Reversible Phase Variation Research Articles

HsdSA regulated extracellular vesicle-associated PLY to protect Streptococcus pneumoniae from macrophage killing via LAPosomes.

S. pneumoniae is a major human pathogen that undergoes a spontaneous and reversible phase variation that allows it to survive in different host environments. Interestingly, we found hsdSA , a gene that manipulated the phase variation, promoted the survival and replication of S. pneumoniae in macrophages by regulating EV production and EV-associated PLY. More importantly, here we provided the first evidence that higher EV-associated PLY (produced by D39) could form LAPosomes that were single membrane compartments containing S. pneumoniae, which are induced by integrin β1/NOX2/ROS pathway. At the same time, EV-associated PLY increased the permeability of lysosome membrane and induced an insufficient acidification to escape the host killing, and ultimately prolonged the survival of S. pneumoniae in macrophages. In contrast, lower EV-associated PLY (produced by D39ΔhsdSA ) activated ULK1 recruitment to form double-layered autophagosomes to eliminate bacteria.

Extending phase-variation voltage zones in P2-type sodium cathodes through high-entropy doping for enhanced cycling stability and rate capability

P2-type layered sodium oxides hold great promise for grid-scale energy storage applications owing to their low-cost merit, while their detrimental structural and chemical transition lead to low reversible capacities and poor cycling stability. Herein, we leverage a high-entropy (HE) doping strategy to stabilize the P2-type Na0.667Mn0.667Ni0.167Co0.167O2 (NMNC) cathode. A novel Na0.667Mn0.667Ni0.167Co0.117Ti0.01Mg0.01Cu0.01Mo0.01Nb0.01O2 (HE-NMNC) composite, with five equal content (1 at %) of cation substitution and exhibiting a pure P63/mmc space group structure, is proposed. Interestingly, a reversible P2-type phase variation is identified for HE-NMNC at high degrees of charge, but such phase variation occurs in a wider voltage region and is much slower than that in the NMNC baseline. It is demonstrated that the HE-NMNC delivers outstanding rate capability and cycling stability under a charging cutoff voltage of 4.5 V, achieving a reversible capacity of 111 mAh/g at 5 C and retaining around 130 mAh/g after 100 cycles at 1 C. Apart from the charge compensation of main transition metals (Ni, Co, and Mn), the reversible redox of lattice oxygen also contributes to the capacity of HE-NMNC upon cycling. The proposed high-entropy doping strategy and reaction mechanism may provide new insights for developing advanced cathode materials.

Inactivation of Transcriptional Regulator FabT Influences Colony Phase Variation of Streptococcus pneumoniae.

ABSTRACTStreptococcus pneumoniae is an opportunistic pathogen that can alter its cell surface phenotype in response to the host environment. We demonstrated that the transcriptional regulator FabT is an indirect regulator of capsular polysaccharide, an important virulence factor of Streptococcus pneumoniae. Transcriptome analysis between the wild-type D39s and D39ΔfabT mutant strains unexpectedly identified a differentially expressed gene encoding a site-specific recombinase, PsrA. PsrA catalyzes the inversion of 3 homologous hsdS genes in a type I restriction-modification (RM) system SpnD39III locus and is responsible for the reversible switch of phase variation. Our study demonstrated that upregulation of PsrA in a D39ΔfabT mutant correlated with an increased ratio of transparent (T) phase variants. Inactivation of the invertase PsrA led to uniform opaque (O) variants. Direct quantification of allelic variants of hsdS derivatives and inversions of inverted repeats indicated that the recombinase PsrA fully catalyzes the inversion mediated by IR1 and IR3, and FabT mediated the recombination of the hsdS alleles in PsrA-dependent and PsrA-independent manners. In addition, compared to D39s, the ΔfabT mutant exhibited reduced nasopharyngeal colonization and was more resistant to phagocytosis and less adhesive to epithelial cells. These results indicated that phase variation in the ΔfabT mutant also affects other cell surface components involved in host interactions.

Did I pick the right colony? Pitfalls in the study of regulation of the phase variable antigen 43 adhesin.

Ag43 is an abundant outer membrane autotransporter adhesin present in most commensal and pathogenic Escherichia coli. Expression of the agn43 gene is characterized by a regulated reversible switch or phase variation between the agn43 ON and agn43 OFF states. Although the agn43 regulatory switch leads to a heterogeneous population of ON and OFF bacteria, studies of Ag43 seldom consider potential biases associated with phase variation. We monitored agn43 ON/OFF phase-variation status genetically and phenotypically and we show that the use of populations with random agn43 ON or OFF status could result in misleading conclusions about Ag43 function or regulation. In particular, we demonstrate that Lrp and MqsR, previously identified as agn43 regulators, do not regulate agn43 expression or ON/OFF switch frequency. We also show that biofilm formation in dynamic flow conditions does not influence agn43 ON/OFF switching but physically selects aggregating agn43 ON cells. This indicates that misinterpretation is possible when studying gene expression within biofilms. Finally, we provide evidence that ignoring the initial agn43 ON/OFF status of the E. coli populations studied is likely to bias analyses of phenotypes associated with other E. coli adhesins. This study therefore emphasizes the importance of monitoring Ag43 phase variation and indicates that caution is required when interpreting experiments using strains that are neither deleted for agn43 nor carefully assessed for agn43 ON/OFF status.

Roles of RseB, σ E , and DegP in Virulence and Phase Variation of Colony Morphotype of Vibrio vulnificus

Vibrio vulnificus is an estuarine bacterium capable of causing serious and often fatal wound infections and primary septicemia. We used alkaline phosphatase insertion mutagenesis to identify genes necessary for the virulence of this pathogen. One mutant had an in-frame fusion of 'phoA to the gene encoding RseB, a periplasmic negative regulator of the alternative sigma factor sigma(E). sigma(E) controls an extensive regulon involved in responding to cell envelope stresses. Colonies of the rseB mutant were less opaque than wild-type colonies and underwent phase variation between translucent and opaque morphologies. rseB mutants were attenuated for virulence in subcutaneously inoculated iron-dextran-treated mice. To obtain insight into the role of rseB and the extracytoplasmic stress response in V. vulnificus, mutants with defined mutations in rseB and two important members of the extracytoplasmic stress regulon, rpoE and degP, were constructed for analysis of virulence, colony morphology, and stress-associated phenotypes. Deletion of rseB caused reversible phase variation in the colony morphotype that was associated with extracellular polysaccharides. Translucent and transparent morphotype strains were attenuated for virulence. rpoE and degP deletion mutants were sensitive to membrane-perturbing agents and heat but were not significantly attenuated for V. vulnificus virulence in mice. These results reveal complex relationships between regulation of the extracytoplasmic stress response, exopolysaccharides, and the virulence of V. vulnificus.

Role of phenotypic diversity in pathogenesis of avian mycoplasmosis

The interactions between avian mycoplasmas and their host cells are far more complex than might be anticipated from their apparent structural and functional simplicity. Phenotypic diversity in the form of reversible phase variation, antigenic variation or size variation is an adaptive mechanism that enables avian mycoplasmas to survive in a hostile and highly evolved host. Despite significant similarities between major membrane antigens of Mycoplasma gallisepticum and Mycoplasma synoviae, the molecular mechanisms that mediate phenotypic variation in these two pathogens are completely different. Throughout the years, these mechanisms have evolved side by side with their host immune system and provided mycoplasmas the capacity to colonize, invade and persist in an intricate host. In this article, recent advances in the understanding of the molecular mechanisms of phenotypic variation are reviewed, and implications of such variation in pathogenesis of the disease and development of vaccines and diagnostic assays are outlined.

Genetic Variation in the Vibrio vulnificus Group 1 Capsular Polysaccharide Operon

Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show "phase variation" to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.

Role of RpoS and MutS in phase variation of Pseudomonas sp. PCL1171

Pseudomonas sp. strain PCL1171 undergoes reversible colony phase variation between opaque phase I and translucent phase II colonies, which is dependent on spontaneous mutations in the regulatory genes gacA and gacS. Mutation of the mutS gene and constitutive expression of rpoS increases the frequency at which gac mutants appear 1000- and 10-fold, respectively. Experiments were designed to study the relationship between gacS, rpoS and mutS. These studies showed that (i) a functional gac system is required for the expression of rpoS, (ii) RpoS suppresses the expression of mutS and therefore increases the frequency of gac mutants, and (iii) upon mutation of rpoS and gacS, the expression of mutS is increased. Mutation of gacS abolishes suppression of mutS expression in stationary growth, suggesting that additional gac-dependent factors are involved in this suppression. In conclusion, inefficient mutation repair via MutS, of which the expression is influenced by gacA/S itself and by rpoS in combination with other factors, contributes to the high frequency of mutations accumulating in gacA/S. The role of RpoS in the growth advantage of a gac mutant was analysed, and mutation of rpoS only reduced the length of the lag phase, but did not affect the growth rate, suggesting a role for both RpoS and a reduction of metabolic load in the growth advantage of a gac mutant.

Reversible phase variation in the phnE gene, which is required for phosphonate metabolism in Escherichia coli K-12.

It is known that Escherichia coli K-12 is cryptic (Phn-) for utilization of methyl phosphonate (MePn) and that Phn+ variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn+ variants are present at the surprisingly high frequency of >10(-2) in K-12 strains. Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions. This revealed that, once selection for growth on MePn is removed, Phn+ revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence. It appears that, in K-12, phnE contains a high-frequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization. The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate. All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences. The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn+ and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources.

Variable surface protein Vmm of Mycoplasma mycoides subsp. mycoides small colony type.

A variable surface protein, Vmm, of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) has been identified and characterized. Vmm was specific for the SC biotype and was expressed by 68 of 69 analyzed M. mycoides SC strains. The protein was found to undergo reversible phase variation at a frequency of 9 x 10(-4) to 5 x 10(-5) per cell per generation. The vmm gene was present in all of the 69 tested M. mycoides SC strains and encodes a lipoprotein precursor of 59 amino acids (aa), where the mature protein was predicted to be 36 aa and was anchored to the membrane by only the lipid moiety, as no transmembrane region could be identified. DNA sequencing of the vmm gene region from ON and OFF clones showed that the expression of Vmm was regulated at the transcriptional level by dinucleotide insertions or deletions in a repetitive region of the promoter spacer. Vmm-like genes were also found in four closely related mycoplasmas, Mycoplasma capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae, Mycoplasma sp. bovine serogroup 7, and Mycoplasma putrefaciens. However, Vmm could not be detected in whole-cell lysates of these species, suggesting that the proteins encoded by the vmm-like genes lack the binding epitope for the monoclonal antibody used in this study or, alternatively, that the Vmm-like proteins were not expressed.

Identification of a group 1-like capsular polysaccharide operon for Vibrio vulnificus.

Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using TnphoA mutagenesis, we identified a V. vulnificus CPS locus, which included an upstream ops element, a wza gene (wza(Vv)), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wza gene product is required for transport of CPS to the cell surface in Escherichia coli. Polar transposon mutations in wza(Vv) eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wza(Vv) was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions in wza(Vv) and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V. vulnificus.

Phase variations of the Mycoplasma penetrans main surface lipoprotein increase antigenic diversity.

Mycoplasma penetrans is a recently identified mycoplasma, isolated from urine samples collected from human immunodeficiency virus (HIV)-infected patients. Its presence is significantly associated with HIV infection. The major antigen recognized during natural and experimental infections is an abundant P35 lipoprotein which, upon extraction, segregates in the Triton X-114 detergent phase and is the basis of M. penetrans-specific serological assays. We report here that the P35 antigen undergoes spontaneous and reversible phase variation at high frequency, leading to heterogeneous populations of mycoplasmas, even when derived from a clonal lineage. This variation was found to be determined at the transcription level, and although this property is not unique among the members of the class Mollicutes, the mechanism by which it occurs in M. penetrans differs from those previously described for other Mycoplasma species. Indeed, the P35 phase variation was due neither to a p35 gene rearrangement nor to point mutations within the gene itself or its promoter. The P35 phase variation in the different variants obtained was concomitant with modifications in the pattern of other expressed lipoproteins, probably due to regulated expression of selected members of a gene family which was found to potentially encode similar lipoproteins. M. penetrans variants could be selected on the basis of their lack of colony immunoreactivity with a polyclonal antiserum against a Triton X-114 extract, strongly suggesting that the mechanisms involved in altering surface antigen expression might allow evasion of the humoral immune response of the infected host.

The major phase-variable outer membrane protein of Escherichia coli structurally resembles the immunoglobulin A1 protease class of exported protein and is regulated by a novel mechanism involving Dam and oxyR.

Here we report the characterization of an Escherichia coli gene (agn43) which encodes the principal phase-variable outer membrane protein termed antigen 43 (Ag43). The agn43 gene encodes a precursor protein of 107 kDa containing a 52-amino-acid signal sequence. Posttranslational processing generates an alpha43 subunit (predicted Mr of 49,789) and a C-terminal domain (beta43) with features typical of a bacterial integral outer membrane protein (predicted Mr of 51, 642). Secondary structure analysis predicts that beta43 exists as an 18-stranded beta barrel and that Ag43 shows structural organization closely resembling that of immunoglobulin A1 protease type of exoprotein produced by pathogenic Neisseria and Haemophilus spp. The correct processing of the polyprotein to alpha43 and beta43 in OmpT, OmpP, and DegP protease-deficient E. coli strains points to an autocatalytic cleavage mechanism, a hypothesis supported by the occurrence of an aspartyl protease active site within alpha43. Ag43, a species-specific antigen, possesses two RGD motifs of the type implicated in binding to human integrins. The mechanism of reversible phase variation was studied by immunochemical analysis of a panel of well-defined regulatory mutants and by analysis of DNA sequences upstream of agn43. Evidence strongly suggests that phase variation is regulated by both deoxyadenosine methylase (Dam) and by OxyR. Thus, oxyR mutants are locked on for Ag43 expression, whereas dam mutants are locked off for Ag43 expression. We propose a novel mechanism for the regulation of phase switching in which OxyR competes with Dam for unmethylated GATC sites in the regulatory region of the agn43 gene.

Nucleotide sequence analysis of the Streptococcus gordonii glucosyltransferase gene, gtfG.

Streptococcus gordonii has an extracellular glucosyltransferase (GTF) that polymerizes the glucose moiety of sucrose to form both water-soluble and water-insoluble glucans. Whereas multiple gtf genes have been identified in strains of mutans streptococci and Streptococcus salivarius, a single gene, designated gtfG, encodes the GTF of S. gordonii Challis. gtfG is also unique among the characterized gtfs in that it has a described regulatory determinant, rgg. Furthermore, the GTF activity in S. gordonii undergoes reversible phase variation between high and low levels. In order to gain insight into this novel GTF system, the nucleotide sequence of gtfG was determined and found to consist of a 4,734 base pair open reading frame encoding a protein with a deduced molecular weight of ca. 174,000. gtfG was similar to other sequenced gtfs with a conserved signal sequence followed by a ca. 600-bp region distinctive for gtfG, a conserved region encoding a putative catalytic active site and a series of six direct repeats in the carboxyl terminal region implicated in glucan binding. Although comparison of gtfG to other gtfs did not show a basis for the primer-independence of the encoded enzyme or the nature of the glucan products, the gtfG sequence data provide an important basis for further studies of these enzymes.

A novel regulatory mechanism for a novel phase-variable outer membrane protein of Escherichia coli.

Antigen 43 (Ag43) is a prominent hetero-oligomeric protein complex in the outer membrane of Escherichia coli. It is composed of two subunits. alpha 43 (M(r) 60, 000) and beta 43 (M(r) 53, 000) in 1:1 stoichiometry. alpha 43 is surface expressed, extends beyond the O-side chains of smooth lipopolysaccharide and is bound to the cell surface through an interaction with beta 43, itself an integral outer membrane protein, alpha 43 shows limited sequence homology with some enterobacterial adhesins. Expression of Ag43 is subject to reversible phase variation, the rates of variation from the Ag43+ve to Ag43-ve states in liquid minimal medium being approximately 2.2 x 10(-3), the corresponding rates from Ag43-ve to Ag43+ve states being approximately 1 x 10(-3). Phase switching of genes encoding Ag43 are transcriptionally regulated by DNA methylation (deoxyadenosine methylase [dam] mutants being "locked OFF") and by OxyR (oxyR mutants being "locked ON"). It is proposed that OxyR acts as a repressor of Ag43 transcription by binding to unmethylated GATC sites in the regulatory region of the gene. Sequencing and mapping has identified Ag43 as the likely product of the metastable flu gene first described in 1980 by Diderichsen and responsible for colony form variation in E. coli.

Phase-variable outer membrane proteins in Escherichia coli

Escherichia coli contains at least two phase-variable proteins in its outer membrane. One, termed antigen 43 (Ag43), is the product of the metastable flu gene located at min 43.6 on the E. coli chromosome and is responsible for colony form variation and for autoaggregation in liquid media. Ag43 is composed of two proteinaceous subunits, α 43 and β 43 in 1:1 stoichiometry. α 43 (apparent M r 60,000) is surface expressed, extends beyond the O-side chains of smooth lipopolysaccharide and is bound to the cell surface through an interaction with β 43 (apparent M r 53,000), itself an integral, heat-modifiable, outer membrane protein. α 43 shows limited N-terminal sequence homology with certain enterobacterial adhesins, and notable sequence homology with AIDA-1, an adhesin of diffuse-adhering E. coli. In addition, α 43 contains an RGD motif and a consensus sequence for an (autoproteolytic?) aspartyl protease active site. Expression of Ag43 is subject to reversible phase variation — in liquid minimal medium, the rates of variation from Ag43 + to Ag43 − states and from Ag43 − to Ag43 + states being ≈2.2 × 10 −3 and ≈1 × 10 −3, respectively. Phase switching of Ag43 is regulated by DNA methylation (deoxyadenosine methylase ( dam) mutants being ‘locked OFF’) and by OxyR ( oxyR mutants being ‘locked ON’). It is proposed that OxyR acts as a repressor of Ag43 transcription by binding to unmethylated GATC sites in the regulatory region of the gene. In some strains, Ag43 may also undergo antigenic variation. A 94 kDa immunocrossreactive outer membrane protein, showing similar rates of phase variation, has additionally been detected for some enteropathogenic and uropathogenic strains of E. coli. This 94 kDa protein can be proteolytically cleaved in situ with trypsin to yield two membrane-bound products with M rs and properties similar to those of α 43 and β 43. Results suggest that Ag43 may represent one of a family of antigenically-related high- M r adhesins which are synthesized as polyprotein precursors. Some members may be processed and presented on the cell surface as bipartite protein complexes (as Ag43). Others can remain uncleaved.

Major membrane proteins and lipoproteins as highly variable immunogenic surface components and strain-specific antigenic markers of Mycoplasma arthritidis

Surface antigenic variation was investigated in Mycoplasma arthritidis, an agent that produces chronic arthritis in rats which shares several features with many mycoplasma-induced diseases and thus defines a well-characterized model system. Hyperimmune rabbit antisera (anti-ISR1, anti-PG6, anti-H606 and anti-158p10) to whole M. arthritidis organisms were used as immunological probes in Western immunoblots of four M. arthritidis prototype strains (ISR1, PG6, H606 and D263) and five rat-passaged substrains (ISR1p1, ISR1p7, ISR1p8, 158p10 and D263p1). Several prominent antigens were identified that varied in expression. By Triton X-114 phase fractionation and treatment of whole cells with trypsin and carboxypeptidase Y, these strain-variant antigens were shown to be integral membrane proteins with C-termini and portions of the polypeptide chains oriented outside the membrane. Western blot immunoscreening of a large number of randomly selected clonal isolates and well-established clonal lineages from stock cultures of M. arthritidis ISR1p7, 158p10, PG6 and H606 revealed an expanded repertoire of variant membrane proteins whose expression was subject to independent, reversible phase variation. Colony immunoblots of these clonal populations with a hyperimmune rabbit antiserum to a gel-purified variant membrane protein (P36) showed that this phase switching occurred at a high frequency (10(-4) to 10(-2) per generation). Detailed immunological and biochemical characterization of the phase-variant membrane proteins demonstrated that they are: (i) antigenically related or distinct; (ii) apparently specific to particular strain populations; (iii) proteins or lipoproteins; (iv) major immunogens of M. arthritidis, recognized by serum antibodies from convalescent rat; and (v) able to undergo variation in expression during in vivo passage. Thus, M. arthritidis possesses a complex system capable of creating large repertoires of cell surface phenotypes which may affect the multiple interactions of this organism with its host and dictate its potential as a successful infectious agent and pathogen.

Phase variation of Enterococcus faecalis pAD1 conjugation functions relates to changes in iteron sequence region.

pAD1 (60 kb) is a conjugative, hemolysin/bacteriocin plasmid in Enterococcus faecalis. It confers a mating response to the peptide sex pheromone cAD1 produced by recipient (plasmid-free) cells, leading to highly efficient plasmid transfer in broth matings. Control of the physiological response to cAD1 can been overridden by a reversible phase variation event at frequencies on the order of 10(-4) to 10(-3) per cell per generation (L. T. Pontius and D. B. Clewell, Plasmid 26:172-185, 1991). The variant forms are designated Dryc and Dry+, which reflects the colony morphologies of cells whose conjugation functions are switched on and off, respectively. Here we show that Dryc variants exhibit a structural change in a region between repA and repB that contains two clusters of 8-bp iterons. The change involved a 31- or 32-bp increase in size of this region. In three or four independent variants examined, one of the iteron clusters increased in size from 13 to 17 iterons. When iteron DNA was placed on a multicopy plasmid and introduced into a wild-type pAD1 derivative, the Dryc phenotype was generated. Since traA, a key negative regulator of conjugation, bears several centrally located iteron-like sequences with the same orientation, we speculate that the protein(s) that normally binds iterons (possibly RepA and/or RepB) blocks traA transcription in Dryc variants.

Phase variation of Haemophilus influenzae lipopolysaccharide: characterization of lipopolysaccharide from individual colonies.

The lipopolysaccharide (LPS) of Haemophilus influenzae expresses a number of core oligosaccharide epitopes on its outer surface. The expression of individual epitopes is subject to frequent (approximately 1% bacteria/generation) reversible phase variation, as determined by colony immunoblots. We have used a microtechnique for the extraction of LPS from individual colonies, whose LPS antigenic phenotype has been identified, so that the LPS can be studied by tricine sodium dodecylsulphate polyacrylamide gel electrophoresis (T-SDS-PAGE). This avoids the introduction of heterogeneous phase-varying LPS which is inevitable if bacteria from colonies are grown in broth culture prior to LPS extraction and analysis. Using these techniques we have investigated the repertoire of LPS phase variation exhibited by H. influenzae strain RM7004 (a serotype b meningitis isolate). This technique will facilitate the study of bacteria in which there is variable LPS expression.

Phase variation of H. influenzae fimbriae: Transcriptional control of two divergent genes through a variable combined promoter region

The expression of H. influenzae fimbriae is subject to reversible phase variation between three expression levels. This phenomenon is controlled at the transcriptional level of two divergently orientated genes, hifA and hifB, encoding the major fimbrial subunit and the fimbrial chaperone, respectively. The hifA and hifB promoter regions were found to be clustered through an almost complete divergent overlap with a variable DNA backbone of repetitive TA units. Variation in the number of units changes the normally strictly constrained spacing between the −35 and −10 sequences and controls the bidirectional transcription initiation, thus forming a novel mechanism directing multiple gene transcription.