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Interactions among CotB, CotG, and CotH during Assembly of the Bacillus subtilis Spore Coat. Rita Zilhão, 2004.Spores formed by wild-type Bacillus subtilis are encased ina multilayered protein structure [called the coat] formed bythe ordered assembly of over 30 polypeptides . One polypeptide[CotB] is a surface-exposed coat component that has been usedas a vehicle for the display of heterologous antigens at thespore surface . The cotB gene was initially identified by reversegenetics as encoding an abundant coat component . cotB is predictedto code for a 43-kDa polypeptide, but the form that prevailsin the spore coat has a molecular mass of about 66 kDa [hereindesignated CotB-66] . Here we show that in good agreement withits predicted size, expression of cotB in Escherichia coli resultsin the accumulation of a 46-kDa protein [CotB-46] . Expressionof cotB in sporulating cells of B . subtilis also results ina 46-kDa polypeptide which appears to be rapidly converted intoCotB-66 . These results suggest that soon after synthesis, CotBundergoes a posttranslational modification . Assembly of CotB-66has been shown to depend on expression of both the cotH andcotG loci . We found that CotB-46 is the predominant form foundin extracts prepared from sporulating cells or in spore coatpreparations of cotH or cotG mutants . Therefore, both cotH andcotG are required for the efficient conversion of CotB-46 intoCotB-66 but are dispensable for the association of CotB-46 withthe spore coat . We also show that CotG does not accumulate insporulating cells of a cotH mutant, suggesting that CotH [ora CotH-controlled factor] stabilizes the otherwise unstableCotG . Thus, the need for CotH for formation of CotB-66 resultsin part from its role in the stabilization of CotG . We alsofound that CotB-46 is present in complexes with CotG at thetime when formation of CotB-66 is detected . Moreover, usinga yeast two-hybrid system, we found evidence that CotB directlyinteracts with CotG and that both CotB and CotG self-interact.We suggest that an interaction between CotG and CotB is requiredfor the formation of CotB-66, which may represent a multimericform of CotB. The Nitrate Reductase and Nitrite Reductase Operons and the narT Gene of Staphylococcus carnosus Are Positively Controlled by the Novel Two-Component System NreBC. I. Fedtke, 2002.In Staphylococcus carnosus, the nreABC (for nitrogen regulation) genes were identified and shown to link the nitrate reductase operon (narGHJI) and the putative nitrate transporter gene narT . An nreABC deletion mutant, m1, was dramatically affected in nitrate and nitrite reduction and growth . Transcription of narT, narGHJI, and the nitrite reductase (nir) operon was severely reduced even when cells were cultivated anaerobically without nitrate or nitrite . nreABC transcripts were detected when cells were grown aerobically or anaerobically with or without nitrate or nitrite . NreA is a GAF domain-containing protein of unknown function . In vivo and in vitro studies showed that NreC is phosphorylated by NreB and that phospho-NreC specifically binds to a GC-rich palindromic sequence to enhance transcription initiation . This binding motif was found at the narGHJI, nir, and narT promoters but not at the moeB promoter . NreB is a cytosolic protein with four N-terminal cysteine residues . The second cysteine residue was shown to be important for NreB function . In vitro autophosphorylation of NreB was not affected by nitrate, nitrite, or molybdate . The nir promoter activity was iron dependent . The data provide evidence for a global regulatory system important for aerobic and anaerobic metabolism, with NreB and NreC forming a classical two-component system and NreB acting as a sensor protein with oxygen as the effector molecule . Elevated Abundance of Bacteriophage Infecting Bacteria in Soil. Kevin E. Ashelford, 2003.Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 x 107 g-1), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts . Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold . So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 x 108 g-1, which is equivalent to 4% of the total population of bacteria . This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria . This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population . Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil .
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