Extreme resistance to thermally induced DNA backbone breaks in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degrees C. It is not conceivable that these organisms could survive with genomic DNA that was subject to thermal destruction, yet the mechanisms protecting the genomes of this and other hyperthermophiles against such destruction are obscure. We have determined the effect of elevated temperatures up to 110 degrees C on the molecular weight of DNA in intact P. furiosus cells, compared with the effect of elevated temperatures on DNA in the mesothermophilic bacterium Escherichia coli. At 100 degrees C, DNA in P. furiosus cells is about 20 times more resistant to thermal breakage than that in E. coli cells, and six times fewer breaks were found in P. furiosus DNA after exposure to 110 degrees C for 30 min than in E. coli DNA at 95 degrees C. Our hypothesis for this remarkable stability of DNA in a hyperthermophile is that this hyperthermophile possesses DNA-binding proteins that protect against hydrolytic damage, as well as other endogenous protective mechanisms and DNA repair enzyme systems.     

Functional implications of the expression of PilC proteins in meningococci

Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co-expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil^- backswitcher isolated from the hyper-adherent variant was PilC⁺ but was non-adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC-expressing variant. The hyper-adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper-adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.     

Induction of DNA-protein crosslinks in melanotic cloudman S91 mouse melanoma cells and EMT6 mouse mammary carcinoma cells by monochromatic 254 and 405 nm light

The rates of induction of DNA-protein crosslinks (DPC) by monochromatic radiation at 254 and 405 nm were compared in melanotic S91 mouse melanoma cells and EMT6 mouse mammary carcinoma cells. At 254 nm, the rates of induction of DPC are the same in the two cell lines, whereas, at 405 nm, the rate of induction of DPC in the melanotic cells is considerably less than that in the nonmelanotic cells. Since the major difference in the two cell lines with respect to absorption is melanin, the latter finding implies that intracellular melanin can protect against this DNA damage caused by a component of environmental carcinogenic solar radiation.     

Inactivation of transforming DNA by ultraviolet light. I. Near-UV action spectrum for marker inactivation

The storage effect is defined as an increase of mutational damage after cessation of treatment. It differs from other kinds of delayed effect (replication errors, replicating instabilities) in not requiring replication of DNA. In Neurospora ad3A 38701 inos 37401 diepoxybutane (DEB) yields a storage effect for adenine reversions, but none for inositol reversions. The storage effect takes place in treated washed spores that are sedimented in a centrifuge tube, but not in spores that are agitated in water. Under the latter conditions, response to storage is gradually lost. The storage effect can be imitated by administering very small amounts of DEB to cells that had been previously treated with a moderately high dose (booster effect). During post-treatment shaking in water, responses to booster and to storage conditions disappear together; response to booster disappears at the same rate in spores that are sedimented in centrifuge tubes. No mutagenic action could be detected in eluates from heavily treated cells. We have concluded that treatment with DEB sensitizes the conidia to further small doses of DEB whether these are administered extraneously as booster or present intracellularly during storage. Sensitization is lost in the course of a few hours in shaken as well as in sedimented spores. Thus, while the storage effect is due to traces of mutagen, its gradual disappearance after treatment is not due to loss of these traces.Correlated with the ability to yield a storage effect, and probably part of the storage effect, is the response to temperature between treatment and plating. Conidia that can give a storage effect yield fewer mutations when spread on cold agar than when inplated into warm agar or heat-shocked before spreading; the excess of mutation under the latter two conditions forms part of the final storage effect. The true base line for calculation of the storage effect is therefore mutation frequency among spread spores.For DEB as mutagen, response to storage by the adenine locus and lack of response by the inositol locus are correlated with the responses of these two loci to dose of DEB and to combination treatment of DEB with UV, or DEB with nitrous acid (NA). This makes it possible to fit all observations into the picture of a general hypothesis on the cellular effects of DEB. Because of the differential response of the two loci, storage and plating procedures offer two additional means for manipulating specificity in this system.     

Metagenomic analysis of a stable trichloroethene-degrading microbial community

Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500 bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides, Desulfovibrio, Methanobacterium, Methanospirillum, as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500 bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductive dechlorination. Besides Dehalococcoides, several other members of this community appear to have genes for complete or near-complete cobalamin biosynthesis pathways. In all, 17 genes for putative reductive dehalogenases were identified, including 11 novel ones, all associated with Dehalococcoides. Genes for hydrogenase components (271 in total) were widespread, highlighting the importance of hydrogen metabolism in this community. PhyloChip analysis confirmed the stability of this microbial community.     

Towards understanding the functional role of the glycosyltransferases involved in the biosynthesis of Moraxella catarrhalis lipooligosaccharide

The glycosyltransferase enzymes (Lgts) responsible for the biosynthesis of the lipooligosaccharide-derived oligosaccharide structures from Moraxella catarrhalis have been investigated. This upper respiratory tract pathogen is responsible for a spectrum of illnesses, including otitis media (middle ear infection) in children, and contributes to exacerbations of chronic obstructive pulmonary disease in elderly patients. To investigate the function of the glycosyltransferase enzymes involved in the biosynthesis of lipooligosaccharide of M. catarrhalis and to gain some insight into the mechanism of serotype specificity for this microorganism, mutant strains of M. catarrhalis were produced. Examination by NMR and MS of the oligosaccharide structures produced by double-mutant strains (2951lgt1/4Delta and 2951lgt5/4Delta) and a single-mutant strain (2951lgt2Delta) of the bacterium has allowed us to propose a model for the serotype-specific expression of lipooligosaccharide in M. catarrhalis. According to this model, the presence/absence of Lgt4 and the Lgt2 allele determines the lipooligosaccharide structure produced by a strain. Furthermore, it is concluded that Lgt4 functions as an N-acetylglucosylamine transferase responsible for the addition of an alpha-D-GlcNAc (1-->2) glycosidic linkage to the (1-->4) branch, and also that there is competition between the glycosyltransferases Lgt1 and Lgt4. That is, in the presence of an active Lgt4, GlcNAc is preferentially added to the (1-->4) chain of the growing oligosaccharide, instead of Glc. In serotype B strains, which lack Lgt4, Lgt1 adds a Glc at this position. This implies that active Lgt4 has a much higher affinity/specificity for the beta-(1-->4)-linked Glc on the (1-->4) branch than does Lgt1.     

Moraxella catarrhalis Lgt2, a galactosyltransferase with broad acceptor substrate specificity

The genetic basis of lipo-oligosaccharide (LOS) biosynthesis for the bacterium Moraxella catarrhalis has been elucidated and functions suggested for each of the glycosyltransferases. In this study we have expressed and characterised one of these enzymes, the putative galactosyltransferase Lgt2_B/C. The lgt2_B/C gene was amplified from M. catarrhalis, expressed in Escherichia coli, and Lgt2_B/C was purified. Analysis of its glycosyltransferase catalytic activity ascertained the pH and temperature optima. The donor specificity and acceptor specificity were examined and they showed that Lgt2_B/C is a galactosyltransferase with relatively broad acceptor specificity with optimal activity in the presence of exogenous Mg²⁺.