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.     

SEROLOGICAL STUDIES OF MALE AND FEMALE STRAINS OF EUDORINA ELEGANS (CHLOROPHYCEAE)1,2

Male and female strains of Eudorina elegans Ehrenberg were examined by serological methods for flagellar and cellular antigenic differences. No significant differences could be detected between the strains by using agglutination, immobilization and diffusion tests. However, differentiated sperm were neither agglutinated nor immobilized by anti-vegetative cell sera.     

Influences of inbreeding and genetics on telomere length in mice

We measured telomere lengths of blood leukocytes in several inbred and outbred mammalian species, using a telomere-specific fluorescent probe and flow cytometry. Humans, non-human primates, and three outbred populations of Peromyscus mice (Peromyscus leucopus, Peromyscus maniculatus, and Peromyscus polionotus) have short telomeres. Two common strains of laboratory mice, C57BL/6J and DBA/2J, have telomeres several times longer than most other mammals surveyed. Moreover, the two inbred laboratory mouse strains display significantly different telomere lengths, suggesting the existence of strain-specific genetic determinants. To further examine the effects of inbreeding, we studied three Peromyscus leucopus inbred lines (GS109, GS16A1, and GS16B), all derived from the outbred P. leucopus stock. Telomeres of all three inbred lines are significantly lengthened relative to outbred P. leucopus, and the three lines display strain-specific significantly different telomere lengths, much like the C57BL/6J and DBA/2J strains of M. musculus. To further characterize the genetic inheritance of telomere length, we carried out several crosses to obtain hybrid F₁ mice between parental strains displaying the phenotype of long and short telomeres. In all F₁ mice assayed, peripheral blood leukocyte telomere length was intermediate to that of the parents. Additionally, we generated F₂ mice from a cross of the (P. leucopus outbred × GS16B)F₁. Based on the distribution of telomere length in the F₂ population, we determined that more than five loci contribute to telomere length regulation in Peromyscus. We concluded that inbreeding, through unknown mechanisms, results in the elongation of telomeres, and that telomere length for a given species and/or sub-strain is genetically determined by multiple segregating loci.     

Strain-Specific Neutralization of Human Cytomegalovirus Isolates by Human Sera

Induction of an effective antibody response against human cytomegalovirus (HCMV) is an important defense mechanism since it is potentially capable of neutralizing infectious viruses. We have analyzed the extent of HCMV strain-specific neutralization capacity in human sera. Nine recent HCMV isolates and their corresponding sera were investigated in cross-neutralization assays. We observed differences, independent of the overall neutralization capacity, in the 50% neutralization titers of the sera against individual strains, differences that ranged from 8-fold to more than 60-fold. For one isolate, complete resistance to neutralization by two human sera was observed. The neutralization capacity of human sera was not influenced by the presence of various concentrations (up to 100-fold excess) of noninfectious envelope glycoproteins, an inherent contamination of virus preparations from recent HCMV isolates. This indicated that the decisive parameter for neutralization is the titer of the neutralizing antibodies and that neutralization is largely independent of the concentration of virus. Analysis with transplant patients revealed that during primary infection strain-specific and strain-common antibodies are produced asynchronously. Thus, our data demonstrate that the induction of strain-specific neutralizing antibodies is a common event during infection with HCMV and that it might have important implications for the course of the infection and the development of anti-HCMV vaccines.     

Intraspecific variation in the response of the cyanobacterium Nodularia spumigena to moderate UV-B radiation

Floating and nodularin-producing strains of Nodularia spumigena from the Baltic Sea are regarded as belonging to one species. However, intraspecific variation in the response of N. spumigena to environmental factors has been commonly overlooked. As blooms of N. spumigena occur in late summer, a period with strong light and stable water-column stratification, the cells can be expected to also be exposed to ultraviolet-B radiation (UV-B, 280–320 nm). The UV-B tolerance of four different strains of N. spumigena, isolated from the Baltic Sea, was investigated in the laboratory for 8 days, by measuring photosynthesis, growth and pigment composition. Variables included maximum quantum yield of photosynthesis (F_v/F_m, PAM fluorometry), growth rate (cell counts) and photosynthetic pigments, as well as mycosporine-like amino acids (HPLC). Intraspecific differences regardless of treatment were found for cell dimension, growth rate, F_v/F_m and pigment concentrations. UV-B related effects differed between strains. By Day 8 one of the four strains showed a lower F_v/F_m when treated with UV-B; in another strain the growth rate and cell numbers were lower. In three strains, UV-B exposure resulted in higher cell concentrations of carotenoids and chlorophyll a. In all strains, the concentrations of total mycosporine-like amino acids were 60–130% higher in the UV-B treated samples compared with samples shielded from UV-B. Although strain-specific differences in UV-B tolerance were observed, it is concluded that N. spumigena is a species that is not generally negatively affected by moderate levels of UV-B radiation.     

Antibody-dependent cellular cytotoxicity detects type- and strain-specific antigens among human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus SIVmac isolates.

Human cell lines were infected with different strains of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) as well as with a simian immunodeficiency virus SIVmac isolate and used as targets in an antibody-dependent cellular cytotoxicity (ADCC) assay. Sera from HIV-1- or HIV-2-infected subjects provided the antibody, and lymphocytes from normal donors provided the effector cells. About 60% of HIV-1 antibody-positive sera mediated ADCC when tested against any given HIV-1 isolate-infected target cell (human T-cell lymphotropic virus type IIIB, B40, A2587), and about 75% of HIV-2 antibody-positive sera mediated ADCC when tested against target cells infected with HIV-2 isolates (lymphadenopathy-associated virus type 2 and SBL-6669) or simian immunodeficiency virus from macaques. Within each type, individual sera showed different reactivity patterns, and the probability that a serum was ADCC positive was higher when it was tested against several strains. When the ADCC reactivity of sera against different strains was compared, diversity as detected by ADCC appeared to be greater among HIV-1 strains than among HIV-2 strains. For HIV-1, 54 to 67% of the sera gave concordant ADCC reactions, whereas for HIV-2 and SIVmac, 91% of the sera gave concordant results. Almost no strain-specific differences were seen between SBL-6669 and lymphadenopathy-associated virus type 2. As we determined previously, HIV-1 and HIV-2 did not cross-react in ADCC. The results indicated that HIV-1 and HIV-2 antibody-positive sera mediate both strain- and type-specific ADCC. HIV-2 antibody-positive sera seem to mediate ADCC with broader reactivity and to a higher frequency compared with HIV-1 antibody-positive sera.     

Alleviation of Toxicity of Poisonous Organic Compounds on Hydrophilic Carrier/Hydrophobic Organic Solvent Interface

Growth tests of type culture strains were done on interfaces between nutrient agar and hydrophobic organic solvents. All strains examined were able to grow against n-paraffin (C₁₁-C₁₅), and many could grow against isooctane, and pentyl- and heptylbenzene. The interface cultivation method was also successful even when the solvent layer contained compounds toxic for microorganism.     

Quantitative trait loci for steady-state platelet count in mice

Platelet count in humans is a strongly genetically regulated trait, with approximately 85% of the interindividual variance in platelet numbers attributable to genetic factors. Inbred mouse strains also have strain-specific platelet count ranges. As part of a project to identify novel factors that regulate platelet count, we identified two inbred mouse strains, CBA/CaH and QSi5, with substantial differences in platelet count (mean values of 581 vs. 1062 × 10⁹/L). An F₂ intercross resource of 1126 animals was bred from these two parental strains for a genomewide scan for quantitative trait loci (QTL) for platelet count. QTL were identified on MMU1 (LOD 6.8, p < 0.0005) and MMU11 (LOD 11.2, p < 0.0005) by selectively genotyping animals from the extremes of the F₂ platelet count distribution. Three other QTL of suggestive statistical significance were also detected on MMU7, 13, and 17. It is noteworthy that no QTL were detected in the vicinity of the genes encoding thrombopoietin (Thpo), and its receptor (c-Mpl), both known to influence platelet production. Comparison of gene expression levels between the parental mouse strains by microarrays also showed little difference in the mRNA levels of these known candidate genes. These results represent the first published use of a genetic linkage-based approach in a mouse model toward the identification of genetic factors that regulate platelet count.     

Acaricidal activity of Metarhizium anisopliae isolated from paddocks in the Mexican tropics against two populations of the cattle tick Rhipicephalus microplus

The acaricidal effects of 55 strains of Metarhizium anisopliae (Metschnikoff) Sorokin, 1883 (Hypocreales: Clavicipitaceae) isolated from paddocks of cattle farms were evaluated in two Rhipicephalus microplus (Canestrini 1887) (Ixodida: Ixodidae) populations, of which one was multi‐resistant and one was susceptible to chemical acaricides. Percentage mortality and reproductive efficiency indices in R. microplus were evaluated by adult immersion tests at a dose of 1 × 10⁸ conidia/mL for each fungal strain. Some strains were selected to calculate lethal concentrations to kill 50% (LC₅₀) and 99% (LC₉₉) of engorged ticks. Strains MaV22, MaV26 and MaV55 induced 100% mortality in R. microplus on day 14. Strains MaV05, MaV09 and MaV22 caused mortality of >90% from day 12 onward in both tick populations. The most effective acaricidal fungal strain, MaV55, inhibited egg laying by 54.86 and 55.86% in acaricide‐resistant and ‐susceptible R. microplus populations, respectively. None of the fungal strains had statistically significant effects on larval hatching. In conclusion, nine strains of M. anisopliae demonstrated high acaricidal effects against R. microplus and reduced its egg laying. No differences in acaricidal effects were observed between the two populations of ticks tested.     

Tracing Streptococcus thermophilus strains in three-component yogurt starters

Three-component starters for yogurt were obtained on the base of starter LBB.BY 5-12 for traditional Bulgarian yogurt, containing strains Lactobacillus delbrueckii ssp. bulgaricus B5 and Streptococcus thermophilus A with the addition of either an exopolysaccharide-producing S. thermophilus strain 6V or the fast acidifying S. thermophilus strain N1. To differentiate between the three strains in the starter cultures, randomly amplified polymorphic DNA (RAPD) technique was applied to develop strain-specific probes. Southern hybridization against dot-blots of chromosomal DNA from the three S. thermophilus strains confirmed that two probes, derived from a 770 bp RAPD product obtained with primer RAPD-4 and a 290 bp sequence obtained with primer OPP-7 were specific for S. thermophilus 6V and S. thermophilus A, respectively, while no hybridization to S. thermophilus N1 DNA was observed. The selected probes were used to differentiate between S. thermophilus colonies on a solid agar medium by colony hybridization. The evaluation of the viable cell counts revealed that the populations of S. thermophilus A and the added S. thermophilus strains 6V or N1 in the three-component starters and in yogurt had nearly equal proportion allowing each strain to contribute to the enriched properties of starter and product.     

Group antigens in slow-growing rhizobia

Summary Internal group antigens of several slow-growing and fast-growing Rhizobium strains were tested by gel-diffusion against antisera to three strains of Rhizobium japonicum. At least one, generally two common antigens were found in 13 strains of R. japonicum, 4 strains of R. lupini, 4 strains isolated from cowpea and two slow-growing strains isolated from Lotus. Forty-six fast-growing rhizobia (including two from Lotus and 4 from Leucaena leucocephala) were clearly distinguished from the slow-growing strains in tests with the same antisera. They were wholly negative (9) or gave a much weaker non-identical line with one antiserum (24 strains), two antisera (8) or three antisera (5). The 5 strains of agrobacteria grouped with the fast-growing rhizobia.     

Strain-specific genes of Helicobacter pylori: distribution, function and dynamics

Whole-genome clustering of the two available genome sequences of Helicobacter pylori strains 26695 and J99 allows the detection of 110 and 52 strain-specific genes, respectively. This set of strain-specific genes was compared with the sets obtained with other computational approaches of direct genome comparison as well as experimental data from microarray analysis. A considerable number of novel function assignments is possible using database-driven sequence annotation, although the function of the majority of the identified genes remains unknown. Using whole-genome clustering, it is also possible to detect species-specific genes by comparing the two H.pylori strains against the genome sequence of Campylobacter jejuni. It is interesting that the majority of strain-specific genes appear to be species specific. Finally, we introduce a novel approach to gene position analysis by employing measures from directional statistics. We show that although the two strains exhibit differences with respect to strain-specific gene distributions, this is due to the extensive genome rearrangements. If these are taken into account, a common pattern for the genome dynamics of the two Helicobacter strains emerges, suggestive of certain spatial constraints that may act as control mechanisms of gene flux.     

Characterization of Guinea Pig Antibody Responses to Salivary Proteins of Triatoma infestans for the Development of a Triatomine Exposure Marker

Background

Salivary proteins of Triatoma infestans elicit humoral immune responses in their vertebrate hosts. These immune responses indicate exposure to triatomines and thus can be a useful epidemiological tool to estimate triatomine infestation. In the present study, we analyzed antibody responses of guinea pigs to salivary antigens of different developmental stages of four T. infestans strains originating from domestic and/or peridomestic habitats in Argentina, Bolivia, Chile and Peru. We aimed to identify developmental stage- and strain-specific salivary antigens as potential markers of T. infestans exposure.

Methodology and Principal Findings

In SDS-PAGE analysis of salivary proteins of T. infestans the banding pattern differed between developmental stages and strains of triatomines. Phenograms constructed from the salivary profiles separated nymphal instars, especially the 5^th instar, from adults. To analyze the influence of stage- and strain-specific differences in T. infestans saliva on the antibody response of guinea pigs, twenty-one guinea pigs were exposed to 5^th instar nymphs and/or adults of different T. infestans strains. Western blot analyses using sera of exposed guinea pigs revealed stage- and strain-specific variations in the humoral response of animals. In total, 27 and 17 different salivary proteins reacted with guinea pig sera using IgG and IgM antibodies, respectively. Despite all variations of recognized salivary antigens, an antigen of 35 kDa reacted with sera of almost all challenged guinea pigs.

Conclusion

Salivary antigens are increasingly considered as an epidemiological tool to measure exposure to hematophagous arthropods, but developmental stage- and strain-specific variations in the saliva composition and the respective differences of immunogenicity are often neglected. Thus, the development of a triatomine exposure marker for surveillance studies after triatomine control campaigns requires detailed investigations. Our study resulted in the identification of a potential antigen as useful marker of T. infestans exposure.     

THE GENETICS AND EVOLUTION OF CANNIBALISM IN FLOUR BEETLES (GENUS TRIBOLIUM)

Cannibalism plays a major role in population regulation in Tribolium confusum, accounting for up to tenfold differences in population size between different genetic strains. I characterized the within- and between-strain genetic variation for cannibalism using standard quantitative-genetic methods. The four laboratory strains studied have similar birth and death rates but differ in their strain-specific cannibalistic tendencies. The cannibalism rates of the strains were stable for more than 60 generations of laboratory husbandry. I found considerable genetic variation for cannibalism within each strain. A genetic analysis of the between-strain differences in each of three types of cannibalism (larvae eating eggs, adults eating eggs, and adults eating pupae) showed that all three cannibalism pathways are autosomally inherited and exhibit minor degrees of dominance. Adult cannibalism of eggs and larval cannibalism of eggs appear to be genetically correlated. The differences between the “high” and “low” cannibalism strains appear to be polygenic for two kinds of cannibalism, larvae eating eggs and adults eating pupae. However, strain differences in adult cannibalism of eggs may be due to only two loci. The stability of the between-strain differences for more than 60 generations, the additive nature of inheritance, and the demonstration of considerable within-strain genetic variation suggest that cannibalism may be selectively neutral or under stabilizing selection with many adaptive peaks.     

Stress Tolerance and Genetic Variability of Phosphate-solubilizing Fluorescent Pseudomonas from the Cold Deserts of the Trans-Himalayas

Nineteen efficient phosphate-solubilizing fluorescent Pseudomonas from the cold deserts of the trans-Himalayas were screened for stress tolerance against temperature, alkalinity, salinity, calcium salts, and desiccation. Phylogenetic analysis based on 16S rRNA gene sequencing placed these bacteria under three groups with fourteen strains in Group I including Pseudomonas trivialis and P. poae, two strains in Group II together with Pseudomonas kilonensis and P. corrugata, and three strains in Group III along with Pseudomonas jessenii and P. moraviensis. Genetic diversity assessed by ERIC and BOX-PCR revealed variability among strains belonging to the same phylogenetic groups. Cluster analysis based on the growth characteristics under regimes of different stress levels placed the strains into three distinct clusters displaying no correlation to their phylogenetic groups. Stress-tolerant strains differed in the level of decline in phosphate solubilization under increasing intensity of various stress parameters. The highest decrease occurred with 5% CaCO_3, followed by 2.5% CaCO₃, pH 11, 5% NaCl, temperature of 37°C, 40% PEG, 5% CaSO₄, 2.5% NaCl, 2.5% CaSO₄, pH 9 and temperature of 15°C. Two strains belonging to Phylogenetic Group I exhibited higher phosphate solubilization at lower temperature. The results revealed that stress-tolerance ability was not limited to any particular phylogenetic group. Knowledge about the genetic variants of phosphate-solubilizing fluorescent Pseudomonas with potential for tolerance to desiccation, alkalinity, temperature, and salinity could be useful in understanding their ecological role under stressful environments of low phosphate availability.     

Recovery of soil streptomycetes from arid habitats in Jordan and their potential to inhibit multi-drug resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> pathogens

A total of 161 different Streptomyces isolates were recovered from 5 soil samples representing the driest habitats of Jordan. These were then characterized and assessed for their antagonistic activity against four clinical multi-drug resistant Pseudomonas aeruginosa test pathogens. Results indicated that only 3 strains out of 139 and 6 out of 22 isolated at 27°C and 45°C, respectively, were active against at least three strains of pathogenic Pseudomonas. However, three Streptomyces strains (J_2b, J₄, and J₁₂) that were isolated at 45°C inhibited all of the tested pathogens with an inhibition zone ranging between 5 and 16 mm in diameter. Data obtained from comparing the inhibition activity of these unique Streptomyces strains toward multi-resistant Pseudomonas pathogens with standard used antibiotics revealed that these isolates produce possible different inhibitory bioactive compounds other than the standard antibiotics.     

Variation in fatty acid composition of <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (Spirulina) strains

A study of the fatty acid composition was made for 35 Arthrospira strains, concentrating on the most abundant fatty acids, the two polyunsaturated C₁₈ acids, linoleic and γ-linolenic acid, and palmitic acid. When grown at 30 ^∘C and low irradiance (10 μmol photon m⁻² s⁻¹), these three acids together formed 88–92% of total fatty acids. There were considerable differences in the composition of the two polyunsaturated acids. Depending on the strain, linoleic acid formed 13.1–31.5% and γ-linolenic acid formed 12.9–29.4% total fatty acids. In contrast, the range for palmitic acid was narrow: 42.3–47.6% of total fatty acids. Repeat experiments on several strains under defined conditions led to closely similar results for any particular environment, suggesting that fatty acid composition can be used as an aid in differentiating between strains. Five additional strains, which had apparently originated from the same original stock cultures as 3 of the 35 in the main study, but from different culture collections, were also assayed. With four strains the results were similar, irrespective of culture source, but with one strain marked differences occurred, especially in the polyunsaturated C₁₈ fatty acid fraction. These differences were independent of the age of the culture. In addition, straight morphotypes derived during repeat subcultures of four strains; each showed a similar fatty acid composition to that of the helical morphotypes of the same strains. A decrease in temperature from 30 to 20 ^∘C, an increase in irradiance (at 30 ^∘C) from 10 to 70 μmol photon m⁻² s⁻¹ and transfer to dark heterotrophy all favoured an increase in polyunsaturated C₁₈ fatty acids. The highest γ-linolenic acid content of any conditions was found for three strains grown heterotrophically on glucose in the dark at 30 ^∘C. A comparative study of six strains of Spirulina confirmed a previous study showing the absence of γ-linolenic acid in all Spirulina strains, thus permitting the separation of these two genera.     

Immunological properties of Rickettsia tsutsugamushi, Kawasaki strain, isolated from a patient in Kyushu

Nine isolates of Rickettsia tsutsugamushi were obtained from patients with Tsutsugamushi disease (scrub typhus) in Miyazaki Prefecture in Kyushu. Immunological analyses of these patients' sera and the isolates were performed by indirect immunofluorescence, indirect immunoperoxidase or immunoblotting techniques. In the analysis of reactions of the patients' sera with the prototype strains Gilliam, Karp, and Kato and with the isolates, sera from two patients, including Kawasaki, showed similar profiles and cross-reaction with the two isolates recovered from the corresponding patients, but reacted only weakly with the prototype strains. With guinea pig polyclonal antibodies against the isolate and prototype strains, Kawasaki strain showed some degree of cross-reaction with Gilliam strain but not with either Karp or Kato strain, nor with Shimokoshi strain which is known to be different antigenically from the prototype strains. Additionally, strain-specific murine monoclonal antibodies against Gilliam, Karp, and Kato strains did not react at all with Kawasaki strain. These results suggest that the Kawasaki strain may be different antigenically from the prototype strains and Shimokoshi strain. The finding two strains of the same antigenic type (Kawasaki) among only nine isolates suggests the presence of Kawasaki-type rickettsiae in Miyazaki Prefecture. Shimokoshi strain also did not react with these strain-specific monoclonal antibodies, suggesting that strains of R. tsutsugamushi antigenically distinct from the prototype strains, such as Kawasaki and Shimokoshi strains, may easily be recognized by their nonreactivity with these monoclonal antibodies.     

Antigenic analysis of three strains of Mycoplasma arginini by two-dimensional immunoelectrophoresis.

The occurrence of species-specific and strain-specific antigens in three strains of Mycoplasma arginini (G-230, leonis and 23243) was studied by two-dimensional immunoelectrophoresis. Approximately 20 antigenic components could be detected in each strain. It was possible to analyze 6 to 7 major and distinct components from each strain by two techniques: "enhancement" where antigen to an additional strain is added to the first phase of the electrophoresis which increases the size of common peaks and "suppression" where antiserum to an additional strain is incorporated in the second phase whereby peak size of components to which both sera have antibody are decreased. A total of 10 distinct antigens were recognized. Electrophoretic mobilities relative to bovine albumin ranged from 0.2 to 1.08. Three components were common to all strains; two of these represented major amounts of material. Four components represented strain-specific components. Unique fast components were found both in strains 23243 and G-230. Three antigens were distributed into only two of the three strains. The electrophoretic mobilities of some common antigens were quite different between strains.