Freshwater Bacteria Can Methylate Selenium through the Thiopurine Methyltransferase Pathway

Involvement of the bacterial thiopurine methyltransferase (bTPMT) in natural selenium methylation by freshwater was investigated. A freshwater environment that had no known selenium contamination but exhibited reproducible emission of dimethyl selenide (DMSe) or dimethyl diselenide (DMDSe) when it was supplemented with an organic form of selenium [(methyl)selenocysteine] or an inorganic form of selenium (sodium selenite) was used. The distribution of the bTPMT gene (tpm) in the microflora was studied. Freshwater bacteria growing on 10 μM sodium selenite and 10 μM sodium selenate were isolated, and 4.5 and 10% of the strains, respectively, were shown by colony blot hybridization to hybridize with a Pseudomonas syringae tpm DNA probe. Ribotyping showed that these strains are closely related. The complete rrs sequence of one of the strains, designated Hsa.28, was obtained and analyzed. Its closest phyletic neighbor was found to be the Pseudomonas anguilliseptica rrs sequence. The Hsa.28 strain grown with sodium selenite or (methyl)selenocysteine produced significant amounts of DMSe and DMDSe. The Hsa.28 tpm gene was isolated by genomic DNA library screening and sequencing. BLASTP comparisons of the deduced Hsa.28 bTPMT sequence with P. syringae, Pseudomonas aeruginosa, Vibrio cholerae, rat, and human thiopurine methyltransferase sequences revealed that the levels of similarity were 52 to 71%. PCR-generated Escherichia coli subclones containing the Hsa.28 tpm open reading frame were constructed. E. coli cells harboring the constructs and grown with sodium selenite or (methyl)selenocysteine produced significant levels of DMSe and DMDSe, confirming that the gene plays a role in selenium methylation. The effect of strain Hsa.28 population levels on freshwater DMSe and DMDSe emission was investigated. An increase in the size of the Hsa.28 population was found to enhance significantly the emission of methyl selenides by freshwater samples supplemented with sodium selenite or (methyl)selenocysteine. These data suggest that bTPMT can play a role in natural freshwater selenium methylation processes.     

The biomechanics of concussion for ice hockey head impact events

The purpose of this research was to conduct reconstructions of concussive and non-concussive impacts in ice hockey to determine the biomechanics and thresholds of concussive injury in ice hockey. Videos of concussive and non-concussive impacts in an elite professional ice hockey league in North America were reconstructed using physical and finite element model methods. Eighty concussive and 45 non-concussive events were studied. Logistic regressions indicate significant thresholds for concussion for linear/rotational acceleration and CSDM_10%. Impacts in ice hockey were mostly long duration events, longer than 15?ms. These results have significant implications for helmet standards and development to prevent concussion.     

Infochemical-mediated preference behavior of the parasitoid Microctonus hyperodae when searching for its adult weevil hosts

Infochemicals are the most important cues used by parasitoids for host location. The attractiveness of infochemicals in a tritrophic context is expected to be determined by the degree of specialization of the parasitoid and its host(s). Microctonus hyperodae Loan (Hymenoptera: Braconidae) is an oligophagous parasitoid that attacks adult Curculionidae of the Brachycerinae subfamily, especially Listronotus bonariensis Kuschel, on Gramineae. In 1996, a new host–parasitoid association between the carrot weevil Listronotus oregonensis LeConte and M. hyperodae was created in the laboratory. In this study, the infochemicals used by M. hyperodae when searching for its adult weevil hosts were determined using a Y‐shaped olfactometer. Three curculionid species (L. oregonensis, Listronotus sparsus Say, and Neydus flavicaudis Boheman) and one bruchid species [Callosobruchus maculatus (Fabricius)], and their feces, were tested. It was expected that hosts phylogenetically and ecologically close to L. bonariensis would be more attractive than species less related but in fact, M. hyperodae responded only to L. oregonensis and its feces. When feces and host insects were tested separately, M. hyperodae responded to the odors emitted by L. oregonensis adults but not to their feces, suggesting that most of the kairomones came from the host itself. Host plants were also tested, but M. hyperodae responded neither to Lolium multiflorum Lamark (Gramineae) nor to Daucus carota L. (Umbelliferae) leaves.     

Distribution and genetic diversity of bacterial thiopurine methyltransferases in soils emitting dimethyl selenide

Dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe) emissions by soil samples spiked with selenite or (methyl)selenocysteine, with or without a supplement of nutrient broth and glucose were measured. DMSe was the main form of volatile Se produced, and was observed for both Se-substrates. DMDSe was only emitted from soils spiked with (methyl)selenocysteine. Two bacterial thiopurine methyltransferases (TPMTs), TPMT-I and TPMT-E, have been reported to be involved in DMSe and DMDSe emissions [J. Bacteriol. 184 (2002) 3146; Appl. Environ. Microbiol. 69 (2003) 3784]. To establish if these TPMTs or other members of their gene family could have contributed to the DMSe emissions observed, the diversity of bTPMT gene (tpm) sequences among the soils of this study was investigated. Total DNAs from these soils were extracted and screened using the tpm PTCF2-PTCR2 consensus primers defined to PCR amplify this gene family. The PCR products obtained from two soils were cloned, analysed by PCR-RFLP, and sequenced. Their analysis showed an important diversity of tpm lineages (around 12) in soils. Phylogenetic analysis of the deduced TPMT sequences of these soils revealed lineages not previously recorded in the databases, sequences closely related or identical to freshwater TPMTs, or sequences encoding TPMTs closely related to those of Pseudomonas fragi TPMT-K, Pseudomonas Hsa.28 TPMT-I, or Colwellia psychrerythraea TPMT-Z. Nested PCRs, allowing detection of about 13 distinct tpm soil and freshwater lineages by PTCF2-PTCR2 PCR screenings, were performed on the soil total DNAs. These PCRs confirmed the sequencing data, and allowed to recover lineages not detected by the cloning strategy. These results indicate that soils, like the freshwater samples, harbour TPMT-I gene sequences but may also have distinct tpm lineages. This study further supports our hypothesis that TPMTs contribute to DMSe soil emissions.     

Genome sequence of the human- and animal-pathogenic strain Nocardia cyriacigeorgica GUH-2

The pathogenic strain Nocardia cyriacigeorgica GUH-2 was isolated from a fatal human nocardiosis case, and its genome was sequenced. The complete genomic sequence of this strain contains 6,194,645 bp, an average G+C content of 68.37%, and no plasmids. We also identified several protein-coding genes to which N. cyriacigeorgica's virulence can potentially be attributed.     

Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K₂TeO₃ was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K₂TeO₃ concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K₂TeO₃-amended medium. The bona fide agrobacterium colonies growing on media amended with K₂TeO₃ were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10³ to 10⁴ agrobacteria · g of dry soil⁻¹ in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.     

Burkholderia cepacia Genomovar III Is a Common Plant-Associated Bacterium

A polyphasic taxonomic study involving DNA-DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis revealed that a group of Burkholderia cepacia-like organisms isolated from the rhizosphere or tissues of maize, wheat, and lupine belong to B. cepacia genomovar III, a genomic species associated with “cepacia syndrome” in cystic fibrosis patients. The present study also revealed considerable protein electrophoretic heterogeneity within this species and demonstrated that the B. cepacia complex consists of two independent phylogenetic lineages.     

Genetic complementation of rhizobial nod mutants with Frankia DNA: artifact or reality?

Two divergent reports have been published on the genetic complementation of rhizobial nod mutants using Frankia DNA. In 1991 putative Frankia cosmid library clones were reported to restore normal nodulation properties to Rhizobium leguminosarum biovar viciaenodD::Tn5, but no supporting sequence data were published. In 1992 a second group reported a failure to find any evidence of functional complementation of various rhizobial nod mutants by Frankia DNA (nodA, nodB and nodC). Complementation tests of nine Nod⁻ R. leguminosarum bv. viciae or Sinorhizobium meliloti Tn5 mutants (nodA ⁻ , nodB ⁻ , nodC ⁻ , nodD ⁻ , nodF  ⁻ , nodL ⁻ , nodH ⁻ ) were thus performed using a Frankia gene library in pLAFR3 to clarify this situation. Rhizobial transconjugants obtained by tri-parental matings were screened for restoration of the nodulation phenotype on their host plants, Vicia sativa subsp. nigra or Medicago sativa. Nodulation was observed on plants inoculated with transconjugants of the R. leguminosarum bv. viciaenodC::Tn5 mutant. The Nod⁺ rhizobial transconjugants were isolated and analysed. The Nod⁺ phenotype of these transconjugants was found to be due to Tn5 excision/transposition. No functional complementation was found with any of the mutants used, suggesting that rhizobial complementation of nod mutants with Frankia DNA is unlikely to occur. Received: 17 April 1998 / Accepted: 22 July 1998     

The effect of the Saccharomyces cerevisiae endo-exonuclease NUD1 gene expression on the resistance of HeLa cells to DNA-damaging agents.

HeLa cells transiently transfected with a mammalian expression DNA vector expressing the Saccharomyces cerevisiae endo-exonuclease (EE) NUD1 gene have exhibited changes in cell survival frequencies after treatment with different DNA-damaging agents as compared to HeLa cells transfected with a control plasmid. The NUD1-transfected cells showed a dose-dependent increase in sensitivity to UV irradiation resulting in up to 58% decrease in cell survival. In response to gamma-irradiation NUD1 transfected cells featured an increased survival at doses equal to and greater than 2.0 Gy, reaching a maximum enhancement in survival frequency of 17%. At the same time, the NUD1-transfectants featured an increase in resistance to 0.25 microM-0.5 microM cis-platin (up to 58% increase in cell survival) and 1.0 mM EMS (11% increase). At higher concentrations of EMS NUD1 expression resulted in a decreased cell survival of the transfected cells (17% decrease for 2.5 mM EMS). No difference in cell survival frequencies between the NUD1-transfectants and the controls was observed after treatment with different concentrations of chlorambucil and mechlorethamine. These results suggest possible roles played by EEs in different DNA repair pathways--being stimulatory for the repair of certain types of DNA lesions, such as double strand breaks (DSBs), and interfering with the endogenous DNA repair systems for the repair of other types of lesions. Furthermore, these results also provide additional indirect evidence for the role of EEs in homologous recombination.