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The Type II Protein Secretion System of Legionella pneumophila Promotes Growth at Low Temperatures. Maria A. Söderberg, 2004.The gram-negative bacterium Legionella pneumophila grows in both natural and man-made water systems and in the mammalian lung as a facultative intracellular parasite . The PilD prepilin peptidase of L . pneumophila promotes type IV pilus biogenesis and type II protein secretion . Whereas pili enhance adherence, Legionella type II secretion is critical for intracellular growth and virulence . Previously, we observed that pilD transcript levels are greater in legionellae grown at 30 versus 37°C . Using a new pilD::lacZ fusion strain, we now show that pilD transcriptional initiation increases progressively as L . pneumophila is grown at 30, 25, and 17°C . Legionella pilD mutants also had a dramatically reduced ability to grow in broth and to form colonies on agar at the lower temperatures . Whereas strains specifically lacking type IV pili were not defective for low-temperature growth, mutations in type II secretion (lsp) genes greatly impaired the capacity of L . pneumophila to form colonies at 25, 17, and 12°C . Indeed, the lsp mutants were completely unable to grow at 12°C . The growth defect of the pilD and lsp mutants was complemented by reintroduction of the corresponding intact gene . Interestingly, the lsp mutants displayed improved growth at 25°C when plated next to a streak of wild-type but not mutant bacteria, implying that a secreted, diffusible factor promotes low-temperature growth . Mutants lacking either the known secreted acid phosphatases, lipases, phospholipase C, lysophospholipase A, or protease grew normally at 25°C, suggesting the existence of a critical, yet-to-be-defined exoprotein(s) . In summary, these data document, for the first time, that L . pneumophila replicates at temperatures below 20°C and that a bacterial type II protein secretion system facilitates growth at low temperatures . Persistence of Mycobacterium paratuberculosis during Manufacture and Ripening of Cheddar Cheese. J. A. Donaghy, 2004.Model Cheddar cheeses were prepared from pasteurized milk artificially contaminated with high 104 to 105 CFU/ml) and low (101 to 102 CFU/ml) inocula of three different Mycobacterium paratuberculosis strains . A reference strain, NCTC 8578, and two strains (806PSS and 796PSS) previously isolated from pasteurized milk for retail sale were investigated in this study . The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar . The survival of M . paratuberculosis cells was monitored over a 27-week ripening period by plating homogenized cheese samples onto HEYM agar medium supplemented with the antibiotics vancomycin, amphotericin B, and nalidixic acid without a decontamination step . A concentration effect was observed in M . paratuberculosis numbers between the inoculated milk and the 1-day old cheeses for each strain . For all manufactured cheeses, a slow gradual decrease in M . paratuberculosis CFU in cheese was observed over the ripening period . In all cases where high levels (>3.6 log10) of M . paratuberculosis were present in 1-day cheeses, the organism was culturable after the 27-week ripening period . The D values calculated for strains 806PSS, 796PSS, and NCTC 8578 were 107, 96, and 90 days, respectively . At low levels of contamination, M . paratuberculosis was only culturable from 27-week-old cheese spiked with strain 806PSS . M . paratuberculosis was recovered from the whey fraction in 10 of the 12 manufactured cheeses . Up to 4% of the initial M . paratuberculosis load was recovered in the culture-positive whey fractions at either the high or low initial inoculum . New Insights into the Developmental History of the Bacterial Cell Division Site. Lawrence Rothfield, 2003. Cloning, Sequencing, and Expression of the Chitinase Gene chiA74 from Bacillus thuringiensis. J. Eleazar Barboza-Corona, 2003.The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5  F' . A sequence of 676 amino acids was deduced when the gene was completely sequenced . A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus . The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%) . Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain . All three domains showed conserved sequences when compared to other bacterial chitinase sequences . A ca . 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain . ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34 . The optimal temperature was estimated at 57.2°C when tested at pH 6 . The potential use of ChiA74 as a synergistic agent, along with the B . thuringiensis insecticidal Cry proteins, is discussed .
Phylogeny and Characterization of Three nifH-Homologous Genes from Paenibacillus azotofixans. Quok-Cheong Choo, 2003.In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation . Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment . This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes . The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively . Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins . NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea .
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