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Studies of the Interaction of Escherichia coli YjeQ with the Ribosome In Vitro. Denis M. Daigle, 2004.Escherichia coli YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of E . coli and Bacillus subtilis . The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity [8h-1] [D . M . Daigle, L . Rossi, A . M . Berghuis, L . Aravind, E.V . Koonin, and E . D . Brown, Biochemistry 41: 11109-11117, 2002].In the work reported here, YjeQ from E . coli was found to copurify with ribosomes from cell extracts . The copy number of the proteinper cell was nevertheless low relative to the number of ribosomes[ratio of YjeQ copies to ribosomes, 1:200] . In vitro, recombinantYjeQ protein interacted strongly with the 30S ribosomal subunit,and the stringency of that interaction, revealed with salt washes,was highest in the presence of the nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate [GMP-PNP] . Likewise, associationwith the 30S subunit resulted in a 160-fold stimulation of YjeQGTPase activity, which reached a maximum with stoichiometricamounts of ribosomes . N-terminal truncation variants of YjeQrevealed that the predicted OB-fold region was essential forribosome binding and GTPase stimulation, and they showed thatan N-terminal peptide [amino acids 1 to 20 in YjeQ] was necessaryfor the GMP-PNP-dependent interaction of YjeQ with the 30S subunit.Taken together, these data indicate that the YjeQ protein participatesin a guanine nucleotide-dependent interaction with the ribosomeand implicate this conserved, essential GTPase as a novel factorin ribosome function. Evidence that the algI/algJ Gene Cassette, Required for O Acetylation of Pseudomonas aeruginosa Alginate, Evolved by Lateral Gene Transfer. Michael J. Franklin, 2004.Pseudomonas aeruginosa strains, isolated from chronically infected patients with cystic fibrosis, produce the O-acetylated extracellular polysaccharide, alginate, giving these strains a mucoid phenotype . O acetylation of alginate plays an important role in the ability of mucoid P . aeruginosa to form biofilms and to resist complement-mediated phagocytosis . The O-acetylation process is complex, requiring a protein with seven transmembrane domains (AlgI), a type II membrane protein (AlgJ), and a periplasmic protein (AlgF) . The cellular localization of these proteins suggests a model wherein alginate is modified at the polymer level after the transport of O-acetyl groups to the periplasm . Here, we demonstrate that this mechanism for polysaccharide esterification may be common among bacteria, since AlgI homologs linked to type II membrane proteins are found in a variety of gram-positive and gram-negative bacteria . In some cases, genes for these homologs have been incorporated into polysaccharide biosynthetic operons other than for alginate biosynthesis . The phylogenies of AlgI do not correlate with the phylogeny of the host bacteria, based on 16S rRNA analysis . The algI homologs and the gene for their adjacent type II membrane protein present a mosaic pattern of gene arrangement, suggesting that individual components of the multigene cassette, as well as the entire cassette, evolved by lateral gene transfer . AlgJ and the other type II membrane proteins, although more diverged than AlgI, contain conserved motifs, including a motif surrounding a highly conserved histidine residue, which is required for alginate O-acetylation activity by AlgJ . The AlgI homologs also contain an ordered series of motifs that included conserved amino acid residues in the cytoplasmic domain CD-4; the transmembrane domains TM-C, TM-D, and TM-E; and the periplasmic domain PD-3 . Site-directed mutagenesis studies were used to identify amino acids important for alginate O-acetylation activity, including those likely required for (i) the interaction of AlgI with the O-acetyl precursor in the cytoplasm, (ii) the export of the O-acetyl group across the cytoplasmic membrane, and (iii) the transfer of the O-acetyl group to a periplasmic protein or to alginate . These results indicate that AlgI belongs to a family of membrane proteins required for modification of polysaccharides and that a mechanism requiring an AlgI homolog and a type II membrane protein has evolved by lateral gene transfer for the esterification of many bacterial extracellular polysaccharides . The ADP-Ribosylating Toxin, AexT, from Aeromonas salmonicida subsp . salmonicida Is Translocated via a Type III Secretion Pathway. Sarah E. Burr, 2003.AexT is an extracellular ADP ribosyltransferase produced by the fish pathogen Aeromonas salmonicida subsp . salmonicida . The protein is secreted by the bacterium via a recently identified type III secretion system . In this study, we have identified a further 12 open reading frames that possess high homology to genes encoding both structural and regulatory components of the Yersinia type III secretion apparatus . Using marker replacement mutagenesis of aopB, the A . salmonicida subsp . salmonicida homologue of yopB in Yersinia, we demonstrate that the bacterium translocates the AexT toxin directly into the cytosol of cultured fish cells via this type III secretion pathway . An acrV mutant of A . salmonicida subsp . salmonicida displays a calcium-blind phenotype, expressing and secreting significant amounts of AexT even in the presence of CaCl2 concentrations as high as 10 mM . This acrV mutant is also unable to translocate AexT into the cytosol of fish cells, indicating AcrV is involved in the translocation process . Inactivation of either the aopB or acrV gene in A . salmonicida subsp . salmonicida (resulting in an inability to translocate AexT) is accompanied by a loss of cytotoxicity that can be restored by trans complementation . Finally, we present data indicating that preincubation of the wild-type bacteria with antibodies directed against recombinant AcrV-His protein provides fish cells protection against the toxic effects of the bacterium . Evaluation of the Kinetic Properties of the Sporulation Protein SpoIIE of Bacillus subtilis by Inclusion in a Model Membrane. Tim Searls, 2004.Starvation induces Bacillus subtilis to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell . The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor  F, and it also has a morphogenic activity required for asymmetric division . An increase in the local concentration of SpoIIE at the polar septum of B . subtilis precedes dephosphorylation of the anti-anti-sigma factor SpoIIAA in the prespore . After closure and invagination of the asymmetric septum, phosphatase activity of SpoIIE increases severalfold, but the reason for this dramatic change in activity has not been determined . The central domain of SpoIIE has been seen to self-associate (I . Lucet et al., EMBO J . 19:1467-1475, 2000), suggesting that activation of the C-terminal PP2C-like phosphatase domain might be due to conformational changes brought about by the increased local concentration of SpoIIE in the sporulating septum . Here we report the inclusion of purified SpoIIE protein into a model membrane as a method for studying the effect of local concentration in a lipid bilayer on activity . In vitro assays indicate that the membrane-bound enzyme maintains dephosphorylation rates similar to the highly active micellar state at all molar ratios of protein to lipid . Atomic force microscopy images indicate that increased local concentration does not lead to self-association .
Mercury Methylation Independent of the Acetyl-Coenzyme A Pathway in Sulfate-Reducing Bacteria. Eileen B. Ekstrom, 2003.Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems . Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production . The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species . To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities . Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway . So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury . Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth . However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition) . Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B12 in some and perhaps many incomplete-oxidizing SRB strains .
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