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Gas Channels for NH3: Proteins from Hyperthermophiles Complement an Escherichia coli Mutant. Eric Soupene, 2002.Ammonium transport (Amt) proteins appear to be bidirectional channels for NH3 . The amt genes of the hyperthermophiles Aquifex aeolicus and Methanococcus jannaschii complement enteric amtB mutants for growth at 25 nM NH3 at 37°C . To our knowledge, Amt proteins are the first hyperthermophilic membrane transport proteins shown to be active in a mesophilic bacterium . Despite low expression levels, His-tagged Aquifex Amt could be purified by heating and nickel chelate affinity chromatography . It could be studied genetically in Escherichia coli . Demonstration and Characterization of a Specific Interaction between Gonococcal Transferrin Binding Protein A and TonB. Christopher D. Kenney, 2002.Iron scavenging by Neisseria gonorrhoeae is accomplished by the expression of receptors that are specific for host iron-binding proteins, such as transferrin and lactoferrin . Efficient transferrin-iron acquisition is dependent on the combined action of two proteins, designated TbpA and TbpB . TbpA is a TonB-dependent outer membrane receptor, whereas TbpB is lipid modified and serves to increase the efficiency of transferrin-iron uptake . Both proteins, together or separately, can be isolated from the gonococcal outer membrane by using affinity chromatography techniques . In the present study, we identified an additional protein in transferrin-affinity preparations, which had an apparent molecular mass of 45 kDa . The ability to copurify this protein by transferrin affinity was dependent upon the presence of TbpA and not TbpB . The amino-terminal sequence of the 45-kDa protein was identical to the amino terminus of gonococcal TonB, indicating that TbpA stably interacted with TonB, without the addition of chemical cross-linkers . Using immunoprecipitation, we could recover TbpA-TonB complexes without the addition of transferrin, suggesting that ligand binding was not a necessary prerequisite for TonB interaction . In contrast, a characterized TonB box mutant of TbpA did not facilitate interaction between these two proteins such that complexes could be isolated . We generated an in-frame deletion of gonococcal TonB, which removed 35 amino acids, including a Neisseria-specific, glycine-rich domain . This mutant protein, like the parental TonB, energized TbpA to enable growth on transferrin . Consistent with the functionality of this deletion derivative, TbpA-TonB complexes could be recovered from this strain . The results of the present study thus begin to define the requirements for a functional interaction between gonococcal TbpA and TonB . Purification and Characterization of the PcrA Helicase of Bacillus anthracis. Asma Naqvi, 2003.PcrA is an essential helicase in gram-positive bacteria, and a gene encoding this helicase has been identified in all such organisms whose genomes have been sequenced so far . The precise role of PcrA that makes it essential for cell growth is not known; however, PcrA does not appear to be necessary for chromosome replication . The pcrA gene was identified in the genome of Bacillus anthracis on the basis of its sequence homology to the corresponding genes of Bacillus subtilis and Staphylococcus aureus, with which it shares 76 and 72% similarity, respectively . The pcrA gene of B . anthracis was isolated by PCR amplification and cloning into Escherichia coli . The PcrA protein was overexpressed with a His6 fusion at its amino-terminal end . The purified His-PcrA protein showed ATPase activity that was stimulated in the presence of single-stranded (ss) DNA (ssDNA) . Interestingly, PcrA showed robust 3'  5' as well as 5'3' helicase activities, with substrates containing a duplex region and a 3' or 5' ss poly(dT) tail . PcrA also efficiently unwound oligonucleotides containing a duplex region and a 5' or 3' ss tail with the potential to form a secondary structure . DNA binding experiments showed that PcrA bound much more efficiently to oligonucleotides containing a duplex region and a 5' or 3' ss tail with a potential to form a secondary structure than to those with ssDNAs or duplex DNAs with ss poly(dT) tails . Our results suggest that specialized DNA structures and/or sequences represent natural substrates of PcrA in biochemical processes that are essential for the growth and survival of gram-positive organisms, including B . anthracis .
ClgR, a Novel Regulator of clp and lon Expression in Streptomyces. Audrey Bellier, 2004.The clp genes encoding the Clp proteolytic complex are widespread among living organisms . Five clpP genes are present in Streptomyces. Among them, the clpP1 clpP2 operon has been shown to be involved in the Streptomyces growth cycle, as a mutation blocked differentiation at the substrate mycelium step . Four Clp ATPases have been identified in Streptomyces coelicolor (ClpX and three ClpC proteins) which are potential partners of ClpP1 ClpP2 . The clpC1 gene appears to be essential, since no mutant has yet been obtained . clpP1 clpP2 and clpC1 are important for Streptomyces growth, and a study of their regulation is reported here . The clpP3 clpP4 operon, which has been studied in Streptomyces lividans, is induced in a clpP1 mutant strain, and regulation of its expression is mediated via PopR, a transcriptional regulator . We report here studies of clgR, a paralogue of popR, in S . lividans. Gel mobility shift assays and DNase I footprinting indicate that ClgR binds not only to the clpP1 and clpC1 promoters, but also to the promoter of the Lon ATP-dependent protease gene and the clgR promoter itself . ClgR recognizes the motif GTTCGC-5N-GCG . In vivo, ClgR acts as an activator of clpC1 gene and clpP1 operon expression . Similarly to PopR, ClgR degradation might be ClpP dependent and could be mediated via recognition of the two carboxy-terminal alanine residues . Genetic Organization and Molecular Analysis of the EcoVIII Restriction-Modification System of Escherichia coli E1585-68 and Its Comparison with Isospecific Homologs. Iwona Mruk, 2003.The EcoVIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp) . The two genes were cloned and characterized . The G+C content of the EcoVIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E . coli genomic DNA (50.8%) . The difference suggests that there is a possibility that the EcoVIII R-M system was recently acquired by the genome . The 921-bp EcoVIII endonuclease (R · EcoVIII) gene (ecoVIIIR) encodes a 307-amino-acid protein with an Mr of 35,554 . The convergently oriented EcoVIII methyltransferase (M · EcoVIII) gene (ecoVIIIM) consists of 912 bp that code for a 304-amino-acid protein with an Mr of 33,930 . The exact positions of the start codon AUG were determined by protein microsequencing . Both enzymes recognize the specific palindromic sequence 5'-AAGCTT-3' . Preparations of EcoVIII R-M enzymes purified to homogeneity were characterized . R · EcoVIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5' protruding ends . M · EcoVIII functions as a monomer and modifies the first adenine residue at the 5' end of the specific sequence to N6-methyladenine . These enzymes are thus functionally identical to the corresponding enzymes of the HindIII (Haemophilus influenzae Rd) and LlaCI (Lactococcus lactis subsp . cremoris W15) R-M systems . This finding is reflected by the levels of homology of M · EcoVIII with M · HindIII and M · LlaCI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m6 N-adenine ß-class methyltransferases . The deduced amino acid sequence of R · EcoVIII shows weak homology with its two isoschizomers, R · HindIII (26%) and R · LlaCI (17%) . A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R · EcoVIII (D108X12DXK123), as well as in the primary structures of R · LlaCI and R · HindIII . Polyclonal antibodies raised against R · EcoVIII did not react with R · HindIII, while anti-M · EcoVIII antibodies cross-reacted with M · LlaCI but not with M · HindIII . R · EcoVIII requires Mg(II) ions for phosphodiester bond cleavage . We found that the same ions are strong inhibitors of the M · EcoVIII enzyme . The biological implications of this finding are discussed .
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