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PubMed1997

Phase variation and conservation of lipooligosaccharide epitopes in Haemophilus somnus.

The bovine-specific pathogen Haemophilus somnus is capable of undergoing structural and antigenic phase variation in its lipooligosaccharide (LOS) components after in vivo and in vitro passage. However, commensal isolates from the reproductive tract have not been observed to vary in phase (T. J. Inzana, R. P. Gogolewski, and L. B. Corbeil, Infect. Immun. 60:2943-2951, 1992). We now report that specific monoclonal antibodies (MAbs) to the LOSs of Haemophilus aegyptius, Neisseria gonorrhoeae, and Haemophilus influenzae, as well as H. somnus, reacted with some phase-variable epitopes in H. somnus LOS. All reactive MAbs bound to LOS components of about 4.3 kDa in the same H. somnus isolates, including a non-phase-varying strain. Following in vitro passage of a clonal variant of strain 738 that was nonreactive with the MAbs, 11.8% of young colonies shifted to a reactive phenotype. A digoxigenin-labelled 5'-CAATCAATCAATCAATCAATCAATCAAT-3' oligonucleotide probe hybridized to genomic DNA from strain 738 but did not react with DNA from a non-phase-varying strain. Sequence analysis of the gene containing 5'-CAAT-3' tandem sequences revealed 48% amino acid homology with the lex-2B gene-encoded protein of H. influenzae type b. Our results indicate that some LOS epitopes are conserved between H. somnus and other Haemophilus and Neisseria species, that LOS phase variation may occur at a high rate in some strains of H. somnus, and that phase variation may, in part, be due to 5'-CAAT-3' tandem sequences present in H. somnus genes.

Amino Acid Sequence↗
PubMed1990

Colonial morphology of staphylococci on Memphis agar: phase variation of slime production, resistance to beta-lactam antibiotics, and virulence.

The growth of Staphylococcus epidermidis sensu stricto and Staphylococcus saprophyticus on Memphis agar yielded up to 6 morphotypes with each strain. With S. epidermidis, one morphotype produced slime (rho) but became non-slime-producing (epsilon) at a high frequency. The slime-producing rho variants were methicillin-resistant and more virulent than methicillin-susceptible epsilon variants in an endocarditis model. With S. saprophyticus, phase variation was of higher frequency. Nitrosoguanidine mutagenesis produced a stable blue epsilon form that was more virulent than the parent in a mouse model of urinary tract infection. Mutants with the blue epsilon phenotype differed from gold epsilon parents in a variety of phenotypic properties, including increased resistance to oxacillin. These staphylococcal species have a high frequency of phase variation: Phase variants differ in antibiotic resistance and virulence, which is only partially correlated with suggested virulence factors such as slime production.

Animals↗
PubMed2000

Phase variation of F165(1) (Prs-like) fimbriae from Escherichia coli causing septicaemia in animals.

Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and synthesize fimbriae involved in virulence, designated as F165(1). F165(1) fimbriae belong to the P fimbrial family and are encoded by the foo gene cluster. The foo regulatory region of strain 5131 possesses characteristics similar to that of members of the P regulatory family, including papI and papB homologues, and two GATC sites separated by 102 bp, targets of differential Dam methylation. In wild-type strains, the synthesis of F165(1) is repressed by leucine and the fimbriae undergo phase variation. Immunofluorescence staining showed that phase variation of F165(1) results in a majority of cells (98%) in the ON phase, in contrast with phase variation of other members of this regulatory family, for which the majority of the cells are in the OFF state. Using a translational fusion in strain 5131 between phoA and fooA, encoding for the major structural subunit of F165(1), it was shown that leucine inhibits the OFF to ON switch and modulates the basal transcription of the foo operon.

Alanine↗
PubMed1995

Identification and characterization of a cell envelope protein of Haemophilus influenzae contributing to phase variation in colony opacity and nasopharyngeal colonization.

Haemophilus influenzae undergoes spontaneous phase variation in colony morphology. Organisms from transparent colonies efficiently colonize the nasopharynx in an infant rat model of H. influenzae carriage, whereas organisms from more opaque colonies are deficient at colonization. A genetic approach relying on the transformability of H. influenzae was used to identify a locus contributing to opacity variation. By screening a library of chomosomal DNA from an opaque variant of strain Rd, it was possible to isolate a single clone capable of transforming a transparent Rd host to a more opaque phenotype. A region containing two genes, designated oapA and oapB, was identified. The deduced amino acid sequence of oapB has similarity to a consensus sequence for bacterial lipoproteins. Genetically defined mutations in oapA were transformed into the transparent Rd to confirm that this gene is required for expression of the transparent colony phenotype. Although oapA lacks a signal sequence, gene fusions to phoA show that OapA is secreted in H. influenzae and undergoes phase variation in expression. Mutagenesis of oapA in strain Rd, and type b strain Eagan, resulted in loss of the ability to colonize the nasopharynx of infant rats. The type b mutant, however, was as virulent as its parent strain when inoculated intraperitoneally. This suggests that the contribution of OapA to pathogenesis is limited to events associated with colonization of the mucosal surface.

Animals↗
PubMed1993

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.

Bacterial Proteins↗
PubMed2003

Phase variation in Bartonella henselae.

Bartonella henselae is a fastidious, Gram-negative bacterial pathogen of cats and humans. Previous workers have shown that serial passage in vitro leads to attenuation of virulence-associated attributes such as expression of pili, invasion of human epithelial cell lines and the stimulation of endothelial cell proliferation. In contrast to the published data, it was found that pilin expression is frequently preserved in organisms which have undergone phase variation in vitro. Transition from a slow-growing, dry agar-pitting (DAP) to a faster-growing, smooth non-agar-pitting (SNP) form appears to occur predictably and may reflect competition between two populations growing at different rates. Better survival of the slower-growing (DAP) form may explain its relatively easy retrieval from piliated SNP populations allowed to age on solid media. Pilin expression is associated with auto-agglutination in liquid suspension or broth cultures, and appears to be necessary but not sufficient for expression of the agar-pitting phenotype and for the formation of biofilms. Outer-membrane protein variation is seen in association with phase variation, but lipopolysaccharide expression is preserved in piliated as well as extensively passaged non-piliated isolates. The EagI/HhaI infrequent restriction site-PCR fingerprint, which has been previously used to discriminate between serotypes Marseille and Houston, is shown to alter with phase variation in vitro, and there is evidence that genetic change accompanies these events. The extent of genetic and phenotypic variability of phase-variant B. henselae has previously been underestimated. It may lead to new insights into the pathogenicity of this organism, and must be considered when interpreting data arising from such studies.

Animals↗
PubMed1995

Using a phase-locked mutant of Myxococcus xanthus to study the role of phase variation in development.

The bacterium Myxococcus xanthus undergoes a primitive developmental cycle in response to nutrient deprivation. The cells aggregate to form fruiting bodies in which a portion of the cells differentiate into environmentally resistant myxospores. During the growth portion of the M. xanthus life cycle, the organism also undergoes a phase variation, in which cells alternate between yellow and tan colony-forming variants. Phase variation occurs in our laboratory strain (M102, a derivative of DK1622) at a frequency high enough that a single colony of either the yellow or the tan phase already contains cells of the alternate phase. In this study we demonstrate that tan cells within a predominantly yellow population of phase variation-proficient cells are preferentially recovered as heat- and sonication-resistant spores. To further investigate the possibility of a differential role of tan and yellow cells during development, a tan-phase-locked mutant was used to compare the developmental phenotypes of a pure tan population with a predominantly yellow, phase variation-proficient population. Pure tan-phase populations did not produce fruiting bodies or mature spores under conditions in which predominantly yellow wild-type populations did so efficiently. Pure populations of tan-phase cells responded to developmental induction by changing from vegetative rod-shaped cells to round forms but were unable to complete the maturation to heat- and sonication-resistant, refractile spores. The developmental defect of a tan-phase-locked mutant was rescued by the addition of phase variation-proficient cells from a predominantly yellow culture. In such mixtures the tan-phase-locked mutant not only completed the process of forming spores but also was again preferentially represented among the viable spores. These findings suggest the intriguing possibility that the tan-phase cells within the vegetative population entering development are the progenitors of spores and implicate a requirement for yellow-phase cells in spore maturation.

Bacterial Adhesion↗
PubMed2001

Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis.

Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC(+) phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.

Animals↗
PubMed2000

Phase variation of Ag43 in Escherichia coli: Dam-dependent methylation abrogates OxyR binding and OxyR-mediated repression of transcription.

It has been shown previously that phase variation of the outer membrane protein Antigen43 (Ag43) of Escherichia coli requires the DNA-methylating enzyme deoxyadenosine methyltransferase (Dam) and the global regulator OxyR. In this study, we analysed the regulation of the Ag43 encoding gene (agn) using isolates containing a fusion of the agn regulatory region to the reporter gene lacZ. Our results indicate that phase variation of Ag43 is regulated at the level of transcription. Repression of agn'-lacZ transcription required OxyR, whereas activation required Dam. The regulatory region of agn contains three GATC sequences, which are target sites for Dam-dependent methylation. In vivo, the methylation state of these GATC sequences correlated with the transcription state of agn'-lacZ. These GATC sequences were not protected from Dam-dependent methylation in an oxyR background, suggesting that OxyR binding results in Dam-dependent methylation protection in OFF cells. In vitro, both oxidized OxyR and OxyR(C199S), which is locked in the reduced conformation, bound to the agn regulatory region, but methylation of the three GATC sequences abrogated this binding. In vivo, OxyR(C199S) was sufficient to repress Ag43 transcription. Our data support a model in which OxyR-mediated repression of transcription is alleviated by methylation of three GATC sequences in its binding site. In addition, we show that, in an oxyR background, Dam was still required for full activation, suggesting that the model concerning the role of Dam in agn regulation is incomplete. These results show that Dam-dependent phase variation in E. coli is not limited to the previously identified regulatory system of the family of pap-like fimbrial operons.

Adhesins, Bacterial↗
PubMed1984

Immunofluorescence study of fimbrial phase variation in Escherichia coli KS71.

An immunofluorescence assay was developed to study fimbrial phase variation in a pyelonephritogenic Escherichia coli strain, KS71. By using fluorochrome-labeled antibodies specific for either P, type-1C, or type-1 fimbriae of strain KS71, it was shown that in a broth culture of strain KS71 the fimbrial types mostly occurred on different cells. Only 9% of the cells carried more than one fimbrial type. The KS71 cell population was fractionated into subpopulations expressing only one of the fimbrial types or lacking fimbriae. Immunofluorescence assay of the subpopulations revealed a rapid phase variation in fimbrial synthesis. Kinetic analyses of a nonfimbriated cell population suggested that a change from one fimbrial phase to another was not totally random.

Escherichia coli↗
PubMed1991

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.

Adult↗
PubMed1993

Relationship between colony morphology and the life cycle of Haemophilus influenzae: the contribution of lipopolysaccharide phase variation to pathogenesis.

Colonies of Haemophilus influenzae are heterogeneous in appearance because of phase variation in opacity. The only cell surface component found to have structural variation correlating with differences in opacity was the lipopolysaccharide (LPS). Changes in LPS structure, seen as migration patterns on tricine-SDS-PAGE, appeared to be independent of a previously described mechanism for generating LPS phase variation. The more transparent variants expressing a higher-molecular-weight LPS were serum sensitive and could efficiently colonize the infant rat nasopharynx after intranasal inoculation. In contrast, the fully opaque variant expressing a smaller-molecular-weight LPS was serum resistant, unable to colonize the nasopharynx, and more virulent when intraperitoneally administered. Organisms disseminating into the blood-stream from the nasopharynx changed phenotype from transparent to opaque. These findings demonstrate the potential importance of LPS structures that determine opacity in pathogenesis and colonization of mucosal surfaces.

Animals↗
PubMed1999

Molecular switches--the ON and OFF of bacterial phase variation.

The expression of most bacterial genes is controlled at the level of transcription via promoter control mechanisms that permit a graded response. However, an increasing number of bacterial genes are found to exhibit an 'all-or-none' control mechanism that adapts the bacterium to more than one environment. One such mechanism is phase variation, traditionally defined as the high-frequency ON<-->OFF switching of phenotype expression. Phase variation events are usually random, but may be modulated by environmental conditions. The mechanisms of phase variation events and their significance within the microbial community are discussed here.

Adaptation, Physiological↗
PubMed2002

Dam-dependent phase variation of Ag43 in Escherichia coli is altered in a seqA mutant.

In Escherichia coli, phase variation of the outer membrane protein Ag43 encoded by the agn43 gene is mediated by DNA methylation and the global regulator OxyR. Transcription of agn43 occurs (ON phase) when three Dam target sequences in the agn43 regulatory region are methylated, which prevents the repressor OxyR from binding. Conversely, transcription is repressed (OFF) when these Dam target sequences are unmethylated and OxyR binds. A change in expression phase requires a concomitant change in the DNA methylation state of these Dam target sequences. To gain insight into the process of inheritance of the expression phase and the DNA methylation state, protein-DNA interactions at agn43 were examined. Binding of OxyR at agn43 was sufficient to protect the three GATC sequences contained within its binding site from Dam-dependent methylation in vitro, suggesting that no other factors are required to maintain the unmethylated state and OFF phase. To maintain the methylated state of the ON phase, however, Dam must access the hemimethylated agn43 region after DNA replication, and OxyR binding must not occur. OxyR bound hemimethylated agn43 DNA, but the affinity was severalfold lower than for unmethylated DNA. This presumably contributes to the maintenance of the methylated state but, at the same time, may allow for infrequent OxyR binding and a switch to the OFF phase. Hemimethylated agn43 DNA was also a binding substrate for the sequestration protein SeqA. Thus, SeqA, OxyR and Dam may compete for the same hemimethylated agn43 DNA that is formed after DNA replication in an ON phase cell. In isolates with a mutant seqA allele, agn43 phase variation rates were altered and resulted in a bias to the OFF phase. In part, this can be attributed to the observed decrease in the level of DNA methylation in the seqA mutant.

Adhesins, Bacterial↗
PubMed1994

Analysis of the fimB promoter region involved in type 1 pilus phase variation in Escherichia coli.

Many strains of Escherichia coli express type 1 pili which undergo phase variation during growth in broth, but become uniformly nonpiliated when passaged on agar media. In a previous analysis of these agar phase locked strains, we demonstrated that fimB and fimE, which mediate the site-specific DNA rearrangement involved in phase variation, are differentially transcribed under the two growth conditions. In this study the fimB promoter region was sequenced and characterized in agar phase locked strain J96. Primer extension analysis of the fimB gene identified three putative transcription initiation sites. Transcription starting from two of the sites (P1 and P2) would produce an mRNA that approximates the transcript size of fimB previously detected by Northern blot hybridizations. Gel mobility shift studies revealed the presence of promoter binding activity in broth-grown cell extracts, but not in extracts from agar-grown cells, that reacted specifically with DNA fragments upstream of the P1 and P2 promoter regions. This putative binding protein may be involved in the regulation of fimB, perhaps by acting as a transcriptional activator.

Bacterial Proteins↗
PubMed2001

Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila.

We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.

Blotting, Southern↗
PubMed1994

Leucine-responsive regulatory protein and deoxyadenosine methylase control the phase variation and expression of the sfa and daa pili operons in Escherichia coli.

The Escherichia coli operons daa and sfa encode F1845 and S pili, respectively. In this paper we show that the expression of these operons is under phase variation control at a transcriptional level. The transcription of both operons is dependent on the global regulator leucine-responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp is required for methylation protection of two GATC sites located within conserved DNA sequences in the regulatory regions of these operons. These GATC sites are differentially methylated, establishing a methylation pattern which is characteristic of either the phase ON or phase OFF state. We also show that Lrp binds to the daa and sfa regulatory regions and that this binding is modulated by the methylation of the GATC sites. These results indicate that the phase variation of the daa and sfa operons is regulated by a mechanism involving differential binding of Lrp owing to methylation of GATC sites in the regulatory region, which is similar to the mechanism that controls phase variation of the pap operon.

Adhesins, Escherichia coli↗
PubMed2000

Stationary-phase variation due to transposition of novel insertion elements in Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. Spontaneous mutants which are deficient for virulence and extracellular polysaccharide (Eps) production accumulate in large numbers in stationary-phase cultures of this bacterium, a phenomenon which we have called stationary-phase variation. A clone (pSD1) carrying the Eps biosynthetic gene (gum) cluster of X. oryzae pv. oryzae restored Eps production and virulence to several spv (for stationary-phase variation) mutants. Data from localized recombination analysis, Southern hybridization, PCR amplification, and sequence analysis showed that the mutations are due to insertion of either one of two novel endogenous insertion sequence (IS) elements, namely, ISXo1 and ISXo2, into gumM, the last gene of the gum gene cluster. The results of Southern analysis indicate the presence of multiple copies of both IS elements in the genome of X. oryzae pv. oryzae. These results demonstrate the role of IS elements in stationary-phase variation in X. oryzae pv. oryzae.

Alleles↗