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Myxococcus xanthus Chemotaxis Homologs DifD and DifG Negatively Regulate Fibril Polysaccharide Production. Wesley P. Black, 2004.The extracellular matrix fibrils of Myxococcus xanthus are essential for the social lifestyle of this unusual bacterium . These fibrils form networks linking or encasing cells and are tightly correlated with cellular cohesion, development, and social (S) gliding motility . Previous studies identified a set of bacterial chemotaxis homologs encoded by the dif locus . It was determined that difA, difC, and difE, encoding respective homologs of a methyl-accepting chemotaxis protein, CheW, and CheA, are required for fibril production and therefore S motility and development . Here we report the studies of three additional genes residing at the dif locus, difB, difD, and difG . difD and difG encode homologs of chemotaxis proteins CheY and CheC, respectively. difB encodes a positively charged protein with limited homology at its N terminus to conserved bacterial proteins with unknown functions . Unlike the previously characterized dif genes, none of these three newly studied dif genes are essential for fibril production, S motility, or development . The difB mutant showed no obvious defects in any of the processes examined . In contrast, the difD and the difG mutants were observed to overproduce fibril polysaccharides in comparison with production by the wild type . The observation that DifD and DifG negatively regulate fibril polysaccharide production strengthens our hypothesis that the M . xanthus dif genes define a chemotaxis-like signal transduction pathway which regulates fibril biogenesis . To our knowledge, this is the first report of functional studies of a CheC homolog in proteobacteria . In addition, during this study, we slightly modified previously developed assays to easily quantify fibril polysaccharide production in M . xanthus . Sigma 54 Levels and Physiological Control of the Pseudomonas putida Pu Promoter. Paola Jurado, 2003.The cellular levels of the alternative sigma factor  54
of Pseudomonas putida have been examined in a variety of
growth stages and culture conditions with a single-chain Fv antibody
tailored for detection of scarce proteins . The levels of
54
were also monitored in P . putida strains with knockout
mutations in ptsO or ptsN, known to be required for the
C-source control of the
54-dependent
Pu promoter of the TOL plasmid . Our results show that
 80
± 26 molecules of
54
exist per cell . Unlike that in relatives of Pseudomonas (e.g.,
Caulobacter), where fluctuations of
54
determine adaptation and differentiation when cells face starvation,
54
in P . putida remains unexpectedly constant at different growth
stages, in nitrogen starvation and C-source repression conditions,
and in the ptsO and ptsN mutant strains analyzed . The
number of
54
molecules per cell in P . putida is barely above the predicted
number of
54-dependent
promoters . These figures impose a framework on the mechanism by which
Pu (and other
54-dependent
systems) may become amenable to physiological control .
Molecular Genetic Analysis of ICEF, an Integrative Conjugal Element That Is Present as a Repetitive Sequence in the Chromosome of Mycoplasma fermentans PG18. Michael J. Calcutt, 2002.Mycoplasma genomes contain compact gene sets that approach the minimal complement necessary for life and reflect multiple evolutionary instances of genomic reduction . Lateral gene transfer may play a critical role in shaping the mobile gene pool in these organisms, yet complex mobile elements have not been reported within this genus . We describe here a large (  23-kb) genetic element with unique features that is present in four copies in the Mycoplasma fermentans PG18 chromosome, accounting for approximately 8% of the genome . These novel elements, designated ICEF (integrative conjugal elements of M . fermentans), resemble conjugative, self-transmissible integrating elements (constins) in that circular, nonreplicative extrachromosomal forms occur in which the left and right termini of the integrated element are juxtaposed and separated by a coupling sequence derived from direct repeats flanking chromosomal copies of ICEF as a result of target site duplication . ICEF contain multiple similarly oriented open reading frames (ORFs), of which some have homology to products of known conjugation genes but others have no known counterparts . Surprisingly, unlike other constins, ICEF lack homologs of known integrases, transposases, or recombinases, suggesting that a novel enzyme may be employed for integration-excision . Skewed distribution and varied sites of chromosomal integration among M . fermentans isolates suggest a role for ICEF in promoting genomic and phenotypic variation in this species . Identification of homologs of terminal ICEF ORFs in two additional mycoplasma species indicates that ICEF is the prototype member of a family of ICE-related elements that may be widespread among pathogenic mycoplasmas infecting diverse vertebrate hosts .
pIIICTX, a Predicted CTX  Minor Coat Protein, Can Expand the Host Range of Coliphage fd To Include Vibrio cholerae.Andrew J. Heilpern, 2003.CTX is a filamentous bacteriophage that encodes cholera toxin . CTX infection of its host bacterium, Vibrio cholerae, requires the toxin-coregulated pilus (TCP) and the products of the V . cholerae tolQRA genes . Here, we have explored the role of OrfU, a predicted CTX minor coat protein, in CTX infection . Prior to the discovery that it was part of a prophage, orfU was initially described as an open reading frame of unknown function that lacked similarity to known protein sequences . Based on its size and position in the CTX genome, we hypothesized that OrfU may function in a manner similar to that of the coliphage fd protein pIII and mediate CTX infection as well as playing a role in CTX assembly and release . Deletion of orfU from CTX dramatically reduced the number of CTX virions detected in supernatants from CTX-bearing cells . This defect was complemented by expression of orfU in trans, thereby confirming a role for this gene in CTX assembly and/or release . To evaluate the requirement for OrfU in CTX infection, we introduced fragments of orfU into gIII in an fd derivative to create OrfU-pIII fusions . While fd is ordinarily unable to infect V . cholerae, an fd phage displaying the N-terminal 274 amino acids of OrfU could infect V . cholerae in a TCP- and TolA-dependent fashion . Since our findings indicate that OrfU functions as the CTX pIII, we propose to rename OrfU as pIIICTX . Our data also provide new evidence for a conserved pathway for filamentous phage infection .
Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase. Sandrine Sauge-Merle, 2003.Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15) . In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content . When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively . We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes . Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the Lactobacillus casei Group. Cathy Provencher, 2003.A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group . Each hybrid primer consisted of a short 3' degenerate core based on four highly conserved amino acids and a longer 5' consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria . The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L . casei, Lactobacillus zeae, and Streptococcus thermophilus . The priming GT gene was detected in the genome of both non-EPS-producing (EPS-) and EPS-producing (EPS+) strains of L . rhamnosus . The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS+ bacteria . Specific primers designed from the L . rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS+ strains of L . rhamnosus . Phylogenetic analysis revealed that Lactobacillus spp . form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date . Moreover, the sequences show a divergence existing among strains of L . rhamnosus with respect to the terminal region of the priming GT gene . Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes .
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