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PubMed1991

[Application of trichloroacetic acid-treated antigen for serodiagnosis of Q fever by indirect immunofluorescence technique].

It is well known that the etiologic agent, Coxiella burnetii, exhibits an antigenic phase variation (phase I to phase II), and the diagnostic significance of the relative antibody titers against phase I and phase II antigens is pointed out. Therefore both phase I and phase II antigens are necessary for the serological examination of Q fever. But it is not so easy to prepare and maintain the phase II antigen by the conventional method. In the present study we tried to prepare the phase II antigen for immunofluorescence test by chemical treatment of the phase I antigen. As a result, treatment of the TK-1 strain of C. burnetii (phase I) with 10% trichloroacetic acid for 4 hr at 4 degrees C modified the antigenicity. The modified antigen reacted strongly with anti-phase II antibody.

Animals↗
PubMed1994

Use of a phase variation-specific promoter of Myxococcus xanthus in a strategy for isolating a phase-locked mutant.

The bacterium Myxococcus xanthus alternates between two colony types distinguished by colony morphology and pigmentation. Because the two phases are interconvertible, this phenomenon has been termed phase variation. In one phase, the colonies are bright yellow, rough, and swarming. In the alternate phase, the colonies are tan and mucoid with smooth edges. During exponential vegetative growth, the populations within a colony reach an equilibrium of approximately 99% yellow and 1% tan cells. Neither the biological function nor the genetic mechanism of phase variation is currently understood. To investigate phase variation, a yellow-phase-specific promoter was identified by Tn5lac mutagenesis. A tan-phase-locked mutant was isolated by a strategy, described in this study, which involved introducing a selectable marker expressed under phase-regulated expression. This was accomplished by a fusion of the cloned yellow-phase-specific promoter to a promoterless kanamycin resistance gene. The defect in the phase-locked mutant, given the designation var-683, caused the rate of switching from the tan to yellow phase to be reduced by at least 10(3)-fold below the wild-type rate of switching. This strain will provide a stable tan population for genetic and biological analysis. Evidence is presented for the existence of a transcriptional regulator which controls the expression of phase-regulated promoters.

Cloning, Molecular↗
PubMed1993

MR flow measurement using RF phase gradients in receiver coil arrays.

Radio frequency (RF) phase gradients in the receiver coil field pattern can encode flow velocity information in magnetic resonance (MR) images in the form of phase variations. These phase variations are not readily observed in MR images because they are relatively small compared to phase variations caused by static magnetic field (B0) inhomogeneities, susceptibility variations, and other sources. However, the phase contributions from these other sources are independent of the receiver coil. Therefore, the RF phase gradient encoded flow information can be recovered by subtracting images obtained simultaneously using arrays of independent receiver coils and a multiple channel receiver. This flow velocity information can be extracted retrospectively from standard imaging sequences, including flow-compensated sequences. No additional time is required for the flow study as the flow measurements are made using sequences chosen for optimal imaging, and the images from each coil are obtained simultaneously. Initial results indicate that sufficient sensitivity is obtained to make flow measurements in the range of velocities commonly found in the carotid arteries and other major vessels. In principle, the method works with only two receiver coils. However, additional elements provide additional phase measurements that can be used to increase accuracy, remove ambiguities in flow direction or velocity calculations, and increase the region over which velocity measurements can be accurately made.

Artifacts↗
PubMed2006

Processing gas chromatographic data and confidence interval calculation for partition coefficients determined by the phase ratio variation method.

The phase ratio variation (PRV) method is widely used for the determination of partition coefficient values (dimensionless Henry's law constants) by headspace gas chromatography. Traditional data processing by linear regression has several drawbacks: potential bias introduced by linearization, absence of quality indicator of the resulting value and, in case of replicate determinations, poor utilisation of the existing measurements leading to unnecessarily large confidence intervals. The paper compares existing PRV data processing methods (linear and nonlinear regression, parametric) and derives confidence intervals for the resulting partition coefficient values. The possibility of using several series of measurements to derive a single partition coefficient value with tighter and more reliable confidence intervals is presented for all three processing methods. The methods are tested on published literature data and new experimental data for 12 volatile organic compounds in water at 25 degrees C. The nonlinear regression based on several series of measurements appears to be the method of choice.

Chromatography, Gas↗
PubMed2005

The role of phenotypic variation in rhizosphere Pseudomonas bacteria.

Colony phase variation is a regulatory mechanism at the DNA level which usually results in high frequency, reversible switches between colonies with a different phenotype. A number of molecular mechanisms underlying phase variation are known: slipped-strand mispairing, genomic rearrangements, spontaneous mutations and epigenetic mechanisms such as differential methylation. Most examples of phenotypic variation or phase variation have been described in the context of host-pathogen interactions as mechanisms allowing pathogens to evade host immune responses. Recent reports indicate that phase variation is also relevant in competitive root colonization and biological control of phytopathogens. Many rhizospere Pseudomonas species show phenotypic variation, based on spontaneous mutation of the gacA and gacS genes. These morphological variants do not express secondary metabolites and have improved growth characteristics. The latter could contribute to efficient root colonization and success in competition, especially since (as shown for one strain) these variants were observed to revert to their wild-type form. The observation that these variants are present in rhizosphere-competent Pseudomonas bacteria suggests the existence of a conserved strategy to increase their success in the rhizosphere.

Bacterial Proteins↗
PubMed1986

Expression of a cloned 987P adhesion-antigen fimbrial determinant in Escherichia coli K-12 strain HB101.

The properties of three independent enterotoxigenic Escherichia coli isolates known to express 987P adhesion fimbriae in a manner subject to phase variation were examined. Phase variation could not be correlated with any major changes in the plasmid DNA content of these strains or with readily detectable changes in any other tested phenotypic markers. The 987P genetic determinant from one of these strains, E. coli 987, was cloned into the non-fimbriated E. coli K-12 strains HB101, and expressed, using the cosmid vector system. 987P fimbriae produced by cells harbouring these recombinant plasmids (987P+ phenotype) could not be distinguished from 987P fimbriae produced by strain 987. Expression of 987P fimbriae from some recombinant plasmids was unstable but none of the recombinants exhibited the phase variation phenotype displayed by the parental strain. One recombinant plasmid, pPM200, contained an insert of strain 987 DNA of ca. 33 kb. The HB101[pPM200] displayed a rather stable 987P+ phenotype, but this was not true for several hosts, since pPM200 acquired approx. 20-kb deletions following transformations of E. coli K-12 strains other than HB101. The deletions mapped to the same region of pPM200 irrespective of the host strain transformed. Cells harbouring the deleted plasmids did not express 987P fimbriae (987P- phenotype).

Cloning, Molecular↗
PubMed1987

Effects of recA mutations on pilus antigenic variation and phase transitions in Neisseria gonorrhoeae.

Intragenic recombination between the single complete pilin gene (expression locus) and multiple, distinct, partial pilin gene copies (silent, storage loci) is thought to account for the generation of pilus antigenic diversity and piliation phase (on-off) changes exhibited by Neisseria gonorrhoeae. The mechanisms operating in the genomic rearrangements associated with these forms of pilus variation were investigated through the study of isogenic strains of gonococci bearing either wild-type or altered recA alleles. Examination of the rates of pilus phase variation and the genetic basis for changes in piliation status displayed by these strains show that recA mediated homologous recombination is required for these high frequency events and confirm that the nonpiliated state results from mutations in the expressed pilin gene. In a strain that is deficient in recA mediated homologous recombination, pilus phase variation occurs at a 100-1000-fold reduced rate and results predominantly from one class of spontaneous frameshift mutations within the pilin structural gene.

Alleles↗
PubMed1995

The genetic basis of colony opacity in Streptococcus pneumoniae: evidence for the effect of box elements on the frequency of phenotypic variation.

Streptococcus pneumoniae undergoes spontaneous phase variation in colony morphology. Differences in colony opacity have previously been shown to correlate with differences in the ability of organisms to colonize the mucosal surface of the nasopharynx in an animal model. The genetic basis of opacity variation was identified in transformation experiments. A DNA library, from a strain that varies at high frequency, was screened to identify a single clone capable of transforming a transparent recipient strain which varies at low frequency to an opaque phenotype. Analysis of this opacity locus revealed two genes, glpD and glpF, with similarity to genes required for glycerol metabolism in other bacteria. Following the pneumococcal glpF, repetitive intergenic elements, boxes A and C, were identified. These stem-loop-forming elements were not present in the same locus of the recipient strain. Although not required for phase variation in colony opacity, the box element was necessary for expression of phase variation at high frequency. Introduction of the box elements during transformation affected colony morphology, possibly by altering expression of a putative regulatory gene downstream from the box element. Mutagenesis within this region confirmed the contribution of the putative regulatory gene to the expression of colony opacity. Growth characteristics of strains generated in this study provide additional evidence for an association of differences in cell wall autolysis and variation in colony opacity.

Amino Acid Sequence↗
PubMedDate not supplied

Influence of environmental cues on transcriptional regulation of foo and clp coding for F165(1) and CS31A adhesins in Escherichia coli.

F165(1) (foo) and CS31A (clp) are bacterial adhesins synthesized by Escherichia coli strains associated with diarrhea and septicemia in piglets and calves. They belong to the P-regulatory family and as such are subject to a phase variation control mediated by Lrp (leucine responsive regulatory protein) and regulators homologous to PapI. Analysis of expression of transcriptional fusions between the fooB or fooI promoters and lacZ showed that Lrp is an activator of foo and fooI transcription, whereas it represses clp transcription. Furthermore, foo phase variation leads to a large majority of phase-ON cells, whereas clp phase variation leads to a majority of phase-OFF cells. We compared the influence of several environmental cues on foo and clp expression, with special attention to the effects of leucine and alanine known to be mediated by Lrp. Inhibition or significant repression of foo and clp transcription was observed at low temperature, in LB medium, and in the presence of glucose, alanine, or leucine. Glucose repression of foo but not of clp was totally relieved by addition of cAMP. Osmolarity and pH had little effect. Alanine but not leucine, and LB medium inhibited foo and clp phase variation, locking cells in the OFF phase. Low temperature inhibited clp phase variation and altered the switch frequency of foo phase variation, leading to more phase-OFF cells. Glucose altered the phase variation of both operons, increasing the number of phase-OFF cells in the population. The regulation pattern of foo and clp is consistent with F165(1) and CS31A production in low nutrient environments, even at moderately acidic pH or high osmolarity.

Adhesins, Escherichia coli↗
PubMed2000

Molecular dissection of Mycoplasma hominis.

M. hominis is commonly found as part of the normal flora in the female genital tract, but several studies have shown that it may be involved in a variety of urogenital infections. The basis for clinical manifestations in some patients has varyingly been attributed to host and M. hominis factors. The host factors involved in the infection process are largely unknown. M. hominis have no cell wall and outer membranes, and at present it seems plausible that M. hominis possesses genetic systems allowing the bacteria in vivo to alter its antigenic structure on the membrane surface and consequently circumvent the host immune system. The studies of M. hominis have shown that the antigenic variation is pronounced between surface exposed membrane proteins from different isolates. The genetic background for this variation has been investigated for three surface exposed membrane proteins: P120, Lmp, and Vaa. P120 and P120' are similar proteins in M. hominis without any homology to other known proteins. A hypervariable region in the otherwise conserved P120 protein seems to be very antigenic in patients with immunologically verified M. hominis infection. The remaining part of P120 as well as the entire P120' protein do not seem to elicit significant antibody formation. Two genes in M. hominis, lmp1 and lmp3, contain numerous highly similar 0.5 kb tandem repeats at their 3'-end. The proteins, Lmp1 and Lmp3, are synthesized from the lmp1 and lmp3 genes, respectively. Lmp1 shows size variation among M. hominis isolates. M. hominis isolates investigated in detail show that the size variation of Lmp1 corresponds to the variation in number of 0.5 kb repeats contained within the lmp1 gene. Lmp3 appears to have a lesser tendency to size variation. M. hominis isolates were found with deletions involving the lmp1 stop codon leading to translation of the downstream gene lmp2 and expression of a chimeric Lmp1-Lmp2 protein. The number of repeated elements in the lmp1 gene of a M. hominis isolate correlates with the extent of anti-Lmp antibody induced agglutination between the bacteria. Vaa is a protein involved in cell adherence. vaa is a single copy gene containing tandem repeated elements like the lmp gene family. The number of repeats in the Vaa protein differs between M. hominis isolates leading to size variation. It has been suggested that the number of repeated elements is of importance in the bacteria-host adhesion process. Beside the size variation Vaa demonstrates phase variation due to frequent frame shift mutation in a specific region near the 5'-end of the structural gene. Based on the investigations of M. hominis and other mycoplasmas several genetic mechanisms seem to be responsible for the antigenic variation of surface exposed membrane proteins in mycoplasmas: 1) variation in protein size due to insertions or deletion of repeated elements in the structural gene, 2) presence of multi-gene families, and 3) phase variation due to mutations in the promotor region or the coding region. The influence of specific antibodies on antigenic variation of membrane proteins has not been studied in greater detail in mycoplasmas. In M. hominis it was investigated whether the presence in the culture medium of monoclonal antibodies directed against the repeated elements in the M. hominis Lmp proteins would affect gene structure and consequently protein expression. The presence of anti-Lmp antibodies resulted in overgrowth of bacteria with specific deletions in the repeated elements of lmp1 leaving the lmp3 gene unchanged. The precise mechanism leading to the dominance of M. hominis isolates with fewer 0. (ABSTRACT TRUNCATED)

Amino Acid Sequence↗
PubMed2004

Phase and antigenic variation in bacteria.

Phase and antigenic variation result in a heterogenic phenotype of a clonal bacterial population, in which individual cells either express the phase-variable protein(s) or not, or express one of multiple antigenic forms of the protein, respectively. This form of regulation has been identified mainly, but by no means exclusively, for a wide variety of surface structures in animal pathogens and is implicated as a virulence strategy. This review provides an overview of the many bacterial proteins and structures that are under the control of phase or antigenic variation. The context is mainly within the role of the proteins and variation for pathogenesis, which reflects the main body of literature. The occurrence of phase variation in expression of genes not readily recognizable as virulence factors is highlighted as well, to illustrate that our current knowledge is incomplete. From recent genome sequence analysis, it has become clear that phase variation may be more widespread than is currently recognized, and a brief discussion is included to show how genome sequence analysis can provide novel information, as well as its limitations. The current state of knowledge of the molecular mechanisms leading to phase variation and antigenic variation are reviewed, and the way in which these mechanisms form part of the general regulatory network of the cell is addressed. Arguments both for and against a role of phase and antigenic variation in immune evasion are presented and put into new perspective by distinguishing between a role in bacterial persistence in a host and a role in facilitating evasion of cross-immunity. Finally, examples are presented to illustrate that phase-variable gene expression should be taken into account in the development of diagnostic assays and in the interpretation of experimental results and epidemiological studies.

Amino Acid Sequence↗
PubMed1995

Susceptibility magnetic resonance imaging using spectral decomposition.

Susceptibility differences of materials in magnetic resonance imaging (MRI) usually lead to the intravoxel spin phase variations. Subsequently, the phase variation in the voxel results in a reduction of the signal intensity. This signal intensity reduction is known as the susceptibility effect in MRI and has been studied extensively. In this paper, a new spectral decomposition technique is proposed with which the signal change due to the susceptibility effect can be analyzed. Further, an NMR pulse sequence for the spectral decomposition of the susceptibility was developed and applied to susceptibility imaging of venous blood possessing paramagnetic properties. The computer simulations of the spectral decomposition method and their corresponding experimental results obtained using both a phantom and human volunteers are reported.

Brain↗
PubMed1992

Neisseria gonorrhoeae PilC expression provides a selective mechanism for structural diversity of pili.

Pili of Neisseria gonorrhoeae undergo both phase and structural variation. Phase variation of gonococcal pili can be caused by a RecA-independent on/off switch in PilC, a protein involved in pilus biogenesis. We show here that spontaneous nonpiliated PilC- derivatives as well as PilC- insertional mutants have also acquired sequence alterations in pilE relative to the pilE gene of the piliated MS11mk(P+)-u parent, so that the pilin produced is processed to soluble S-pilin and can be released into the medium. It is proposed that pilin alterations are selected for in PilC- bacteria if the parental nonassembled pilin is toxic to the cells--i.e., is not degradable to S-pilin at rates sufficient to allow viability of the cells. Toxicity is indicated by the extreme instability of certain unassembled pilin sequences and by the low frequency of nonpiliated, pilin+, PilC- variants that emerge from piliated recA- cells. The presence of a point mutation changing leucine-39 to phenylalanine at the cleavage site for S-pilin in one nonpiliated, PilC-, recA- variant relative to its piliated parent is a further argument for a selective mechanism of structural diversity of the gonococcal pilin.

Amino Acid Sequence↗