Porin from the halophilic species Ectothiorhodospira vacuolata: cloning, structure of the gene and comparison with other porins

The gene coding for the anion-specific porin of the halophilic eubacterium Ectothiorhodospira (Ect.) vacuolata was cloned and sequenced, the first such gene so analyzed from a purple sulfur bacterium. It encodes a precursor protein consisting of 374 amino acid (aa)-residues including a signal peptide of 22-aa residues. Comparison with aa sequences of porins from several other members of the Proteobacteria revealed little homology. Only two regions showed local homology with the previously sequenced porins of Neisseria species, Comamonas acidovorans, Bordetella pertussis, Alcaligenes eutrophus, and Burkholderia cepacia. Genomic Southern blot hybridization studies were carried out with a probe derived from the 5′ end of the gene coding for the porin of Ect. vacuolata. Two related species, Ect. haloalkaliphila and Ect. shaposhnikovii, exhibited a clear signal, while the extremely halophilic bacterium Halorhodospira (Hlr.) halophila (formerly Ect. halophila) did not show any cross-hybridization even at low stringency. This result is in good accordance with a recently proposed reassignment within the family Ectothiorhodospiraceae, which included the separation of the extremely halophilic species into the new genus Halorhodospira

Insight into the induction mechanism of the GntR/HutC bacterial transcription regulator YvoA

YvoA is a GntR/HutC transcription regulator from Bacillus subtilis implicated in the regulation of genes from the N-acetylglucosamine-degrading pathway. Its 2.4-Å crystal structure reveals a homodimeric assembly with each monomer displaying a two-domain fold. The C-terminal domain, which binds the effector N-acetylglucosamine-6-phosphate, adopts a chorismate lyase fold, whereas the N-terminal domain contains a winged helix–turn–helix DNA-binding domain. Isothermal titration calorimetry and site-directed mutagenesis revealed that the effector-binding site in YvoA coincides with the active site of related chorismate lyase from Escherichia coli. The characterization of the DNA- and effector-binding properties of two disulfide-bridged mutants that lock YvoA in two distinct conformational states provides for the first time detailed insight into the allosteric mechanism through which effector binding modulates DNA binding and, thereby regulates transcription in a representative GntR/HutC family member. Central to this allosteric coupling mechanism is a loop-to-helix transition with the dipole of the newly formed helix pointing toward the phosphate of the effector. This transition goes in hand with the emergence of internal symmetry in the effector-binding domain and, in addition, leads to a 122° rotation of the DNA-binding domains that is best described as a jumping-jack-like motion

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder–order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates

A Functional mobA Gene for Molybdopterin Cytosine Dinucleotide Cofactor Biosynthesis Is Required for Activity and Holoenzyme Assembly of the Heterotrimeric Nicotine Dehydrogenases of Arthrobacter nicotinovorans

Two Arthrobacter nicotinovorans molybdenum enzymes hydroxylate the pyridine ring of nicotine. Molybdopterin cytosine dinucleotide (MCD) was determined to be a cofactor of these enzymes. A mobA gene responsible for the formation of MCD could be identified and its function shown to be required for assembly of the heterotrimeric molybdenum enzymes

Genes Involved in Anaerobic Metabolism of Phenol in the Bacterium Thauera aromatica

Genes involved in the anaerobic metabolism of phenol in the denitrifying bacterium Thauera aromatica have been studied. The first two committed steps in this metabolism appear to be phosphorylation of phenol to phenylphosphate by an unknown phosphoryl donor (“phenylphosphate synthase”) and subsequent carboxylation of phenylphosphate to 4-hydroxybenzoate under release of phosphate (“phenylphosphate carboxylase”). Both enzyme activities are strictly phenol induced. Two-dimensional gel electrophoresis allowed identification of several phenol-induced proteins. Based on N-terminal and internal amino acid sequences of such proteins, degenerate oligonucleotides were designed to identify the corresponding genes. A chromosomal DNA segment of about 14 kbp was sequenced which contained 10 genes transcribed in the same direction. These are organized in two adjacent gene clusters and include the genes coding for five identified phenol-induced proteins. Comparison with sequences in the databases revealed the following similarities: the gene products of two open reading frames (ORFs) are each similar to either the central part and N-terminal part of phosphoenolpyruvate synthases. We propose that these ORFs are components of the phenylphosphate synthase system. Three ORFs showed similarity to the ubiD gene product, 3-octaprenyl-4-hydroxybenzoate carboxy lyase; UbiD catalyzes the decarboxylation of a 4-hydroxybenzoate analogue in ubiquinone biosynthesis. Another ORF was similar to the ubiX gene product, an isoenzyme of UbiD. We propose that (some of) these four proteins are involved in the carboxylation of phenylphosphate. A 700-bp PCR product derived from one of these ORFs cross-hybridized with DNA from different Thauera and Azoarcus strains, even from those which have not been reported to grow with phenol. One ORF showed similarity to the mutT gene product, and three ORFs showed no strong similarities to sequences in the databases. Upstream of the first gene cluster, an ORF which is transcribed in the opposite direction codes for a protein highly similar to the DmpR regulatory protein of Pseudomonas putida. DmpR controls transcription of the genes of aerobic phenol metabolism, suggesting a similar regulation of anaerobic phenol metabolism by the putative regulator