Resistance to trifluoroperazine, a calmodulin inhibitor, maps to the fabD locus in Escherichia coli.

A mutant, tfpA1, resistant to the calmodulin inhibitor trifluoroperazine (TFP) at 30 degrees C, was isolated in Escherichia coli. The mutant showed a reduced growth rate at 30 degrees C and was temperature sensitive (ts) at 42 degrees C for growth, forming short filaments. The mutation was mapped to the 24 min region of the chromosome and the gene was cloned by complementation of the ts defect. Subsequent subcloning, complementation analysis, marker rescue mapping and sequencing, identified tfpA as fabD, encoding the 35 kDa, malonyl-coenzyme A transacylase (MCT) enzyme, required for the initial step in the elongation cycle for fatty acid biosynthesis. Resistance to TFP may result from altered permeability of the cell envelope, although the mutant remained sensitive to other calmodulin inhibitors and to other antibacterial agents. Alternatively, resistance may be more indirect, resulting from alterations in intracellular Ca++ levels which affect the activity of the TFP target in some way.

Cloning and analysis of the entire Escherichia coli ams gene. ams is identical to hmp1 and encodes a 114 kDa protein that migrates as a 180 kDa protein.

We have used an antibody to a previously identified 180 kDa (Hmp1) protein in Escherichia coli to clone the corresponding gene, which encodes a polypeptide of 114 kDa that has a mobility equivalent to 180 kDa in SDS/PAGE. We have demonstrated that the 180 kDa polypeptide is the primary gene product and not due to aggregation with other molecules. Moreover, our data indicate that the highly charged C-terminal region of the protein is responsible for its anomalous behaviour when analysed by SDS/PAGE. The hmp1 gene is in fact identical to ams (abnormal mRNA stability), also designated rne (RnaseE), and reported to have an ORF of 91 kDa. This discrepancy with the data in this paper can be ascribed to the omission of two bases in the previously reported sequence, generating an apparent stop codon. We previously demonstrated that the 180 kDa Hmp1/Ams protein cross reacted with both a polyclonal antibody and a monoclonal antibody raised against a yeast heavy chain myosin. However, we could detect no homology with myosin genes in the ams/hmp1 sequence. From the DNA sequence data, we identified a putative nucleotide binding site and a transmembrane domain in the N-terminal half of the molecule. In the C-terminal half, which appears to constitute a separate domain dominated by proline and charged amino acids, we also identified a region homologous to the highly conserved 70 kDa snRNP protein, involved in RNA splicing in eukaryotes. This feature would be consistent with reports that ams encodes RNaseE, an enzyme required for the processing of several stable RNAs in E. coli.

The mdoA locus of Escherichia coli consists of an operon under osmotic control.

In Escherichia coli, the 5 kb mdoA locus is involved in the osmotically controlled biosynthesis of periplasmic membrane-derived oligosaccharides (MDOs). The structure of this locus was analysed by in vitro cassette insertion, transposon mutagenesis, and gene-fusion analysis. A 'neo' cassette, derived from the neomycin phosphotransferase II region of transposon Tn5, was inserted into mdoA, borne by a multicopy plasmid. This plasmid was shown to complement two previously described mdoA mutations, depending on the orientation of the exogenous gene. Thus, the gene altered by these mutations could be expressed under the control of the exogenous promoter. Moreover, the 'neo' cassette inactivated another, uncharacterized, mdo gene, because when this insertion was transferred into the chromosome MDO synthesis was abolished. The existence of a second gene was confirmed by complementation analysis with a collection of Tn1000 insertions into mdoA. Two groups were defined, and the two genes are organized into an operon (mdoGH). This conclusion was reached because Tn1000 insertions in the first gene displayed a polar effect on the expression of the second gene. An active gene fusion was obtained on a multicopy plasmid between the beginning of mdoH and lacZ. The hybrid beta-galactosidase activity followed the same osmotically controlled response as that described for of MDO synthesis. This regulation was unaffected by the presence, or absence, of MDOs in the periplasm. Finally, the amount of mdoA-specific mRNAs, determined by dot blot hybridization, decreased when the osmolarity of the growth medium increased.

Analysis of a myosin-like protein and the role of calcium in the E. coli cell cycle.

For a number of years now, we have argued that current models for the control of initiation of DNA synthesis, chromosomal partitioning and septum formation in Escherichia coli are unsatisfactory. Indeed, we could argue that despite considerable efforts, with the possible exception of dnaA and ftsZ, no genes specifically implicated in these control processes have been identified. In the cases of DnaA and FtsZ, no evidence has appeared to indicate how such molecules might be regulated to act once per cycle. In 1988, we formulated a specific proposal that the timing of cell cycle events in E. coli might be determined by a Ca++ flux, mediated by calcium-binding proteins and protein kinases and culminating, in the case of chromosome segregation and division, in the action of force-generating proteins such as myosin (Norris et al., 1988). In formulating this proposal, we took the view that the fundamental elements of cell cycle regulation are likely to be highly conserved across all species including prokaryotes. In this presentation, we shall describe the approaches we have been taking in order to test this hypothesis and to summarize the data obtained, in particular in relation to new genes identified which may play a role in the E. coli cell cycle. We shall also briefly indicate recent data from other laboratories consistent with our general hypothesis.

Molecular cloning and expression of a locus (mdoA) implicated in the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

Mutants of Escherichia coli defective in the mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides. The mdoA locus has now been cloned into multicopy plasmids. A 5 kb DNA fragment is necessary to complement mdoA mutations. Cells harbouring the mdoA+ plasmid produced three to four times more MDO than wild-type cells. MDO overproduction did not affect the degree of MDO substitution with sn-1-phosphoglycerol residues. The biosynthesis of MDO remained under osmotic control in overproducing strains.

Regulation of expression of the gene for vitamin B12 receptor cloned on a multicopy plasmid in Escherichia coli.

The btuB gene of Escherichia coli codes for a protein (BtuB) located in the outer membrane. BtuB is the receptor for vitamin B12 (cyanocobalamin). We have cloned the btuB gene into pUC8 using transposon Tn5 as the marker to first isolate several parts of the relevant DNA fragment from the specialized transducing phage lambda darg13. After reconstitution of the gene, Tn5 was removed by selecting for spontaneous excision. The partial nucleotide sequence and transcriptional start of the btuB gene were determined. The BtuB+ plasmid allowed a large amplification of the synthesis of BtuB, resulting in a 65-fold increased level of vitamin B12 binding. The level of vitamin B12 binding was reduced by a factor of 22 when cells were grown in the presence of high concentrations of vitamin B12. The regulation of the gene was studied in more detail by the use of a protein fusion between the extreme amino-terminus of BtuB and beta-galactosidase of E. coli. The kinetics of repression and derepression were consistent with the presence in the cells of a large amount of a regulatory molecule exhibiting an apparent Km for vitamin B12 of 3 microM.

[A case of overlapping reading frames in Escherichia coli]. / Un cas de chevauchement de cadres de lecture chez Escherichia coli.

The two promoters and the first 65 nucleotides of a gene related to the rrnB operon are localized in the coding sequence of the btuB gene.