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At least 19 recordsLinked to original sources
PubMed1985

Role of chromosomal rearrangement in N. gonorrhoeae pilus phase variation.

N. gonorrhoeae undergoes pilus phase and antigenic variation. During phase variation, the pilin gene is turned on and off at high frequencies. Two loci on the gonococcal chromosome from strain MS11 function as expression sites for the pilin gene (pilE1 and pilE2); many other sites apparently contain silent variant pilin sequences. We reported previously that during pilus phase variation, when cells switch from the pilus expressing state (P+) to the nonexpressing state (P-), genome rearrangement occurs. We have examined phase variation in more detail, and we report that in most P+ to P- switches a deletion of pilin gene information occurs in one or both expression sites. This deletion is due to either a simple or a multiple-step recombination event involving directly repeated sequences in the expression loci. The deletion explains the state of some P- cells, but not all. In the latter cells pilin expression is probably controlled by an undefined regulator.

Bacterial Outer Membrane Proteins↗
PubMed1990

Characterization of repetitive sequences controlling phase variation of Haemophilus influenzae lipopolysaccharide.

Phase variation of lipopolysaccharide epitopes of an Haemophilus influenzae serotype b strain (strain RM.7004) occurs through a mechanism which depends on multiple tandem repeats of the DNA sequence 5'-CAAT-3' situated within the chromosomal locus lic1. We report here that the same tetranucleotide repeats are also found in two other genomic loci (lic2 and lic3) of RM.7004. Similar to lic1, there are multiple tandem repeats of 5'-CAAT-3' present at the 5' ends of long open reading frames in lic2 and lic3. Variation in the number of repeats of CAAT, by shifting the upstream initiation codons in or out of phase with the remainder of the open reading frame, could switch on or off the translation of downstream genes. Similar to previously reported findings for lic1, site-directed mutations in the open reading frame downstream (3') from the repeats of CAAT in lic2 abolished phase variation and identified DNA sequences required for the expression of additional oligosaccharide epitopes. When we used an oligonucleotide comprising five repeats of CAAT or DNA sequences specific for lic1, lic2, and lic3 as probes, a survey of other encapsulated H. influenzae strains (serotypes a through f) and nontypable H. influenzae strains (including biotype aegyptius) showed that the chromosome of H. influenzae can have from two to five regions which contain multiple tandem repeats of CAAT in addition to other sequences which hybridize to lic1 and lic2.

Base Sequence↗
PubMed1985

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.

Escherichia coli↗
PubMed1987

Integration host factor is required for the DNA inversion that controls phase variation in Escherichia coli.

The on-and-off expression (phase variation) of type 1 fimbriae, encoded by fimA, in Escherichia coli is controlled by the inversion of a promoter-containing 314-base-pair DNA element. This element is flanked on each side by a 9-base-pair inverted, repeat sequence and requires closely linked genes for inversion. Homology analysis of the products of these genes, fimB and fimE, reveals a strong similarity with the proposed DNA binding domain of lambda integrase, which mediates site-specific recombination in the presence of integration host factor. Integration host factor, encoded by himA and hip/himD, binds to the sequence 5' TNYAANNNRTTGAT 3', where Y = pyrimidine and R = purine, in mediating integration-excision. In analyzing the DNA flanking the fim 314-base-pair inversion sequence, we found the adjacent sequence 5' TTTAACTTATTGAT 3', which corresponds perfectly with the consensus integration host factor binding site. To characterize the role of himA in phase variation, we transduced either a deletion of himA or an insertionally inactivated hip/himD gene into an E. coli strain with a fimA-lacZ operon fusion. We found the rate of phase variation decreases sharply from 10(-3) to less than 10(-5) per cell per generation. Southern hybridization analysis demonstrates that the himA mutation results in a failure of the switch-generated genetic rearrangement. When the transductant was transformed with a himA+ plasmid, normal switching returned. Thus integration host factor is required for normal type 1 fimbriae phase variation in E. coli.

Bacterial Adhesion↗
PubMed1991

Phase variation of pili of Corynebacterium pilosum.

Whether or not (?) phase variation occurs in the pili of Corynebacterium pilosum was biologically examined using the colony enzyme-linked immunosorbent assay (ELISA) blot test with anti-pili immune serum. From the densely piliated clone (35P+) of C. pilosum 35, non-piliated variants were isolated at a frequency of 2.45 x 10(-3). From one of the non-piliated variants (designated as P(-11)/35P+), a piliated variant was isolated at a frequency of 4.68 x 10(-4), one log less frequently than the non-piliated variant. From this piliated variant as designated (P+4/P(-11)/35P+), a non-piliated variant was isolated at a frequency of 3.86 x 10(-3). C. pilosum was thus alternated between piliated and non-piliated at a fairly high frequency, suggesting that the pili may undergo phase variation. This is the first finding of phase variation of the pili of Gram-positive bacteria.

Corynebacterium↗
PubMed1989

The molecular mechanism of phase variation of H. influenzae lipopolysaccharide.

Multiple carbohydrate structures on the outer-membrane lipopolysaccharide (LPS) of the gram-negative pathogen H. influenzae undergo high frequency, reversible loss, indicative of phase variation. Characterization of a genetic locus, lic-1, responsible for expression of two LPS epitopes displaying phase variation, showed it to comprise four genes. The first gene mediates phase variation. At its 5' end, within the open reading frame, are a variable number of tandem repeats of the tetramer CAAT. By shifting upstream initiation codons in or out of frame, these 4 bp units create a translational switch. The phenotype of organisms corresponds to the number of 4 bp units. Phase variation between three levels of expression ( +, +, and -) of lic-1-derived epitopes is caused by differences in the three phases of translation of the 5' terminus of this gene. Phase variation also allows for selection of organisms displaying certain LPS epitopes in vivo.

Amino Acid Sequence↗
PubMed2025

Quorum sensing and DNA methylation play active roles in clinical Burkholderia phase variation.

Phenotypic diversity in bacteria often results from adaptation to changing environmental conditions, exemplified by variable colony morphotypes. In Burkholderia pseudomallei, discrete genomic alterations and modulation of gene expression facilitate adaptation. Adapted variants of species within the Burkholderia cepacia complex (Bcc) often lose the pC3 virulence megaplasmid, impacting their colony morphology and their production of virulence factors. In this study, we characterize variants arising in Burkholderia ambifaria clinical isolates using proteomics and phenotypic tests and show that some of them have retained the pC3, indicating a distinct phase variation mechanism at play in this Bcc species. Interestingly, variants of B. ambifaria strains CEP0996 (pC3-null) and HSJ1 (pC3-positive) still share similarities in phenotypes controlled by the Cep quorum-sensing (QS) system. We further investigated the role of QS in B. ambifaria HSJ1 phase variation and confirmed that the Cep QS system is important for the emergence of variants. Given that DNA methylation is a key epigenetic factor regulating virulence factors in Burkholderia cenocepacia, we hypothesized that adenosine DNA methylation also governs phase variation in B. ambifaria HSJ1. By deleting the genes encoding putative adenosine DNA methyltransferases, we discovered that an orphan type II DNA methyltransferase inhibits the emergence of phase variants. This study is the first to demonstrate that quorum sensing and adenosine DNA methylation are two antagonistic systems independently controlling phase variation in B. ambifaria.IMPORTANCESome Burkholderia species are pathogenic to plants, animals, or humans. In immunocompromised individuals, and people suffering from cystic fibrosis, infection from the Burkholderia cepacia complex (Bcc) can lead to "cepacia syndrome." In northern Australia and southeast Asia, melioidosis caused by Burkholderia pseudomallei is prevalent among native population, particularly among people with diabetes, chronic lung or kidney disease or alcoholism. Burkholderia's phenotypic plasticity, including colony morphotype variation (CMV), enables rapid adaptation to diverse environments, enhancing its survival and pathogenicity. This study reveals phase variation as a new CMV mechanism within the Bcc group and is the first to report that quorum sensing and DNA methylation are involved in phase variation. Understanding the underlying mechanisms of CMV could lead to the development of targeted therapies against these highly antibiotic-tolerant bacteria.

Quorum Sensing↗
PubMed1988

The vir locus and phase-variation in Bordetella pertussis.

By a phenomenon known as phase-variation Bordetella pertussis is capable of changing between a virulent-phase in which multiple virulence-associated determinants are expressed, and an avirulent-phase in which the virulence-associated determinants are not expressed. Mutations in the vir locus of B. pertussis have a similar effect. We have examined the state of the vir locus in each of a series of strains derived one from the other by phase-variation. We have found that a single base-pair change is associated with the change between the virulent and avirulent phases. This single base-pair change corresponds to a frameshift mutation in the vir locus.

Bordetella pertussis↗
PubMed1989

Phase variation of gonococcal protein II: regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence.

Expression of outer membrane protein II (P.II) of Neisseria gonorrhoeae is subject to reversible phase variation at a rate of 10(-3)-10(-4)/cell/generation. The signal peptide coding regions of P.II genes contain variable numbers of tandem repeats of the sequence CTCTT. Changes in the number of CTCTT units, leading to frameshifting within the gene, are responsible for changes in P.II expression. Phase variation mediated by the CTCTT repeat also occurred in E. coli, as assayed with a P.II-alkaline phosphatase (phoA) gene fusion. Phase variation in both the gonococcus and E. coli was recA-independent, occurred at similar rates, and involved insertions or deletions of one or more repeat units. The characteristics of the phase variation process were consistent with a model in which expression of P.II genes is regulated by slipped-strand mispairing of the DNA in the CTCTT repeat region.

Bacterial Outer Membrane Proteins↗
PubMed1986

Bordetella bronchiseptica phase variation induced by crystal violet.

A method for effective induction of phase variation in Bordetella bronchiseptica by treatment with crystal violet (CV) is presented. When grown in CV-broth, phase I cells dissociated into three serial phases. Appearance of variant cells was observed simultaneously with the beginning of cell multiplication. The maximum effect of CV was obtained at a concentration of 8 micrograms/ml, when the proportion of variants in the population reached 100%. The main factors which affected phase variation were concentration of CV, culture age, and temperature of treatment. The phase variants obtained were phenotypically stable upon serial passages on Bordet-Gengou agar plates. By this treatment, no reversion of phase descendants to former phases was observed.

Agglutination Tests↗
PubMed1991

Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in Escherichia coli.

Transcription of the pyelonephritis-associated pilus (pap) operon of Escherichia coli is subject to regulation by a phase variation control mechanism in which the pap pilin gene alternates between transcriptionally active (phase-on) and inactive (phase-off) states. Pap phase variation appears to involve differential inhibition of deoxyadenosine methylase (Dam) methylation of two pap GATC sites, GATC1028 and GATC1130, located in the regulatory region upstream of the papBA promoter. DNA from phase-on cells contains an unmethylated adenosine in the GATC1028 site, whereas DNA from phase-off cells contains an unmethylated adenosine in the GATC1130 site. papI and papB are two regulatory genes in the pap operon. Analysis of pap deletion mutants suggests that papI is required for methylation inhibition at the GATC1028 site; however, neither papI nor papB is required for inhibition of methylation at the GATC1130 site. We have identified a chromosomal locus, mbf (methylation-blocking factor), that is required for methylation protection of both the pap GATC1028 and GATC1130 sites. The mbf locus was identified after transposon mTn10 mutagenesis and mapped to 19.6 min on the E. coli chromosome. The effect of transposon mutations within mbf on pap pilin transcription was determined by using a papBAp-lac operon fusion which places lacZ under control of the papBA promoter. E. coli containing mbf::mTn10 and phase-off mbf+ E. coli cells both expressed beta-galactosidase levels about 30-fold lower than the beta-galactosidase level measured for phase-on mbf+ E. coli cells. These results indicated that mbf was necessary for pap pilin transcription and were supported by Northern (RNA) blotting and primer extension analyses. Moreover, transposon insertion within mbf greatly reduced Pap pilus expression. The mbf locus was isolated on a low-copy-number cosmid, pMBF1. Complementation analysis indicated that each of seven mbf::mTn10 mutants isolated contained a transposon insertion within the same gene or operon. The identification of the mbf locus, required for pap transcription, supports the hypothesis that pap phase variation is controlled by a mechanism involving alternation between different methylation states.

Amino Acid Sequence↗
PubMed1985

Studies on phase variation in Bordetella pertussis.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. The spontaneous process was studied in pathogenic B. pertussis strains Tohama, 165 and 18323 by isolating spontaneous variants, selected for their ability to grow on synthetic and semi-synthetic solid media. In strains Tohama and 165, the frequency of variants able to grow on synthetic and semi-synthetic media was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried pertussis toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin was related quantitatively to the other traits. These results suggest that phase variation in B. pertussis is a non random process, involving ordered disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. Since all the variant strains were phenotypically stable upon further passaging in vitro, they represent the stable, final outcome of the variation process which may have occurred in "Phase I" colonies. In contrast, 300 variants of B. pertussis 18323, which were able to grow on selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral virulence.

Adaptation, Physiological↗
PubMed1985

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.

Animals↗
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↗
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↗
PubMed1991

A phase variation event that activates conjugation functions encoded by the Enterococcus faecalis plasmid pAD1.

Enterococcus faecalis cells carrying the conjugative plasmid pAD1 undergo several related changes when induced by the sex pheromone cAD1. Included are the production of novel surface proteins, the formation of cellular aggregates in broth cultures, the ability to transfer the plasmid at high frequency in broth matings, and the change from a soft to a "dry" colony morphology. Spontaneous, constitutively dry colony (Dryc) variants of E. faecalis (pAD1) were found to arise at a frequency of 10(-4)-10(-2). Dryc phase variants constitutively expressed aggregation and plasmid transfer functions typically expressed only under cAD1-inducing conditions. Reversion of Dryc variants to a cAD1-inducible phenotype (Dry+) occurred at a similar frequency. Tn917-lac mutagenesis of regions of pAD1 previously shown to be involved in plasmid transfer revealed that in Dry+ cells these regions were transcribed only when the inducer, cAD1, was present. In Dryc variants the regions were transcribed constitutively. A pAD1 miniplasmid containing determinants regulating cAD1 inducible plasmid transfer and a cAD1-inducible lacZ transcriptional fusion displayed phase variation in LacZ expression at a rate similar to the Dry+/Dryc phase variation. These results suggest that the site of mutation(s) resulting in the Dryc phenotype is within the regulation-related region of pAD1. Complementation tests showed that this region, when supplied in trans, complemented the Dryc phenotype. Phase variation affecting mating functions represents an alternative (pheromone independent) method of regulating pAD1 transfer.

Conjugation, Genetic↗
PubMed1985

Is phase variation in Bordetella caused by mutation and selection?

In vitro growth conditions of Bordetella bronchiseptica led to an enrichment of phase variants. The frequency of phase variation was about 10(-6) per cell per generation. Therefore phase variation in Bordetella may result from a random mutation in a controlling region, followed by selection.

Adenylyl Cyclases↗
PubMed1990

Regulation of pap pilin phase variation by a mechanism involving differential dam methylation states.

Transcription of the pap pilin (papA) gene in Escherichia coli is subject to control by a heritable phase variation mechanism in which alternation between transcriptionally active (phase on) and inactive (phase off) states occurs. Our results suggest that phase switching occurs without DNA rearrangement of pap DNA sequences, distinguishing this system from those described for E. coli type 1 pili and Salmonella flagellar phase variation. Analysis of the regulatory region upstream of papA in DNAs isolated from phase off and phase on cell populations showed that two deoxyadenosine methylase (Dam) sites, GATC1028 and GATC1130, were present. Southern blot analysis of MboI and DpnI restriction digests of DNAs showed that the GATC1028 site was unmethylated only in DNA isolated from phase on populations. Conversely, GATC1130 sites were unmethylated in DNA isolated from phase off populations. The presence of unmethylated GATC sites in E. coli is unusual and to our knowledge has not been previously reported. These results suggest that the methylation states of GATC1028 and GATC1130 may regulate pap transcription. Consistent with this hypothesis, Dam methylase levels affected the regulation of pap transcription; papA transcription was absent in dam- E. coli. Moreover, transition from the phase off to phase on state was not observed in E. coli expressing aberrantly high levels of Dam. A basic model is presented which outlines a possible mechanism by which alternation between phase off and phase on methylation states could occur.

Amino Acid Sequence↗