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Interaction of PomB with the Third Transmembrane Segment of PomA in the Na+-Driven Polar Flagellum of Vibrio alginolyticus. Toshiharu Yakushi, 2004.The marine bacterium Vibrio alginolyticus has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na+-driven flagellar rotation . PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex . First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent . Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined . Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins . The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants . Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it . Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent . The strain with this combination showed a lower affinity for Na+ than the wild-type combination . PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product . From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na+ conduction to flagellar rotation . Combined Phospholipid Biomarker-16S rRNA Gene Denaturing Gradient Gel Electrophoresis Analysis of Bacterial Diversity and Physiological Status in an Intertidal Microbial Mat. Laura Villanueva, 2004. Mutational Evidence for a Functional Connection between Two Domains of 23S rRNA in Translation Termination. Alexey L. Arkov, 2002.Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center . The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality . Defects in translation termination caused by this mutation have also been demonstrated in vitro . To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A . Here we report the isolation and characterization of such a secondary mutation . The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center . The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG . In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A . An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes . Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time . These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination . Natural Transformation of Campylobacter jejuni Requires Components of a Type II Secretion System. Rebecca S. Wiesner, 2003.The human pathogen Campylobacter jejuni is one of more than 40 naturally competent bacterial species able to import macromolecular DNA from the environment and incorporate it into their genomes . However, in C . jejuni little is known about the genes involved in this process . We used random transposon mutagenesis to identify genes that are required for the transformation of this organism . We isolated mutants with insertions in 11 different genes; most of the mutants are affected in the DNA uptake stage of transformation, whereas two mutants are affected in steps subsequent to DNA uptake, such as recombination into the chromosome or in DNA transport across the inner membrane . Several of these genes encode proteins homologous to those involved in type II secretion systems, biogenesis of type IV pili, and competence for natural transformation in gram-positive and gram-negative species . Other genes identified in our screen encode proteins unique to C . jejuni or are homologous to proteins that have not been shown to play a role in the transformation in other bacteria . Structural Model for 12-Helix Transporters Belonging to the Major Facilitator Superfamily. Teruhisa Hirai, 2003.The major facilitator superfamily includes a large collection of evolutionarily related proteins that have been implicated in the transport of a variety of solutes and metabolites across the membranes of organisms ranging from bacteria to humans . We have recently reported the three-dimensional structure, at 6.5 Å resolution, of the oxalate transporter, OxlT, a representative member of this superfamily . In the oxalate-bound state, 12 helices surround a central cavity to form a remarkably symmetrical structure that displays a well-defined pseudo twofold axis perpendicular to the plane of the membrane as well as two less pronounced, mutually perpendicular pseudo twofold axes in the plane of the membrane . Here, we combined this structural information with sequence information from other members of this protein family to arrive at models for the arrangement of helices in this superfamily of transport proteins . Our analysis narrows down the number of helix arrangements from about a billion starting possibilities to a single probable model for the relative spatial arrangement for the 12 helices, consistent both with our structural findings and with the majority of previous biochemical studies on members of this superfamily . Terminal Restriction Fragment Length Polymorphism Data Analysis for Quantitative Comparison of Microbial Communities. Christopher B. Blackwood, 2003.Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community . Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities . These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors . Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared . Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers . Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height . Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples . Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles . Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units . It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand . It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers . Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units .
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