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Genes Required for Intrinsic Multidrug Resistance in Mycobacterium avium. Julie S. Philalay, 2004.Genes required for intrinsic multidrug resistance by Mycobacterium avium were identified by screening a library of transposon insertion mutants for the inability to grow in the presence of ciprofloxacin, clarithromycin, and penicillin at subinhibitory concentrations . Two genes, pks12 and Maa2520, were disrupted in multiple drug-susceptible mutants . The pks12 gene (Maa1979), which may be cotranscribed with a downstream gene (Maa1980), is widely conserved in the actinomycetes . Its ortholog in Mycobacterium tuberculosis is a polyketide synthase required for the synthesis of dimycocerosyl phthiocerol, a major cell wall lipid . Mutants of M . avium with insertions into pks12 exhibited altered colony morphology and were drug susceptible, but they grew as well as the wild type did in vitro and intracellularly within THP-1 cells . A pks12 mutant of M . tuberculosis was moderately more susceptible to clarithromycin than was its parent strain; however, susceptibility to ciprofloxacin and penicillin was not altered . M . avium complex (MAC) and M . tuberculosis appear to have different genetic mechanisms for resisting the effects of these antibiotics, with pks12 playing a relatively more significant role in MAC . The second genetic locus identified in this study, Maa2520, is a conserved hypothetical gene with orthologs in M . tuberculosis and Mycobacterium leprae . It is immediately upstream of Maa2521, which may code for an exported protein . Mutants with insertions at this locus were susceptible to multiple antibiotics and slow growing in vitro and were unable to survive intracellularly within THP-1 cells . Like pks12 mutants, they exhibited increased Congo red binding, an indirect indication of cell wall modifications . Maa2520 and pks12 are the first genes to be linked by mutation to intrinsic drug resistance in MAC . Validation of a PCR-Based Method for Detection of Food-Borne Thermotolerant Campylobacters in a Multicenter Collaborative Trial. M. H. Josefsen, 2004. Patterns of Sequence Conservation in the S-Layer Proteins and Related Sequences in Clostridium difficile. Emanuela Calabi, 2002.Clostridium difficile is the etiological agent of antibiotic-associated diarrhea . Among the factors that may play a role in infection are S-layer proteins (SLPs) . Previous work has shown these to consist mainly of two components, resulting from the cleavage of a precursor encoded by the slpA gene . The high-molecular-weight (MW) subunit is related both to amidases from B . subtilis and to at least another 28 gene products in C . difficile strain 630 . To gain insight into the functions of the SLPs and related proteins, we have further investigated the pattern of variability both at the slpA locus and at six nearby paralogs . Sequencing of the slpA gene from an S-layer group II strain and a variant S-layer group strain confirms a high degree of divergence in the low-MW SLP, which may result from diversifying selection . A highly conserved motif, however, is found at the C terminus in all low-MW subunits and may be essential for SlpA precursor cleavage . In strain 167, a variant cleavage product is present, suggesting a secondary processing site . Southern blotting analysis shows slpA-like open reading frames (ORFs) 2 to 7 to be conserved in all nine strains tested, with one exception: ORF2, which encodes a 66-kDa polypeptide coextracted at low pH with the main SLPs in strain 630, may be partially deleted in strain 167 . Polymorphism within the slpA-ORF7 cluster may be more pronounced in the region proximal to the slpA gene . Unexpectedly, a high-MW subunit probe cross hybridizes to sequences outside the slpA locus, which appear to vary in number in different strains . Crystal Structures of the Quinone Oxidoreductase from Thermus thermophilus HB8 and Its Complex with NADPH: Implication for NADPH and Substrate Recognition. Yoshimitsu Shimomura, 2003.The crystal structures of the  -crystalline-like soluble quinone oxidoreductase from Thermus thermophilus HB8 (QORTt) and of its complex with NADPH have been determined at 2.3- and 2.8-Å resolutions, respectively . QORTt is composed of two domains, and its overall fold is similar to the folds of Escherichia coli quinone oxidoreductase (QOREc) and horse liver alcohol dehydrogenase . QORTt forms a homodimer in the crystal by interaction of the ßF-strands in domain II, forming a large ß-sheet that crosses the dimer interface . High thermostability of QORTt was evidenced by circular dichroic measurement . NADPH is located between the two domains in the QORTt-NADPH complex . The disordered segment involved in the coenzyme binding of apo-QORTt becomes ordered upon NADPH binding . The segment covers an NADPH-binding cleft and may serve as a lid . The 2'-phosphate group of the adenine of NADPH is surrounded by polar and positively charged residues in QORTt, suggesting that QORTt binds NADPH more readily than NADH . The putative substrate-binding site of QORTt, unlike that of QOREc, is largely blocked by nearby residues, permitting access only to small substrates . This may explain why QORTt has weak p-benzoquinone reduction activity and is inactive with such large substrates of QOREc as 5-hydroxy-1,4-naphthoquinone and phenanthraquinone .
Competence-Induced Cells of Streptococcus pneumoniae Lyse Competence-Deficient Cells of the Same Strain during Cocultivation. Hilde Steinmoen, 2003.Several streptococcal species are able to take up naked DNA from the environment and integrate it into their genomes by homologous recombination . This process is called natural transformation . In Streptococcus pneumoniae and related streptococcal species, competence for natural transformation is induced by a peptide pheromone through a quorum-sensing mechanism . Recently we showed that induction of the competent state initiates lysis and release of DNA from a subfraction of the bacterial population and that the efficiency of this process is influenced by cell density . Here we have further investigated the nature of this cell density-dependent release mechanism . Interestingly, we found that competence-induced pneumococci lysed competence-deficient cells of the same strain during cocultivation and that the efficiency of this heterolysis increased as the ratio of competent to noncompetent cells increased . Furthermore, our results indicate that the lysins made by competent pneumococci are not released into the growth medium . More likely, they are anchored to the surface of the competent cells by choline-binding domains and cause lysis of noncompetent pneumococci through cell-to-cell contact . Mitogen-Activated Protein Kinase in Pfiesteria piscicida and Its Growth Rate-Related Expression. Senjie Lin, 2003.A full-length cDNA (1,434 bp) of mitogen-activated protein kinase (MAPK), a key molecule of a signal transduction cascade, was isolated from the estuarine heterotrophic dinoflagellate Pfiesteria piscicida . This cDNA (Ppmapk1) encoded a protein (PpMAPK1) of 428 amino acid residues that shared about 30 to 40% amino acid similarity with MAPKs in other organisms . Phylogenetic analysis indicated that PpMAPK1 was tightly clustered with MAPK3 in protozoans . Using reverse transcription-PCR, expression of this gene was evaluated for P . piscicida cultures grown under different conditions . While salinity shock, heat shock, starvation, and a subsequent encounter with prey did not appear to affect expression of this gene, Ppmapk1 expression level was correlated with growth rate, suggesting involvement of this gene in the regulation of cell proliferation in the organism . Characterization of the Microbial Community in Indoor Environments: a Chemical-Analytical Approach. Aleksandra Sebastian, 2003.An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust . A hydrolysate of 13C-labeled cyanobacterial cells is used as an internal standard for the first three markers . These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol . The method may be used to characterize microbial communities in environmental samples .
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