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Identification and Localization of Flagellins FlaA and FlaB3 within Flagella of Methanococcus voltae. Sonia L. Bardy, 2002.Methanococcus voltae possesses four flagellin genes, two of which (flaB1 and flaB2) have previously been reported to encode major components of the flagellar filament . The remaining two flagellin genes, flaA and flaB3, are transcribed at lower levels, and the corresponding proteins remained undetected prior to this work . Electron microscopy examination of flagella isolated by detergent extraction of whole cells revealed a curved, hook-like region of varying length at the end of a long filament . Enrichment of the curved region of the flagella resulted in the identification of FlaB3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal sequencing, and the localization of this flagellin to the cell-proximal portion of the flagellum was confirmed through immunoblotting and immunoelectron microscopy with FlaB3-specific antibodies, indicating that FlaB3 likely composes the curved portion of the flagella . This could represent a unique case of a flagellin performing the role of the bacterial hook protein . FlaA-specific antibodies were used in immunoblotting to determine that FlaA is found throughout the flagellar filament. M . voltae cells were transformed with a modified flaA gene containing a hemagglutinin (HA) tag introduced into the variable region . Transformants that had replaced the wild-type copy of the flaA gene with the HA-tagged version incorporated the HA-tagged version of FlaA into flagella which appeared normal by electron microscopy . The Histone-Like Protein HU Does Not Obstruct Movement of T7 RNA Polymerase in Escherichia coli Cells but Stimulates Its Activity. Pilar Morales, 2002.In vivo, RNA polymerases (RNAPs) do not transcribe naked DNA but do transcribe protein-associated DNA . Studies with the model enzyme T7 RNAP have shown that, in eukaryotic cells or in vitro, nucleosomes can inhibit both transcription initiation and elongation . We examine here whether the presence of HU, one of the major histone-like proteins in Escherichia coli cells (the genuine milieu for T7 RNAP) affects its activity . An engineered lac operon fused to the T7 late promoter was introduced into the chromosome of T7 RNAP-producing strains that either overexpress HU or lack it . The flows of RNAP that enter and exit this operon were compared with regard to the content of HU . We found that the fraction of T7 RNAP molecules that do not reach the end of the lac operon (ca . 15%) is the same whether the host cells overexpressed HU or lacked it: thus, the enzyme either freely displaces HU or transcribes through it . However, in these cells, the transcript yield was increased when HU is overexpressed and decreased in the hup mutants, presumably reflecting changes in DNA supercoiling . Thus, in contrast to eukaryotic nucleosomes, HU does not impair T7 RNAP activity but has a stimulatory effect . Finally, our results suggest that HU can also influence mRNA stability in vivo . Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression. Eric Soupene, 2003.Escherichia coli strain MG1655 was chosen for sequencing because the few mutations it carries (ilvG rfb-50 rph-1) were considered innocuous . However, it has a number of growth defects . Internal pyrimidine starvation due to polarity of the rph-1 allele on pyrE was problematic in continuous culture . Moreover, the isolate of MG1655 obtained from the E . coli Genetic Stock Center also carries a large deletion around the fnr (fumarate-nitrate respiration) regulatory gene . Although studies on DNA microarrays revealed apparent cross-regulation of gene expression between galactose and lactose metabolism in the Stock Center isolate of MG1655, this was due to the occurrence of mutations that increased lacY expression and suppressed slow growth on galactose . The explanation for apparent cross-regulation between galactose and N-acetylglucosamine metabolism was similar . By contrast, cross-regulation between lactose and maltose metabolism appeared to be due to generation of internal maltosaccharides in lactose-grown cells and may be physiologically significant . Lactose is of restricted distribution: it is normally found together with maltosaccharides, which are starch degradation products, in the mammalian intestine . Strains designated MG1655 and obtained from other sources differed from the Stock Center isolate and each other in several respects . We confirmed that use of other E . coli strains with MG1655-based DNA microarrays works well, and hence these arrays can be used to study any strain of interest . The responses to nitrogen limitation of two urinary tract isolates and an intestinal commensal strain isolated recently from humans were remarkably similar to those of MG1655 . Leucine-Responsive Regulatory Protein-Mediated Repression of clp (Encoding CS31A) Expression by L-Leucine and L-Alanine in Escherichia coli. Cécile Crost, 2003.CS31A produced by septicemic and diarrheic Escherichia coli belongs to the Pap-regulatory family of adhesive factors, which are under methylation-dependent transcriptional regulation . Common features of operons encoding members of this family include two conserved GATC sites in the upstream regulatory region, and transcriptional regulators homologue to the PapB and PapI proteins . Methylation protection of GATC sites was previously shown to be dependent on the leucine-responsive regulatory protein (Lrp) . Lrp and ClpB, the PapB equivalent, repressed clp basal transcription . A PapI homologue (AfaF) was required together with Lrp to establish the phase variation control, which gave rise to phase-ON cells that expressed CS31A and phase-OFF cells that did not express CS31A . In phase-OFF cells, the GATCdist site was methylated and the GATCprox site was protected from methylation, whereas in phase-ON cells, the inverse situation was found . Unlike Pap fimbriae, CS31A synthesis was dramatically reduced in media containing L-alanine or L-leucine . L-Alanine prevented the OFF-to-ON switch, locking clp expression in the OFF phase, whereas L-leucine repressed transcription without obvious effect on the switch frequency of phase variation . In phase-variable cells, leucine and alanine promoted methylation of GATCdist and methylation protection of GATCprox, increasing the methylation pattern characteristic of repressed cells . Furthermore, alanine prevented the AfaF-dependent methylation protection of GATCdist and thus the appearance of phase-ON cells . In addition, analysis of clp expression in a Lrp-negative background indicated that alanine and leucine also repressed clp transcription by a methylation-independent mechanism . Motility of Colwellia psychrerythraea Strain 34H at Subzero Temperatures. Karen Junge, 2003.We examined the Arctic bacterium Colwellia psychrerythraea strain 34H for motility at temperatures from -1 to -15°C by using transmitted-light microscopy in a temperature-controlled laboratory . The results, showing motility to -10°C, indicate much lower temperatures to be permissive of motility than previously reported (5°C), with implications for microbial activity in frozen environments .
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