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Pyelonephritis-associated pili (pap) allow uropathogenic Escherichia coli to bind to epithelial cells and play an important role in urinary tract infection. Expression of pap is controlled by a phase-variation mechanism, based on the two distinct heritable states that are the result of adenine N6-methylation in either of the two GATC sequences in its regulatory region. The methylation status of these two sequences is sensed by the action of two proteins, Lrp and PapI, and they play a central role in determining pap gene expression in both phase-ON and phase-OFF cells. We used modern NMR techniques to determine the solution structure and backbone dynamics of PapI. We found its overall fold resembles closely that of the winged helix-turn-helix family of DNA-binding proteins. We determined that PapI possesses its own DNA-binding activity, albeit non-sequence-specific, independent of Lrp. PapI appears to bind to DNA with a K(d) in the 10 microM range. Possible mechanisms by which PapI might participate in the regulation of the pap operon are discussed in light of these new findings. 相似文献
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Growth phase variation of integration host factor level in Escherichia coli. 总被引:17,自引:3,他引:14
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We have measured the intracellular abundance of integration host factor (IHF), a site-specific, heterodimeric DNA-binding protein, in exponential- and stationary-phase cultures of Escherichia coli K-12. Western immunoblot analysis showed that cultures that had been growing exponentially for several generations contained 0.5 to 1.0 ng of IHF subunits per microgram of total protein and that this increased to 5 to 6 ng/microgram in late-stationary-phase cultures. IHF is about one-third to one-half as abundant in exponentially growing cells as HU, a structurally related protein that binds DNA with little or no site specificity. Wild-type IHF is metabolically stable, but deletion mutations that eliminated one subunit reduced the abundance of the other when cells enter stationary phase. We attribute this reduction to the loss of stabilizing interactions between subunits. A mutation that inactivates IHF function but not subunit interaction increased IHF abundance, consistent with results of previous work showing that IHF synthesis is negatively autoregulated. We estimate that steady-state exponential-phase cultures contain about 8,500 to 17,000 IHF dimers per cell, a surprisingly large number for a site-specific DNA-binding protein with a limited number of specific sites. Nevertheless, small reductions in IHF abundance had significant effects on several IHF-dependent functions, suggesting that the wild-type exponential phase level is not in large excess of the minimum required for occupancy of physiologically important IHF-binding sites. 相似文献
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The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function. 相似文献
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The assembly of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli requires the processing of CFA/III major pilin (CofA) by a peptidase, likely another type IV pilus formation system. Western blot analysis of CofA reveals that CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to 20.5-kDa mature pilin by a prepilin peptidase. This processing is essential for exportation of the CofA from the cytoplasm to the periplasm. In this experiment, the structural gene, cofP, encoding CFA/III prepilin peptidase which cleavages at the Gly-30-Met-31 junction of CofA was identified, and the nucleotide sequence of the gene was determined. CofP consists of 819 bp encoding a 273-amino acid protein with a relative molecular mass of 30,533 Da. CofP is predicted to be localized in the inner membrane based on its hydropathy index. The amino acid sequence of CofP shows a high degree of homology with other prepilin peptidases which play a role in the assembly of type IV pili in several gram-negative bacteria. 相似文献
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Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle.
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A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle. 相似文献
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Isolation and characterization of Escherichia coli phase variants and mutants deficient in type 1 pilus production.
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L M Swaney Y P Liu C M To C C To K Ippen-Ihler C C Brinton 《Journal of bacteriology》1977,130(1):495-505
Type 1 pili of Escherichia coli are the prototype of the somatic class of pili found on many strains of bacteria. As a first step in the genetic analysis of type 1 piliation, an extensive series of nonpiliated derivatives of E. coli K-12 strain AW405, was characterized to produce attached or free pili when examined in the antiserum or appeared to produce attached or free pili when examined in the electron microscope. The derivatives fell into two classes; phase variants and mutants. Phase variants that formed colonies of two distinctive types, one associated with a predominantly piliated (P+), and the other associated with a nonpiliated (P-) phase, were obtained. Each phase could give rise to the other at a relatively high rate, which was greater in the P- to P+ direction during culture in unshaken liquid medium. In addition, 77 Pil- mutants were selected on the basis of a subtle difference in colonial morphology. The mutants reverted, if at all, at a much lower rate than that of the P- to P+ change. The stability of Pil- derivatives grown in unshaken liquid medium was used as a criterion for distinguishing between phase variants and mutants, Phase variation also effected colonial morphology and chemotactic swarming. These properties did not directly depend upon piliation since Pil- mutants were only slightly altered in colonial form and unaltered in chemotactic swarming. Piliation of Pil+ bacteria was quantitatively affected by growth conditions. 相似文献
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murH, a new genetic locus in Escherichia coli involved in cell wall peptidoglycan biosynthesis. 总被引:3,自引:2,他引:1
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A temperature-sensitive mutant of Escherichia coli defective in peptidoglycan synthesis was characterized. The incorporation of radiolabeled meso-diaminopimelate into peptidoglycan by the mutant was inhibited at the restrictive growth temperature, resulting in autolysis. The defective step appeared to be part of the terminal stage in peptidoglycan synthesis involving the incorporation of disaccharide peptide units into the wall peptidoglycan. The mutation was assigned to a new locus, designated murH, at 99.2 min on the E. coli linkage map. 相似文献
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Feng L Liu B Liu Y Ratiner YA Hu B Li D Zong X Xiong W Wang L 《Journal of bacteriology》2008,190(13):4470-4477
The occurrence of unilateral flagellar phase variation was previously demonstrated in Escherichia coli strains carrying the non-fliC flagellin-specifying locus flk. In this study, we investigated the mechanism involved in this process. By using sequencing and sequence analysis, the flk region between the chromosomal genes yhaC and rnpB was characterized in all described flk-positive E. coli strains, including the H35 strain identified in this study (the other strains used are H3, H36, H47, and H53 strains), and this region was found to contain a putative integrase gene and flanking direct repeats in addition to the flk flagellin-specifying gene flkA and a fliC repressor gene, flkB, indicating that there is a typical genomic islet (GI), which was designated the flk GI. The horizontal transfer potential of the flk GI was indicated by detection of the excised extrachromosomal circular form of the flk GI. By generating fliC-expressing variants of H3 and H47 strains, unilateral flagellar phase variation in flk-positive strains was shown to be mediated by excision of the flk GI. The function of the proposed integrase gene was confirmed by deletion and a complementation test. The potential integration sites of the flk GI were identified. A general model for flagellar phase variation in flk-positive E. coli strains can be expressed as fliC(off) + flkA(on) --> fliC(on) + flkA(none). This is the first time that a molecular mechanism for flagellar phase variation has been reported for E. coli. 相似文献
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Inversion-independent phase variation of type 1 fimbriae in Escherichia coli. 总被引:10,自引:5,他引:5
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M S McClain I C Blomfield K J Eberhardt B I Eisenstein 《Journal of bacteriology》1993,175(14):4335-4344
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Chromosomal location of a gene (nmpA) involved in expression of a major outer membrane protein in Escherichia coli. 总被引:6,自引:6,他引:0
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The phenotypic expression of protein E, a recently described major outer membrane protein, is associated with a mutation at a locus on the Escherichia coli chromosome that we call nmpA. nmpA is located between rbsK and uncA at 82.7 min on the E. coli linkage map. The nmpA locus is also the site of the mutations which lead to the formation of major outer membrane proteins Ic or e. It is likely proteins E, Ic, and e are closely related or identical. The mutant nmpA allele is dominant. 相似文献
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I. Loubens L. Debarbieux A. Bohin J.-M. Lacroix J.-P. Bohin 《Molecular microbiology》1993,10(2):329-340
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