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FlrA, a  54-Dependent
Transcriptional Activator in Vibrio fischeri, Is Required for Motility
and Symbiotic Light-Organ Colonization.Deborah S. Millikan, 2003.Flagellum-mediated motility of Vibrio fischeri is an essential factor in the bacterium's ability to colonize its host, the Hawaiian squid Euprymna scolopes . To begin characterizing the nature of the flagellar regulon, we have cloned a gene, designated flrA, from V . fischeri that encodes a putative 54-dependent
transcriptional activator . Genetic arrangement of the flrA locus
in V . fischeri is similar to motility master-regulator operons
of Vibrio cholerae and Vibrio parahaemolyticus . In addition,
examination of regulatory regions of a number of flagellar operons
in V . fischeri revealed apparent
54
recognition motifs, suggesting that the flagellar regulatory
hierarchy is controlled by a similar mechanism to that described in
V . cholerae . However, in contrast to its closest known
relatives, flrA mutant strains of V . fischeri ES114
were completely abolished in swimming capability . Although flrA
provided in trans restored motility to the flrA mutant,
the complemented strain was unable to reach wild-type levels of
symbiotic colonization in juvenile squid, suggesting a possible role
for the proper expression of FlrA in regulating symbiotic
colonization factors in addition to those required for motility .
Comparative RNA arbitrarily primed PCR analysis of the flrA
mutant and its wild-type parent revealed several differentially
expressed transcripts . These results define a regulon that includes
both flagellar structural genes and other genes apparently not
involved in flagellum elaboration or function . Thus, the transcriptional
activator FlrA plays an essential role in regulating motility,
and apparently in modulating other symbiotic functions, in V .
fischeri .
Rotavirus Virus-Like Particles as Surrogates in Environmental Persistence and Inactivation Studies. Santiago Caballero, 2004.Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios . Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used . After 1 month in seawater at 20°C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate . In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20°C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times . However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles . The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus . Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water . The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs . In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus . Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced . Proteins Released by Helicobacter pylori In Vitro. Nayoung Kim, 2002.Secretion of proteins by Helicobacter pylori may contribute to gastric inflammation and epithelial damage . An in vitro analysis was designed to identify proteins released by mechanisms other than nonspecific lysis . The radioactivity of proteins in the supernatant was compared with that of the intact organism by two-dimensional gel phosphorimaging following a 4-h pulse-chase . The ratio of the amount of UreB, a known cytoplasmic protein, in the supernatant to that in the pellet was found to be 0.25, and this was taken as an index of lysis during the experiments (n = 6) . Ratios greater than that of UreB were used to distinguish proteins that were selectively released into the medium . Thus, proteins enriched more than 10-fold in the supernatant compared to UreB were identified by mass spectrometry . Sixteen such proteins were present in the supernatant: VacA; a conserved secreted protein (HP1286); putative peptidyl cis-trans isomerase (HP0175); six proteins encoded by HP0305, HP0231, HP0973, HP0721, HP0129, and HP0902; thioredoxin (HP1458); single-stranded-DNA-binding 12RNP2 precursor (HP0827); histone-like DNA-binding protein HU (HP0835); ribosomal protein L11 (HP1202); a putative outer membrane protein (HP1564); and outer membrane proteins Omp21 (HP0913) and Omp20 (HP0912) . All except HP0902, thioredoxin, HP0827, HP0835, and HP1202 had a signal peptide . When nalidixic acid, a DNA synthesis inhibitor, was added to inhibit cell division but not protein synthesis, to decrease possible contamination due to outer membrane shedding, two outer membrane proteins (Omp21 and Omp20) disappeared from the supernatant, and the amount of VacA also decreased . Thus, 13 proteins were still enriched greater than 10-fold in the medium after nalidixic acid treatment, suggesting these were released specifically, possibly by secretion . These proteins may be implicated in H . pylori-induced effects on the gastric epithelium . Identification of Methyl Coenzyme M Reductase A (mcrA) Genes Associated with Methane-Oxidizing Archaea. Steven J. Hallam, 2003.Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation . Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically . In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments . Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries . Four out of five novel mcrA types identified from these sources were associated with ANME-1 or ANME-2 group members . Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences . Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids . These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea .
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