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Amplification of the Tetracycline Resistance Determinant of pAM  1 in Enterococcus faecalis Requires a Site-Specific Recombination Event Involving Relaxase.M. Victoria Francia, 2002.The small multicopy plasmid pAM 1 (9.75 kb) encoding tetracycline resistance in Enterococcus faecalis is known to generate tandem repeats of a 4.1-kb segment carrying tet(L) when cells are grown extensively in the presence of tetracycline . Here we show that the initial (rate-limiting) step involves a site-specific recombination event involving plasmid-encoded relaxase activity acting at two recombination sequences (RS1 and RS2) that flank the tet determinant . We also present the complete nucleotide sequence of pAM1 .
Interactions of FliJ with the Salmonella Type III Flagellar Export Apparatus. Gillian M. Fraser, 2003.FliJ, a 17-kDa protein, is a soluble component of the Salmonella type III flagellar protein export system that has antiaggregation properties and several other characteristics that suggest it may have a chaperone-like function . We have now examined this protein in detail . Ten-amino-acid scanning deletions covering the entire 147-amino-acid sequence were tested for complementation of a fliJ null strain; only the first and last deletions complemented . A few of the deletions, especially towards the C terminus, exerted a dominant negative effect on wild-type cells, indicating that they were actively interfering with function . Two truncated versions of FliJ, representing its N- and C-terminal halves, failed to complement and were not dominant . We tested for FliJ self-association by several techniques . Size-exclusion chromatography (Superdex 200) indicated an apparent molecular mass of around 50 kDa, which could reflect either multimerization or an elongated shape or both . Multiangle light scattering gave a peak value of 20 kDa, close to the molecular mass of the monomer . Analytical ultracentrifugation gave evidence for weak self-association as a trimer or tetramer . It was known from previous studies that FliJ interacts with the N-terminal region of FliH, a negative regulator of the ATPase FliI . Using both truncation and deletion versions of FliJ, we now show that it is its C-terminal region that is responsible for this interaction . We also show that FliJ interacts with the soluble cytoplasmic domain of the largest membrane component of the export apparatus, FlhA; although small deletions in FliJ did not interfere with the association, both truncated versions failed to associate, indicating that a substantial amount of the central region of the FliJ sequence participates in the association . We present a model summarizing these multiple interactions . New Thermosensitive Delivery Vector and Its Use To Enable Nisin-Controlled Gene Expression in Lactobacillus gasseri. T. Neu, 2003.Derivatives of a cryptic plasmid from Lactobacillus curvatus showed temperature-sensitive replication in thermophilic lactobacilli . The thermosensitive replicon was used to construct the new delivery vector pTN1, which allows site-specific replacement of chromosomal DNA sequences . pTN1 carries an erythromycin resistance marker suitable for selection of single-copy integrants and replicates readily at 35°C, whereas replication is efficiently shut down at 42°C . To demonstrate the functionality of pTN1, the signal transduction genes (nisRK) of the nisin-controlled expression system were integrated downstream of the pepN gene into the chromosome of Lactobacillus gasseri . In the resulting strain, UKLbg1, expression of nisRK was likely driven by cotranscription with pepN and enabled nisin-dependent induction of a fusion of a reporter gene (pepI) to the nisA promoter . The induction rates were correlated with the amount of nisin used, and maximum pepI expression was achieved with nisin concentrations (above 25 ng/ml) at which growth of the bacteria was already inhibited . Catabolism of Arylboronic Acids by Arthrobacter nicotinovorans Strain PBA. Ana C. Negrete-Raymond, 2003.Arthrobacter sp . strain PBA metabolized phenylboronic acid to phenol . The oxygen atom in phenol was shown to be derived from the atmosphere using 18O2 . 1-Naphthalene-, 2-naphthalene-, 3-cyanophenyl-, 2,5-fluorophenyl-, and 3-thiophene-boronic acids were also transformed to monooxygenated products . The oxygen atom in the product was bonded to the ring carbon atom originally bearing the boronic acid substituent with all the substrates tested .
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