Phase Variation Of Type Research Articles

Excision-reintegration at a pneumococcal phase-variable restriction-modification locus drives within- and between-strain epigenetic differentiation and inhibits gene acquisition.

Phase-variation of Type I restriction-modification systems can rapidly alter the sequence motifs they target, diversifying both the epigenetic patterns and endonuclease activity within clonally descended populations. Here, we characterize the Streptococcus pneumoniae SpnIV phase-variable Type I RMS, encoded by the translocating variable restriction (tvr) locus, to identify its target motifs, mechanism and regulation of phase variation, and effects on exchange of sequence through transformation. The specificity-determining hsdS genes were shuffled through a recombinase-mediated excision-reintegration mechanism involving circular intermediate molecules, guided by two types of direct repeat. The rate of rearrangements was limited by an attenuator and toxin-antitoxin system homologs that inhibited recombinase gene transcription. Target motifs for both the SpnIV, and multiple Type II, MTases were identified through methylation-sensitive sequencing of a panel of recombinase-null mutants. This demonstrated the species-wide diversity observed at the tvr locus can likely specify nine different methylation patterns. This will reduce sequence exchange in this diverse species, as the native form of the SpnIV RMS was demonstrated to inhibit the acquisition of genomic islands by transformation. Hence the tvr locus can drive variation in genome methylation both within and between strains, and limits the genomic plasticity of S. pneumoniae.

Pleiotropic Roles of uvrY on Biofilm Formation, Motility and Virulence in Uropathogenic Escherichia coli CFT073

Urinary tract infections primarily caused by uropathogenic strains of Escherichia coli (E. coli) remain a significant public health problem in both developed and developing countries. An important virulence determinant in uropathogenesis is biofilm formation which requires expression of fimbriae, flagella, and other surface components such as lipopolysaccharides. In this study, we explored the regulation of uvrY and csrA genes in biofilm formation, motility and virulence determinants in uropathogenic E. coli. We found that mutation in uvrY suppressed biofilm formation on abiotic surfaces such as polyvinyl chloride, polystyrene and glass, and complementation of uvrY in the mutant restored the biofilm phenotype. We further evaluated the role of uvrY gene in expression of type 1 fimbriae, an important adhesin that facilitates adhesion to various abiotic surfaces. We found that phase variation of type 1 fimbriae between fimbriated and afimbriated mode was modulated by uvrY at its transcriptional level. Deletion mutant of uvrY lowered expression of fimbrial recombinase genes, such as fimB, fimE, and fimA, a gene encoding major fimbrial subunit. Furthermore, transcription of virulence specific genes such as papA, hlyB and galU was also reduced in the deletion mutant. Swarming motility and expression of flhD and flhC was also diminished in the mutant. Taken together, our findings unravel a possible mechanism in which uvrY facilitates biofilm formation, persistence and virulence of uropathogenic E. coli.

SlyA Protein Activates fimB Gene Expression and Type 1 Fimbriation in Escherichia coli K-12

We have demonstrated that SlyA activates fimB expression and hence type 1 fimbriation, a virulence factor in Escherichia coli. SlyA is shown to bind to two operator sites (OSA1 and OSA2), situated between 194 and 167 base pairs upstream of the fimB transcriptional start site. fimB expression is derepressed in an hns mutant and diminished by a slyA mutation in the presence of H-NS only. H-NS binds to multiple sites in the promoter region, including two sites (H-NS2 and H-NS3) that overlap OSA1 and OSA2, respectively. Mutations that disrupt either OSA1 or OSA2 eliminate or reduce the activating effect of SlyA but have different effects on the level of expression. We interpret these results as reflecting the relative competition between SlyA and H-NS binding. Moreover we show that SlyA is capable of displacing H-NS from its binding sites in vitro. We suggest SlyA binding prevents H-NS binding to H-NS2 and H-NS3 and the subsequent oligomerization of H-NS necessary for full inhibition of fimB expression. In addition, we show that SlyA activates fimB expression independently of two other known regulators of fimB expression, NanR and NagC. It is demonstrated that the rarely used UUG initiation codon limits slyA expression and that low SlyA levels limit fimB expression. Furthermore, Western blot analysis shows that cells grown in rich-defined medium contain ∼1000 SlyA dimers per cell whereas those grown in minimal medium contain >20% more SlyA. This study extends our understanding of the role that SlyA plays in the host-bacterial relationship.

Distant cis-active sequences and sialic acid control the expression of fimB in Escherichia coli K-12.

The phase variation of type 1 fimbriation in Escherichia coli is controlled by the inversion of a 314 bp element of DNA, determined by FimB (switching in both directions) or FimE (switching from the ON-to-OFF orientation predominantly), and influenced by auxiliary factors IHF, Lrp and H-NS. The fimB gene is separated from the divergently transcribed yjhATS operon by a large (1.4 kbp) intergenic region of unknown function. Here, we show that fimB expression is regulated by multiple cis-active sequences that lie far upstream (>600 bp) of the transcription start sites for the recombinase gene. Two regions characterized further (regions 1 and 2) show sequence identity, and each coincides with a methylation-protected Dam (5'-GATC) site. Regions 1 and 2 apparently control fimB expression by an antirepression mechanism that involves additional sequences proximal to yjhA. Region 1 encompasses a 27 bp DNA sequence conserved upstream of genes known (nanAT ) or suspected (yjhBC) to be involved in sialic acid metabolism, and we show that FimB expression and recombination are suppressed by N-acetylneuraminic acid. We propose that E. coli recognizes the amino sugars as a harbinger of potential host defence activation, and suppresses the expression of type 1 fimbriae in response.

PapB paralogues and their effect on the phase variation of type 1 fimbriae in Escherichia coli.

Recent work has demonstrated that expression of type 1 fimbriae is repressed by PapB, a regulator of pyelonephritis-associated pili (P-pili). PapB belongs to family of related adhesin regulators, for which consensus residues required for DNA binding and oligomerization have been identified. Of the regulators tested in this study, PapB, SfaB (S-fimbriae) and PefB (Salmonella enterica serovar Typhimurium--plasmid-encoded fimbriae) repressed FimB-promoted off-to-on inversion of the fim switch, although complete repression was only demonstrated by PapB. DaaA, FaeB, FanA, FanB and ClpB had no effect on fim switching. In addition, only PapB stimulated FimE-promoted on-to-off inversion. Deletion analysis demonstrated that this specificity resides in the carboxy terminal of the protein, and not the amino terminal, with the central region being homologous among the family members. Exchange of Leu(82) and Ile(83) of PapB for the equivalent residues from the DaaA protein (Phe and Gln) within the carboxy terminal virtually abolished cross-talk activity. Whereas PapB can bind to a region around the left inverted repeat of the fim switch, DaaA and the PapB double mutant were effectively unable to bind this region. A previously characterized PapB DNA binding mutant also failed to bind to this region and failed to inhibit FimB activity at the fim switch. Thus, repression of fim expression appears unique to PapB and SfaB within E. coli and requires DNA binding involving amino acid residues located both within the homologous core and in the heterogeneous carboxy terminus. The variation in the carboxy terminus between the PapB family members explains their differential effects on fim. This mechanism of cross-talk seems restricted to the P and S family adhesins with type 1 fimbriae and may ensure variable and sequential expression of adhesins during urinary tract infections.

The molecular basis for the specificity of fimE in the phase variation of type 1 fimbriae of Escherichia coli K-12.

The expression of type 1 fimbriae in Escherichia coli is phase variable, with cells switching between fimbriate (ON) and afimbriate (OFF) phases. The phase variation is dependent on the orientation of a 314 bp DNA element (the switch) that undergoes DNA inversion. DNA inversion requires either fimB or fimE, site-specific recombinases that differ in both specificity and activity. Whereas fimB promotes recombination with little orientational bias, fimE promotes recombination in the ON-to-OFF direction exclusively. In wild-type cells, fimE activity predominates and, hence, most bacteria are afimbriate. Here, it is shown that fimE specificity is caused by two different, but complementary, mechanisms. First, FimE shows a strong preference for the switch in the ON orientation as a substrate for recombination. Differences in the nucleotide sequence of the recombinase binding sites is a key factor in determining FimE specificity, although one or more additional cis-active sites that flank the fim switch also appear to be involved. Secondly, the orientation of the switch controls fimE in cis, most probably to control recombinase expression.

Orientation-dependent enhancement by H-NS of the activity of the type 1 fimbrial phase switch promoter in Escherichia coli.

Phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a 314-bp DNA element, positioned proximal to and upstream of fimA, which encodes the major type 1 fimbrial subunit. This DNA switch region contains a promoter that drives transcription of fimA only when the switch is in the ON orientation. Using chromosomal and plasmid-borne lacZ reporter cassettes, we show here how the global regulator H-NS affects the activity of the fimA promoter. In phase-locked reporter cassettes the activity of the fimA promoter was found to be enhanced in a hns-positive background, but only when the switch was in the ON orientation. Also, the number of fimbriae produced by a phase-locked ON strain was significantly higher in a hns-positive background. By means of competitive gel retardation and DNase I protection assays, H-NS binding to DNA segments adjacent to and within the phase switch region was demonstrated.

Differential temperature modulation by H-NS of the fimB and fimE recombinase genes which control the orientation of the type 1 fimbrial phase switch

Phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a 314-bp DNA element. This DNA switch directs transcription of fimA, encoding the major type 1 fimbrial subunit, in the on orientation but not in the off orientation. Inversion of the DNA element requires either FimB (confers both on and off orientations) or FimE (confers off orientation). Here we show, by chromosomally located fimB- and fimE- lacZ cassettes in isogenic strain sets differing only in the hns locus, how the global regulator H-NS affects the expression of type 1 fimbriae. H-NS was found to downregulate fimB and fimE in a temperature-dependent manner which affected the genes inversely at 30°C and 37°C. By gel-retardation assays H-NS binding was demonstrated to the regions containing the fimB promoter and the fimE promoter, respectively. In vitro recombination analysis suggested no direct involvement of H-NS in the inversion of the phase switch. Rather than directly affecting the switching process per se, it appeared that the orientation of this element was affected by the differential temperature modulation of H-NS of the fimB and fimE genes. Taken together the results suggest that H-NS modulates expression of type 1 fimbriae in a way which seems to favor a fimbriate state at the mammalian body temperature.

Differential temperature modulation by H-NS of the fimB and fimE recombinase genes which control the orientation of the type 1 fimbrial phase switch.

Phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a 314-bp DNA element. This DNA switch directs transcription of fimA, encoding the major type 1 fimbrial subunit, in the on orientation but not in the off orientation. Inversion of the DNA element requires either FimB (confers both on and off orientations) or FimE (confers off orientation). Here we show, by chromosomally located fimB- and fimE-lacZ cassettes in isogenic strain sets differing only in the hns locus, how the global regulator H-NS affects the expression of type 1 fimbriae. H-NS was found to downregulate fimB and fimE in a temperature-dependent manner which affected the genes inversely at 30 degrees C and 37 degrees C. By gel-retardation assays H-NS binding was demonstrated to the regions containing the fimB promoter and the fimE promoter, respectively. In vitro recombination analysis suggested no direct involvement of H-NS in the inversion of the phase switch. Rather than directly affecting the switching process per se, it appeared that the orientation of this element was affected by the differential temperature modulation of H-NS of the fimB and fimE genes. Taken together the results suggest that H-NS modulates expression of type 1 fimbriae in a way which seems to favor a fimbriate state at the mammalian body temperature.

Leucine alters the interaction of the leucine-responsive regulatory protein (Lrp) with the fim switch to stimulate site-specific recombination in Escherichia coli.

The leucine-responsive regulatory protein (Lrp) is a global regulator that controls the expression of numerous operons in Escherichia coli. Lrp can act as a repressor or as an activator of transcription with its effects being potentiated, repressed or unaffected by the presence of exogenous leucine. The phase variation of type 1 fimbria in E. coli provides a unique system in which to investigate the effects of leucine on Lrp, as it is the only known example in which Lrp is a positive regulator and leucine potentiates this effect. Previous studies determined that Lrp binds with high affinity to two sites within the fim switch (fim sites 1 and 2), and binding to these sites stimulates recombination. Here, it is shown that, even though leucine stimulates the fim switch in vivo, it nevertheless causes a slight decrease in Lrp binding to the fim switch in vitro. These contradictory results are explicable by the finding that Lrp binding to a third region adjacent to fim sites 1 and 2 inhibits recombination. According to this model, leucine stimulates recombination by selectively disrupting Lrp binding to this newly characterized region, while having little or no effect on Lrp binding to fim sites 1 and 2.

Lrp stimulates phase variation of type 1 fimbriation in Escherichia coli K-12

The phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a short DNA element. This element (switch) acts in cis to control transcription of fimA, the major fimbrial subunit gene. Thus, fimA is transcribed when the switch is in one orientation (the on orientation) but not the other (the off orientation). The fim inversion requires either fimB (on-to-off or off-to-on inversion) or fimE (on-to-off inversion only), as well as integration host factor, and is also influenced by the abundant DNA-binding protein H-NS. Here we report that an additional gene, lrp, a factor known to influence the expression of both Pap and K99 fimbriae, is also required for normal activity of the fim switch. The frequencies of both fimB-promoted and fimE-promoted inversions, and consequently the phase variation of type 1 fimbriation, are lower in lrp mutants. Lrp affects slightly the transcription of both fimB (which is increased) and fimE (which is decreased). We believe that these alterations in fimB and fimE transcription alone are unlikely to account for the sharp reduction in switching found in lrp mutants.

Growth conditions mediate differential transcription of fim genes involved in phase variation of type 1 pili.

Type 1 pili in Escherichia coli undergo phase variation in which individual cells in a population reversibly switch between piliated (Pil+) and nonpiliated (Pil-) states. The switching process is mediated by an invertible DNA fragment which contains the promoter for fimA, the gene encoding the major structural subunit of type 1 pili. Although type 1 pili randomly phase vary in broth cultures, many clinical isolates of E. coli do not express type 1 pili when cultured on agar media. We investigated the role of the invertible element and the upstream genes, fimB and fimE, in the agar-mediated suppression of pili in an agar-negative clinical isolate, strain 149. Southern hybridization and polymerase chain reaction analyses of the fimA promoter region in broth-grown 149 cells indicated that the invertible element was present in orientations corresponding to both Pil+ and Pil- phenotypes. In contrast, only one orientation of the invertible element, corresponding to the Pil- phenotype, was observed in strain 149 cells cultured on agar. A second clinical isolate, strain 2-7, which expresses type 1 pili on agar was also examined; the invertible element was found in both the Pil+ and Pil- orientations during growth of this strain on agar as well as in broth. The introduction of the fim gene cluster from strain J96 on a multicopy plasmid into agar-negative strain 149 resulted in the production of both J96 and 149 pili during growth on agar. Experiments with subclones of the J96 genes indicated that the presence of an intact fimB gene allowed strain 149 pili to be produced on agar. Differences in pilus production between agar and broth cultures appear to be the result of differential transcription of fimB and fimE under the two growth conditions. In contrast, the pattern of expression of these genes in agar phase-variable strain 2-7 did not differ between broth- and agar-grown cells.

Roles of fimB and fimE in site-specific DNA inversion associated with phase variation of type 1 fimbriae in Escherichia coli

Evidence obtained with an improved in vivo assay of fimbrial phase variation in Escherichia coli supported a revised understanding of the roles of fimB and fimE in the site-specific DNA rearrangement with which they are associated. A previously proposed model argued that fimB and fimE play antagonistic, unidirectional roles in regulating the orientation of the invertible DNA element located immediately upstream of fimA, the gene encoding the major subunit of type 1 fimbriae. This conclusion, though, is based on an in vivo DNA inversion assay using recombinant plasmid substrates under conditions that, among other things, were incapable of detecting recombination of the fim invertible element from the on to the off orientation. Using a modified system that overcome this and several additional technical problems, we confirmed that fimB acts independently of fimE on the invertible element and that the additional presence of fimE results in the preferential rearrangement of the element to the off orientation. It is now demonstrated that fimE can act in the absence of fimB in this recombination to promote inversion primarily from on to off. In contrast to the previous studies, the effect of fimB on a substrate carrying the invertible element in the on orientation could be examined. It was found that fimB mediates DNA inversion from on to off, as well as from off to on, and that, contrary to prior interpretations, the fimB-associated inversion occurs with only minimal orientational preference to the on phase.

Potential role of phase variation of type 1 pili in urinary tract infection and bacterial prostatitis

Adherence of Escherichia coli to uroepithelial cells is an important step in the pathogenesis of urinary tract infection. The process is frequently mediated by pili which undergo phase variation in response to changes in environmental growth conditions in vitro. It is possible that phase variation of pili and other bacterial virulence factors may play a role in the pathogenesis of bacterial prostatitis. The obvious differences in the environmental conditions of urethra, bladder, and prostate could favor strains that have the ability to adapt. For example, adhesion to the urethral and bladder mucosa could be facilitated by pili and their expression could be advantageous in the early phase of prostatitis. However, after the prostate ducts are colonized, pili may be detrimental since they can render E. coli more susceptible to phagocytosis. Further studies on phase variation of virulence factors of bacteria that cause prostatitis appear warranted.

Change in degree of type 1 piliation of Escherichia coli during experimental peritonitis in the mouse.

To determine whether expression of type 1 pili varies during the course of Escherichia coli infection in vivo, mice were injected intraperitoneally with 5 X 10(7) CFU of piliated or nonpiliated phase variants per ml, and the degree of piliation was measured in peritoneal exudate by an enzyme-linked immunosorbent assay inhibition method. In the animals challenged with the piliated bacteria, the numbers of organisms increased a log over 9 h and the amount of pilus antigen decreased from 3 to 0.075 micrograms/10 bacteria. After a 4-h delay, nonpiliated bacteria also increased by one log over 9 h; however, the amount of piliation remained virtually undetectable. Piliated E. coli were more virulent than nonpiliated variants in this model (50% lethal dose of 7.5 X 10(6) versus 3 X 10(7), respectively). The difference was significantly reduced by prior passive immunization with rabbit serum containing high titers of antipili antibody. Piliated bacteria adhered in significantly greater numbers to isolated mouse peritoneal membranes than did nonpiliated variants (15,400 +/- 2,700 versus 1,300 +/- 700 bacteria/mm2, respectively; P = 0.05). Adherence was inhibited by the presence of 0.1 M alpha methyl mannose (1,500 +/- 1,800 bacteria/mm2, P = 0.01). These results confirm the results of previous qualitative studies showing that phase variation of type 1 pili occurs in vivo and suggest that these pili may confer an initial advantage for growth of E. coli in the peritoneal cavity, presumably by fostering colonization of the peritoneal serosal surface.

Role of type 1 pili and effects of phase variation on lower urinary tract infections produced by Escherichia coli.

Phase variation of type 1 pili (fimbriae) was studied during the in vivo growth of Escherichia coli in two animal models. In the first, a heavily piliated urinary tract isolate (strain 149) was placed in 1-cm polypropylene chambers sealed with 0.22-micron-pore-size filters. The chambers were surgically implanted intraperitoneally in mice and recovered at various times. Piliation, as determined by electron microscopy and by measuring the minimum number of bacteria needed to produce mannose-sensitive hemagglutination, gradually decreased, and by day 5, most of the organisms were nonpiliated. In the second model, piliated and nonpiliated E. coli phase variants were inoculated into the bladders of BALB/c mice via urinary catheters, and their fate in the lower urinary tract was studied. Viable counts of bladder homogenates revealed that piliated phase variants were significantly more effective in colonizing the bladder urothelium than were their nonpiliated counterparts. Specific antibody to type 1 pili prevented colonization by the piliated organisms. After inoculation of piliated variants, the bladder-associated bacteria gave rise to approximately 80% mannose-sensitive hemagglutination-positive colonies, and immunocytochemistry of bladder lavages revealed large numbers of type 1 piliated bacteria adhering to the bladder transitional cells. Electron microscopy confirmed the presence of piliated bacteria in association with the bladder urothelium. The urine of these mice, whose bladders were colonized with piliated bacteria, frequently showed no growth, and when bacteria were present, strain 149 yielded less than 30% hemagglutination-positive colonies. The results suggest that for some E. coli strains, phase variation may be a factor in determining the fate of the E. coli in the urinary tract and that the urine may not necessarily reflect the bacteriologic state of the bladder mucosa.

Genetic analysis of the phase variation control of expression of type 1 fimbriae in Escherichia coli.

Expression of type 1 fimbriae in Escherichia coli exhibits phase variation, whereby individual cells can alternate between states of organelle expression (Fim+) and nonexpression (Fim-). Strains with a fimD-lac operon fusion, in which lac, rather than fimD, expression is under the control of the fimD promoter, undergo Lac+ in equilibrium Lac- phase variation, instead. After positioning a lambda prophage adjacent to the operon fusion, we were able to isolate specialized lambda phage carrying both the fimD-lac fusion and the phase variation control region. Introduction of such phage into an Fim+ strain resulted in construction of a strain with a double, independently switching phenotype (Fim+ in equilibrium Fim- and Lac+ in equilibrium Lac-), demonstrating that the region controlling phase variation is contiguous with the fimD-lac operon fusion and is cis acting. When the specialized lambda phage was propagated on a delta lac delta fim strain, phase variation occurred within the plaques, confirming that the phase variation control region is carried on the specialized transducing phage. All lysogens acquired the Lac+ in equilibrium Lac- phenotype, except for two nonswitching Lac+ recombinants, which acquired Lac+ in equilibrium Lac- phase variation only by trans complementation with fim. Phase variation of type 1 fimbriae, therefore, appears to involve both a cis-active element, which is cloned on a specialized lambda phage, and a trans-active permissive factor, which is not present on the phage, but rather must be supplied by the recipient strain in the transduction.