Iridium acetohutylicum in continuous culture: strain differences. Journal o/' Applied Bacteriology 69, 71 8-728.Several strains of Clostridium acetobutylicum, including strains ATCC 824 and DSM I73 I, continue to produce solvents during prolonged periods of chemostat culture. In such cultures, dominance is established by asporogenous mutant(s) that retain the ability to produce solvents. Strain NCIB 8052 (which is not identical with ATCC 824) behaved differently in that its chemostat cultures invariably became acidogenic due to ultimate selection of asporogenous mutant(s) unable to produce solvents, incapable of synthesizing granulose, and demonstrating enhanced sensitivity to environmental stresses of various types. These mutants spontaneously reverted. at a low but measurable frequency, to the parental phenotype, indicating that their multiple loss of capacities was the pleiotropic consequence of a lesion in some global regulatory gene. Their resemblance to previously described cls mutants of strain P262 and the possible nature of the affected regulatory gene are discussed. A simple tetrazolium blue plate assay procedure is described which allows visual discrimination between solvent-producing and non-solventogenic colonies of CI. acetobutylicum When Clostridiurn acetobutylicurn NCIB 8052 was grown in chemostat culture under conditions that were unsuitable for solvent production, asporogenous mutant strain@) incapable of producing acetone and butan-1-01 in normal batch culture were quickly selected (Gottschal & Morris 1981). Even when chemostat culture was performed under conditions that were propitious for solvent formation, it was impossible to establish a steady state, and ultimately, irrespective of the nature of the limiting nutrient, solvent production ceased (Stephens et al. 1985). Other strains of CI. acetobutylicurn, e.g. ATCC 824 and DSM 1731, sustained their solvent productivity during prolonged chemostat culture despite the emergence and eventual preponderance of asporogenous mutants (Meinecke et a/. 1984). In contrast, when strain
Herein, we identify vceC as a component of a vceCAB operon, which codes for the Vibrio cholerae VceAB multiple-drug resistance (MDR) efflux pump, and vceR, which codes for a transcriptional autoregulatory protein that negatively regulates the expression of the vceCAB operon and is modulated by some of the substrates of this MDR efflux pump.MDR efflux pumps contribute to the intrinsic and acquired antibiotic resistance in bacteria and significantly impact the emergence of drug-resistant pathogens (9,10,16,18,24,25). These efflux systems can be amplified in resistant cells (9,11,17) and can alter their substrate profiles with mutation (6, 21), making them a major threat to the successful application of antibiotic therapy.In an earlier study, we cloned a 6.6-kb DNA fragment from Vibrio cholerae (pVC2) that encoded an MDR efflux pump that functioned in an Escherichia coli tolC mutant (3). In that study, we identified two genes, vceA and vceB, that code for a membrane fusion protein (MFP) and a cytoplasmic membrane translocase (CMT), respectively. VceB belongs to the major facilitator superfamily of proton antiporters, and the VceAB pump components share homology with the E. coli EmrAB efflux MDR pump. The substrates of this pump included hydrophobic agents, such as deoxycholate (DOC), antibiotics, such as chloramphenicol and nalidixic acid, and the proton motive force uncoupler, cyanide carbonyl m-chlorophenylhydrazone (CCCP). However, we did not identify the outer membrane efflux (channel) protein (OEP) of this MDR system. In this report, we identify VceC (4) as the OEP of this pump and demonstrate that its gene resides in an operon with vceAB (i.e., vceCAB). We also identify VceR, a transcriptional autoregulatory protein that negatively regulates the expression of the vceCAB operon and whose function is modulated by some of the substrates of this MDR system.Identification of the OEP component of vceAB MDR pump. Examination of the V. cholerae El Tor N16961 genome sequence (The Institute for Genomic Research) revealed an open reading frame (ORF) designated VC1409 (accession no. Q9KS51) which lies just upstream of vceA and codes for a putative OEP. To determine if VC1409 is essential for the functioning of the VceAB pump we examined the ability of this ORF (pVC91) to complement vceAB (pVC4) for the production of a functional VceAB pump in a tolC mutant. pVC4 has previously been described (3) and pVC91 contains the V. cholerae DNA fragment of pVC9 (3) cloned into a plasmid (pACYC184) which is compatible with pBR322 (i.e., pVC4). As expected, neither pVC4 nor pVC91 transformants could provide a tolC mutant with a functional VceAB pump. However, double transformants carrying both pVC4 and pVC91 conferred resistance to CCCP and DOC but not sodium dodecyl sulfate (SDS) or novobiocin. This substrate specificity is consistent with that of the VceAB MDR efflux pump (3) and indicates that pVC91 codes for a transacting factor(s) necessary for the functioning of VceAB. It also demonstrates that the OEP of this pump cannot repla...
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