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Archives of Microbiology (v.169, #5)

The oxygen-responsive NIFL-NIFA complex: a novel two-component regulatory system controlling nitrogenase synthesis in γ-Proteobacteria by R. Dixon (pp. 371-380).

The high energetic requirements for nitrogen fixation and the extreme oxygen sensitivity of the nitrogenase enzyme impose physiological constraints on diazotrophy that necessitate stringent control of nitrogen fixation (nif) gene expression at the transcriptional level. In the γ-subdivision of the Proteobacteria, this control is maintained by a regulatory complex comprising an enhancer-binding protein (NIFA), which activates transcription at σ^N-dependent nif (nitrogen fixation)promoters, and a sensor protein (NIFL), which inhibits NIFA activity in response to fixed nitrogen and external concentrations of molecular oxygen. Inhibition of NIFA activity by NIFL apparently requires stoichiometric amounts of the two proteins, implying direct protein-protein interaction rather than catalytic modulation of NIFA activity. NIFL contains FAD as a prosthetic group and is a novel type of flavoprotein in which the oxidation state of the bound flavin acts as a molecular switch to control transcriptional activation by NIFA. The FAD-binding domain of NIFL contains a motif common to a large family of redox sensory proteins. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP, suggesting that formation of the inhibitory complex might be regulated by the ATP/ ADP ratio. Proposed mechanisms for the inhibition of NIFA activity by NIFL are beginning to emerge.

Keywords: Key words Nitrogen fixation; Transcriptional; activation; Flavoprotein; Redox response; Oxygen; control; Nucleotides; Protein-protein complex; Domain; structure; nif regulation

Characterization of a new Agrobacterium tumefaciens strain from alfalfa (Medicago sativa L.) by Jeffrey D. Palumbo; Donald A. Phillips; C. I. Kado (pp. 381-386).

Agrobacterium tumefaciens strain 1D1609 is reported here as the first field isolate from alfalfa (Medicago sativa L.). Unlike well-characterized A. tumefaciens strains such as C58 and Ach5, strain 1D1609 is highly virulent on alfalfa and has a distinctive host range. Interestingly, strain 1D1609 is naturally resistant to kanamycin and spectinomycin. The Ti plasmid in strain 1D1609 is an octopine-type; thus, tumors formed by strain 1D1609 synthesize octopine, which is utilized by the bacterium as a sole carbon source. Reciprocal exchange of Ti plasmids between strains 1D1609 and C58 showed that both chromosomal and Ti plasmid genes in strain 1D1609 contribute specifically to tumor formation on alfalfa. In addition, the nondormant CUF101 alfalfa cultivar from which strain 1D1609 was isolated was significantly more susceptible to all Agrobacterium strains tested than was the dormant Agate cultivar.

Keywords: Key words Agrobacterium tumefaciens; Alfalfa; Medicago sativa; Ti plasmid; Virulence; Octopine

Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region by José Manuel Guillamón; Josepa Sabaté; Eladio Barrio; Josep Cano; A. Querol (pp. 387-392).

In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation.

Keywords: Key words Wine yeast identification; rRNA gene; RFLP-PCR; Anamorph state; Teleomorph state

The Helicobacter felis ftsH gene encoding an ATP-dependent metalloprotease can replace the Escherichia coli homologue for growth and phage λ lysogenization by Klaus Melchers; Thomas Wiegert; Anita Buhmann; Stefan Postius; Klaus P. Schäfer; W. Schumann (pp. 393-396).

Cloning and sequencing of an approximately 6.0-kb chromosomal DNA fragment from Helicobacter felis revealed five complete open reading frames. The deduced amino acid sequence of one ORF exhibited sequence similarity to the FtsH protein, an ATP-dependent metalloprotease, from various bacterial species. The encoded protein consists of 638 amino acid residues with a molecular mass of 70.2 kDa. The hydropathy profile of the FtsH protein predicted two N-terminal transmembrane regions that were confirmed experimentally. Insertion of ftsH into a new versatile expression vector resulted in overexpression of FtsH protein in Escherichia coli. In addition, the E. coli ftsH gene could be replaced by the H. felis homologue to allow reduced growth and tenfold increased lysogenization by temperate phage λ.

Keywords: Key words ftsH; copA; copP; Expression vector; Transmembrane domains; Bacteriophage λ; Hydropathy

Phylogenetic analysis of Methanobrevibacter isolated from feces of humans and other animals by Chuzhao Lin; T. L. Miller (pp. 397-403).

Comparative 16S rRNA gene sequence and genomic DNA reassociation analyses were used to assess the phylogenetic relationships of Methanobrevibacter fecal isolates. The 16S rRNA gene sequences of Methanobrevibacter smithii strain PS and the human fecal isolates B181 and ALI were essentially identical, and their genomic DNA reassociated at values greater than 94%. The analysis of 16S rRNA sequences of the horse, pig, cow, rat, and goose fecal isolates confirm that they are members of the genus Methanobrevibacter. They had a high degree of sequence similarity (97–98%) with the 16S rRNA gene of M. smithii, indicating that they share a common line of descent. The 16S rRNA genes of the horse and pig isolates had 99.3% sequence similarity. Sequence analysis of the 16S rRNA gene of the sheep fecal isolate showed that it formed a separate line of descent in the genus Methanobrevibacter. Genomic DNA reassociation studies indicate that the horse, pig, cow, and goose fecal isolates represent at least three new species. The horse and pig isolates were the only animal isolates that had > 70% genomic DNA reassociation and represent strains of a single species. The cow, goose, and sheep isolates had little or no genomic DNA reassociation with M. smithii or with each other. The relationship of the rat isolate to the other animal isolates was not determined. An evaluation of the relationship of 16S rRNA gene sequence similarity and genomic DNA reassociation of Methanobrevibacter and other methanogenic archaea indicated that genomic DNA reassociation studies are necessary to establish that two methanogenic organisms belong to the same species.

Keywords: Key words Archaea; Methanogen; Methanobrevibacter; 16S rRNA gene; Genomic DNA reassociation

Genetic evidence for a role of thioesterase domains, integrated in or associated with peptide synthetases, in non-ribosomal peptide biosynthesis in Bacillus subtilis by Axel Schneider; M. A. Marahiel (pp. 404-410).

Next to almost all prokaryotic operons encoding peptide synthetases, which are involved in the nonribosomal synthesis of peptide antibiotics, distinct genes have been detected that encode proteins with strong sequence similarity to type II fatty acid thioesterases of vertebrate origin. Furthermore, sequence analysis of bacterial and fungal peptide synthetases has revealed a region at the C-terminal end of modules that are responsible for adding the last amino acid to the peptide antibiotics; that region also exhibits significant similarities to thioesterases. In order to investigate the function of these putative thioesterases in non-ribosomal peptide synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis, srfA fragments encoding the thioesterase domain of the surfactin synthetase 3 and the thioesterase-like protein SrfA-TE were deleted. This led to a 97 and 84% reduction of the in vivo surfactin production, respectively. In the double mutant, however, no surfaction production was detectable. These findings demonstrate for the first time that the C-terminal thioesterase domains and the SrfA-TE protein are directly involved in nonribosomal peptide biosynthesis.

Keywords: Key words Nonribosomal peptide biosynthesis; Thioesterase domain; Thioesterase-like protein; Bacillus subtilis; Surfactin

Urea uptake and urease activity in Corynebacterium glutamicum by Ruth M. Siewe; Brita Weil; Andreas Burkovski; Lothar Eggeling; R. Krämer; Thomas Jahns (pp. 411-416).

When Corynebacterium glutamicum is grown with a sufficient nitrogen supply, urea crosses the cytoplasmic membrane by passive diffusion. A permeability coefficient for urea diffusion of 9 × 10^–7 cm s^–1 was determined. Under conditions of nitrogen starvation, an energy-dependent urea uptake system was synthesized. Carrier-mediated urea transport was catalyzed by a secondary transport system linked with proton motive force. With a K _m for urea of 9 μM, the affinity of this uptake system was much higher than the affinity of urease towards its substrate (K _m approximately 55 mM urea). The maximum uptake velocity depended on the expression level and was relatively low [2–3.5 nmol min^–1 (mg dry wt.)^–1].

Keywords: Key wordsCorynebacterium glutamicum; Urea uptake; Secondary transport; Urease activity

Characterization of HetR protein turnover in Anabaena sp. PCC 7120 by Ruanbao Zhou; Zongxun Cao; J. Zhao (pp. 417-426).

The hetR gene plays an important role in heterocyst development and pattern formation in heterocystous cyanobacteria. The hetR gene from Anabaena sp. PCC 7120 was overexpressed in Escherichia coli. Antibodies raised against the recombinant HetR protein (rHetR) were used to characterize metabolism of the HetR of Anabaena sp. PCC 7120 in vivo. HetR was present at a low level when Anabaena sp. PCC 7120 was grown in the presence of combined nitrogen. Shifting from nitrogen repletion conditions to nitrogen depletion conditions led to a two fold increase of HetR in total cell extracts, and most of HetR was located in heterocysts. The amount of HetR in total cellular extracts increased rapidly after shifting to nitrogen depletion conditions and reached a maximum level 3 h after the shift. Isoelectrofocusing electrophoresis revealed that the native HetR had a more acidic isoelectric point than did rHetR. After combined nitrogen was added to the nitrogen-depleted cultures, the degradation of HetR depended on culture conditions: before heterocysts were fully developed, HetR was rapidly degraded; after heterocysts were fully developed, HetR was degraded much more slowly. The distribution of HetR in other species of cyanobacteria was also studied.

Keywords: Key wordsAnabaena; Cyanobacteria; Heterocyst; HetR protein; Immunoblot; Overexpression; Pattern; formation

A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens by Jörg Simon; Roland Gross; Oliver Klimmek; Michael Ringel; A. Kröger (pp. 424-433).

During growth with fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic fumarate reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a ΔfccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens fumarate reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens fumarate reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The ΔfccABC mutant of W. succinogenes grew with fumarate and formate or sulfide, suggesting that the deleted proteins were not required for fumarate respiration with either electron donor.

Keywords: Key words Fumarate reduction; Sulfide oxidation; Wolinella succinogenes; Shewanella putrefaciens

Molecular genetic evidence for extracytoplasmic localization of sulfur globules in Chromatium vinosum by Kobchai Pattaragulwanit; Daniel C. Brune; Hans G. Trüper; C. Dahl (pp. 434-444).

Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. We cloned the genes sgpA, sgpB, and sgpC, which encode the three different proteins that constitute the sulfur globule envelope of Chromatium vinosum D (DSMZ 180^T). Southern hybridization analyses and nucleotide sequencing showed that these three genes are not clustered in the same operon. All three genes are preceded by sequences resembling σ⁷⁰-dependent promoters, and hairpin structures typical for rho-independent terminators are found immediately downstream of the translational stop codons of sgpA, sgpB, and sgpC. Insertional inactivation of sgpA in Chr. vinosum showed that the presence of only one of the homologous proteins SgpA and SgpB suffices for formation of intact sulfur globules. All three sgp genes encode translation products which – when compared to the isolated proteins – carry amino-terminal extensions. These extensions meet all requirements for typical signal peptides indicating an extracytoplasmic localization of the sulfur globule proteins. A fusion of the phoA gene to the sequence encoding the proposed signal peptide of sgpA led to high specific alkaline phosphatase activities in Escherichia coli, further supporting the envisaged targeting process. Together with electron microscopic evidence these results provide strong indication for an extracytoplasmic localization of the sulfur globules in Chr. vinosum and probably in other Chromatiaceae. Extracytoplasmic formation of stored sulfur could contribute to the transmembranous Δp that drives ATP synthesis and reverse electron flow in Chr. vinosum.

Keywords: Key words Chromatium vinosum; Phototrophic; sulfur bacteria; Sulfur globules; Extracytoplasmic; localization; Sulfide oxidation; Sulfur deposition; Thiocapsa roseoperscina; Interposon mutagenesis; phoA fusion

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions by L. S. Green; Dale B. Karr; David W. Emerich (pp. 445-451).

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min^–1 (mg protein)^–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent K _m of isocitrate lyase for d,l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V _max nor the d,l-isocitrate K _m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum.

Keywords: Key words Isocitrate lyase; Malate synthase; Glyoxylate cycle; Bradyrhizobium japonicum; Isocitrate dehydrogenase; Bacteroids

Two new motile phototrophic consortia: “Chlorochromatium lunatum” and “Pelochromatium selenoides” by C. A. Abella; X. P. Cristina; A. Martinez; I. Pibernat; X. Vila (pp. 452-459).

Two new phototrophic consortia, “Chlorochromatium lunatum” and “Pelochromatium selenoides”, were observed and collected in the hypolimnion of several dimictic lakes in Wisconsin and Michigan (USA). The two consortia had the same morphology but different pigment composition. The cells of the photosynthetic components of the consortia were half-moon-shaped. This morphology was used to differentiate them from the previously described motile phototrophic consortia “Chlorochromatium aggregatum” and “Pelochromatium roseum”. These phototrophic cells did not resemble any described unicellular green sulfur bacteria. The predominant pigments detected were bacteriochlorophyll d and chlorobactene for the green-colored “Clc. lunatum”, and bacteriochlorophyll e and isorenieratene for the brown-colored “Plc. selenoides”. Their pigment compositions and the presence of chlorosomes attached to the inner face of the cytoplasmic membrane in both kinds of photosynthetic cells confirmed this new half-moon-shaped morphotype as a green sulfur bacterium. Both consortia were found thriving in lakes with low concentrations of sulfide (< 60 μM), below the layers of “Clc. aggregatum” and “Plc. roseum”. The green consortia were observed in lakes where the oxic-anoxic interface was located at shallow depths (2–7 m), while the brown consortia were found at greater depths (8–16 m). The two newly described consortia were never detected together at the same depth in any lake.

Keywords: Key words Phototrophic consortia; “Chlorochromatium lunatum”; “Pelochromatium selenoides”; “Chlorochromatium aggregatum”; “Pelochromatium; roseum”; Green sulfur bacteria; Syntrophic associations

Fractionation of sulfur isotopes during thiosulfate reduction by Desulfovibrio desulfuricans by Andrea M. Smock; M. E. Böttcher; H. Cypionka (pp. 460-463).

Sulfur isotope fractionation during reduction of thiosulfate was investigated with growing batch cultures of Desulfovibrio desulfuricans CSN (DSM 9104) at 30 °C. The sulfide produced was depleted in ³⁴S by 10‰ as compared to total thiosulfate sulfur. The depletion was equal to that during sulfate reduction under similar conditions. The two sulfur atoms of the thiosulfate molecule were affected differently by fractionation. Sulfide produced from sulfonate sulfur was depleted by 15.4‰, sulfide produced from sulfane sulfur by 5.0‰.

Keywords: Key words Sulfate-reducing bacteria; Thiosulfate; reduction; Sulfate reduction; Stable isotopes; Sulfur isotope fractionation; Mass spectrometry

The rnf gene products in Rhodobacter capsulatus play an essential role in nitrogen fixation during anaerobic DMSO-dependent growth in the dark by K. Saeki; Hirotaka Kumagai (pp. 464-467).

The rnf genes in Rhodobacter capsulatus are essential for nitrogen fixation in the light. Because R. capsulatus grows readily on N₂ in the dark by anaerobic respiration with dimethylsulfoxide, the diazotrophic capacities of various strains in the dark were examined. No rnf mutants tested grew diazotrophically, and a nonpolar fdxN-null mutant showed decreased diazotrophic growth in the dark, suggesting that the Rnf and FdxN proteins form the primary electron donor pathway to nitrogenase in the dark as well as in the light. Nonphotosynthetic mutants lacking the component of cyclic electron transport grew diazotrophically and the levels of Rnf proteins were similar to those of the wild-type. These results indicate that rnf gene products play an essential role in nitrogen fixation without any functional link to the cyclic electron transport system.

Keywords: Key words Rhodobacter capsulatus; Nitrogen; fixation; Anaerobic respiration; Cyclic electron; transport; rnf gene; fdxN gene; Dimethylsulfoxide

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Archives of Microbiology (v.169, #5)