Regulation of the expression of the traM gene of the F sex factor of Escherichia coli.

Conjugative F-plasmid transfer is mediated by the transfer (tra) region which encodes nearly 40 genes, 25 of which are essential for this process in Escherichia coli. TraM is required for conjugation and is encoded on a separate operon between the origin of transfer and the traJ gene. The traJ gene product is the positive regulator of transcription of the 30 kb tra operon, the first gene of which is traY. Using primer-extension assays and immunoblots on the F plasmid itself and its derivatives, we demonstrate that F TraM regulates its own expression from two promoters and that it requires TraY as well as expression of the tra operon for maximal traM transcription. traY is the first gene in the tra operon under the control of the TraJ regulator, which is in turn negatively regulated by the antisense RNA, FinP, and the FinO protein. Thus, a control circuit has been established whereby traM is negatively regulated by the FinOP fertility inhibition system through its repression of TraJ expression, which adversely affects transcription of the traY gene.

Stationary phase expression of a novel Escherichia coli outer membrane lipoprotein and its relationship with mammalian apolipoprotein D. Implications for the origin of lipocalins.

We report a novel outer membrane lipoprotein of Escherichia coli. DNA sequencing between ampC and sugE at the 94.5 min region of the E. coli chromosome revealed an open reading frame specifying 177 amino acid residues. Primer extension analysis demonstrated that the promoter is activated at the transition between exponential and stationary growth phases under control of the rpoS sigma factor gene, and this was confirmed in vivo by monitoring expression of beta-galactosidase activity from a lacZ translational fusion. The amino acid sequence exhibited 31% identity with human apolipoprotein D (apoD), which is a component of plasma high density lipoprotein and belongs to the eukaryotic family of lipocalins. The bacterial lipocalin (Blc) contained a short deletion of 7 amino acid residues corresponding to a hydrophobic surface loop that is thought to facilitate the physical interaction between apoD and high density lipoprotein. However, Blc exhibited a typical prokaryotic lipoprotein signal peptide at its amino terminus. Overexpression, membrane fractionation, and metabolic labeling with [3H]palmitate demonstrated that Blc is indeed a globomycin-sensitive outer membrane lipoprotein. Blc represents the first bacterial member of the family of lipocalins and may serve a starvation response function in E. coli.

The nature of the traK4 mutation in the F sex factor of Escherichia coli.

The sequence of traK gene of the F sex factor of Escherichia coli is presented; the traK gene product is predicted to be a protein of 25,627 Da with a signal sequence of 21 amino acids to give a mature protein of 23,307 Da. The traK4 mutation is an extremely polar mutation in the F plasmid that affects F pilus synthesis and plasmid transfer. traK genes carrying the traK4 mutation and a nonpolar mutation traK105 were cloned, sequenced, and identified as an amber nonsense and a frameshift mutation, respectively. The traK4 mutation occurred within one predicted rho-dependent transcription termination element (TTE) and immediately upstream of another, while the traK105 mutation occurred after the two potential TTEs within the traK gene. S1 nuclease protection analysis and Northern (RNA) blot analysis were used to confirm that the traK4 mutation, but not the traK105 mutation, caused premature termination of transcription. Computer analysis of the F transfer region suggested the presence of TTE motifs at regular intervals throughout the 33.4-kb sequence.