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Translational Coupling to an Upstream Gene Promotes Folding of the Mycobacterial Plasmid pAL5000 Replication Protein RepB and Thereby Its Origin Binding Activity. Abhijit Basu, 2004.In the mycobacterial plasmid pAL5000 replication region, the replication genes repA and repB are organized in an operon . Earlier, a RepB-dependent origin binding activity was detected in Escherichia coli cells expressing the repA-repB operon . This activity was maximal when expression of the two genes was coupled (A . Basu, M . Chawla-Sarkar, S . Chakrabarti, and S . K . Das Gupta, J . Bacteriol . 184:2204-2214, 2002) . In this study we have shown that translational coupling makes a significant difference in the structure and function of RepB . When repB expression was coupled to repA, the polypeptide folded into an active structure (referred to as RepB*), which possessed higher helical content than RepB expressed independently . RepB* could also be distinguished from the less active RepB on the basis of sensitivity to OmpT, an outer membrane protease of E . coli: RepB* was sensitive to the protease, whereas RepB was resistant . Similar conformational differences between RepB* and RepB could be observed when repA was replaced with an unrelated gene, malE (encoding maltose binding protein) . These results show that translational coupling of repB to an upstream gene is necessary for better folding and origin binding activity . It is speculated that in coupled systems where translation machinery is passed on from the upstream to the downstream open reading frame, cotranslational folding of the polypeptide expressed from the downstream open reading frame is enhanced due to increased folding competence of translationally primed ribosomes . New Recombination Methods for Sinorhizobium meliloti Genetics. Brent L. House, 2004.The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes . A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative  -proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system . Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S . meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense . In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes . Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S . meliloti, predicted to code for a denitrification pathway . The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots .
Rapid Estimation of Numbers of Fecal Bacteroidetes by Use of a Quantitative PCR Assay for 16S rRNA Genes. Linda K. Dick, 2004. IntI2 Integron Integrase in Tn7. Karin Hansson, 2002.Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination . Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21 . A second class of integron is found on transposon Tn7 and its relatives . We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon . This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes . The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1 . In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid . The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons . We also observed a fourth excisable cassette downstream of those described previously in Tn7 . The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids . IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies . The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7 . However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E . In Vivo Evidence for TonB Dimerization. Annette Sauter, 2003.TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and  80 . To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx . ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (Pctx::lacZ) . Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated Pctx::lacZ transcription . Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation . The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system . This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition . Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined . The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail . They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors .
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