential and stationary phases of growth in a bolA null mutant suggest a possible role for the BolA protein in the induction of the expression of penicillin‐binding protein 6 (PBP6) in the transition to the stationary phase. Supporting this hypothesis is the presence of a putative DNA‐binding domain within the bolA coding sequence.

Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT‐‐‐‐GC transition at position 2251 of the gene, which produces a Trp751‐‐‐‐Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell‐free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double‐strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well‐defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17‐resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme‐DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.

A newly found morphogene of Escherichia coli, boLA, mapping at min 10 of the genetic map, was cloned in a 7.2-kilobase BamHI fragment and identified by its ability to produce osmotically stable spherical cells when overexpressed. This gene codes for a polypeptide of 13 kilodaltons. Overexpression of boUl+ was achieved in low-copy-number vectors with operon fusions to the tet and lac promoters, indicating a clockwise direction of transcription. While no modification of any of the penicillin-binding proteins was observed, morphological effects due to overexpression of boUl+ were shown to be dependent on the presence of an active ftsZ gene product. Our results suggest the existence of a mechanism mediated by FtsZ for modifying the conformation of nascent murein in the early steps of septum formation.

The synthesis of the peptide antibiotic microcin B17 was shown to occur as the cells entered the stationary phase of growth. This type of growth phase regulation is commonly observed in the production of a number of different bacterial products such as toxins and antibiotics. Microcin B17 synthesis is also dependent on the product of the ompR gene. To determine the role of transcription in this double regulation of microcin B17 production, operon fusions with Mu dl (Ap lac) were constructed. Insertions were obtained in all four plasmid genes involved in production of microcin B17 (mcbA-D) and in the immunity region. Three classes of fusions were obtained. Fusions into mcbA, mcbB, and mcbC (first class) exhibited an increase in their transcription as the cells approached the stationary phase. These increases as well as basal levels of transcription were dependent on OmpR. Expression of fusions in mcbD and in the immunity region (second class) was also dependent on OmpR, but their expression remained constant throughout growth. One fusion in mcbC (third class) was obtained which was transcribed in the opposite direction than the others. It showed no growth phase regulation and no OmpR dependence. The implications of these results in terms of the transcriptional organization of the mbc genes are discussed.

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains harbouring plasmid pMccB17. We have isolated two mutations that strongly reduce the production of MccB17. These mutations, which map at 96 min on the E. coli chromosome, define a new gene that we have called pmbA. A chromosomal DNA fragment of about 13 kb, including the wild-type pmbA allele, was cloned into a mini-Mu plasmid vector. pmbA was located within the cloned DNA fragment by insertional mutagenesis and deletion analysis. The nucleotide sequence of a 1.7 kb DNA region containing the gene was determined. pmbA encodes a hydrophilic protein of 450-amino-acid residues with a predicted molecular size of 48375D, which was visualized in polyacrylamide gels. Protein profiles of cellular envelope and soluble fractions from cells with plasmids overproducing PmbA indicated that it is cytoplasmic. Physiological experiments suggested that pmbA mutants synthesize a molecule (pro-MccB17) able to inhibit DNA replication but unable to be released from cells. We propose that PmbA facilitates the secretion of the antibiotic by completing its maturation.