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Purpose. We investigated the transcription of adhesin-encoding genes sabA, hopZ and labA in Helicobacter pylori strain J99. Each possesses a repeating homopolymeric nucleotide tract within their promoter regions, and sabA and hopZ possess repeats within their 5′ coding regions. Methodology. We altered the repeat lengths associated with the adhesin genes and quantified mRNA levels by real-time quantitative PCR. Using adherence to AGS cells and IL-8 assays, we examined the effects of altered transcript levels. We assessed the role of ArsRS in transcription using an arsS null mutant and by examining ArsR binding to promoter regions via electrophoretic mobility shift assays. Results. Extensions or truncations of promoter region repeats in hopZ and labA increased transcript levels, mirroring results shown by our lab and others for mutations in the sabA promoter. Altered lengths of the poly-cytosine thymine tract within the 5′ coding region of sabA demonstrated that switching from phase-off to phase-on significantly increased mRNA levels. However, mutations in the poly-thymine tract of sabA, which increased mRNA levels, do not behave synergistically with phase-on mutations. Phase-on mutations of sabA resulted in increased H. pylori adherence to AGS cells, but only a modest effect on IL-8. hopZ and labA, and sabA paralogue sabB, transcript levels were increased in an arsS mutant and ArsR bound the promoter regions for each of these genes in vitro. Conclusion. This work highlights the complex nature of adhesin regulation, its impact on H. pylori attachment and the pervasive role of ArsRS in adhesin expression. Such regulation may help facilitate the decades-long persistence of infection.

We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc²155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites.

The pathogenesis of diseases elicited by the gastric pathogen Helicobacter pylori is partially determined by the effectiveness of adaptation to the variably acidic environment of the host stomach. Adaptation includes appropriate adherence to the gastric epithelium via outer membrane protein adhesins such as SabA. The expression of sabA is subject to regulation via phase variation in the promoter and coding regions as well as repression by the two-component system ArsRS. In this study, we investigated the role of a homopolymeric thymine [poly(T)] tract −50 to −33 relative to the sabA transcriptional start site in H. pylori strain J99. We quantified sabA expression in H. pylori J99 by quantitative reverse transcription-PCR (RT-PCR), demonstrating significant changes in sabA expression associated with experimental manipulations of poly(T) tract length. Mimicking the length increase of this tract by adding adenines instead of thymines had similar effects, while the addition of other nucleotides failed to affect sabA expression in the same manner. We hypothesize that modification of the poly(T) tract changes DNA topology, affecting regulatory protein interaction(s) or RNA polymerase binding efficiency. Additionally, we characterized the interaction between the sabA promoter region and ArsR, a response regulator affecting sabA expression. Using recombinant ArsR in electrophoretic mobility shift assays (EMSA), we localized binding to a sequence with partial dyad symmetry −20 and +38 relative to the sabA +1 site. The control of sabA expression by both ArsRS and phase variation at two distinct repeat regions suggests the control of sabA expression is both complex and vital to H. pylori infection.

Helicobacter pylori is a genetically diverse bacterial species that colonizes the stomach in about half of the human population. Most persons colonized by H. pylori remain asymptomatic, but the presence of this organism is a risk factor for gastric cancer. Multiple populations and subpopulations of H. pylori with distinct geographic distributions are recognized. Genetic differences among these populations might be a factor underlying geographic variation in gastric cancer incidence. Relatively little is known about the genomic features of African H. pylori strains compared to other populations of strains. In this study, we first analyzed the genomes of H. pylori strains from seven globally distributed populations or subpopulations and identified encoded proteins that exhibited the highest levels of sequence divergence. These included secreted proteins, an LPS glycosyltransferase, fucosyltransferases, proteins involved in molybdopterin biosynthesis, and Clp protease adaptor (ClpS). Among proteins encoded by the cag pathogenicity island, CagA and CagQ exhibited the highest levels of sequence diversity. We then identified proteins in strains of Western African origin (classified as hspWAfrica by MLST analysis) with sequences that were highly divergent compared to those in other populations of strains. These included ATP-dependent Clp protease, ClpS, and proteins of unknown function. Three of the divergent proteins sequences identified in West African strains were characterized by distinct insertions or deletions up to 8 amino acids in length. These polymorphisms in rapidly evolving proteins represent robust genetic signatures for H. pylori strains of West African origin.

Phase variation is frequently utilized by bacterial species to affect gene expression such that phenotypic variants are maintained within populations, ensuring survival as environmental or host conditions change. Unusual among Helicobacter pylori phase variable or contingency genes is arsS, encoding a sensory histidine kinase involved in the acid acclimation of the organism. The presence of a 3′ homopolymeric cytosine tract of variable length in arsS among Helicobacter pylori strains allows for the expression of various functional ArsS isoforms, differing in carboxy-terminal protein domains. In this study, we analyzed this 3′ arsS region via amplified fragment length polymorphism (AFLP) and sequencing analyses for H. pylori populations from 3 different gastric sites of 12 patients. Our data indicate the presence of multiple arsS alleles within each population of H. pylori derived from the gastric antrum, cardia, or corpus of these patients. We also show that H. pylori, derived from the same anatomical site and patient, are predicted to express multiple ArsS isoforms in each population investigated. Furthermore, we identify a polymorphic deletion within arsS that generates another alternate ArsS C-terminal end. These findings suggest that four C-terminal variations of ArsS adds to the complexity of the ArsRS acid adaptation mechanism as a whole and may influence the ability of H. pylori to persist in the gastric niche for decades.

Summary To identify Helicobacter pylori genes with expression that is enhanced under low pH conditions, we used subtractive hybridization methodology. We identified 28 acid-induced genes, of which 18 have known or putative functions. Six pairs of genes were co-transcribed. Primer extension analysis identified single or multiple transcriptional start points (tsp) for 14 of the 22 loci. Sequence analysis of the −10 regions upstream of the tsps revealed consensus motifs for multiple RNA polymerase sigma factors present in H. pylori (σ80, σ54 and σ28). No sequences resembling the −35 Escherichia coli consensus sequence (TTGACA) were present upstream of any of the genes. Both increased gene transcription and decreased mRNA decay contribute to the observed increase in H. pylori transcript abundance at acid pH. These studies document the complex response of H. pylori to environmental pH changes, and provide insight into mechanisms used for intragastric survival.

We demonstrate here that the arbitrary primer polymerase-chain-reaction-based DNA fingerprinting method (also termed random amplified polymorphic DNA or RAPD) can be used to distinguish among strains of the avian pathogen Mycoplasma gallisepticum. Ten base oligonucleotide primers were used individually to prime DNA synthesis from genomic DNAs. Strain-specific arrays of DNA fragments were generated, which allowed us to identify and group isolates. Isolates of M. synoviae, M. gallinarum and M. iners yielded arrays of DNA fragments that differed markedly from those generated from the M. gallisepticum isolates using the same arbitrary primers. These results show that the RAPD fingerprinting method distinguishes genetically different strains of M. gallisepticum and indicates that it should be valuable for monitoring transmission of this pathogen.

Mycoplasma pneumoniae-specific monoclonal antibodies were constructed for the purpose of developing reagents for clinical diagnostics. The monoclonal antibodies, designated Mp2B12 and Mp3A11, recognize surface exposed antigens of 28 kDa and 170 kDa, respectively. Mp3A11 recognizes an epitope on a cytadhesin molecule, P1, which is not shared on the analogous cytadhesin of M. genitalium. Both monoclonal antibodies are capable of detecting 1 x 10(5) M. pneumoniae in a standard ELISA test.

Summary A64kDa lipoprotein (LP 64) haemagglutinin (pI 4.9–5.0) was isolated from the membrane of Mycoplasma gallisepticum. Triton X-114 phase partitioning has demonstrated that the hydrophobic nature of this haemagglutinin is due to a lipid portion of the molecule. Autoradiography of [3H]-palmitate-labelled M. gallisepticum revealed the presence of several additional lipoproteins. Immunoelectron microscopy demonstrated the localization of LP 64 to the base of the terminal structure. Densitometric scans of stained polyacrylamide gels of M. gallisepticum showed that LP 64 constitutes 1.7% of the total protein. Scans of immunoblots of M. gallisepticum indicate that LP 64 is highly immunogenic in chickens, accounting for 7.4% of the total serum IgG response at four weeks post-infection. A quantitative value for the IgG response to LP 64, relative to the percentage of total protein (the Relative Immunogenicity Index) was 4.4. LP 64 is conserved among several strains of M. gallisepticum, but its presence could not be detected in Mycoplasma synoviae. Antiserum raised to electroeluted LP 64 reacted specifically with this lipoprotein when assessed on either one-or two-dimensional immunoblots of M. gallisepticum. This antiserum, as well as Fab fragments, inhibited haemagglutination of chicken erythrocytes and inhibited the attachment of 14Clabelled M. gallisepticum to chicken tracheal epithelium in vitro by 62%.

The H. pylori cag type 4 secretion system (T4SS) translocates CagA into host cells which can lead to NF-κβ activation. The T4SS component CagY is essential for CagA translocation. cagY harbors two repeat motifs which engender considerable genetic diversity to this locus. We used Mongolian gerbils to select for Hp derivative strains that differed in intensity of host interactions to define the biology of long-term colonization and genetic alteration of virulence factors within the gastric niche. Gerbils were initially challenged with the carcinogenic Hp strain 7.13 for 12 weeks; two derivative isolates, 7.13-1 and 7.13-2, were then obtained from 2 independent gerbils. In vitro, strain 7.13-1 induced significantly higher levels of NF-κβ activation and CagA translocation in AGS gastric epithelial cells compared to strain 7.13-2 (fold/uninfected: 100±7 vs. 26±7, respectively; p

Screening a Helicobacter pylori genomic library with antisera raised against H. pylori broth culture supernatant resulted in the identification of six antigens: urease, HspB, Lpp20, DnaK, MsrA, and a cysteine-rich 28-kDa protein (designated HcpA). H. pylori antigens may be released into the extracellular space by multiple mechanisms, including specific secretion pathways, autolysis, and membrane vesicle formation.

To identify putative members of a regulon controlled by the H. pylori sensory histidine kinase HP0164 (HK0164), we constructed HK0164 null mutant H. pylori strains and analyzed bacterial gene transcription using DNA arrays. Seven genes were differentially expressed in multiple HK0164 mutant strains compared to their expression in control strains. Strain-dependent variations in differential expression were also detected. These results indicate that the signal transduction circuit utilizing HK0164 controls the transcription of at least seven genes in H. pylori .

Analysis of 12 Helicobacter pylori promoters indicates the existence of a consensus −10 hexamer (TAtaaT) but little conservation of −35 sequences. In this study, mutations in either the H. pylori vacA −10 region or the −35 region resulted in decreased vacA transcription and suggested that an extended −10 motif is utilized. Thus, despite the lack of a −35 consensus sequence for H. pylori promoters, the −35 region plays a functional role in vacA transcription.

Helicobacter pylori colonizes the human gastric mucosa and causes gastritis, ulceration, or gastric cancer. A previously uncharacterized region of the H. pylori genome was identified and sequenced. This region includes a putative operon containing three open reading frames termed gidA (1,866 bp), dapE (1,167 bp), and orf2 (753 bp); the gidA and dapE products are highly homologous to other bacterial proteins. In E. coli, dapE encodes N-succinyl-L-diaminopimelic acid desuccinylase, which catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to L-diaminopimelic acid (L-DAP) and succinate. When wild-type H. pylori strains were transformed to select for dapE mutagenesis, mutants were present when plates were supplemented with DAP but not with lysine; orf2 mutants were selected without DAP supplementation. Consistent with the finding that GidA is essential in Escherichia coli, we were unable to obtain a gidA mutant in H. pylori despite evidence that insertional mutagenesis had occurred. The positions of gidA, dapE, and orf2 suggest that they form an operon, which was supported by slot blot RNA hybridization and reverse transcriptase PCR studies. The data imply that the H. pylori dapE mutant may be useful as a conditionally lethal vaccine.

The repetitive element Rep MP 1 has been previously described as a 300-bp element present within the chromosome of Mycoplasma pneumoniae in at least 10 copies. Sequence found flanking Rep MP 1 in lambda clone 5B52 has been demonstrated to exist in multiple copies within the genome of M. pneumoniae. A HindIII M. pneumoniae fragment containing the lambda MP 5B52 insert and its flanking sequences was cloned into pBluescript. Sequence comparisons of this clone, designated pMPH 624-20, with nucleotide database entries showed that in addition to the 300-bp Rep MP 1 element, flanking sequence blocks were conserved within several other cloned sequences. These short repeated sequences, approximately 80, 56, and 71 nucleotides long, are termed sRep A, sRep B, and sRep C, respectively. These sRep regions were used as probes in the selection of cloned sequences containing these repetitive flanking regions. Comparison of these sequences demonstrated that the 300-bp Rep MP 1 elements is actually the core element within a larger, variable repetitive mosaic. The repetitive regions surrounding the core element are found in various combinations, arrangements, and distances from the core in a mosaic pattern. These newly identified portions of the mosaic do not exist independently of the core element. The core appears to be the only invariant portion of this repetitive mosaic.

The gyrAB genes encoding the entire B and a portion of the A subunit of DNA gyrase (E.C. 5.99.1.3) from Mycoplasma gallisepticum (Mg), strain S6, were cloned and sequenced. These gyrAB genes are co-transcribed as a single, polycistronic mRNA transcript. The Mg gyrB appears unique among prokaryotic gyrB in its use of GUG as a start codon.