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PubMed1995

Mycoplasma hyorhinis vlp gene transcription: critical role in phase variation and expression of surface lipoproteins.

Mycoplasma hyorhinis contains clustered vlp genes encoding variable lipoproteins (Vlps), the major coat proteins and surface antigens of this wall-less prokaryotic pathogen. vlp genes are subject to discrete, high frequency mutations independently affecting the size or the expression of variant Vlp products. Change in Vlp size occurs by mutations altering the number of tandem intragenic repeats at the 3' end of each single-copy vlp gene. In this report, phase-variant Vlp expression is shown to result from altered vlp gene transcription. vlpA, vlpB and vlpC transcripts were monitored in a clonal lineage selected to display various Vlp phenotypes. Each vlp gene was expressed as a distinct transcript, which was subject to drastic ON/OFF switches associated with random insertion/ deletion mutations in a homopolymeric tract of adenine residues in the promoter region of all vlp genes. Unexpectedly, the level of vlp transcripts appeared to depend on the length of the corresponding genes in the ON configuration. Higher proportional levels of shorter vlp transcripts were shown to reflect a greater abundance of short Vlp lipoproteins present in L-[35S]-cysteine-labelled membrane protein preparations. The vlp cluster provides a heritable, and highly mutable, locus for the generation of surface diversity through random promoter mutations affecting the expression of genes, whose products also vary in length and abundance, by virtue of separate mutations in structural regions of the genes.

Antigens, Bacterial↗
PubMed2001

Growth phase variation in cell and nucleoid morphology in a Bacillus subtilis recA mutant.

The major role of RecA is thought to be in helping repair and restart stalled replication forks. During exponential growth, Bacillus subtilis recA cells exhibited few microscopically observable nucleoid defects. However, the efficiency of plating was about 12% of that of the parent strain. A substantial and additive defect in viability was also seen for addB and recF mutants, suggesting a role for the corresponding recombination paths during normal growth. Upon entry into stationary phase, a subpopulation (approximately 15%) of abnormally long cells and nucleoids developed in B. subtilis recA mutants. In addition, recA mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation.

Bacillus subtilis↗
PubMed1982

High performance gel permeation chromatography of bovine thyrotropin (TSH): effect of column stability and mobile phase variation.

Because of previously observed variability in the elution patterns of radiolabeled bovine TSH (bTSH) and bTSH bioactivity (in a particular bTSH preparation) on gel permeation high performance liquid chromatography (GPC), studies were conducted to examine the effect of different conditions on the elution of this material. Continued use of a Waters' I-125 column at pH 7.2 demonstrated progressive retardation in the elution of bioactivity and radioactivity in this highly-purified bTSH preparation, with progressive but incomplete separation of these two functions. With decreasing mobile phase pH the elution positions of TSH bioactivity and radioactivity advanced to a coincident peak near to the void volume (pH 4). Addition to the mobile phase of the ion pair reagents tetrabutyl-ammonium phosphate (PIC-A) and pentane sulfonic acid (PIC B5) produced marked alterations in the elution positions of TSH radioactivity and bioactivity, with separation of these functions in the presence of the former reagent. These data indicate the present limitations of GPC for the purification of TSH.

Animals↗
PubMed2003

Phase variation of the multiple banded protein in Ureaplasma urealyticum and Ureaplasma parvum.

Ureaplasma urealyticum and U. parvum are common commensals and, possibly, pathogens of the human urogenital tract. Like other Mycoplasmatales they possess variable surface proteins. The multiple banded (MB) protein shows a striking variability of its molecular weight. This is caused by changes of the number of C-terminal repeating units. In this study, selective pressure was imposed against cytadherence of U. urealyticum and U. parvum. Ureaplasmas were co-incubated with either erythrocytes or HeLa cells and the cell-bound fraction was removed. Additionally, U. urealyticum populations were transferred serially through broth containing specific polyclonal antibodies. Both approaches led to the emergence of escape variants in which no MB protein was detectable. PCR studies with several primers on different parts of the mba gene indicated major differences between wild-type strains and MB-negative escape variants. In experiments with clonal lineages, however, the loss of the MB protein was shown to be reversible. Therefore, it is proposed that the multiple banded proteins of U. urealyticum and U. parvum are subjected to a phase-switching mechanism as it has already been described for several other Mycoplasmatales.

Antibodies, Monoclonal↗
PubMed1998

Phase variation of hemoglobin utilization in Neisseria gonorrhoeae.

Most Neisseria gonorrhoeae isolates are unable to use human hemoglobin as the sole source of iron for growth (Hgb-), but a minor population is able to do so (Hgb+). This minor population grows luxuriously on hemoglobin, expresses two outer membrane proteins of 42 kDa (HpuA) and 89 kDa (HpuB), and binds hemoglobin under iron-stressed conditions. In addition to the previously reported HpuB, we identified and characterized HpuA, which is encoded by the gene hpuA, located immediately upstream of hpuB. Expression of both proteins was found to be controlled at the translational level by frameshift mutations in a run of guanine residues within the hpuA sequence encoding the mature HpuA protein. The "on-phase" hemoglobin-utilizing variants contained 10 G's, while the "off-phase" variants contained 9 G's. Insertional hpuB mutants of FA19 Hgb+ and FA1090 Hgb+ no longer expressed HpuB but still produced HpuA. A polar insertional mutation of the upstream hpuA gene in FA1090 Hgb+ eliminated production of both HpuA and HpuB, whereas a nonpolar insertional mutant expressed HpuB only. Insertional mutagenesis of either hpuA or hpuB or both substantially decreased the hemoglobin binding ability of the FA1090 Hgb+ variant and prevented growth on hemoglobin plates. Therefore, both HpuA and HpuB were required for the utilization of hemoglobin for growth.

Amino Acid Sequence↗
PubMed1996

Invasion of primary nasopharyngeal epithelial cells by Neisseria meningitidis is controlled by phase variation of multiple surface antigens.

We have investigated bacterial factors required for the entry of Neisseria meningitidis serogroup B into mucosal cells using a novel in vitro infection model of primary cultures of human nasopharyngeal epithelium. An invasive meningococcal phenotype was obtained after several cycles of selection for intracellular bacteria with gentamicin. Invasive bacteria differed from those in the initial inoculum in that they lacked a capsule and pili, exhibited a nonsialylated low-molecular-weight type of lipopolysaccharide (LPS), and produced a new 28-kDa opacity outer membrane protein. LPS revertants of the selected meningococci expressed a nonsialylated L3,(7,9) type of LPS and were also invasive, while after LPS sialylation bacterial entry was inhibited. Variants lacking the 28-kDa opacity protein were poorly invasive. Coexpression of the outer membrane protein Opc and the 28-kDa opacity protein strongly inhibited microbial invasion into the primary cultured nasopharyngeal cells. Conversely, meningococcal internalization by cells of various epithelia] cell lines was correlated with the expression of Opc rather than the 28-kDa opacity protein. Our data indicate that a concurrent phase switching of multiple phase-variable bacterial surface components may be a prerequisite for meningococcal invasion into nasopharyngeal epithelium and that meningococcal class 5 proteins (Opa and Opc) may promote tissue tropism.

Antigens, Bacterial↗
PubMed1989

Phase-variation of pyelonephritis-associated pili in Escherichia coli: evidence for transcriptional regulation.

The regulation of pyelonephritis-associated pili (pap) pilin gene transcription has been examined using two operons (pap-17 and pap-21) isolated from the pyelonephritogenic Escherichia coli strain C1212. DNA sequence analysis and E. coli minicell analysis were used to map two genes (papB and papI) within the pilin regulatory regions of both pap-17 and pap-21, and the protein products of these genes were identified. Pilin transcription, initiated at the papBA promoter, was monitored by constructing single copy operon fusions with lacZYA in E. coli K-12. Inoculation of E. coli (pap'-lac) strains onto solid M9 minimal medium containing glycerol and the Lac indicator X-gal (M9-Glycerol) yielded both Lac+ and Lac- colony phenotypes. The Lac+ ("phase on') and Lac- ("phase off') phenotypes were heritable since reinoculation of M9-Glycerol with bacteria picked from Lac+ colonies gave rise to a much higher fraction of Lac+ colonies than reinoculation of M9-Glycerol with bacteria picked from Lac- colonies. Measurement of phase transition rates for E. coli (pap17'-lac) inoculated onto M9-Glycerol showed that the Lac(-)----Lac+ transition frequency (1.57 X 10(-4)/cell/generation) was reduced 35-fold when cells were inoculated onto minimal medium containing glucose (M9-Glucose). However, the Lac+----Lac-transition frequency obtained using M9-Glycerol (2.60 X 10(-2)/cell/generation) was 1.4-fold lower compared to results obtained with M9-Glucose. In contrast, lowering the incubation temperature of E. coli (pap17'-lac) cultures from 37 degrees C to 23 degrees C caused all cells to shift to the Lac- state.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Amplitude and phase variations of harmonic components in human achromatic and chromatic visual evoked potentials.

Occipital visual evoked potentials (VEPs) were recorded in response to low-contrast, low spatial-frequency chromatic, and achromatic gratings. Fast Fourier Transform (FFT) and time-domain analysis were used to reveal differences in harmonic content and amplitude of chromatic and achromatic response components over a wide range of temporal frequencies. The chromatic ON/OFF VEP is dominated by the fundamental component indicating that onset and offset responses are different. This type of response is typical of neurons with sustained type response characteristics. Conversely, the achromatic onset VEP contains a predominant second harmonic component in addition to the fundamental. This similarity between onset and offset responses suggests that transient mechanisms are responsible for the generation of achromatic components. Frequency analysis of VEPs elicited by phase-reversing stimuli reveals that all of the response energy is concentrated at the second harmonic of the stimulating frequency. The magnitude of the second harmonic component is maximal for achromatic stimuli and undergoes a distinct minimum for isoluminant, chromatic stimuli. This behavior indicates that under the stimulus conditions used, magnocellular neurons with transient characteristics dominate the reversal VEP.

Adult↗
PubMed1994

Phase variation in streptococci of serological group B. Characteristic properties of isolates from human and bovine infection.

Encapsulation is thought to be a critical virulence factor in streptococci of serological group B. In the present study two encapsulated low-density variants could be separated from their unencapsulated original strains by Percoll gradient centrifugation. The original strains had been isolated from human endocarditis and bovine mastitis. Type antigen preparations of the encapsulated human and bovine group B streptococcus reacted with type III- and type IV-specific antiserum, respectively. No comparable reactions could be observed with their unencapsulated parent strains. In contrast to the original strains, the encapsulated variants grew with uniform turbidity in fluid medium and formed diffuse colonies in soft agar. The original strains grew as granular sediment and formed compact colonies in soft agar. In addition, the original strains appeared to have a more hydrophobic surface and showed significantly greater adherence to epithelial cells. In contrast to the nonencapsulated parent strains, the encapsulated variants were less phagocytosed by polymorphonuclear leukocytes. These findings may help our understanding of the pathogenic importance of phase variants in infections with this bacterial organism.

Animals↗
PubMed1997

Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates.

Staphylococcus epidermidis is a common cause of catheter-associated infections and septicemia in immunocompromised patients. To answer the question whether S. epidermidis skin isolates differ from isolates causing septicemic diseases, 51 strains obtained from blood cultures, 1 strain from shunt-associated meningitis, and 36 saprophytic isolates were characterized. The study demonstrates that most of the blood culture strains formed a multilayered biofilm on plastic material, whereas skin and mucosal isolates did not. Moreover, biofilm-producing strains were found to generate large bacterial autoaggregates in liquid culture. Autoaggregation and biofilm formation on polymer surfaces was associated with the presence of a DNA sequence encoding an intercellular adhesion gene cluster (ica) that mediates the production of a polysaccharide intercellular adhesin. The presence of the intercellular adhesion genes in blood culture isolates was also found to be correlated with the exhibition of black colonies on Congo red agar, whereas the adhesin-negative strains formed red colonies. Upon subcultivation on Congo red agar, the black colony forms of the blood culture strains exhibited red colony variants which were biofilm and autoaggregation negative and occurred at a frequency of 10(-5). The DNA analysis of these S. epidermidis variants by pulsed-field gel electrophoresis and Southern hybridization with an ica-specific gene probe revealed no detectable difference between the black and red colony types. Moreover, after repeated passage, the phenotype of the parent strain could be restored. Therefore, these colony forms were regarded as phase variants. This phenotypic change was observed exclusively in adhesin-positive clinical isolates and not in adhesin-negative saprophytic strains of S. epidermidis.

Bacteremia↗
PubMed1991

Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells.

Neisseria gonorrhoeae is a facultative intracellular bacterium capable of penetrating into certain human epithelial cell types. In order to identify gonococcal factors essential for invading Chang human conjunctiva cells, a gentamicin selection assay for the quantification of viable intracellular bacteria was used in conjunction with microscopy. The results demonstrate a correlation between the invasive behaviour of gonococci and the expression of Opa proteins, a family of variable outer membrane proteins present in all pathogenic Neisseria species. However, only particular Opa proteins supported invasion into Chang cells as indicated by the use of two unrelated gonococcal strains. Invasion was sensitive to cytochalasin D, and strong adherence mediated by the Opa proteins appeared to be essential for the internalization of gonococci. In contrast pili, which also conferred binding to Chang conjunctiva cells, did not support cellular invasion but rather were inhibitory.

Bacterial Adhesion↗
PubMed1987

Hydrophobic adherence and phase variation in Bordetella pertussis.

The hydrophobicity of Bordetella pertussis was assayed by measuring the ability of cells in suspension to adhere to a polystyrene surface. The quantity of adhered bacteria was measured by the binding of enzyme-conjugated anti B. pertussis antibodies. Hydrophobic adherence of non-pathogenic variant strains was about 20% of that exhibited by pathogenic strains. Hydrophobicity was a stable trait as it did not change with passaging or storage. Assays of a series of characterized stable variants suggested that the Filamentous Hemagglutinin (FHA) is the cell surface moiety responsible for hydrophobic adherence in B. pertussis.

Bacterial Adhesion↗
PubMed2003

Phase-variation of the truncated lipo-oligosaccharide of Neisseria meningitidis NMB phosphoglucomutase isogenic mutant NMB-R6.

The detection of antibodies specific to meningococcal lipo-oligosaccharides (LOSs; outer-core-->inner-core-->lipid A) in sera of patients convalescent from meningococcal infection suggests the potential use of LOS as a vaccine to combat pathogenic Neisseria spp. Removal of the outer-core region, which expresses glycans homologous to human blood-group antigens, is a required first-step in order to avoid undesirable immunological reactions following vaccination. To this end, we describe here the structural makeup of the LOS produced by serogroup B N. meningitidis NMB isogenic phosphoglucomutase (Pgm) mutant (NMB-R6). The dominant LOS types produced by NMB-R6 expressed a deep-truncated inner-core region, GlcNAc-(1-->2)-LDHepII-(1-->3)-LDHepI-(1-->5)-[Kdo-2-->4]-Kdo-->lipid A, with one PEA unit attached at either O-6 or O-7 of LDHepII, or with two simultaneously PEA moieties attached at O-3 and O-6 or O-3 and O-7 of the same unit. Unexpectedly, this mutation did not completely deactivate the production of Glc, as some LOS molecules were observed to carry Glc at O-4 of LDHepI and at O-3 of LDHepII. A glycoconjugate vaccine comprised of NMB-R6 LOSs is currently being evaluated in our laboratory.

Acetylglucosamine↗