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PubMed1995

Analysis of the cbbF genes from Alcaligenes eutrophus that encode fructose-1,6-/sedoheptulose-1,7-bisphosphatase.

The cbbF genes of the facultative chemoautotroph Alcaligenes eutrophus H16 are part of two highly homologous cbb operons. Both the chromosomal and the megaplasmid pHG1-borne copy of cbbF were cloned and sequenced. Subsequent analyses including comparison with known sequences from other organisms and heterologous expression in Escherichia coli revealed that each of the genes encodes fructose-1,6-bisphosphatase (FBPase). A closely related activity likewise operating in the Calvin carbon reduction cycle, sedoheptulose-1,7-bisphosphatase, was also catalyzed by the two isoenzymes which were purified from autotrophically grown cells of A. eutrophus. Two-dimensional gel electrophoresis allowed the separation of the cbbF gene products. Preliminary physical evidence by Southern hybridization with a heterologous gene probe was obtained for the existence of a third FBPase gene, fbp, on the chromosome of the organism. Its product is probably involved in the heterotrophic carbon metabolism.

Alcaligenes↗

PubMed1995

Analysis of the genes forming the distal parts of the two cbb CO2 fixation operons from Alcaligenes eutrophus.

In the facultative chemoautotroph Alcaligenes eutrophus H16, most of the genes (cbb genes) encoding enzymes of the Calvin carbon reduction cycle are organized within two highly homologous cbb operons, one located on the chromosome and the other on the megaplasmid pHG1. Nucleotide sequencing of the promoter-distal part of the operons revealed three open reading frames, designated cbbG, cbbK, and cbbA. Similarity searches in databases and heterologous expressions of the subcloned genes in Escherichia coli identified them as genes encoding the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and a class II fructose-1,6-bisphosphate aldolase, respectively. The aldolase could be grouped together with the enzymes from Rhodobacter sphaeroides and Bacillus subtilis as a new subtype of class II aldolases. A phenotypic complementation analysis with a cbb operon mutant of A. eutrophus showed that the cbbG product is essential for autotrophic growth of the organism, whereas the products of cbbK and cbbA can apparently be substituted by isoenzymes encoded elsewhere on the chromosome. No or only low constitutive promoter activity was associated with cbbK and cbbA, respectively, confirming the two genes as parts of the cbb operon. Downstream of cbbA, the very high overall nucleotide sequence identity (about 94%) prevailing throughout the two cbb operons discontinues, suggesting that cbbA is the most promoter-distal gene of the operon.

Alcaligenes↗

PubMed1992

Metabolic pathways in Paracoccus denitrificans and closely related bacteria in relation to the phylogeny of prokaryotes.

Denitrification and methylotrophy in Paracoccus denitrificans are discussed. The properties of the enzymes of denitrification: the nitrate-nitrite antiporter, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase are described. The genes for none of these proteins have yet been cloned and sequenced from P. denitrificans. A number of sequences are available for enzymes from Escherichia coli, Pseudomonas stutzeri and Pseudomonas aeruginosa. It is concluded that pathway specific c-type cytochromes are involved in denitrification. At least 40 genes are involved in denitrification. In methanol oxidation at least 20 genes are involved. In this case too pathway specific c-type cytochromes are involved. The sequence homology between the quinoproteins methanol dehydrogenase, alcoholde-hydrogenase and glucose dehydrogenase is discussed. This superfamily of proteins is believed to be derived from a common ancestor. The moxFJGI operon determines the structural components of methanol dehydrogenase and the associated c-type cytochrome. Upstream of this operon 3 regulatory proteins were found. The moxY protein shows the general features of a sensor protein and the moxX protein those of a regulatory protein. Thus a two component regulatory system is involved in both denitrification and methylotrophy. The phylogeny of prokaryotes based on 16S rRNA sequence is discussed. It is remarkable that the 16S rRNA of Thiosphaera pantotropha is identical to that of P. denitrificans. Still these bacteria show a number of differences. T. pantotropha is able to denitrify under aerobic circumstances and it shows heterotrophic nitrification. Nitrification and heterotrophic nitrification are found in species belonging to the beta-and gamma-subdivisions of purple non-sulfur bacteria. Thus the occurrence of heterotrophic nitrification in T. pantotropha, which belongs to the alpha-subdivision of purple non-sulfur bacteria is a remarkable property. Furthermore T. pantotropha contains two nitrate reductases of which the periplasmic one is supposed to be involved in aerobic denitrification. The nitrite reductase is of the Cu-type and not of the cytochrome cd1 type as in P. denitrificans. Also the cytochrome b of the Qbc complex of T. pantotropha is highly similar to its counterpart in P. denitrificans. It is hypothesized that the differences between these two organisms which both contain large megaplasmids is due to a combination of loss of genetic information and plasmid-coded properties. The distribution of a number of complex metabolic systems in eubacteria and in a number of species belonging to the alpha-group of purple non sulphur bacteria is reviewed.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteria↗

PubMed2003

IS Rm31, a new insertion sequence of the IS 66 family in Sinorhizobium meliloti.

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti p SymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, IS Rm31. IS Rm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that IS Rm31 is a member of the IS 66 family. IS Rm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying IS Rm31 suggested that IS Rm31 inserts randomly into S. meliloti genomes. Moreover, analysis of IS Rm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, IS Rm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.

Base Composition↗

PubMed1996

Identification of cbbBc as an additional distal gene of the chromosomal cbb CO2 fixation operon from Ralstonia eutropha.

Ralstonia eutropha (formerly Alcaligenes eutrophus) strain H16 possesses two highly homologous cbb operons encoding most of the Calvin cycle enzymes. One copy of the operon is located on the chromosome, the other on the megaplasmid pHG1 of the organism. Sequence analysis of the region downstream of the presumptive 3'-terminal gene (cbbAc) of the chromosomal operon revealed the presence of an open reading frame comprising 2,274 bp. Evidence is presented that this open reading frame is an additional distal gene (designated cbbBc) of the operon. In contrast to the other genes of the operon, cbbBc is not duplicated in the plasmid-borne operon. The deduced amino acid sequence of the cbbBc product (757 residues, molecular mass 83.17 kDa) showed the highest similarity to the large catalytic subunits of various bacterial formate dehydrogenases (FDH), suggesting that cbbBc might represent a structural FDH gene of R. eutropha. However, the properties of a cbbBc mutant strain indicated that the potential gene product is not related to known FDH of the organism. Transcriptional analysis in the homologous host and heterologous expression in Escherichia coli demonstrated that cbbBc is an active gene, which apparently has no essential function in the autotrophic metabolism of R. eutropha. The gene is a novel member of cbb operons in autotrophic bacteria.

Alcaligenes↗

PubMed1997

Autotrophic growth on carbon disulfide is a property of novel strains of Paracoccus denitrificans.

Three distinct strains (KL1, KS1, and KS2) of facultatively chemolitho-autotrophic bacteria able to use carbon disulfide or carbonyl sulfide as sole energy substrates were identified as novel strains of Paracoccus denitrificans. Evidence for their identity as biovars of P. denitrificans and as close relatives of Paracoccus versutus is based on their DNA composition, total sequencing of the genes for their 16S rRNA, muropeptide profiles, amino acid composition of peptidoglycan, kinetics of murein degradation by lysozyme, possession of large plasmids (91-98 kb) and megaplasmids (> 450 kb), and plasmid transfer between the strains and with P. denitrificans and P. versutus. No functions have been identified for the 91- to 98-kb plasmids of strains KL1 and KS2, but curing strain KL1 of its plasmid did not affect growth on carbon disulfide, thiosulfate or succinate. Emendation of the formal description of Paracoccus denitrificans is presented. Autotrophic growth on carbon disulfide and thiosulfate was confirmed by 14CO2 fixation. Evidence is presented for initiation of carbon disulfide oxidation by an NADH-dependent oxygenase. Cell-free extracts catalyzed (1) NADH-stimulated uptake of oxygen in the presence of carbon disulfide, and (2) carbon-disulfide-stimulated oxidation of NADH. The activity was not sedimented at 50,000 x g. Intermediates in aerobic carbon disulfide metabolism were shown by GC and GC/MS to include carbonyl sulfide and hydrogen sulfide, but anaerobic production of COS and H2S from carbon disulfide did not occur. SDS-PAGE of cell-free extracts showed polypeptides that were unique to growth on carbon disulfide, common to carbon disulfide and carbonyl sulfide, or found after growth on carbon disulfide, carbonyl sulfide or thiosulfate. The possible identity of these as proteins involved in sulfur compound metabolism is discussed.

Bacterial Proteins↗

PubMed1998

Duplication of hyp genes involved in maturation of [NiFe] hydrogenases in Alcaligenes eutrophus H16.

Alcaligenes eutrophus H16 harbors seven hyp genes (hypA, B, F, C, D, E, and X) as part of the hydrogenase gene cluster on megaplasmid pHG1. Here we demonstrate that three of the hyp genes (hypA, B, and F) are duplicated in A. eutrophus, which explains the lack of a phenotypic change in single-site mutants impaired in one of the two copies. Mutants with lesions in both copies showed clear alterations in hydrogenase activities. Deletions in hypF1 and hypF2 completely abolished activities of the soluble hydrogenase and of the membrane-bound hydrogenase, mutations in hypA1 and hypA2 totally blocked the membrane-bound hydrogenase activity, while residual soluble hydrogenase activity accounted for the extremely slow growth of the strain on H2. Both hydrogenase activities of mutants defective in hypB1 and hypB2 were partially restored by elevating the concentration of nickel chloride in the medium. Reduction of hydrogenase activities in the double mutants correlated with varying degrees of maturation deficiency based upon the amount of unprocessed nickel-free hydrogenase precursor. Despite a high identity between the two copies of hyp gene products, substantial structural differences were identified between the two copies of hypF genes. HypF1, although functionally active, is a truncated version of HypF2, whose structure resembles HypF proteins of other organisms. Interestingly, the N-terminus of HypF2, which is missing in the HypF1 counterpart, contains a putative acylphosphatase domain in addition to a potential metal binding site.

Bacterial Proteins↗

PubMed2006

The repetitive DNA elements called CRISPRs and their associated genes: evidence of horizontal transfer among prokaryotes.

We have found direct DNA repeats 21-47 bp in length interspersed with nonrepetitive sequences of similar length, or clustered regularly interspaced short palindromic repeats (CRISPRs) in a wide range of diverse prokaryotes, including many Archaeal and Eubacterial species. A number of cas, CRISPR-associated genes have also been characterized in many of the same organisms. Phylogenetic analysis of these cas genes suggests that the CRISPR loci have been propagated via HGT, horizontal gene transfer. We suggest a mechanism by which this HGT has occurred, namely, that the CRISPR loci can be carried between cells on megaplasmids > or = 40 kb in length.

Base Sequence↗

PubMed2002

Cloning and characterization of a FAD-monooxygenase gene ( cadA) involved in degradation of chloranilic acid (2,5-dichloro-3,6-dihydroxybenzo-1,4-quinone) in Pseudomonas putida TQ07.

A bacterium culture was isolated on the basis of its ability to degrade chloranilic acid, and was later identified as Pseudomonas putida (TQ07). Several transposon insertion mutants unable to degrade chloranilic acid were selected. The characterization of the site of insertion of one of these mutants led to the identification of the cadA gene encoding an enzyme with significant homology with FAD-monooxygenases involved in the degradation of aromatic and chloroaromatic compounds. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of "halo" formation (a zone of clearing color on agar plates around TQ07 colonies that degrade chloranilic acid) and degradation of chloranilic acid, unequivocally assigned cadA a function in the metabolism of this compound. We also found that most of the transposon insertion mutants unable to degrade chloranilic acid are clustered in a 10-kb region of the P. putidagenome that is encoded in a megaplasmid or in an unstable chromosomal region.

Amino Acid Sequence↗

PubMed2004

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24.

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of approximately 340 and approximately 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the approximately 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the approximately 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The Km of TID for indene was 380 microM. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The Km of TID for naphthalene was 28 microM. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.

Chromatography, High Pressure Liquid↗

PubMed1996

Molecular and Regulatory Properties of the Nitrate Reducing Systems of Rhodobacter

Phototrophic bacteria of the genus Rhodobacter possess several forms of nitrate reductase including assimilatory and dissimilatory enzymes. Assimilatory nitrate reductase from Rhodobacter capsulatus E1F1 is cytoplasmic, it uses NADH as the physiological electron donor and reduced viologens as artificial electron donors, and it is coupled to an ammonium-producing nitrite reductase. Nitrate reductase induction requires a high C/N balance and the presence of nitrate, nitrite, or nitroarenes. A periplasmic 47-kDa protein facilitates nitrate uptake, thus increasing nitrate reductase activity. Two types of dissimilatory nitrate reductases have been found in strains from Rhodobacter sphaeroides. One of them is coupled to a complete denitrifying pathway, and the other is a periplasmic protein whose physiological role seems to be the dissipation of excess reducing power, thus improving photoanaerobic growth. Periplasmic nitrate reductase does not use NADH as the physiological electron donor and is a 100-kDa heterodimeric hemoprotein that receives electrons through an electron transport chain spanning the plasma membrane. This nitrate reductase is regulated neither by the intracellular C/N balance nor by O2 pressure. The enzyme also exhibits chlorate reductase activity, and both reaction products, nitrite and chlorite, are released almost stoichiometrically into the medium; this accounts for the high resistance to chlorate or nitrite exhibited by this bacterium. Nitrate reductases from both strains seem to be coded by genes located on megaplasmids.

Journal Article↗

PubMed1999

The eff-482 locus of Sinorhizobium meliloti CXM1-105 that influences symbiotic effectiveness consists of three genes encoding an endoglycanase, a transcriptional regulator and an adenylate cyclase.

The mutant T482 of Sinorhizobium meliloti CXM1-105, which carries a Tn5 insertion on megaplasmid 1, exhibits an enhanced symbiotic efficiency phenotype. Three genes, eglC, cya3 and syrB2, were identified in the eff-482 region tagged by the Tn5 insertion in T482. The eglC gene encodes an endoglycanase which contributes to the depolymerization of the exo-polysaccharide succinoglycan. The N-terminal region of the predicted cya3 gene product was similar to eukaryotic-type adenylate cyclases from Brevibacterium liquefaciens and Streptomyces coelicolor. Four contiguous tetratricopeptide repeats which are known to mediate protein-protein interactions were identified in the C-terminal portion of Cya3. Complementation analysis demonstrated that cya3 indeed encodes a functional adenylate cyclase. A central helix-turn-helix DNA-binding motif and a putative C-terminal coiled-coil structure implicated in protein oligomerization were found in SyrB2. Extra copies of the syrB2 gene negatively affect transcription of both syrB2 itself and cya3. The Tn5 insertion in T482 was localized between the divergently transcribed genes eglC and syrB2. It eliminated eglC function and slightly stimulated transcription of both syrB2 and cya3, which lies downstream of syrB2. Mutants carrying insertions of the lacZ-Gm interposon in the genes eglC, syrB2 and cya3 exhibit the same phenotype as mutant T482, indicating that these three genes influence symbiotic efficiency.

Adenylyl Cyclases↗

PubMed1986

Isolation of hydrogen-oxidation gene from Alcaligenes hydrogenophilus and its expression in Pseudomonas oxalaticus.

A gene bank of a megaplasmid encoding the hydrogen-oxidizing enzyme system (Hox) in Alcaligenes hydrogenophilus was constructed using a broad host range cosmid vector pVK102, and established in Escherichia coli. Hybrid cosmids containing hox genes were identified by transferring the bank into Pseudomonas oxalaticus OX1 and screening colonies for the ability of H2-dependent autotrophic growth. About 800 colonies were formed under autotrophic conditions. One of the Hox+ transconjugants was isolated and its hydrogenases activities were measured. Although soluble hydrogenase was not detected, the Hox+ transconjugant had four times the membrane-bound hydrogenase activity of A. hydrogenophilus.

Alcaligenes↗

PubMed1991

Construction and properties of a triprotein containing the high-affinity nickel transporter of Alcaligenes eutrophus.

The high-affinity nickel transporter of Alcaligenes eutrophus H16 is encoded by gene hoxN, which maps within the hydrogenase gene cluster of megaplasmid pHG1. A tripartite gene fusion was constructed, consisting of (i) the Escherichia coli lacZ gene for beta-galactosidase, (ii) a segment encoding an endoproteolytically cleavable peptide, and (iii) the A. eutrophus gene hoxN. An E. coli strain harboring this construct (plasmid pCH307) efficiently produced the corresponding triprotein upon induction. A broad-host-range derivative of pCH307 was shown to complement an A. eutrophus HoxN- mutant.

Alcaligenes↗

PubMed1986

At least two nodD genes are necessary for efficient nodulation of alfalfa by Rhizobium meliloti.

A Rhizobium meliloti DNA region (nodD1) involved in the regulation of other early nodulation genes has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicate a large open reading frame with opposite polarity to nodA, -B and -C, coding for a protein of 308 (or 311) amino acid residues. Tn5 insertion within the gene caused a delay in nodulation of Medicago sativa from four to seven days. Hybridization of nodD1 to total DNA of Rhizobium meliloti revealed two additional nodD sequences (nodD2 and nodD3) and both were localized on the megaplasmid pRme41b in the vicinity of the other nod genes. Genetic and DNA hybridization data, combined with nucleotide sequencing showed that nodD2 is a functional gene, while requirement of nodD3 for efficient nodulation of M. sativa could not be detected under our experimental conditions. The nodD2 gene product consists of 310 amino acid residues and shares 86.4% homology with the nodD1 protein. Single nodD2 mutants had the same nodulation phenotype as the nodD1 mutants, while a double nodD1-nodD2 mutant exhibited a more severe delay in nodulation. These results indicate that at least two functional copies of the regulatory gene nodD are necessary for the optimal expression of nodulation genes in R. meliloti.

Amino Acid Sequence↗

PubMed1989

A novel exopolysaccharide can function in place of the calcofluor-binding exopolysaccharide in nodulation of alfalfa by Rhizobium meliloti.

We have found that R. meliloti strain Rm1021, which is known to synthesize a Calcofluor-binding exopolysaccharide (EPS I), also has a cryptic capacity to synthesize a second exopolysaccharide (EPS II). Structural analysis of EPS II has shown that it differs in many respects from EPS I. Genetic analysis indicates that EPS II synthesis requires the products of at least seven loci on the second symbiotic megaplasmid of R. meliloti, and is induced by a mutation, expR101, which causes increased transcription of these genes. Synthesis of EPS II suppresses the symbiotic defects of EPS I-deficient strains on Medicago sativa (alfalfa), but not on four other plants that are normally hosts for Rm1021. These observations suggest that structural features of bacterial exopolysaccharides are involved in the determination of host range. The implications of these results for models of exopolysaccharide function, such as serving as signals to the plant or shielding the bacteria from plant defense responses, are discussed.

Benzenesulfonates↗

PubMed1990

Genetic analyses of Rhizobium meliloti exopolysaccharides.

We have recently obtained strong genetic evidence that the acidic Calcofluor-binding exopolysaccharide (EPS I) of Rhizobium meliloti Rm1021 is required for nodule invasion and possibly for later events in nodule development. Thirteen loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. exoH mutants fail to succinylate their EPS I and form empty Fix- nodules. We have identified two unlinked regulatory loci, exoR and exoS, whose products play negative roles in the regulation of expression of the exo genes. We have recently discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for Rhizobium exopolysaccharides in nodulation are discussed.

Carbohydrate Sequence↗

PubMed1993

Genetics of Paracoccus denitrificans.

In bioenergetic research Paracoccus denitrificans has been used as an interesting model to elucidate the mechanisms of bacterial energy transduction. Genes for protein complexes of the respiratory chain and for proteins which are involved in periplasmic electron transport have been cloned and sequenced. Conjugational gene transfer has allowed the construction of site-specific mutant strains. Complementation experiments did not only open the field for site-directed mutagenesis and investigation of the structure/function relationship of the various electron-transport proteins, but also allowed first insights into processes like oxygen-dependent gene regulation or the assembly of electron-transport complexes. Also data will be presented that characterize two restriction-/modification systems, the codon usage and the promoter sequences of Paracoccus. Details will be given about the extrachromosomal localization of a duplicated cytochrome oxidase subunit I gene on one of the Paracoccus megaplasmids.

Base Sequence↗