Isolation of a New Orientia tsutsugamushi Serotype

Orientia tsutsugamushi, the etiological agent of scrub typhus, is an antigenically diverse organism and many serologically distinct strains have been identified. The 56 kDa protein of O. tsutsugamushi, a major protein in the outer membrane, has been thought to be responsible for this antigenic variability. A strain of O. tsutsugamushi isolated in Korea cross-reacted with both Gilliam strain-specific and Karp strain-specific monoclonal antibodies. When its 56 kDa protein gene was cloned and analyzed, its sequence showed variation especially between 1,200 and 1,250 bp, showing that this isolate is a new O. tsutsugamushi strain.     

Development of IRAP-SCAR marker for strain identification in Lentinula edodes

Rapid and correct authentication of commercial strains is still important in today’s mushroom industry. Here for the first time we reported the using of sequence characterized amplified region (SCAR) marker developed from inter-retrotransposon amplified polymorphism (IRAP) marker to identify Lentinula edodes strain. Genomic polymorphism among 44 shiitake cultivars in China were surveyed by 24 IRAP primer combinations, from which primer combination LTR1L/MarY1R could generate a unique and reproducible 1712 bp fragment to distinguish strain No. 4 from other 43 strains. Based on this strain-specific fragment, a SCAR primer pair was designed and its validity was verified by correctly amplifying a single strain-specific fragment from DNA samples of 100 L. edodes strains. Our study lays the foundation for developing strain-specific SCAR marker by retrotransposon-based marker technique in fungi.     

Comparison of the metabolic activities of four wild-type <Emphasis Type="Italic">Clostridium perfringens</Emphasis> strains with their gatifloxacin-selected resistant mutants

The production of short-chain fatty acids, reductive enzymes, and hydrolytic enzymes by four gatifloxacin-selected, fluoroquinolone-resistant, mutant strains of C. perfringens, with stable mutations either in DNA gyrase or in both DNA gyrase and topoisomerase IV, was compared with that produced by the wild-type parent strains to investigate the effect of mutations associated with the selection of gatifloxacin resistance on bacterial metabolic activities. The mutants differed from their respective wild-type parent strains in the enzymatic activities of azoreductase, nitroreductase, and β-glucosidase and in the ratio of butyric acid to acetic acid production. Microarray analysis of one wild type and the corresponding mutant revealed different levels of mRNA expression for the enzymes involved in short-chain fatty acid (SCFA) synthesis and for β-glucosidase and oxidoreductases. In addition to mutations in the target genes, selection of resistance to gatifloxacin resulted in strain-specific physiological changes in the resistant mutants of C. perfringens that affected their metabolic activities.     

Monoclonal antibodies to surface glycoconjugates in Chlamydomonas eugametos recognize strain-specific O-methyl sugars

Monoclonal antibodies are described that are directed against cell surface components of the unicellular green alga Chlamydomonas eugametos. These antibodies recognize strain-specific epitopes which occur at the surface of vegetative and gametic cells. Two different groups of epitopes are distinguished that are never detectable together in one clonal cell culture. Evidence is presented showing that the antigenicity of cell surface molecules is a consequence of the presence of particular O-methylated sugars. Monoclonal antibodies reacting with one group of epitopes were studied in more detail, and immunoprecipitation and Western-blot studies showed that these epitopes can be arranged into four classes. The use of these monoclonal antibodies as strain-specific markers in light- and electron-microscopical techniques is illustrated.Abbreviations ELISA enzyme-linked immunosorbent assay - mt ^+/- mating type plus or minus - PAS periodic acid Schiff - Mab monoclonal antibody - PBS phosphate-buffered saline     

Genotypic diversity in Oenococcus oeni by high-density microarray comparative genome hybridization and whole genome sequencing

Many bacteria display substantial intra-specific genomic diversity that produces significant phenotypic variation between strains of the same species. Understanding the genetic basis of these strain-specific phenotypes is especially important for industrial microorganisms where these characters match individual strains to specific industrial processes. Oenococcus oeni, a bacterium used during winemaking, is one such industrial species where large numbers of strains show significant differences in commercially important industrial phenotypes. To ascertain the basis of these phenotypic differences, the genomic content of ten wine strains of O. oeni were mapped by array-based comparative genome hybridization (aCGH). These strains comprised a genomically diverse group in which large sections of the reference genome were often absent from individual strains. To place the aCGH results in context, whole genome sequence was obtained for one of these strains and compared with two previously sequenced, unrelated strains. While the three strains shared a core group of conserved ORFs, up to 10% of the coding potential of any one strain was specific to that isolate. The genome of O. oeni is therefore likely to be much larger than that present in any single strain and it is these strain-specific regions that are likely to be responsible for differences in industrial phenotypes.     

Stage-specific antigens of Physarum polycephalum

Antisera were prepared in isogenic F₁ hybrid rats against three amoebal strains and against two genetically related plasmodial strains of Physarum polycephalum. Differences in specificities between the antisera were studied using immunofluorescence tests and Ouchterlony double diffusion tests. There were no strain-specific differences between any of the three anti-amoebal sera, nor were any strain-specific differences found between the two anti-plasmodial sera. However, both ubiquitous and stage-specific antigens were detected.     

Development and application of RAPD-SCAR markers to identify intra-species hybrids of industrial Saccharomyces cerevisiae

To overcome the drawbacks of protoplast fusion in industrial breeding, strain-specific molecular markers were applied to select hybrids of industrial Saccharomyces cerevisiae strains. Random Amplified Polymorphic DNA (RAPD) analysis was used to generate strain-specific RAPD markers for two industrial yeast strains, Z8 and Z9. For industrial and technical controls, two RAPD markers with non-coding regions were converted into stable Sequence Characterized Amplified Region (SCAR) markers. Hybrids of Z8 and Z9 were obtained by protoplast fusion in combination with SCAR markers and were found to increase ethanol production by 4.3–8.1%. Results suggested that protoplast fusion could be combined with RAPD-SCAR molecular markers and applied in industrial breeding instead of auxotrophic markers.     

Comparative Genome Analysis of Helicobacter pylori Strains

DNA macroarrays were used to characterize 17 Helicobacter pylori strains isolated in four geographic regions of Russia (Moscow, St. Petersburg, Kazan, and Novosibirsk). Of all genes, 1272 (81%) proved to occur in all strains and to constitute a functional core of the genome, and 293 (18.7%) were strain-specific and greatly varied among the H. pylori strains. Most (71%) of the latter had unknown functions; the remainder included restriction–modification genes (3–9%), transposition genes (2–4%), and genes coding for outer membrane proteins (2–4%). The Russian H. pylori strains did not differ in genome organization or in the number and distribution of strain-specific genes from strains isolated in other countries.     

Comparative proteome analysis of Hansenula polymorpha DL1 and A16

Proteomic responses of methylotrophic yeasts (Hansenula polymorpha DL1 and A16) to growth medium tuning by carbon source shift (glycerol-->methanol) were monitored and analyzed by two-dimensional gel electrophoresis. Through comparative analyses of two-dimensional gels, intracellular yeast proteins with complex expression patterns were systematically sorted into: (1) proteins that are commonly expressed with comparable high abundance in both strains; (2) strain-specific proteins that are expressed at high level only in a particular strain; (3) strain-specific and methanol-induced proteins that are expressed only in the presence of methanol; and (4) strain-specific and constitutively-expressed proteins that are expressed consistently irrespective of carbon source shift without extreme change in expression level. Among the DL1-specific proteins belonging to group four, the four proteins showing the highest expression levels in the course of the fermentation process were identified as: glucose-6-phosphate dehydrogenase, isocitrate lyase, succinyl-CoA synthetase, and glycerol-3-phosphate dehydrogenase. From these results, it is suggested that DL1 has distinct metabolic characteristics including enhanced metabolic activities both in glycerol uptake and the glyoxylate bypass cycle, as compared to A16. This is likely to explain why the DL1 strain shows a significantly higher rate of glycerol and methanol consumption during the fermentation process. Our systematic approach to the analysis of proteomic responses and the detailed analysis results reported here will be useful to better understand the global physiology of H. polymorpha, as proteome databases for various methylotrophic yeasts are established.     

Strain-specific differences in pili formation and the interaction of <Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> with host cells

Background  

Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated strain-specific differences in adhesion, invasion and intracellular survival and analyzed formation of pili in different isolates.     

Margaret Constance Helen Blackler (1902–81)

Cyanobacteria are effective producers of bioactive metabolites, including both acute toxins and potential pharmaceuticals. In the current work, the biological activity of 27 strains of Baltic cyanobacteria representing different taxonomic groups and chemotypes were tested in a wide variety of assays. The cyanobacteria showed strain-specific differences in the induced effects. The extracts from Nodularia spumigena CCNP1401 were active in the highest number of tests, including protease and phosphatase inhibition assays. Four strains from Nostocales and four from Oscillatoriales increased proliferation of mitogen-stimulated human T cells. In antimicrobial assays, Phormidium sp. CCNP1317 (Oscillatoriales) strongly inhibited the growth of six fouling Gammaproteobacteria. The growth of monocotyl Sorghum saccharatum was inhibited by both toxin-producing and ‘non-toxic’ strains. The Baltic cyanobacteria were also found to be a rich source of commercially important enzymes. Among the 19 enzymatic activities tested, alkaline phosphatase, acid phosphatase, esterase (C4 and C8), and naphthol-AS-BI-phosphohydrolase were particularly common. In the cyanobacterial extracts, different peptides which may have been responsible for the observed effects were identified using LC-MS/MS. Their structures were classified to microcystins, nodularins, anabaenopeptins, cyanopeptolins, aeruginosins, spumigins and nostocyclopeptides.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.     

A comparative electrophoretic light scattering study of various strains of Thiobacillus ferrooxidans

Summary No significant strain-specific differences were found in the shape and position of the pH-dependent electrophoretic mobility curve obtained for various strains of Thiobacillus ferrooxidans under equal conditions of growth, suggesting similarities in their surface charge development.     

A Method for Structure–Activity Analysis of Quorum-Sensing Signaling Peptides from Naturally Transformable Streptococci

Many species of streptococci secrete and use a competence-stimulating peptide (CSP) to initiate quorum sensing for induction of genetic competence, bacteriocin production, and other activities. These signaling molecules are small, unmodified peptides that induce powerful strain-specific activity at nano-molar concentrations. This feature has provided an excellent opportunity to explore their structure–function relationships. However, CSP variants have also been identified in many species, and each specifically activates its cognate receptor. How such minor changes dramatically affect the specificity of these peptides remains unclear. Structure–activity analysis of these peptides may provide clues for understanding the specificity of signaling peptide–receptor interactions. Here, we use the Streptococcus mutans CSP as an example to describe methods of analyzing its structure–activity relationship. The methods described here may provide a platform for studying quorum-sensing signaling peptides of other naturally transformable streptococci.     

Modulation of <Emphasis Type="Italic">Escherichia coli</Emphasis> biofilm growth by cell-free spent cultures from lactobacilli

E. coli biofilms cause serious problems in medical practice by contaminating surfaces and indwelling catheters. Due to the rapid development of antibiotic resistance, alternative approaches to biofilm suppression are needed. This study addresses whether products released by antagonistic bacteria — Lactobacillus isolates from vaginal and dairy-product samples could be useful for controlling E. coli biofilms. The effects of diluted cell-free supernatants (CFS) from late-exponential Lactobacillus cultures on the growth and biofilm production of Escherichia coli were tested. Most of the CFS applied as 10⁻² had no impact on bacterial growth, biofilm development however was influenced even by 10⁻⁴ of CFS. Initial screening by crystal violet assay showed that biofilm modulation varied between different CFS and E. coli combinations from inhibition to activation; however three of the tested CFS showed consistency in biofilm suppression. This was not due to antibacterial activity since Live/Dead fluorescence labeling showed insignificant differences in the amount of dead cells in control and treated samples. Some E. coli strain-specific mechanisms of response to the three CFS included reduction in hydrophobicity and motility. Released exoploysaccharides isolated from the three CFS stimulated sessile growth, but proteinase K reduced their inhibitory activities implying participation of protein or peptide biofilm suppression factor(s).     

Comparative genomic analysis reveals significant enrichment of mobile genetic elements and genes encoding surface structure-proteins in hospital-associated clonal complex 2 Enterococcus faecalis

Background  

Enterococci rank among the leading causes of nosocomial infections. The failure to identify pathogen-specific genes in Enterococcus faecalis has led to a hypothesis where the virulence of different strains may be linked to strain-specific genes, and where the combined endeavor of the different gene-sets result in the ability to cause infection. Population structure studies by multilocus sequence typing have defined distinct clonal complexes (CC) of E. faecalis enriched in hospitalized patients (CC2, CC9, CC28 and CC40).     

Plasmid content and homology of 16 strains of filamentous,nonheterocystous cyanobacteria

We have developed several strain-specific, rapid, small-scale plasmid isolation procedures in order to characterize the plasmid profiles of 16 filamentous, nonheterocstous cyanobacteria. At least one distinct plasmid was found in eight strains, with seven of these containing two or more different plasmids. Eight strains were found to be without plasmid DNA. Both the large, 12.9 kb, and the small, 1.6 kb, plasmids fromPlectonema boryanum 581 were isolated, purified, and cloned. Southern blots of plasmid DNAs from the eight strains were probed with these cloned DNAs and also with ultra-pure plasmid DNA fromPhormidium liridum 426. Four strains ofP. boryanum (485, 581, 594, 1542) andP. luridum 426 have identical plasmid profiles, and plasmid homology is extensive.