Aconitase B is required for optimal growth of Xanthomonas campestris pv. vesicatoria in pepper plants.

The aerobic plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) colonizes the intercellular spaces of pepper and tomato. One enzyme that might contribute to the successful proliferation of Xcv in the host is the iron-sulfur protein aconitase, which catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle and might also sense reactive oxygen species (ROS) and changes in cellular iron levels. Xcv contains three putative aconitases, two of which, acnA and acnB, are encoded by a single chromosomal locus. The focus of this study is aconitase B (AcnB). acnB is co-transcribed with two genes, XCV1925 and XCV1926, encoding putative nucleic acid-binding proteins. In vitro growth of acnB mutants was like wild type, whereas in planta growth and symptom formation in pepper plants were impaired. While acnA, XCV1925 or XCV1926 mutants showed a wild-type phenotype with respect to bacterial growth and in planta symptom formation, proliferation of the acnB mutant in susceptible pepper plants was significantly impaired. Furthermore, the deletion of acnB led to reduced HR induction in resistant pepper plants and an increased susceptibility to the superoxide-generating compound menadione. As AcnB complemented the growth deficiency of an Escherichia coli aconitase mutant, it is likely to be an active aconitase. We therefore propose that optimal growth and survival of Xcv in pepper plants depends on AcnB, which might be required for the utilization of citrate as carbon source and could also help protect the bacterium against oxidative stress.

Unexpected oligomeric structure of the FocA formate channel of Escherichia coli : a paradigm for the formate-nitrite transporter family of integral membrane proteins.

FocA is a predicted formate channel with a deduced mass of 31 kDa that catalyzes the bidirectional movement of formate across the cytoplasmic membrane of Escherichia coli and is the archetype of the formate-nitrite transporter (FNT) family. Overproduced FocA variants with either an N- or a C-terminal Strep-tag increased formate import into anaerobic E. coli cells as determined by the enhanced activity of a single-copy formate-dependent fdhF::lacZ fusion. Using anti-FocA antibodies, we could show that both FocA variants were integrated into the cytoplasmic membrane. Circular dichroism spectroscopy of purified FocA(Strep-N) revealed a high alpha-helical content of 56% consistent with the predicted six transmembrane helices present in the protein. Analysis of the oligomeric state by blue-native polyacrylamide gel electrophoresis revealed FocA to have an unexpected pentameric quaternary structure. This study reports the first isolation of an FNT family member.

Pseudonocardia tetrahydrofuranoxydans sp. nov.

A Gram-positive, rod-shaped, non-endospore-forming but mycelium-forming actinobacterium (strain K1(T)) was isolated from an enrichment culture containing tetrahydrofuran (THF) as the sole source of carbon. On the basis of its G+C content (71.3 mol%) and of 16S rRNA gene sequence similarity studies, strain K1(T) was shown to belong to the family Pseudonocardiaceae, most closely related to Pseudonocardia hydrocarbonoxydans (99.3 %), P. benzenivorans (98.8 %) and P. sulfidoxydans (98.3 %). The 16S rRNA gene sequence similarity to other Pseudonocardia species was less than 97 %. Chemotaxonomic data [major menaquinone MK-8(H(4)); major fatty acids C(16 : 0) iso, C(15 : 0) iso and C(17 : 1)omega6c] supported the affiliation of strain K1(T) to the genus Pseudonocardia. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain K1(T) from the three species P. benzenivorans, P. sulfidoxydans and P. hydrocarbonoxydans, although all four organisms utilized THF. Strain K1(T) represents a novel species, for which the name Pseudonocardia tetrahydrofuranoxydans sp. nov. is proposed, with the type strain K1(T) (=DSM 44239(T)=CIP 109050(T)).

Cloning and characterization of a gene cluster involved in tetrahydrofuran degradation in Pseudonocardia sp. strain K1.

A gene cluster involved in the utilization of tetrahydrofuran by Pseudonocardia sp. strain K1 was cloned and sequenced. Analysis of a 9.2-kb DNA fragment revealed eight ORFs. The genes designated as thmADBC encode the components of a putative monooxygenase exhibiting a high similarity to different binuclear-iron-containing multicomponent monooxygenases. thmA encodes the derived 545-amino-acid oxygenase alpha-subunit, thmD the 360-amino-acid reductase component, thmB the 346-amino-acid oxygenase beta-subunit, and thmC the 117-amino-acid coupling protein. Upstream of the thm genes, an additional ORF ( sad) was identified coding for a protein with high similarity to various aldehyde dehydrogenases. A succinate semialdehyde dehydrogenase activity was specifically expressed in tetrahydrofuran-grown cells. N-terminal sequence analysis of the purified protein revealed that it is encoded by sad. Northern blot analysis indicated that transcription of the thm genes and sad was specifically induced during growth on tetrahydrofuran. Mono-, di- and polycistronic transcripts of these genes were detected. Primer-extension analysis identified transcriptional start sites 37, 61, and 41 bp upstream of the translation start of sad, thmA, and thmB, respectively. Additional ORFs were identified upstream ( orfY) and downstream ( orfZ and aldH) of the thm genes. Furthermore, the data indicated that the analyzed gene cluster was present as a single copy and located on a plasmid.