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Gupta RS. Molecular signatures (unique proteins and conserved indels) that are specific for the epsilon proteobacteria (Campylobacterales). BMC Genomics 2006; 7:167. [PMID: 16817973 PMCID: PMC1557499 DOI: 10.1186/1471-2164-7-167] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2006] [Accepted: 07/04/2006] [Indexed: 11/28/2022] Open
Abstract
Background The epsilon proteobacteria, which include many important human pathogens, are presently recognized solely on the basis of their branching in rRNA trees. No unique molecular or biochemical characteristics specific for this group are known. Results Comparative analyses of proteins in the genomes of Wolinella succinogenes DSM 1740 and Campylobacter jejuni RM1221 against all available sequences have identified a large number of proteins that are unique to various epsilon proteobacteria (Campylobacterales), but whose homologs are not detected in other organisms. Of these proteins, 49 are uniquely found in nearly all sequenced epsilon-proteobacteria (viz. Helicobacter pylori (26695 and J99), H. hepaticus, C. jejuni (NCTC 11168, RM1221, HB93-13, 84-25, CF93-6, 260.94, 11168 and 81-176), C. lari, C. coli, C. upsaliensis, C. fetus, W. succinogenes DSM 1740 and Thiomicrospira denitrificans ATCC 33889), 11 are unique for the Wolinella and Helicobacter species (i.e. Helicobacteraceae family) and many others are specific for either some or all of the species within the Campylobacter genus. The primary sequences of many of these proteins are highly conserved and provide novel resources for diagnostics and therapeutics. We also report four conserved indels (i.e. inserts or deletions) in widely distributed proteins (viz. B subunit of exinuclease ABC, phenylalanyl-tRNA synthetase, RNA polymerase β '-subunit and FtsH protein) that are specific for either all epsilon proteobacteria or different subgroups. In addition, a rare genetic event that caused fusion of the genes for the largest subunits of RNA polymerase (rpoB and rpoC) in Wolinella and Helicobacter is also described. The inter-relationships amongst Campylobacterales as deduced from these molecular signatures are in accordance with the phylogenetic trees based on the 16S rRNA and concatenated sequences for nine conserved proteins. Conclusion These molecular signatures provide novel tools for identifying and circumscribing species from the Campylobacterales order and its subgroups in molecular terms. Although sequence information for these signatures is presently limited to Campylobacterales species, it is likely that many of them will also be found in other epsilon proteobacteria. Functional studies on these proteins and conserved indels should reveal novel biochemical or physiological characteristics that are unique to these groups of epsilon proteobacteria.
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Affiliation(s)
- Radhey S Gupta
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, L8N 3Z5, Canada.
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Newnham E, Chang N, Taylor DE. Expanded genomic map of Campylobacter jejuni UA580 and localization of 23S ribosomal rRNA genes by I-CeuI restriction endonuclease digestion. FEMS Microbiol Lett 1996; 142:223-9. [PMID: 8810506 DOI: 10.1111/j.1574-6968.1996.tb08434.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
The genomic map of Campylobacter jejuni UA580 was expanded and more precisely constructed using I-CeuI, Sal/I and SmaI restriction endonucleases in conjunction with pulsed-field gel electrophoresis (PFGE). The presence of three fragments after digestion with I-CeuI confirmed the presence of three copies of the 23S ribosomal rRNA (rrl) gene. The genome size of Campylobacter jejuni UA580 was determined to be 1725 +/- 5.9 kbp by I-CeuI with fragment sizes of 1053 +/- 4.4, 361 +/- 2.7 and 311 +/- 3.6 kbp. Analysis of a PCR product from C. jejuni UA580 23S rRNA gene showed that I-CeuI did cut within the gene. The precise locations of the three genes were determined using I-CeuI with two copies of the 23S and 5S rRNA genes located separately from the 16S rRNA gene whereas the third copy of the 23S and 5S rRNA genes had a closer linkage to a 16S rRNA gene copy. Homologous gene probes were used to map additional genes and allowed the realignment of a few previously mapped genes on the chromosome. Other strains of C. jejuni were also cut into three fragments with I-CeuI, which generated variable PFGE patterns.
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Affiliation(s)
- E Newnham
- Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada
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4
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Vandamme P, Pot B, Gillis M, de Vos P, Kersters K, Swings J. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 1996; 60:407-38. [PMID: 8801440 PMCID: PMC239450 DOI: 10.1128/mr.60.2.407-438.1996] [Citation(s) in RCA: 367] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Over the last 25 years, a much broader range of taxonomic studies of bacteria has gradually replaced the former reliance upon morphological, physiological, and biochemical characterization. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them in a consensus type of classification, framed in a general phylogeny derived from 16S rRNA sequence analysis. In some cases, the consensus classification is a compromise containing a minimum of contradictions. It is thought that the more parameters that will become available in the future, the more polyphasic classification will gain stability. In this review, the practice of polyphasic taxonomy is discussed for four groups of bacteria chosen for their relevance, complexity, or both: the genera Xanthomonas and Campylobacter, the lactic acid bacteria, and the family Comamonadaceae. An evaluation of our present insights, the conclusions derived from it, and the perspectives of polyphasic taxonomy are discussed, emphasizing the keystone role of the species. Taxonomists did not succeed in standardizing species delimitation by using percent DNA hybridization values. Together with the absence of another "gold standard" for species definition, this has an enormous repercussion on bacterial taxonomy. This problem is faced in polyphasic taxonomy, which does not depend on a theory, a hypothesis, or a set of rules, presenting a pragmatic approach to a consensus type of taxonomy, integrating all available data maximally. In the future, polyphasic taxonomy will have to cope with (i) enormous amounts of data, (ii) large numbers of strains, and (iii) data fusion (data aggregation), which will demand efficient and centralized data storage. In the future, taxonomic studies will require collaborative efforts by specialized laboratories even more than now is the case. Whether these future developments will guarantee a more stable consensus classification remains an open question.
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Affiliation(s)
- P Vandamme
- Laboratorium voor Microbiologie, Universiteit Gent, Belgium
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Kim NW, Gutell RR, Chan VL. Complete sequences and organization of the rrnA operon from campylobacter jejuni TGH9011 (ATCC43431). Gene 1995; 164:101-6. [PMID: 7590296 DOI: 10.1016/0378-1119(95)00471-h] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
The rrnA ribosomal RNA (rRNA) operon of Campylobacter jejuni (Cj) TGH9011 (ATCC43431) was cloned and sequenced to completion. rRNAs were then characterized by primer extension and S1 nuclease mapping analysis. The secondary structure models of Cj 16S and 23S rRNAs were constructed, and the models were compared to the corresponding models from other eubacterial rRNA. The analysis presented a typical 5'-promoter-16S-tRNAs-23S-5S-terminator-3' prokaryotic rRNA operon structure. However, an unusual organization of the intercistronic tRNAs was observed where the two tRNAs, tRNA(Ala) and tRNA(Ile), were present in the order 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', which is opposite of the typical 5'-16S-tRNA(Ile)-tRNA(Ala)-23S-3' structure observed in other bacteria.
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MESH Headings
- Base Sequence
- Campylobacter jejuni/genetics
- Cloning, Molecular
- Genes, Bacterial
- Models, Molecular
- Molecular Sequence Data
- Nucleic Acid Conformation
- Operon
- RNA, Ribosomal, 16S/chemistry
- RNA, Ribosomal, 16S/genetics
- RNA, Ribosomal, 23S/chemistry
- RNA, Ribosomal, 23S/genetics
- RNA, Transfer, Ala/genetics
- RNA, Transfer, Ile/genetics
- Sequence Analysis, DNA
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Affiliation(s)
- N W Kim
- Department of Microbiology, University of Toronto, Ontario, Canada
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Salama SM, Newnham E, Chang N, Taylor DE. Genome map of Campylobacter fetus subsp. fetus ATCC 27374. FEMS Microbiol Lett 1995. [DOI: 10.1111/j.1574-6968.1995.tb07840.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
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Haddad A, Camacho F, Durand P, Cary SC. Phylogenetic characterization of the epibiotic bacteria associated with the hydrothermal vent polychaete Alvinella pompejana. Appl Environ Microbiol 1995; 61:1679-87. [PMID: 7544093 PMCID: PMC167429 DOI: 10.1128/aem.61.5.1679-1687.1995] [Citation(s) in RCA: 125] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Alvinella pompejana is a polychaetous annelid that inhabits active deep-sea hydrothermal vent sites along the East Pacific Rise, where it colonizes the walls of actively venting high-temperature chimneys. An abundant, morphologically diverse epibiotic microflora is associated with the worm's dorsal integument, with a highly integrated filamentous morphotype clearly dominating the microbial biomass. It has been suggested that this bacterial population participates in either the nutrition of the worm or in detoxification of the worm's immediate environment. The primary goal of this study was to phylogenetically characterize selected epibionts through the analysis of 16S rRNA gene sequences. Nucleic acids were extracted from bacteria collected from the dorsal surface of A. pompejana. 16S rRNA genes were amplified with universal bacterial primers by the PCR. These genes were subsequently cloned, and the resulting clone library was screened by restriction fragment length polymorphism analysis to identify distinct clone types. The restriction fragment length polymorphism analysis identified 32 different clone families in the library. Four of these families were clearly dominant, representing more than 65% of the library. Representatives from the four most abundant clone families were chosen for complete 16S rRNA gene sequencing and phylogenetic analysis. These gene sequences were analyzed by a variety of phylogenetic inference methods and found to be related to the newly established epsilon subdivision of the division Proteobacteria. Secondary structural model comparisons and comparisons of established signature base positions in the 16S rRNA confirmed the placement of the Alvinella clones in the epsilon subdivision of the Proteobacteria.
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Affiliation(s)
- A Haddad
- Department of Microbiology, Oregon State University, Corvallis 97331, USA
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8
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Trust TJ, Logan SM, Gustafson CE, Romaniuk PJ, Kim NW, Chan VL, Ragan MA, Guerry P, Gutell RR. Phylogenetic and molecular characterization of a 23S rRNA gene positions the genus Campylobacter in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in Campylobacter spp. J Bacteriol 1994; 176:4597-609. [PMID: 8045890 PMCID: PMC196280 DOI: 10.1128/jb.176.15.4597-4609.1994] [Citation(s) in RCA: 60] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
The nucleotide sequence of a 23S rRNA gene of Campylobacter coli VC167 was determined. The primary sequence of the C. coli 23S rRNA was deduced, and a secondary-structure model was constructed. Comparison with Escherichia coli 23S rRNA showed a major difference in the C. coli rRNA at approximately position 1170 (E. coli numbering) in the form of an extra sequence block approximately 147 bp long. PCR analysis of 31 other strains of C. coli and C. jejuni showed that 69% carried a transcribed spacer of either ca. 147 or ca. 37 bp. Comparison of all sequenced Campylobacter transcribed spacers showed that the Campylobacter inserts were related in sequence and percent G+C content. All Campylobacter strains carrying transcribed spacers in their 23S rRNA genes produced fragmented 23S rRNAs. Other strains which produced unfragmented 23S rRNAs did not appear to carry transcribed spacers at this position in their 23S rRNA genes. At the 1850 region (E. coli numbering), Campylobacter 23S rRNA displayed a base pairing signature most like that of the beta and gamma subdivisions of the class Proteobacteria, but in the 270 region, Campylobacter 23S rRNA displayed a helix signature which distinguished it from the alpha, beta, and gamma subdivisions. Phylogenetic analysis comparing C. coli VC167 23S rRNA and a C. jejuni TGH9011 (ATCC 43431) 23S rRNA with 53 other completely sequenced (eu)bacterial 23S rRNAs showed that the two campylobacters form a sister group to the alpha, beta, and gamma proteobacterial 23S rRNAs, a positioning consistent with the idea that the genus Campylobacter belongs to the epsilon subdivision of the class Proteobacteria.
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Affiliation(s)
- T J Trust
- Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada
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9
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Gebhart CJ, McOrist S, Lawson GH, Collins JE, Ward GE. Specific in situ hybridization of the intracellular organism of porcine proliferative enteropathy. Vet Pathol 1994; 31:462-7. [PMID: 7941236 DOI: 10.1177/030098589403100409] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The identity of the intracellular bacteria found in the enterocytes of pigs with proliferative enteropathy was investigated using specific DNA probes to various Campylobacter species and to a novel organism, ileal symbiont intracellularis. The ilea from pigs (Nos. 1-7) that were diagnosed by routine histopathology as having proliferative enteropathy were used. Diagnosis was made on the basis of proliferation of the enterocytes on hematoxylin and eosin-stained sections and the presence of large numbers of intracellular curved organisms on Warthin-Starry silver-stained sections. Four of these pigs (Nos. 1-4) had the chronic form of the disease, porcine intestinal adenomatosis, and three (Nos. 5-7) had the acute form, proliferative hemorrhagic enteropathy. An additional three normal pigs (Nos. 8-10) were obtained from three separate farms with no history of proliferative enteropathy. Frozen ileal sections were examined by in situ hybridization with DNA probes specific for ileal symbiont intracellularis and the three porcine intestinal Campylobacter species, C. coli, C. hyointestinalis, and C. mucosalis. In all seven pigs with either the intestinal adenomatosis or hemorrhagic enteropathy form of the disease, a DNA probe specific for ileal symbiont intracellularis hybridized to localized foci in the apical cytoplasm of ileal enterocytes. These hybridization sites corresponded to the location of intracellular bacteria in silver-stained sections of adjacent tissue. Sections from the three normal pigs tested with this probe and from all pigs tested with the Campylobacter species-specific DNA probes showed no specific hybridization reactions. The identity of the intracellular organism in these diseased pigs is ileal symbiont intracellularis.
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Affiliation(s)
- C J Gebhart
- Department of Veterinary PathoBiology, University of Minnesota, St. Paul
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Desai M, Linton D, Owen RJ, Cameron H, Stanley J. Genetic diversity of Helicobacter pylori indexed with respect to clinical symptomatology, using a 16S rRNA and a species-specific DNA probe. THE JOURNAL OF APPLIED BACTERIOLOGY 1993; 75:574-82. [PMID: 8294306 DOI: 10.1111/j.1365-2672.1993.tb01597.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
DNA probes are described which identify group and fingerprint strains of the human gastric pathogen Helicobacter pylori, on the basis of well-defined band homologies. A 544 bp internal fragment of the 16S ribosomal RNA gene was generated by polymerase chain reaction (PCR) with primers derived from the Escherichia coli rRNA gene sequence. In genomic Southern blots this probe detected restriction site variation around these loci, generating simple but strain-specific molecular fingerprints. A small conserved chromosomal fragment of 1.2 kbp, Hps, species-specific for H. pylori, was obtained by cloning random HindIII fragments into pUC19. It was useful for dot-blot identification, and also separated isolates into one major and two minor groups. When results for these two probes were combined, a baseline characterization of genotype was obtained. A band-matching database of molecular fingerprints for the type strain and 63 clinical isolates of H. pylori from asymptomatic, ulcer and gastritis contexts is presented. No significant association between the genotypes at this level of definition and the associated clinical symptomatology of the isolates was detected.
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Affiliation(s)
- M Desai
- National Collection of Type Cultures, Central Public Health Laboratory, London
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11
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Structure of 16S and 23S Ribosomal RNA Genes in Campylobacter Species: Phylogenetic Analysis of the Genus Campylobacter and Presence of Internal Transcribed Spacers. Syst Appl Microbiol 1993. [DOI: 10.1016/s0723-2020(11)80266-3] [Citation(s) in RCA: 27] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Identification of Enteropathogenic Campylobacter Species by Oligonucleotide Probes and Polymerase Chain Reaction Based on 16S rRNA Genes. Syst Appl Microbiol 1993. [DOI: 10.1016/s0723-2020(11)80248-1] [Citation(s) in RCA: 26] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
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Linton D, Moreno M, Owen RJ, Stanley J. 16S rrn gene copy number in Helicobacter pylori and its application to molecular typing. THE JOURNAL OF APPLIED BACTERIOLOGY 1992; 73:501-6. [PMID: 1490911 DOI: 10.1111/j.1365-2672.1992.tb05012.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The copy number of the genes encoding 16S ribosomal RNA was analysed for the genomes of geographically diverse strains of Helicobacter pylori, and restriction site variation within and around the genes was characterized. A DNA probe of 550 bp was amplified by the polymerase chain reaction from genomic DNA of the type strain NCTC 11637. This probe constituted a sequence internal to the 3' end of the 16S rrn gene. Homology profiles were compared for genomic Southern blots made with four restriction enzymes cutting within and outside the probe sequence. A copy number of two was established for all 12 strains analysed. This approach yielded significantly simpler data than does conventional 'ribotyping ' of H. pylori. It was equally discriminatory, however, and provided strain-specific 16S rrn gene 'signatures'. These represent both fundamental physical-genetic information and a novel approach to typing this gastric pathogen.
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Affiliation(s)
- D Linton
- Molecular Genetics and Campylobacter Unit, National Collection of Type Cultures, Central Public Health Laboratory, London
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14
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Comparative systematic study on ?Spirillum? 5175, Campylobacter and Wolinella species. Arch Microbiol 1992. [DOI: 10.1007/bf00245247] [Citation(s) in RCA: 77] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Abstract
Little information concerning the genome of either Campylobacter jejuni or Campylobacter coli is available. Therefore, we constructed genomic maps of C. jejuni UA580 and C. coli UA417 by using pulsed-field gel electrophoresis. The genome sizes of C. jejuni and C. coli strains are approximately 1.7 Mb, as determined by SalI and SmaI digestion (N. Chang and D. E. Taylor, J. Bacteriol. 172:5211-5217, 1990). The genomes of both species are represented by single circular DNA molecules, and maps were constructed by partial restriction digestion and hybridization of DNA fragments extracted from low-melting-point agarose gels. Homologous DNA probes, encoding the flaAB and 16S rRNA genes, as well as heterologous DNA probes from Escherichia coli, Bacillus subtilis, and Haemophilus influenzae, were used to identify the locations of particular genes. C. jejuni and C. coli contain three copies of the 16S and 23S rRNA genes. However, they are not located together within an operon but show a distinct split in at least two of their three copies. The positions of various housekeeping genes in both C. jejuni UA580 and C. coli UA417 have been determined, and there appears to be some conservation of gene arrangement between the two species.
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Affiliation(s)
- D E Taylor
- Department of Medical Microbiology, University of Alberta, Edmonton, Canada
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Vandamme P, Goossens H. Taxonomy of Campylobacter, Arcobacter, and Helicobacter: a review. ZENTRALBLATT FUR BAKTERIOLOGIE : INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY 1992; 276:447-72. [PMID: 1611203 DOI: 10.1016/s0934-8840(11)80671-7] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- P Vandamme
- Laboratorium voor Microbiologie en microbiële Genetica, University of Gent, Belgium
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Lane DJ, Harrison AP, Stahl D, Pace B, Giovannoni SJ, Olsen GJ, Pace NR. Evolutionary relationships among sulfur- and iron-oxidizing eubacteria. J Bacteriol 1992; 174:269-78. [PMID: 1729214 PMCID: PMC205705 DOI: 10.1128/jb.174.1.269-278.1992] [Citation(s) in RCA: 190] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Some 37 reverse transcriptase, partial 16S rRNA sequences from sulfur- and/or iron-oxidizing eubacteria, including sequences from species of the genera Thiobacillus, Thiothrix, Thiomicrospira, Acidophilium, "Leptospirillum," Thiovulum, and Chlorobium, have been determined. In addition, 16S sequences from a number of unnamed sulfur- and/or iron-oxidizing bacteria from hydrothermal vent sites, from invertebrate-bacterial endosymbioses, and from various mineral recovery operations also have been determined. The majority of sequences place their bacterial donors in one or another of the subdivisions of the Proteobacteria. However, three unnamed facultatively thermophilic iron-oxidizing isolates, Alv, BC, and TH3, are affiliated with the gram-positive division. One H2S-oxidizer, from the genus Thiovulum, is affiliated with Campylobacter, Wolinella, and other genera in what appears to be a new subdivision of the Proteobacteria. Three "Leptospirillum"-helical vibrioid isolates, BU-1, LfLa, and Z-2, exhibit no clear phylum level affiliation at all, other than their strong relationship to each other. A picture is emerging of an evolutionary widespread capacity for sulfur and/or iron oxidation among the eubacteria.
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Affiliation(s)
- D J Lane
- Department of Biology, Indiana University, Bloomington 47405
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Ho SA, Hoyle JA, Lewis FA, Secker AD, Cross D, Mapstone NP, Dixon MF, Wyatt JI, Tompkins DS, Taylor GR. Direct polymerase chain reaction test for detection of Helicobacter pylori in humans and animals. J Clin Microbiol 1991; 29:2543-9. [PMID: 1723072 PMCID: PMC270370 DOI: 10.1128/jcm.29.11.2543-2549.1991] [Citation(s) in RCA: 186] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
We designed a polymerase chain reaction (PCR) for amplifying the Helicobacter pylori gene encoding 16S rRNA. Primers for the specific detection of H. pylori were designed for areas of the 16S rRNA gene in which there is the least sequence homology between H. pylori and its closest relatives. The specificity of detection was confirmed by ensuring that the primers did not amplify DNA extracts from the campylobacters H. cinaedi, H. mustelae, and Wolinella succinogenes, which are the closest relatives of H. pylori, as determined by 16S rRNA sequencing. Serial dilution experiments revealed the detection of as little as 0.1 pg of DNA by PCR and 0.01 pg by nested PCR. H. pylori DNA was detected successfully in clinical paraffin-embedded and fresh gastric biopsy specimens from patients positive for the bacterium and also in fecal suspensions seeded with the organism. The DNA from the nonculturable coccoid form of H. pylori was also identified by the primers. Universal primers designed for highly conserved areas on the 16S rRNA gene enabled large amplification products to be produced for direct sequencing analysis. Gastric bacteria resembling H. pylori have been isolated from animals. DNA of these animal gastric bacteria amplified with H. pylori-specific primers yielded PCR products identical to those from human isolates of H. pylori, as confirmed by the use of a 20-base radiolabelled probe complementary to an internal sequence flanked by the H. pylori-specific primers. The results of PCR amplification and partial 16S rRNA gene sequence analysis strongly support the contention that the gastric organisms previously recovered from a pig, a baboon, and rhesus monkeys are H. pylori.
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Affiliation(s)
- S A Ho
- Department of Pathology, University of Leeds, United Kingdom
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Wesley IV, Wesley RD, Cardella M, Dewhirst FE, Paster BJ. Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequences. J Clin Microbiol 1991; 29:1812-7. [PMID: 1723076 PMCID: PMC270216 DOI: 10.1128/jcm.29.9.1812-1817.1991] [Citation(s) in RCA: 46] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Deoxyoligonucleotide probes were constructed for the identification of Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequence data. Probes were targeted to hypervariable regions of 16S rRNA. Specificity of oligonucleotide probes was tested in a colony blot assay with type strains of 15 Campylobacter and Arcobacter species as well as in a slot blot format using genomic DNA extracted from field strains of C. fetus and C. hyointestinalis. Two oligonucleotides were constructed for C. fetus that hybridized with equal specificity with each of 57 biochemically confirmed isolates of C. fetus but not with any other Campylobacter species. The C. hyointestinalis probe reacted with 47 of 48 biochemically confirmed field isolates of C. hyointestinalis. In Southern blot hybridization of BglII digests of genomic DNA, the respective probes reacted within three restriction fragments of either C. hyointestinalis (7.2, 8.2, and 10.1 kb) or C. fetus (7.0, 7.7, and 9.0 kb). This suggests multiple copies of genes encoding 16S rRNA.
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Affiliation(s)
- I V Wesley
- National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 50010
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Höök-Nikanne J, Solin ML, Kosunen TU, Kaartinen M. Comparison of Partial 16S rRNA Sequences of Different Helicobacter pylori Strains, Helicobacter mustelae and a Gastric Campylobacter-like Organism (GCLO). Syst Appl Microbiol 1991. [DOI: 10.1016/s0723-2020(11)80380-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/14/2022]
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Chemical characterization of Campylobacter jejuni lipopolysaccharides containing N-acetylneuraminic acid and 2,3-diamino-2,3-dideoxy-D-glucose. J Bacteriol 1991; 173:618-26. [PMID: 1987154 PMCID: PMC207052 DOI: 10.1128/jb.173.2.618-626.1991] [Citation(s) in RCA: 81] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Lipopolysaccharides (LPS) of four nonencapsulated strains of the human enteric pathogen Campylobacter jejuni were chemically characterized. When applied to two of the strains, extraction by a modified phenol-chloroform-petroleum ether method (H. Brade and C. Galanos, Eur. J. Biochem. 122:233-237, 1982) gave better yields of LPS than did extraction by the conventional hot phenol-water technique. Constituents common to all LPS were D-glucose, D-galactose, L-glycero-D-manno-heptose, 3-deoxy-D-manno-2-octulosonic acid, D-glucuronic acid, D-galactosamine, and phosphorylethanolamine. Phosphate was present in a relatively high amount. In addition, the LPS of three strains contained N-acetylneuraminic acid, whereas the LPS of the strain lacking this component contained 3-amino-3,6-dideoxy-D-glucose. The lipid A component contained phosphate with D-glucosamine and 2,3-diamino-2,3-dideoxy-D-glucose as the major amino sugars. Ethanolamine-phosphate was present also. The major fatty acids were ester- and amide-bound 3-hydroxytetradecanoic and ester-bound hexadecanoic acids, with a minor amount of ester-bound tetradecanoic acid. This is the first report of N-acetylneuraminic acid in the oligosaccharide moiety and diaminoglucose in the lipid A of C. jejuni LPS.
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Multivariate analyses of cellular fatty acids in Bacteroides, Prevotella, Porphyromonas, Wolinella, and Campylobacter spp. J Clin Microbiol 1991; 29:183-9. [PMID: 1993755 PMCID: PMC269725 DOI: 10.1128/jcm.29.1.183-189.1991] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
The genera Bacteroides, Wolinella, and Campylobacter contain several similar species that require taxonomic revision. Fatty acid profiles of whole bacterial cells have proven useful for taxonomy. In this study, cellular fatty acids from Bacteroides, Prevotella, Porphyromonas, Wolinella, and Campylobacter spp. were identified and quantitated by gas chromatography and gas chromatography-mass spectrometry, and the data were subjected to principal component analyses. Bacteroides fragilis, the type species of the genus Bacteroides, was distinct from the other organisms. While Bacteroides gracilis, Wolinella succinogenes, Wolinella curva, Wolinella recta, and Campylobacter fetus subsp. venerealis were close to each other, Prevotella (Bacteroides) buccae, Prevotella oralis, Prevotella oris, Prevotella disiens, Prevotella veroralis, Prevotella heparinolyticus, Porphyromonas (Bacteroides) endodontalis, and Bacteroides ureolyticus could be distinguished. B. fragilis was characterized by the presence of C3OH-i-1-, Ca-15, and Ci-15 and the absence of C12:0 and unsaturated fatty acids. For comparison, B. gracilis, B. ureolyticus, W. succinogenes, W. curva, W. recta, and Campylobacter fetus subsp. venerealis contained C12:0, C16:1, C18:1, and C3-OH-14 acids but lacked branched hydroxy and branched nonhydroxy acids. B. gracilis and B. ureolyticus are not "true" bacteroides.
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Neefs JM, Van de Peer Y, Hendriks L, De Wachter R. Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res 1990; 18 Suppl:2237-317. [PMID: 1692117 PMCID: PMC331875 DOI: 10.1093/nar/18.suppl.2237] [Citation(s) in RCA: 309] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Affiliation(s)
- J M Neefs
- Departement Biochemie, Universiteit Antwerpen, Belgium
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Abstract
Campylobacter pylori is a newly described, spiral-shaped, gram-negative bacillus that is oxidase positive, catalase positive, and urease positive and grows slowly in culture. Although observed in human tissue at the beginning of the century, it was not cultured until 1982. Because there are significant morphological and genetic differences between this organism and other species of Campylobacter, it will probably be reclassified in a new genus. Current information indicates that the organism primarily resides in the stomach tissue of humans and nonhuman primates and may occasionally spread to the esophagus or other parts of the alimentary tract under appropriate conditions. Significant evidence has accumulated in the last several years to show that it causes gastritis, and there is mounting evidence that it may participate in the development of duodenal ulcers. It may also be associated with gastric ulcers and nonulcer dyspepsia. It can be detected in patients by culture of biopsy specimens or histological staining of biopsy tissue. Indirect evidence for the presence of the organism can be obtained by detection of urease in a tissue biopsy specimen, by urea breath tests, or by detection of specific antibody. It may not be necessary to implement these procedures for routine use, however, until the role of the organism can be defined better. Ultimately, the discovery of this organism may lead to radical changes in the diagnosis and treatment of gastric disease.
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Dunn BE, Perez-Perez GI, Blaser MJ. Two-dimensional gel electrophoresis and immunoblotting of Campylobacter pylori proteins. Infect Immun 1989; 57:1825-33. [PMID: 2722241 PMCID: PMC313362 DOI: 10.1128/iai.57.6.1825-1833.1989] [Citation(s) in RCA: 61] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Whole-cell, outer-membrane protein, flagellum-associated antigens and partially purified urease of Campylobacter pylori were analyzed by two-dimensional gel electrophoresis. C. pylori strains were readily distinguished from strains of Campylobacter jejuni, C. coli, and C. fetus by absence of major outer membrane proteins with Mrs of 41,000 to 45,000. C. pylori strains also lacked the acidic surface-array proteins at Mr 100,000 to 149,000 identified previously in serum-resistant strains of C. fetus. Surface labeling of intact C. pylori cells with 125I revealed two common major proteins, which we have designated protein 2 (pI 5.6 to 5.8, Mr 66,000) and protein 3 (pI 5.2 to 5.5, Mr 63,000). Proteins 2 and 3 were also the major components (subunits) observed in partially purified urease. Partially purified preparations of flagella consistently contained proteins 2 and 3. Thus, urease appears to be associated with both outer membranes and flagella of C. pylori. C. pylori strains also possessed an antigen at Mr 59,000 which was cross-reactive with antiserum against flagella of C. jejuni. However, the antigen did not appear to be associated with flagella per se in C. pylori. Protein 2 was unique to C. pylori among the Campylobacter species studied. It was not recognized by antibody against whole cells of C. jejuni or C. fetus or flagella of C. jejuni. Protein 3 was cross-reactive with antiserum against whole cells of C. jejuni and C. fetus, as were several other major protein antigens. Because protein 2 is a major outer membrane protein that is apparently unique to C. pylori, development of monospecific antibodies against this antigen may be useful for the identification of C. pylori in tissues, and purified antigen may be useful for serologic tests for specific diagnosis of C. pylori infections.
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Affiliation(s)
- B E Dunn
- Laboratory Service, Denver Veterans Administration Medical Center, Colorado 80220
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Owen RJ, Costas M, Morgan DD, On SL, Hill LR, Pearson AD, Morgan DR. Strain variation in Campylobacter pylori detected by numerical analysis of one-dimensional electrophoretic protein patterns. Antonie Van Leeuwenhoek 1989; 55:253-67. [PMID: 2757368 DOI: 10.1007/bf00393854] [Citation(s) in RCA: 29] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
A total of 21 clinical isolates of Campylobacter pylori from Peru and the United Kingdom and two reference strains (from Australia), including the type strain (NCTC 11637T), were characterized by high resolution one-dimensional SDS-polyacrylamide gel electrophoresis of cellular proteins. The protein patterns contained more than 40 discrete bands and the approximate molecular weights of the major bands were 22, 27, 46, 57, 60, 65 and 93 kD. The total patterns were used as the basis of numerical analysis. Most strains were clustered in four phenons at 91% similarity with the exception of six ungrouped strains. Overall similarity was high with all strains linked in the phenogram at greater than or equal to 81%. Variation among strains was attributable principally to qualitative and quantitative band differences in the 47 to 56 kD (hypervariable) region of the C. pylori protein profile. From the analysis, ten different electropherotypes (EP-types) were identified. We demonstrated that differences were detectable among isolates from widely separated geographical locations as well as from the same location, although multiple isolates from two Peruvian patients had the same electropherotype. Our results indicate that determination of protein profiles provides the basis of a reproducible method for characterization of C. pylori isolates.
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Affiliation(s)
- R J Owen
- National Collection of Type Cultures, Central Public Health Laboratory, London, UK
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Béji A, Mégraud F, Vincent P, Gavini F, Izard D, Leclerc H. GC content of DNA of Campylobacter pylori and other species belonging or related to the genus Campylobacter. ANNALES DE L'INSTITUT PASTEUR. MICROBIOLOGY 1988; 139:527-34. [PMID: 3252903 DOI: 10.1016/0769-2609(88)90152-4] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
DNA of type strain Campylobacter pylori NCTC 11637 and 32 other strains of C. pylori recovered from gastric biopsy specimens was examined by thermal denaturation for its guanine-plus-cytosine (GC) content. The GC content of strain NCTC 11637 was 35.6 mol % (standard deviation (SD) 0.3), and the GC content of the 32 other C. pylori strains ranged from 34.1 to 37.5 mol % (average value 35.2, SD 1.0). A total of 14 type strains of other Campylobacter and Wolinella species were included in this study and the results obtained were compared with those cited in the literature.
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Affiliation(s)
- A Béji
- Unité INSERM 146, Villeneuve d'Ascq, France
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Raué HA, Klootwijk J, Musters W. Evolutionary conservation of structure and function of high molecular weight ribosomal RNA. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 1988; 51:77-129. [PMID: 3076243 DOI: 10.1016/0079-6107(88)90011-9] [Citation(s) in RCA: 126] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
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Dams E, Hendriks L, Van de Peer Y, Neefs JM, Smits G, Vandenbempt I, De Wachter R. Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res 1988; 16 Suppl:r87-173. [PMID: 2453029 PMCID: PMC340911 DOI: 10.1093/nar/16.suppl.r87] [Citation(s) in RCA: 218] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Affiliation(s)
- E Dams
- Departement Biochemie, Universiteit Antwerpen (UIA), Belgium
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