Anaerobic control of colicin E1 production

Abstract

Expression of the cea gene, which is carried by the ColEl plasmid and which encodes colicin El, was found to be greatly increased when the cells were grown anaerobically. By using cea-lacZ fusions to quantitate expression, aerobic levels were found to be only a few percent of the anaerobic levels. The anaerobic increase in expression was observed both in protein and in operon fusions, indicating that its regulation occurred at the level of transcription. It was also found to require a functionalfitr gene and to occur when the cea-lacZ fusion was present as a single copy in the bacterial chromosome instead of in the multicopy ColEl plasmid. Anaerobic expression was regulated by the SOS response and catabolite repression as is aerobic expression. The start site of the mRNA produced under anaerobic conditions was mapped by primer extension and found to be the same as the start for mRNA produced under aerobic conditions. These observations show that the cea gene is Regulation of the cea-kil operon is complex. Previous studies reported effects of the SOS system (9, 14), catabolite repression (10, 28, 41), the stringent response (23), and anaerobiosis (27,28). The repressor of the SOS response, LexA, is the repressor of the cea-kil operon (9, 11). When LexA is inactivated in the RecA-dependent response to DNA damage (22, 49), cea and kil are expressed. However, cea and kl expression shows a pronounced lag compared with expression of other SOS response genes (34, 56). Catabolite repression, mediated by binding of the cyclic Amp (CAMP) receptor protein (CRP)-cAMP complex upstream from the cea-kil promoter (32), stimulates transcription and affects the lag in induction (32,34). An effect of the stringent response has also been observed in vitro (23).Results of Nakazawa et al. (27,28) suggested that maximum production of colicin El occurred in minimal media when cells were grown anaerobically with glucose as a carbon source. We have reinvestigated this effect, using cea-lacZ protein and operon fusions to quantitate expression. Here we report that cea expression is much higher when cells are grown anaerobically than when they are grown aerobically. A 45-fold increase was observed in anaerobically growing cells. This stimulation of expression was due to transcriptional activation, since both protein and operon fusions showed the increase in expression. The anaerobic conditions started at the same site which had been previously reported for cea mRNA produced under aerobic conditions. MATERIALS AND METHODSMedia and chemicals. MCC medium was M63 salts supplemented with 0.4% glucose or glycerol, 0.4% vitamin-free Casamino Acids (Difco Laboratories, Detroit, Mich.), and 0.1 mg of thiamine per ml; 40 mM potassium fumarate was added as an electron acceptor for the anaerobic cultures. Other media were as described previously (42). Ampicillin and kanamycin were used at 150 and 25 ,ug/ml, respectively. 5-Bromo-4-chloro-3-indolyl-,-D-galactoside (X-Gal) was used to score the LacZ phenotype. Mitomycin C (MMC) was used at a final con...

Characterization of the Bacillus subtilis YxdJ response regulator as the inducer of expression for the cognate ABC transporter YxdLM

“…For this purpose, fragments of various lengths of the yxdL regulatory region were cloned in plasmid pGE593 (Eraso & Weinstock, 1992), creating a series of transcriptional fusions with the lacZ reporter gene (plasmids pGE-A, pGE-B, pGE-C and pGE-D). Each of the recombinant plasmids was introduced into a DlacZ E. coli strain (DH5a) bearing the compatible pBAD-yxdJ plasmid that contains the yxdJ gene under the control of an arabinose-inducible promoter.…”

“…Deletion analysis of the yxdLMyxeA promoter. DNA fragments were PCR-amplified using genomic B. subtilis DNA as template and were cloned upstream of lacZ in plasmid pGE593 (Eraso & Weinstock, 1992). The PCR products were generated using a common primer (YxdKL_right) and one of the following specific primers: for fragment A (2281, 159), YxdKL_left_A; for fragment B (294, 159), YxdKL_left_B; for fragment C (269, 159), YxdKL_left_C; and for fragment D (230, 159), YxdKL_left_D.…”

Colicin Biology

“…The lag is more or less significant according to growth conditions and to the bacterial strain used. Colicin synthesis has been shown to be stimulated in various cases by thymine starvation (589), stringent response (419), catabolite repression (172,535), ompR mutation (543), the stationary phase of growth (185,569), anaerobiosis (184), high temperatures (85,86,333), or nutrient depletion (368). In contrast, it is significantly reduced by low temperatures and in pldA null mutants (85,88).…”

Structural and functional organization of the Yersinia pestis bacteriocin pesticin gene cluster

“…The first region resides in the SOS promoter of the pst gene, indicating a similar mechanism of regulation of the pesticin to the other colicins (Fig. 3) (Lotz, 1978;Ebina & Nakazawa, 1983;Eraso & Weinstock, 1992). The other region of high homology can be identified upstream of the pim gene.…”