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Candida glabrata erg1 Mutant with Increased Sensitivity to Azoles and to Low Oxygen Tension. Huei-Fung Tsai, 2004.A Candida glabrata erg1 (Cgerg1) mutant, CgTn201S, was identified by transposon mutagenesis and by increased fluconazole susceptibility . CgERG1 encodes a 489-amino-acid protein which, on the basis of its homology with Saccharomyces cerevisiae ERG1, is a squalene epoxidase essential for ergosterol synthesis . Interruption following codon 475 of CgErg1p decreased the ergosterol content by 50%; caused accumulation of the squalene precursor; increased the levels of susceptibility to fluconazole, itraconazole, and terbinafine; increased the level of resistance to amphotericin B; increased the levels of rhodamine 6G and [3H]-fluconazole uptake; reduced the level of growth; and blocked growth under conditions of low oxygen tension . In addition, CgTn201S efficiently took up exogenous cholesterol from cholesterol-containing serum . Cholesterol constituted 34% of the extractable sterols in CgTn201S when it was grown aerobically on serum-containing medium . Under the same conditions, C . albicans contained only 0.1 to 1.2% cholesterol . Exogenous sterols also restored growth under conditions of low oxygen tension . Finally, complementation of the Cgerg1 mutation restored the levels of [3H]fluconazole uptake and drug susceptibility to wild-type levels . An Antisense RNA-Mediated Transcriptional Attenuation Mechanism Functions in Escherichia coli. Sabine Brantl, 2002.Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501 . In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids . In this work we asked whether such a mechanism can be supported by Escherichia coli . The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host . In vivo lacZ reporter gene assays showed that the antisense RNAs of these plasmids can inhibit lacZ expression and that most of this effect can be accounted for by reduced mRNA readthrough . Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate antisense RNA, as expected for this mechanism . Similarly, both antisense RNAs induced premature termination of their cognate target RNAs in an E . coli in vitro transcription system, whereas the noncognate antisense RNAs had no effect . Thus, this report shows that antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g., Bacillus subtilis . Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed . Role of the RecBCD Recombination Pathway in Salmonella Virulence. David A. Cano, 2002.Mutants of Salmonella enterica lacking the RecBC function are avirulent in mice and unable to grow inside macrophages (N . A . Buchmeier, C . J . Lipps, M . Y . H . So, and F . Heffron, Mol . Microbiol . 7:933–936, 1993) . The virulence-related defects of RecBC- mutants are not suppressed by sbcB and sbcCD mutations, indicating that activation of the RecF recombination pathway cannot replace the virulence-related function(s) of RecBCD . Functions of the RecF pathway such as RecJ and RecF are not required for virulence . Since the RecBCD pathway, but not the RecF pathway, is known to participate in the repair of double-strand breaks produced during DNA replication, we propose that systemic infection by S . enterica may require RecBCD-mediated recombinational repair to prime DNA replication inside phagocytes . Mutants lacking both RecD and RecJ are also attenuated in mice and are unable to proliferate in macrophages, suggesting that exonucleases V and IX provide alternative functions for RecBCD-mediated recombinational repair during Salmonella infection . Polar Targeting of DivIVA in Bacillus subtilis Is Not Directly Dependent on FtsZ or PBP 2B. Leendert W. Hamoen, 2003.DivIVA is involved in Bacillus subtilis cell division and is located at the cell poles . Previous experiments suggested that the cell division proteins FtsZ and PBP 2B are required for polar targeting of DivIVA . By using outgrowing spores, we show that DivIVA accumulates at the cell poles independent of the presence of FtsZ or PBP 2B . Transcription of Clostridium cellulovorans Cellulosomal Cellulase and Hemicellulase Genes. Sung Ok Han, 2003.Transcription of the cellulosomal cellulase/hemicellulase genes of Clostridium cellulovorans has been investigated by Northern blot, reverse transcriptase PCR (RT-PCR), primer extension, and S1 nuclease analysis . Northern hybridizations revealed that the cellulosomal cbpA gene cluster is transcribed as polycistronic mRNAs of 8 and 12 kb . The 8-kb mRNA coded for cbpA and exgS, and the 12-kb mRNA coded for cbpA, exgS, engH, and engK . The sizes of the mRNAs were about 3 kb for engE, 1.8 kb for manA, 2.7 kb for xynA, and 4 kb for pelA, indicating monocistronic transcription of these genes . Primer extension and S1 nuclease analysis of C . cellulovorans RNA showed that the transcriptional start sites of cbpA, engE, manA, and hbpA were located 233, 97, 64, and 61 bp upstream from the first nucleotide of each of the respective translation initiation codons . Alignment of the cbpA, engE, manA, and hbpA promoter regions provided evidence for highly conserved sequences that exhibited strong similarity to the  A consensus promoter sequences of gram-positive bacteria .
Role of  B in Regulating the Compatible Solute Uptake Systems of Listeria monocytogenes: Osmotic Induction of opuC Is
B Dependent.Katy R. Fraser, 2003.The regulation of the compatible solute transport systems in Listeria monocytogenes by the stress-inducible sigma factor B was investigated . Using wild-type strain 10403S and an otherwise isogenic strain carrying an in-frame deletion in sigB, we have examined the role of
B in regulating the ability of cells to utilize betaine and carnitine during growth under conditions of hyperosmotic stress . Cells lacking
B were defective for the utilization of carnitine but retained the ability to utilize betaine as an osmoprotectant . When compatible solute transport studies were performed, the initial rates of uptake of both betaine and carnitine were found to be reduced in the sigB mutant; carnitine transport was almost abolished, whereas betaine transport was reduced to approximately 50% of that of the parent strain . Analysis of the cytoplasmic pools of compatible solutes during balanced growth revealed that both carnitine and betaine steady-state pools were reduced in the sigB mutant . Transcriptional reporter fusions to the opuC (which encodes an ABC carnitine transporter) and betL (which encodes an a secondary betaine transporter) operons were generated by using a promoterless copy of the gus gene from Escherichia coli . Measurement of ß-glucuronidase activities directed by opuC-gus and betL-gus revealed that transcription of opuC is largely
B dependent, consistent with the existence of a potential
B consensus promoter motif upstream from opuCA . The transcription of betL was found to be sigB independent . Reverse transcriptase PCR experiments confirmed these data and indicated that the transcription of all three known compatible solute uptake systems (opuC, betL, and gbu), as well as a gene that is predicted to encode a compatible solute transporter subunit (lmo1421) is induced in response to elevated osmolarity . The osmotic induction of opuCA and lmo1421 was found to be strongly
B dependent . Together these observations suggest that
B plays a major role in the regulation of carnitine utilization by L . monocytogenes but is not essential for betaine utilization by this pathogen .
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