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Genetic Analysis of the Cell Division Protein FtsI (PBP3): Amino Acid Substitutions That Impair Septal Localization of FtsI and Recruitment of FtsN. Mark C. Wissel, 2004.FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of several proteins that localize to the septal ring . FtsI comprises a small cytoplasmic domain, a transmembrane helix, a noncatalytic domain of unknown function, and a catalytic (transpeptidase) domain . The last two domains reside in the periplasm . We used PCR to randomly mutagenize ftsI, ligated the products into a green fluorescent protein fusion vector, and screened  7,500
transformants for gfp-ftsI alleles that failed to complement
an ftsI null mutant . Western blotting and penicillin-binding
assays were then used to weed out proteins that were unstable,
failed to insert into the cytoplasmic membrane, or were defective in
catalysis . The remaining candidates were tested for septal
localization and ability to recruit another division protein, FtsN,
to the septal ring . Mutant proteins severely defective in
localization to the septal ring all had lesions in one of three amino
acids—R23, L39, or Q46—that are in or near the transmembrane helix
and implicate this region of FtsI in septal localization . Mutant FtsI
proteins defective in recruitment of FtsN all had lesions in one of
eight residues in the noncatalytic domain . The most interesting of
these mutants had lesions at G57, S61, L62, or R210 . Although
separated by
150
residues in the primary sequence, these amino acids are close
together in the folded protein and might constitute a site of
FtsI-FtsN interaction .
A Two-Component Regulator Mediates Population-Density-Dependent Expression of the Bradyrhizobium japonicum Nodulation Genes. John Loh, 2002.Bradyrhizobium japonicum nod gene expression was previously shown to be population density dependent . Induction of the nod genes is highest at low culture density and repressed at high population densities . This repression involves both NolA and NodD2 and is mediated by an extracellular factor found in B . japonicum conditioned medium . NolA and NodD2 expression is maximal at high population densities . We demonstrate here that a response regulator, encoded by nwsB, is required for the full expression of the B . japonicum nodYABC operon . In addition, NwsB is also required for the population-density-dependent expression of both nolA and nodD2 . Expression of nolA and nodD2 in the nwsB mutant remained at a basal level, even at high culture densities . The nwsB defect could be complemented by overexpression of a second response regulator, NodW . Consistent with the fact that NolA and NodD2 repress nod gene expression, the expression of a nodY-lacZ fusion in the nwsB mutant was unaffected by culture density . In plant assays with GUS fusions, nodules infected with the wild type showed no nodY-GUS expression . In contrast, nodY-GUS expression was not repressed in nodules infected with the nwsB mutant . Nodule competition assays between the wild type and the nwsB mutant revealed that the addition of conditioned medium resulted in a competitive advantage for the nwsB mutant . Measurement of Adhesive Forces between Individual Staphylococcus aureus MSCRAMMs and Protein-Coated Surfaces by Use of Optical Tweezers. Kathryn H. Simpson, 2003.The force required to rupture bonds between individual Staphylococcus aureus MSCRAMMs and surfaces coated with extracellular matrix molecules has been quantified by using optical tweezers . The observed binding forces between fibrinogen or fibronectin and S . aureus MSCRAMMs occurred as an approximate integer multiple of 20 or 25 pN, respectively . Application of the shsp Gene, Encoding a Small Heat Shock Protein, as a Food-Grade Selection Marker for Lactic Acid Bacteria. Hassan A. M. El Demerdash, 2003.Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A . Geis, H . A . M . El Demerdash, and K . J . Heller, Plasmid 50:53-69, 2003) . Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature . Transformation of the cloned shsp gene into S . thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60°C or pH 3.5 and in the ability to grow at 52°C . A food-grade cloning system for S . thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed . This approach allowed selection after transfer of native and recombinant shsp plasmids into different S . thermophilus and Lactococcus lactis strains . Using a recombinant plasmid carrying an erythromycin resistance (Emr) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells . The average transformation rates in S . thermophilus (when we were selecting for heat resistance) were determined to be 2.4 x 104 and 1.0 x 104 CFU/0.5 µg of DNA, with standard deviations of 0.54 x 104 and 0.32 x 104, for shsp and Emr selection, respectively . When we selected for pH resistance, the average transformation rates were determined to be 2.25 x 104 and 3.8 x 103 CFU/0.5 µg of DNA, with standard deviations of 0.63 x 104 and 3.48 x 103, for shsp and Emr selection, respectively . The applicability of shsp as a selection marker was further demonstrated by constructing S . thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S . thermophilus plasmid as a functional trait .
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