Cellular control of conjugation in Escherichia coli K12. Effect of chromosomal cpx mutations on F-plasmid gene expression.
ABSTRACT DNA donor activity and surface exclusion of Escherichia coli F+ and Hfr strains require expression of both F-plasmid and chromosomal genes. The plasmid genes are contained in the 35,000 base tra region, where most of them are organized as a co-regulated gene block designated the traY → Z operon. The chromosomal genes have been identified among chromosomal mutants that fail to express donor activity and surface exclusion, even when they carry normal F-plasmid DNA. We show here that mutations in two chromosomal genes, cpxA and cpxB, together reduce the abundance of tra operon mRNA to 15% or less of the value in otherwise isogenic cpxA+cpxB+ cells. The cpxB1 mutation alone had no effect on the tra operon messenger RNA level, in agreement with previous evidence that this allele by itself is cryptic. We attribute the effect of both cpx mutations on tra operon mRNA to a transcriptional defect resulting from the inability of mutant cells to accumulate the traJ gene product, a 24,000 Mr outer membrane protein that is also required for efficient tra operon expression in vivo. Ultraviolet light-irradiated cpxA2 cpxB1 mutant cells infected with a λp(traJ) transducing bacteriophage that contains an intact traJ gene and its normal control sequences failed to accumulate the TraJ protein as a 24,000 Mr polypeptide, whereas cpxA+ cpxB1 cells, otherwise isogenic, did. In the same experimental system, both cpxA2 cpxB1 and cpxA+cpxB1 cells infected with the λp(traJ) bacteriophage accumulated comparable levels of RNA complementary to traJ. Moreover, cpxA2 cpxB1 and cpxA+ cpxB1 cells synthesized comparable levels of β-galactosidase from a traJ-lacZ protein fusion. These results show that the cpx mutations do not reduce transcription or translation initiation at traJ sequences. They define a new cellular contribution to conjugation, which we propose is related to the translocation of the TraJ protein to the outer membrane.
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ABSTRACT: The Escherichia coli CpxAR two-component signal transduction system senses and responds to extracytoplasmic stress. The cpxA101* allele was previously found to reduce F plasmid conjugation by post-transcriptional inactivation of the positive activator TraJ. Microarray analysis revealed upregulation of the protease-chaperone pair, HslVU, which was shown to degrade TraJ in an E. coli C600 cpxA101* background. Double mutants of cpxA101* and hslV or hslU restored TraJ and F conjugation to wild-type levels. The constitutive overexpression of nlpE, an outer membrane lipoprotein that induces the Cpx stress response, also led to HslVU-mediated degradation of TraJ and repression of F transfer. However, Cpx-mediated TraJ degradation appears to be growth phase-dependent, as induction of nlpE in mid-log phase cells did not appreciably alter TraJ levels. Further, His6-TraJ was sensitive to HslVU degradation in vitro only when it was purified from cells overexpressing nlpE. Thus, TraJ appears to become resistant to HslVU during normal growth, with this resistance mapping to the F transfer region. Extracytoplasmic stress prevents this modification of TraJ, leaving it susceptible to HslVU. Thus, the CpxAR stress response indirectly controls the synthesis of the F mating apparatus, a complex transenvelope type IV secretion system, by degrading TraJ.Molecular Microbiology 03/2008; 67(3):516-27. · 4.96 Impact Factor
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ABSTRACT: Conjugative plasmids are involved in the dissemination of important traits such as antibiotic resistance, virulence determinants and metabolic pathways involved in adapting to environmental niches, a process termed horizontal or lateral gene transfer. Conjugation is the process of transferring DNA from a donor to a recipient cell with the establishment of the incoming DNA and its cargo of genetic traits within the transconjugant. Conjugation is mediated by self-transmissible plasmids as well as phage-like sequences that have been integrated into the bacterial chromosome, such as integrative and conjugative elements (ICEs) that now include conjugative transposons. Both conjugative plasmids and ICEs can mediate the transfer of mobilizable elements by sharing their conjugative machinery. Conjugation can either be induced, usually by small molecules or peptides or by excision of the ICE from the host chromosome, or it can be tightly regulated by plasmid- and host-encoded factors. The transfer potential of these transfer regions depends on the integration of many signals in response to environmental and physiological cues. This review will focus on the mechanisms that influence transfer potential in these systems, particularly those of the IncF incompatibility group.Future Microbiology 07/2010; 5(7):1057-71. · 4.02 Impact Factor
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ABSTRACT: The Cpx envelope stress response is induced by the misfolding of periplasmic proteins and restores envelope homeostasis by upregulating several periplasmic protein folding and degrading factors. The Cpx response also regulates the expression of a variety of envelope-spanning protein complexes, including flagella, secretion systems and pili, which play an important role in pathogenesis. In a previous study, we inactivated the Cpx response in enteropathogenic Escherichia coli (EPEC), a causative agent of infant diarrhoea, and observed decreased expression of its major adhesin, the bundle-forming pilus (BFP). Here, we examined the mechanism underlying this BFP expression defect, and found that this phenotype can be attributed to insufficient expression of periplasmic folding factors, such as DsbA, DegP and CpxP. Hence, a low level of Cpx pathway activity promotes BFP synthesis by upregulating factors important for folding of BFP component proteins. Conversely, we found that full induction of the Cpx response inhibits BFP expression, mainly by repressing transcription of the bfp gene cluster. In combination with a previous report examining EPEC type III secretion, our results demonstrate that the Cpx response co-ordinates the repression of cell-surface structures during periods of envelope stress.Molecular Microbiology 04/2010; 76(5):1095-110. · 4.96 Impact Factor