RNA colony blot hybridization method for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria.

A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of V. cholerae and V. mimicus was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse V. cholerae-V. mimicus strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by Grimontia (Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent V. cholerae strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when G. hollisae was not present. When the protocol was applied to laboratory microcosms containing V. cholerae attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of V. cholerae per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable V. cholerae bacteria 1 to 2 months prior to observed increases in pathogenic V. cholerae and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of V. cholerae in the environment. The RNA blot hybridization protocol can also be applied to detection of other gram-negative bacteria for taxon-specific enumeration.

Serogroup, virulence, and genetic traits of Vibrio parahaemolyticus in the estuarine ecosystem of Bangladesh.

Forty-two strains of Vibrio parahaemolyticus were isolated from Bay of Bengal estuaries and, with two clinical strains, analyzed for virulence, phenotypic, and molecular traits. Serological analysis indicated O8, O3, O1, and K21 to be the major O and K serogroups, respectively, and O8:K21, O1:KUT, and O3:KUT to be predominant. The K antigen(s) was untypeable, and pandemic serogroup O3:K6 was not detected. The presence of genes toxR and tlh were confirmed by PCR in all but two strains, which also lacked toxR. A total of 18 (41%) strains possessed the virulence gene encoding thermostable direct hemolysin (TDH), and one had the TDH-related hemolysin (trh) gene, but not tdh. Ten (23%) strains exhibited Kanagawa phenomenon that surrogates virulence, of which six, including the two clinical strains, possessed tdh. Of the 18 tdh-positive strains, 17 (94%), including the two clinical strains, had the seromarker O8:K21, one was O9:KUT, and the single trh-positive strain was O1:KUT. None had the group-specific or ORF8 pandemic marker gene. DNA fingerprinting employing pulsed-field gel electrophoresis (PFGE) of SfiI-digested DNA and cluster analysis showed divergence among the strains. Dendrograms constructed using PFGE (SfiI) images from a soft database, including those of pandemic and nonpandemic strains of diverse geographic origin, however, showed that local strains formed a cluster, i.e., "clonal cluster," as did pandemic strains of diverse origin. The demonstrated prevalence of tdh-positive and diarrheagenic serogroup O8:K21 strains in coastal villages of Bangladesh indicates a significant human health risk for inhabitants.

Major tdh(+)Vibrio parahaemolyticus serotype changes temporally in the Bay of Bengal estuary of Bangladesh.

Vibrio parahaemolyticus is responsible for seafood-related gastroenteritis worldwide. In Bangladesh, diarrhea is endemic and diarrheagenic V. parahaemolyticus serotypes occur naturally in the coastal and estuarine aquatic environment. V. parahaemolyticus strains, isolated from estuarine surface water of the Bay of Bengal villages of Bangladesh during 2006-2008, were tested for the presence of virulence and pandemic-marker genes, serodiversity, and phylogenetic relatedness. PCR analysis of V. parahaemolyticus (n=175) showed 53 (30.3%) strains to possess tdh, the major virulence gene encoding thermostable direct hemolysin. Serotyping results revealed the tdh(+)V. parahaemolyticus strains to belong to 10 different serotypes, of which the O8:K21 (30.2%) and O3:K6 (24.5%) were predominantly non-pandemic and pandemic serotypes, respectively; while O5:K30 and O9:KUT were new. The pandemic markers, orf8 and toxRS(variant), were present only in the pandemic serotype O3:K6 (n=13) and its serovariant O4:K68 (n=2). Temporal distribution of the tdh(+) serotypes revealed the O8:K21 to be predominant in 2006 and 2007, while O3:K6 was the predominant tdh(+) serotype in 2008. Pulsed-field gel electrophoresis (PFGE) of SfiI-digested genomic DNA revealed high genetic diversity among the V. parahaemolyticus strains, while dendrogram constructed with the PFGE patterns formed two major clusters separating the tdh(+) O3:K6 and its pandemic serovariants from the tdh(+) non-pandemic (O8:K21) strains, suggesting different lineages for them. The potential health risk related to the prevalent tdh(+) strains, including the observed temporal change of the predominant tdh(+) serotype, from O8:K21 to the pandemic serotype O3:K6 in estuarine surface waters serving as the major source of drinking water suggests the need for routine environmental monitoring to prevent V. parahaemolyticus infection in Bangladesh.

The translation elongation factor eEF1B plays a role in the oxidative stress response pathway.

The multi-subunit guanine nucleotide exchange factor eEF1B for Saccharomyces cerevisiae Translation Elongation Factor 1A (eEF1A) has catalytic (eEF1Balpha) and noncatalytic (eEF1Bgamma) subunits. Deletion of the two nonessential genes encoding eEF1Bgamma has no dramatic effects on total protein synthesis or translational fidelity. Instead, loss of each gene gives resistance to oxidative stress, and loss of both is additive. The level of stress resistance is similar to overexpression of the Yap1p stress transcription factor and is dependent on the presence of the YAP1gene. Cells lacking the catalytic eEF1Balpha subunit show even greater resistance to CdSO(4), with or without eEF1Bgamma present. Thus, the loss of guanine nucleotide exchange activity promotes the resistance. As nucleotide exchange is a critical regulator of most G-proteins, these results indicate a new mechanism in the growing list of examples of post-transcriptional responses to cellular stress.

1.15†Šresolution structure of the proteasome-assembly chaperone Nas2 PDZ domain.

The 26S proteasome is a 2.5 MDa protease dedicated to the degradation of ubiquitinated proteins in eukaryotes. The assembly of this complex containing 66 polypeptides is assisted by at least nine proteasome-specific chaperones. One of these, Nas2, binds to the proteasomal AAA-ATPase subunit Rpt5. The PDZ domain of Nas2 binds to the C-terminal tail of Rpt5; however, it does not require the C-terminus of Rpt5 for binding. Here, the 1.15 Šresolution structure of the PDZ domain of Nas2 is reported. This structure will provide a basis for further insights regarding the structure and function of Nas2 in proteasome assembly.

Interaction between 25S rRNA A loop and eukaryotic translation initiation factor 5B promotes subunit joining and ensures stringent AUG selection.

In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes.

Random mutagenesis of yeast 25S rRNA identify bases critical for 60S subunit structural integrity and function.

In yeast Saccharomyces cerevisiae, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During translation initiation, the 60S subunit joins the 40S initiation complex, producing the 80S initiation complex. During elongation, the 60S subunit binds the CCA-ends of aminoacyl- and peptidyl-tRNAs at the A-loop and P-loop, respectively, transferring the peptide onto the α-amino group of the aminoacyl-tRNA. To study the role of 25S rRNA in translation in vivo, we randomly mutated 25S rRNA and isolated and characterized seven point mutations that affected yeast cell growth and polysome profiles. Four of these mutations, G651A, A1435U, A1446G and A1587G, change a base involved in base triples crucial for structural integrity. Three other mutations change bases near the ribosomal surface: C2879U and U2408C alter the A-loop and P-loop, respectively, and G1735A maps near a Eukarya-specific bridge to the 40S subunit. By polysome profiling in mmslΔ mutants defective in nonfunctional 25S rRNA decay, we show that some of these mutations are defective in both the initiation and elongation phases of translation. Of the mutants characterized, C2879U displays the strongest defect in translation initiation. The ribosome transit-time assay directly shows that this mutation is also defective in peptide elongation/termination. Thus, our genetic analysis not only identifies bases critical for structural integrity of the 60S subunit, but also suggests a role for bases near the peptidyl transferase center in translation initiation.

Sequential eukaryotic translation initiation factor 5 (eIF5) binding to the charged disordered segments of eIF4G and eIF2ß stabilizes the 48S preinitiation complex and promotes its shift to the initiation mode.

During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2ß bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2ß-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2ß-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2ß mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2ß and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA(i)(Met):mRNA binding to the P site.

Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.