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Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma, which develops within a hypochlorhydric environment. We sequentially isolated H. pylori (strain J99) from a patient who developed corpus-predominant gastritis and hypochlorhydia over a 6-year interval. Archival J99 survived significantly better under acidic conditions than recent J99 strains. H. pylori arsRS encodes a 2-component system critical for stress responses; recent J99 isolates harbored 2 nonsynonymous arsS mutations, and arsS inactivation abolished acid survival. In vivo, acid-resistant archival, but not recent J99, successfully colonized high-acid-secreting rodents. Thus, genetic evolution of arsS may influence progression to hypochlorhydia and gastric cancer.

Broth culture supernatants from Tox + Helicobacter pylori strains induce vacuolation of HeLa cells in vitro and contain VacA in concentrations that are higher than those found in supernatants from Tox − H. pylori strains. To investigate the basis for this phenomenon, we analyzed the transcription of the vacuolating cytotoxin gene ( vacA ) in eight Tox + strains (each with a type s1/m1 vacA genotype) and nine Tox − strains (each with a type s2/m2 vacA genotype). Most of the Tox + and Tox − strains tested used the same vacA transcriptional start point, but Tox + strains yielded significantly stronger primer extension signal intensities than did Tox − strains (mean densitometry values of 15.8 ± 1.9 versus 8.9 ± 1.7, P = 0.0016). Correspondingly, when we introduced vacA :: xylE transcriptional fusions into the chromosomes of a Tox + strain (60190) and a Tox − strain (86-313), the level of XylE activity in 60190 vacA :: xylE was about 30-fold higher than that in 86-313 vacA :: xylE . Sequence analysis and promoter exchange experiments indicated that the different levels of vacA transcription in these two strains cannot be explained solely by a difference in promoter strength. These data indicate that Tox + and Tox − H. pylori strains typically differ not only in the VacA amino acid sequence but also in the level of vacA transcription.

Bilateral conjunctivitis and episcleritis were identified in an adult European starling (Sturnus vulgaris). A novel mycoplasma species, Mycoplasma sturni, was isolated in pure culture from the conjunctiva of both eyes. The clinical presentation was similar to that of conjunctivitis in house finches (Carpodacus mexicanus) caused by Mycoplasma gallisepticum. However, the histologic lesions were distinct, by the presence of ulceration and by the absence of epithelial hyperplasia and lymphoplasmacytic infiltration [corrected]. Mycoplasma sturni ferments glucose, does not hemadsorb or hemagglutinate chicken erythrocytes, and grows rapidly at 37 C in comparison to other Mycoplasma spp. The role of M. sturni in conjunctivitis in other passerine species is presently unknown.

Strain UCMF(T) (T = type strain) was isolated from the conjunctiva of a European starling (Sturnus vulgaris) with conjunctivitis. Colonies grown on conventional mycoplasma agar possessed the typical fried-egg appearance observed with many mycoplasmal species. Electron micrographs of ultrathin sections of UCMF(T) revealed a pleomorphic cellular morphology; the cells ranged from spherical to elliptical or flask shaped. The cell size ranged from 0.3 to 0.5 microns. Strain UCMF(T) grows well in a variety of mycoplasma broth formulations at 25 degrees C, with rapid and heavy growth at 37 degrees C. No growth occurs at 42 degrees C. This organism ferments glucose but does not hydrolyze urea or arginine and has an absolute requirement for sterol from growth. Strain UCMF(T) does not hemagglutinate or hemadsorb chicken erythrocytes. The genome size is 870 kbp, and the guanine-plus-cytosine content is 31 mol%. Sequence analysis of the 16S rRNA gene demonstrated that this organism is unique and has not been described previously. Serological analysis confirmed that strain UCMF(T) is distinct from all previously identified Mycoplasma, Acholeplasma, Spiroplasma, Entomoplasma, and Mesoplasma species. This organism represents a new species, for which we propose the name Mycoplasma sturni. Strain UCMF (= ATCC 51945) is the type strain of M. sturni sp. nov.

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

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

Individual bacteria of numerous species can communicate and coordinate their actions via the production, release, and detection of extracellular signaling molecules. In this study, we used the Vibrio harveyi luminescence bioassay to determine whether Helicobacter pylori produces such a factor. Cell-free conditioned media from H. pylori strains 60190 and 26695 each induced >100-fold-greater luminescence in V. harveyi than did sterile culture medium. The H. pylori signaling molecule had a molecular mass of H. pylori 26695 does not contain any gene predicted to encode an acyl homoserine lactone synthase but does contain an orthologue of luxS , which is required for production of autoinducer-2 (AI-2) in V. harveyi . To evaluate the role of luxS in H. pylori , we constructed luxS null mutants derived from H. pylori 60190 and 26695. Conditioned media from the wild-type H. pylori strains induced >100-fold-greater luminescence in the V. harveyi bioassay than did conditioned medium from either mutant strain. Production of the signaling molecule was restored in an H. pylori luxS null mutant strain by complementation with a single intact copy of luxS placed in a heterologous site on the chromosome. In addition, Escherichia coli DH5α produced autoinducer activity following the introduction of an intact copy of luxS from H. pylori . Production of the signaling molecule by H. pylori was growth phase dependent, with maximal production occurring in the mid-exponential phase of growth. Transcription of H. pylori vacA also was growth phase dependent, but this phenomenon was not dependent on luxS activity. These data indicate that H. pylori produces an extracellular signaling molecule related to AI-2 from V. harveyi . We speculate that this signaling molecule may play a role in regulating H. pylori gene expression.

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 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.