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Characterization of bcsA Mutations That Bypass Two Distinct Signaling Requirements for Myxococcus xanthus Development. John K. Cusick, 2002.The BsgA protease is required for starvation-induced development in Myxococcus xanthus. Bypass suppressors of a bsgA mutant were isolated to identify genes that may encode additional components of BsgA protease-dependent regulation of development . Strain M951 was isolated following Tn5 mutagenesis of a bsgA mutant and was capable of forming fruiting bodies and viable spores in the absence of the BsgA protease . The Tn5  951 insertion was localized to a gene, bcsA, that encodes a protein that has significant amino acid similarity to a group of recently described flavin-containing monooxygenases involved in styrene catabolism . Mutations in bcsA bypassed the developmental requirements for both extracellular B and C signaling but did not bypass the requirement for A signaling . Bypass of the B-signaling requirement by the bcsA mutation was accompanied by restored expression of a subset of developmentally induced lacZ fusions to the BsgA protease-deficient strain . bcsA mutant cells developed considerably faster than wild-type cells at low cell density and altered transcriptional levels of a developmentally induced, cell-density-regulated gene (4427), suggesting that the bcsA gene product may normally act to inhibit development in a cell-density-regulated fashion . Bypass of the requirements for both B and C signaling by bcsA mutations suggests a possible link between these two genetically, biochemically, and temporally distinct signaling requirements .
Influence of Temperature on tRNA Modification in Archaea: Methanococcoides burtonii (Optimum Growth Temperature [Topt], 23°C) and Stetteria hydrogenophila (Topt, 95°C). Kathleen R. Noon, 2003.We report the first study of tRNA modification in psychrotolerant archaea, specifically in the archaeon Methanococcoides burtonii grown at 4 and 23°C . For comparison, unfractionated tRNA from the archaeal hyperthermophile Stetteria hydrogenophila cultured at 93°C was examined . Analysis of modified nucleosides using liquid chromatography-electrospray ionization mass spectrometry revealed striking differences in levels and identities of tRNA modifications between the two organisms . Although the modification levels in M . burtonii tRNA are the lowest in any organism of which we are aware, it contains more than one residue per tRNA molecule of dihydrouridine, a molecule associated with maintenance of polynucleotide flexibility at low temperatures . No differences in either identities or levels of modifications, including dihydrouridine, as a function of culture temperature were observed, in contrast to selected tRNA modifications previously reported for archaeal hyperthermophiles . By contrast, S . hydrogenophila tRNA was found to contain a remarkable structural diversity of 31 modified nucleosides, including nine methylated guanosines, with eight different nucleoside species methylated at O-2' of ribose, known to be an effective stabilizing motif in RNA . These results show that some aspects of tRNA modification in archaea are strongly associated with environmental temperature and support the thesis that posttranscriptional modification is a universal natural mechanism for control of RNA molecular structure that operates across a wide temperature range in archaea as well as bacteria . Uptake of N,N'-Diacetylchitobiose [(GlcNAc)2] via the Phosphotransferase System Is Essential for Chitinase Production by Serratia marcescens 2170. Taku Uchiyama, 2003.The chiR gene of Serratia marcescens 2170, encoding a LysR-type transcriptional activator, was identified previously as an essential factor for expression of chitinases and a chitin-binding protein, CBP21 . To identify other genes that are essential for chitinase production, transposon mutagenesis with mini-Tn5Km1 was carried out, and 25 mutants that were unable to produce chitinases and CBP21 were obtained . Analysis of the mutated gene of one of the mutants, N22, revealed the presence of a pts operon in this bacterium, and a mutation was found in ptsI in the operon . In addition to its inability to produce chitinase, N22 did not grow well on N-acetyl-D-glucosamine (GlcNAc), (GlcNAc)2, and some other carbon sources, most of which were phosphotransferase system (PTS) sugars . Thus, the inability to produce chitinase was assumed to be caused by the defect in uptake of (GlcNAc)2 via the PTS, considering that (GlcNAc)2 is the minimal substrate for chitinase induction and the major product of chitin hydrolysis by chitinases of this bacterium . To confirm this assumption, the chb operon, encoding the (GlcNAc)2-specific enzyme II permease, was cloned by reference to its Escherichia coli counterpart, and the Serratia chb operon was shown to comprise chbB, chbC, bglA, chbR, and chbG . Disruption of chbC drastically reduced production of chitinases and CBP21 and impaired growth on colloidal chitin . These results indicate that uptake of (GlcNAc)2 is mediated by the PTS and that the (GlcNAc)2-specific enzyme II permease constitutes its major pathway . Since (GlcNAc)2 uptake is essential for induction of chitinases and CBP21 production, (GlcNAc)2 appears to be the key molecule in recognition and utilization of chitin by S . marcescens . Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution. Thomas Bulter, 2003.Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae . Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter) . Together with a 22-fold increase in kcat, the total activity was enhanced 170-fold . Specific activities of MtL mutants toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations . The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host . The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold . Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability . Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity . The molecular mass of MtL expressed in S . cerevisiae is 30% higher than that of the same enzyme expressed in M . thermophila (110 kDa versus 85 kDa) . Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase . This S . cerevisiae expression system makes MtL available for functional tailoring by directed evolution .
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