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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.

Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA ) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct "cross-talk" between HpArsRS and HpNikR at neutral pH has been demonstrated.

Five newly isolated mycobacteriophages -Angelica, CrimD, Adephagia, Anaya, and Pixie - have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them - with the exception of TM4 - form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species. © 2011 Pope et al. Fil: Pope, Welkin H.. University of Pittsburgh; Estados Unidos Fil: Ferreira, Christina M.. University of Pittsburgh; Estados Unidos Fil: Jacobs Sera, Deborah. University of Pittsburgh; Estados Unidos Fil: Benjamin, Robert C.. University of North Texas; Estados Unidos Fil: Davis, Ariangela J.. Calvin College; Estados Unidos Fil: DeJong, Randall J.. Calvin College; Estados Unidos Fil: Elgin, Sarah C. R.. Washington University in St. Louis; Estados Unidos Fil: Guilfoile, Forrest R.. University of Pittsburgh; Estados Unidos Fil: Forsyth, Mark H.. The College Of William And Mary; Estados Unidos Fil: Harris, Alexander D.. Calvin College; Estados Unidos Fil: Harvey, Samuel E.. The College Of William And Mary; Estados Unidos Fil: Hughes, Lee E.. University of North Texas; Estados Unidos Fil: Hynes, Peter M.. Washington University in St. Louis; Estados Unidos Fil: Jackson, Arrykka S.. The College Of William And Mary; Estados Unidos Fil: Jalal, Marilyn D.. University of North Texas; Estados Unidos Fil: MacMurray, Elizabeth A.. The College Of William And Mary; Estados Unidos Fil: Manley, Coreen M.. University of North Texas; Estados Unidos Fil: McDonough, Molly J.. The College Of William And Mary; Estados Unidos Fil: Mosier, Jordan L.. University of North Texas; Estados Unidos Fil: Osterbann, Larissa J.. Calvin College; Estados Unidos Fil: Rabinowitz, Hannah S.. Washington University in St. Louis; Estados Unidos Fil: Rhyan, Corwin N.. Washington University in St. Louis; Estados Unidos Fil: Russell, Daniel A.. University of Pittsburgh; Estados Unidos Fil: Saha, Margaret S.. The College Of William And Mary; Estados Unidos Fil: Shaffer, Christopher D.. Washington University in St. Louis; Estados Unidos Fil: Simon, Stephanie E.. University of North Texas; Estados Unidos Fil: Sims, Erika F.. Washington University in St. Louis; Estados Unidos Fil: Tovar, Isabel G.. University of North Texas; Estados Unidos Fil: Weisser, Emilie G.. Washington University in St. Louis; Estados Unidos Fil: Wertz, John T.. Calvin College; Estados Unidos Fil: Weston-Hafer, Kathleen A.. Washington University in St. Louis; Estados Unidos Fil: Williamson, Kurt E.. The College Of William And Mary; Estados Unidos Fil: Zhang, Bo. Washington University in St. Louis; Estados Unidos Fil: Cresawn, Steven G.. James Madison University; Estados Unidos Fil: Jain, Paras. Albert Einstein College Of Medicine Of Yeshiva University; Estados Unidos Fil: Piuri, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Pittsburgh; Estados Unidos Fil: Jacobs, William R.. Albert Einstein College Of Medicine Of Yeshiva University; Estados Unidos Fil: Hendrix, Roger W.. University of Pittsburgh; Estados Unidos Fil: Hatfull, Graham F.. University of Pittsburgh; Estados Unidos

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

In an analysis of Helicobacter pylori genomic DNA by macroarray methodology, genomic DNA from a panel of cag pathogenicity island (PAI)-negative H. pylori clinical isolates failed to hybridize with 27 genes located outside the cag PAI in a cag PAI-positive reference strain. PCR analyses confirmed that HP0217 (encoding a lipopolysaccharide biosynthetic protein) and HP1079 (encoding a protein of unknown function) were present significantly more frequently in cagA -positive strains than in cagA -negative strains. A low G+C content of these two genes suggests they were acquired by horizontal transfer events.

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.