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TeiR, a LuxR-Type Transcription Factor Required for Testosterone Degradation in Comamonas testosteroni. José Luis Pruneda-Paz, 2004.We have identified a new steroid-inducible gene [designatedteiR [testosterone-inducible regulator]] in Comamonas testosteroni that is required for testosterone degradation . Nucleotide sequence analysis of teiR predicts a 391-amino-acid protein which shows homology between residues 327 and 380 [C-terminal domain] tothe LuxR helix-turn-helix DNA binding domain and between residues192 and 227 to the PAS sensor domain . This domain distributionresembles that described for TraR, a specific transcriptionalregulator involved in quorum sensing in Agrobacterium tumefaciens.Analysis of the gene expression indicated that teiR is tightlycontrolled at the transcriptional level by the presence of testosteronein the culture medium . A teiR-disrupted mutant strain was completely unable to use testosterone as the sole carbon and energy source.In addition, the expression of several steroid-inducible geneswas abolished in this mutant . Northern blot assays revealedthat teiR is required for full expression of sip48-ß-HSDgene mRNA [encoding a steroid-inducible protein of 48 kDa and 3ß-17ß-hydroxysteroid dehydrogenase] andalso of other steroid degradation genes, including those encoding3alpha-hydroxysteroid dehydrogenase, delta5-3-ketoisomerase, 3-oxo-steroid delta1-dehydrogenase, and 3-oxo-steroid delta4-[5alpha]-dehydrogenase enzymes.Moreover, when teiR was provided to the teiR-disrupted strainin trans, the transcription level of these genes was restored.These results indicate that TeiR positively regulates the transcriptionof genes involved in the initial enzymatic steps of steroiddegradation in C . testosteroni. Discovery of the Novel Candidate Phylum "Poribacteria" in Marine Sponges. Lars Fieseler, 2004.Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl . We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid . In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent . Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named "Poribacteria", to acknowledge the affiliation of the new bacterium with sponges . The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA . Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner . PCR screening with "Poribacteria"-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative . In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes . Protease-Deficient DegP Suppresses Lethal Effects of a Mutant OmpC Protein by Its Capture. Maria CastilloKeller, 2003.The expression of assembly-defective outer membrane proteins can confer lethality if they are not degraded by envelope proteases . We report here that the expression of a mutant OmpC protein, OmpC2Cys, which forms disulfide bonds in the periplasm due to the presence of two non-native cysteine residues, is lethal in cells lacking the major periplasmic protease, DegP . This lethality is not observed in dsbA strains that have diminished ability to form periplasmic disulfide bonds . Our data show that this OmpC2Cys-mediated lethality in a degP::Kmr dsbA+ background can be reversed by a DegP variant, DegPS210A, that is devoid of its proteolytic activity but retains its reported chaperone activity . However, DegPS210A does not reverse the lethal effect of OmpC2Cys by correcting its assembly but rather by capturing misfolded mutant OmpC polypeptides and thus removing them from the assembly pathway . Displacement of OmpC2Cys by DegPS210A also alleviates the negative effect that the mutant OmpC protein has on wild-type OmpF . Why Is Carbonic Anhydrase Essential to Escherichia coli?. Christophe Merlin, 2003.The can (previously yadF) gene of Escherichia coli encodes a ß-class carbonic anhydrase (CA), an enzyme which interconverts CO2 and bicarbonate.Various essential metabolic processes require either CO2 or bicarbonate and, although carbon dioxide and bicarbonate spontaneously equilibrate in solution, the low concentration of CO2 in air and its rapid diffusion from the cell mean that insufficient bicarbonate is spontaneously made in vivo to meet metabolic and biosynthetic needs . We calculate that demand for bicarbonate is 103- to 104-fold greater than would be provided by uncatalyzed intracellular hydration and that enzymatic conversion of CO2 to bicarbonate is therefore necessary for growth . We find that can expression is ordinarily required for growth in air . It is dispensable if the atmospheric partial pressure of CO2 is high or during anaerobic growth in a closed vessel at low pH, where copious CO2 is generated endogenously . CynT, the single E . coli Can paralog, can, when induced with azide, replace Can; also, the  -CA from Methanosarcina thermophila can at least partially replace it . Expression studies showed that can transcription does not appear to respond to carbon dioxide concentration or to be autoregulated . However, can expression is influenced by growth rate and the growth cycle; it is expressed best in slow-growing cultures and at higher culture densities . Expression can vary over a 10-fold range during the growth cycle and is also elevated during starvation or heat stress .
Assembly of the MexAB-OprM Multidrug Efflux System of Pseudomonas aeruginosa: Identification and Characterization of Mutations in mexA Compromising MexA Multimerization and Interaction with MexB. Dominic Nehme, 2004.The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of P . aeruginosa is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer . A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization . Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N- and C-terminal halves of the protein . Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization) . In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of Escherichia coli, AcrA, that is implicated in binding to its RND component, AcrB (C . A . Elkins and H . Nikaido, J . Bacteriol . 185:5349-5356, 2003) . Given the noted specificity of MFP-RND interaction in this family of pumps, residues unique to MexA may well be important for and define the MexA interaction with its RND component, MexB . Still, all but one of the MexA mutations studied compromised MexA-MexB association, suggesting that native structure and/or proper assembly of the protein may be necessary for this . Cyanobacteria Associated with Coral Black Band Disease in Caribbean and Indo-Pacific Reefs. Jorge Frias-Lopez, 2003.For 30 years it has been assumed that a single species of cyanobacteria, Phormidium corallyticum, is the volumetrically dominant component of all cases of black band disease (BBD) in coral . Cyanobacterium-specific 16S rRNA gene primers and terminal restriction fragment length polymorphism analyses were used to determine the phylogenetic diversity of these BBD cyanobacteria on coral reefs in the Caribbean and Indo-Pacific Seas . These analyses indicate that the cyanobacteria that inhabit BBD bacterial mats collected from the Caribbean and Indo-Pacific Seas belong to at least three different taxa, despite the fact that the corals in each case exhibit similar signs and patterns of BBD mat development . PAC1, a pH-Regulatory Gene from Fusarium verticillioides. Joseph E. Flaherty, 2003.Fumonisins are a group of mycotoxins that contaminate maize and cause leukoencephalomalacia in equine, pulmonary edema in swine, and promote cancer in mice . Fumonisin biosynthesis in Fusarium verticillioides is repressed by nitrogen and alkaline pH . We cloned a PACC-like gene (PAC1) from F . verticillioides . PACC genes encode the major transcriptional regulators of several pH-responsive pathways in other filamentous fungi . In Northern blot analyses, a PAC1 probe hybridized to a 2.2-kb transcript present in F . verticillioides grown at alkaline pH . A mutant of F . verticillioides with a disrupted PAC1 gene had severely impaired growth at alkaline pH . The mutant produced more fumonisin than the wild type when grown on maize kernels and in a synthetic medium buffered at an acidic pH, 4.5 . The mutant, but not the wild type, also produced fumonisin B1 when mycelia were resuspended in medium buffered at an alkaline pH, 8.4 . Transcription of FUM1, a gene involved in fumonisin biosynthesis, was correlated with fumonisin production . We conclude that PAC1 is required for growth at alkaline pH and that Pac1 may have a role as a repressor of fumonisin biosynthesis under alkaline conditions .
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