RecA Expression in Response to Solar UVR in the Marine Bacterium Vibrio natriegens

Solar ultraviolet radiation may produce daily stress on marine and estuarine communities as cells are damaged and repair that damage. Reduction in the earth's stratospheric ozone layer has increased awareness of the potential effects that ultraviolet radiation may have in the environment, including how marine bacteria respond to changes in solar radiation. We examined the use of the bacterial RecA protein as an indicator of the potential of bacteria to repair DNA damage caused by solar UV irradiation using the marine bacterium Vibrio natriegens as a model. RecA is universally present in bacteria and is a regulator protein for the so-called Dark Repair Systems, which include excision repair, postreplication recombinational repair, and mutagenic or SOS repair. Solar UVB and UVA both reduced V. natriegens viability in seawater microcosms. After exposure to unfiltered solar radiation or radiation in which UVB was blocked, survival dropped below 1%, whereas visible light from which UVA and UVB had been filtered had no effect on survival. Using a RecA-specific antibody for detection, RecA protein was induced by solar radiation in a diel pattern in marine microcosms conducted in the Gulf of Mexico. Peak induction was observed at dusk each day. Although RecA expression was correlated with the formation of UVB-induced cyclobutyl pyrimidine dimers, longer wavelength UVA radiation also induced recA gene expression. Our results demonstrate that RecA-regulated, light-independent repair is an important component in the ability of marine bacteria to survive exposure to solar ultraviolet radiation and that RecA expression is a useful monitor of bacterial repair after exposure to solar UVR.     

Application of the zona-free manipulation technique to porcine somatic nuclear transfer

The recent demonstration of a successful zona-free manipulation technique for bovine somatic nuclear transfer (NT) that is both simpler and less labor intensive is of considerable benefit to advance the applications of this technology. Here, we describe that this method is also applicable to porcine somatic NT. Porcine cumulus oocyte complexes were matured in TCM-199 medium before sequential removal of the cumulus and zonae. Zona-free oocytes were bisected using a microknife, and the halves containing the metaphase plate (as determined by Hoechst 33342 staining) were discarded. Each half cytoplast was agglutinated to a single granulosa cell (primary cultures grown in 0.5% serum for 2-5 days prior to use) in phytohaemagglutinin-P. Subsequently, each half cytoplast-granulosa cell couplet was simultaneously electrofused together and to another half cytoplast. Reconstructed embryos were activated in calcium ionophore A23187 followed by DMAP and were then individually cultured in microwells in NCSU-23 medium. On day 7 after activation, blastocyst yield and total cell numbers were counted. Of 279 attempted reconstructed NT embryos, 85.0 +/- 2.8% (mean +/- SEM; n = 5 replicates) successfully fused and survived activation. The blastocyst rate (per successfully fused and surviving embryo) was 4.8 +/- 2.3% (11/236; range, 0-12.8%). Total blastocyst cell count was 36.0 +/- 4.5 (range, 18-58 cells). The blastocyst rate and total cell numbers of parthenogenetically activated and zona-free control oocytes propagated under the same conditions was 11.6 +/- 3.9% (35/335 embryos; n = 3 replicates) and 36.8 +/- 5.2, respectively. Developmentally halted embryos that could still be evaluated on day 7 possessed 54.4 +/- 2.3% (53/96 embryos; n = 3 replicates) anucleate blastomeres, the latter representing 53.5 +/- 6.6% of the blastomeres in such embryos. In conclusion, blastocyst yield was independent of activation efficiency and was likely reduced by insufficient nuclear remodeling, reprogramming, imprinting, or other effects. The data also suggest that fragmentation was a considerable problem that could conceivably contribute to halted development in a high proportion of embryos. The results indicate that the zona-free manipulation technique can be successfully applied to pig somatic NT. Although such zona-free early cleavage stage embryos cannot be transferred to recipients at present, this technique permits simplification of the NT technique for application in basic research, until pig nonsurgical blastocyst transfer becomes a realistic option.     

A mechanism of regulating transmembrane potassium flux through a ligand-mediated conformational switch

The regulation of cation content is critical for cell growth. However, the molecular mechanisms that gate the systems that control K+ movements remain unclear. KTN is a highly conserved cytoplasmic domain present ubiquitously in a variety of prokaryotic and eukaryotic K+ channels and transporters. Here we report crystal structures for two representative KTN domains that reveal a dimeric hinged assembly. Alternative ligands NAD+ and NADH block or vacate, respectively, the hinge region affecting the dimer's conformational flexibility. Conserved, surface-exposed hydrophobic patches that become coplanar upon hinge closure provide an assembly interface for KTN tetramerization. Mutational analysis using the KefC system demonstrates that this domain directly interacts with its respective transmembrane constituent, coupling ligand-mediated KTN conformational changes to the permease's activity.     

The CXCR3 binding chemokine IP-10/CXCL10: structure and receptor interactions

The structure of IP-10 was solved by NMR spectroscopy and represents the first structure from the class of agonists toward the receptor CXCR3. CXCR3 binding chemokines are unique in their ability to bind receptors from both the CC and CXC classes of chemokine receptors. An unusual structural feature of IP-10 was identified that may provide the basis for the ability of IP-10 to bind both CXCR3 and CCR3. The surface of IP-10 that interacts with the N-terminus of CXCR3 was defined by monitoring changes in the NMR spectrum of IP-10 upon addition of a CXCR3 N-terminal peptide. These studies indicated that the interaction involves a hydrophobic cleft, formed by the N-loop and 40s-loop region of IP-10, similar to the interaction surface observed for other chemokines such as IL-8. An additional region of interaction was observed that consists of a hydrophobic cleft formed by the N-terminus of IP-10 and 30s-loop of IP-10.     

Analysis of the kefA2 mutation suggests that KefA is a cation-specific channel involved in osmotic adaptation in Escherichia coli

Mechanosensitive channels play an essential role in the regulation of turgor pressure in bacteria. In Escherichia coli, there are multiple mechanosensitive channels that have been characterized genetically: MscL, YggB and KefA. In this report, we describe the cloning of the kefA gene, the organization of the KefA protein and the phenotype of a missense mutation, kefA, which affects the KefA mechanosensitive channel. The altered function of the channel is manifest through increased sensitivity to K+ during growth at low osmolarity and complete inhibition of growth in media containing high K+ concentrations (0.6 M) in the presence of betaine or proline. Growth in high Na+ medium (0.6 M NaCl plus 20 mM K+) is normal. Analysis of the cytoplasmic pools shows that the mutant cannot regulate the K+ content of the cytoplasm when grown in high K+ medium. However, regulation of pools of amino acids is essentially normal and the mutant can accumulate high pools of proline during growth inhibition. The mutant shows increased sensitivity to acid hypo-osmotic shock (transition from neutral to acid pH combined with a reduction in osmolarity). The data are consistent with abnormal regulation of KefA in the presence of high K+ concentrations and either betaine or proline.     

Modulation of the F-actin cytoskeleton by c-Abl tyrosine kinase in cell spreading and neurite extension

The nonreceptor tyrosine kinase encoded by the c-Abl gene has the unique feature of an F-actin binding domain (FABD). Purified c-Abl tyrosine kinase is inhibited by F-actin, and this inhibition can be relieved through mutation of its FABD. The c-Abl kinase is activated by physiological signals that also regulate the actin cytoskeleton. We show here that c-Abl stimulated the formation of actin microspikes in fibroblasts spreading on fibronectin. This function of c-Abl is dependent on kinase activity and is not shared by c-Src tyrosine kinase. The Abl-dependent F-actin microspikes occurred under conditions where the Rho-family GTPases were inhibited. The FABD-mutated c-Abl, which is active in detached fibroblasts, stimulated F-actin microspikes independent of cell attachment. Moreover, FABD-mutated c-Abl stimulated the formation of F-actin branches in neurites of rat embryonic cortical neurons. The reciprocal regulation between F-actin and the c-Abl tyrosine kinase may provide a self-limiting mechanism in the control of actin cytoskeleton dynamics.     

Carbon source utilization profiles as a method to identify sources of faecal pollution in water

AIMS: Carbon source utilization profiles as a phenotypic fingerprinting methodology for determining sources of faecal pollution in water were evaluated. METHODS AND RESULTS: Three hundred and sixty-five Enterococcus isolates were collected from known faecal sources in four different geographical regions and were identified to species with the commercial Biolog system. Discriminant analysis (DA) was used to identify the substrate-containing wells that best classified the 365 isolates by source. By using 30 of the 95 wells for the analysis, the average rate of correct classification (ARCC) by source was 92.7% for a human vs non-human two-way classification when isolates from all regions were combined into one library. Corresponding ARCCs for other classification schemes were 81.9% for a four-way classification of human vs livestock vs wildlife vs domestic pets, and 85.7% for a three-way classification without human isolates. When three individual libraries were made based on classification of sources within Enterococcus species, the ARCC was 95.3% for the Ent. faecalis library, 95.8% for the Ent. gallinarum library and 94.7% for the Ent. mundtii library. Thirty Enterococcus isolates (unknown sources) were obtained from each of three stream sites where a specific source of pollution was apparent; 90.0% of the isolates from a human-suspected source were classified as human, 86.6% were classified as livestock from a livestock-suspected site, and 93.3% were classified as wildlife from a wildlife-suspected site. CONCLUSIONS: Phenotypic fingerprinting with carbon source utilization profiles provided levels of correct classification by sources from an Enterococcus library that were in the upper range of those reported in the literature. ARCCs for three Enterococcus species-specific libraries were very high and may be the best approach for further developing this concept and methodology. SIGNIFICANCE ANC IMPACT OF THE STUDY: The results, based on a modest Enterococcus library and a preliminary field validation test, demonstrated the potential for carbon source utilization profiles to be employed as a phenotypic method for determining sources of faecal pollution in water.