Xanthobacter flavus strain UE15 was isolated in wastewater obtained from the Ulsan industrial complex, Korea. This strain functions as a 1,2-dichloroethane (1,2-DCA) degrader, via a mechanism of hydrolytic dechlorination, under aerobic conditions. The UE15 strain was also capable of dechlorinating other chloroaliphatics, such as 2-chloroacetic acid and 2-chloropropionic acid. The dhlA gene encoding 1,2-DCA dechlorinase was cloned from the genomic DNA of the UE15 strain, and its nucleotide sequence was determined to consist of 933 base pairs. The deduced amino acid sequence of the DhlA dechlorinase exhibited 100% homology with the corresponding enzyme from X. autotrophicus GJ10, but only 27 to 29% homology with the corresponding enzymes from Rhodococcus rhodochrous, Pseudomonas pavonaceae, and Mycobacterium sp. strain GP1, which all dechlorinate haloalkane compounds. The UE15 strain has an ORF1 (1,356 bp) downstream from the dhlA gene. The OFR1 shows 99% amino acid sequence homology with the transposase reported from X. autotrophicus GJ10. The transposase gene was not found in the vicinity of the dhlA in the GJ10 strain, but rather beside the dhlB gene coding for haloacid dechlorinase. The dhlA and dhlB genes were confirmed to be located at separate chromosomal loci in the Xanthobacter flavus UE15 strain as well as in X. autotrophicus GJ10. The dhlA and transposase genes of the UE15 strain were found to be parenthesized by a pair of insertion sequences, IS1247, which were also found on both sides of the transposase gene in the GJ10 strain. This unique structure of the dhlA gene organization in X. flavus strain UE15 suggested that the dechlorinase gene, dhlA, is transferred with the help of the transposase gene.

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.

The genes encoding fructosebisphosphatase and phosphoribulokinase present on a 2.5 kb SalI fragment from Xanthobacter flavus H4-14 were sequenced. Two large open reading frames (ORFs) were identified, preceded by plausible ribosome-binding sites. The ORFs were transcribed in the same direction and were separated by 39 base pairs. They encoded proteins of 364 and 291 amino acids, with molecular masses of 38739 and 33409 Da, respectively. The ORFs were identified as the genes encoding FBPase and PRK, respectively, on the basis of similarity with FBPase and PRK sequences from other sources.

The genes encoding the large (cfxL) and small (cfxS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisC/O) from Xanthobacter flavus H4-14 were identified and characterized. The RuBisC/O genes are separated by 11 bp and cotranscribed in Escherichia coli from the lac promoter in the order cfxLS. Primer extension and R-loop experiments with RNA isolated from autotrophically grown X. flavus H4-14 showed that transcription of cfxL and cfxS initiated 22 bp upstream from cfxL and resulted in a mRNA of at least 2.3 kb. DNA sequence analysis identified the start of an open reading frame transcribed divergently from cfxL, and displaying significant similarities with genes belonging to the lysR family of transcriptional activators. Downstream from cfxS an additional open reading frame was identified with unknown function. Expression studies showed that the genes encoding fructosebisphosphatase (cfxF) and phosphoribulokinase (cfxP) are located downstream from cfxLS. The cfxF and cfxP genes are cotranscribed in the same direction as cfxLS in the order cfxFP.

The expression of the cbb and gap-pgk operons of Xanthobacter flavus encoding enzymes of the Calvin cycle is regulated by the transcriptional regulator CbbR. In order to identify other genes involved in the regulation of these operons, a mutant was isolated with a lowered activity of a fusion between the promoter of the cbb operon and the reporter gene lacZ. This mutant was unable to grow autotrophically and had a reduced growth rate on medium supplemented with gluconate or succinate. The regulation of the gap-pgk operon in the mutant was indistinguishable from the wild-type strain, but induction of the cbb operon upon transition to autotrophic growth conditions was delayed. Complementation of the mutant with a genomic library of X. flavus resulted in the isolation of a 1.1 kb ApaI fragment which restored autotrophic growth of the mutant. One open reading frame (ORF) was present on the ApaI fragment, which could encode a protein highly similar to triosephosphate isomerase proteins from other bacteria. Cell extracts of the mutant grown under glycolytic or gluconeogenic conditions had severely reduced triosephosphate isomerase activities. The ORF was therefore identified as tpi, encoding triosephosphate isomerase. The tpi gene is not linked to the previously identified operons encoding Calvin cycle enzymes and therefore represents a third transcriptional unit required for autotrophic metabolism.

Xanthobacter flavus is able to grow autotrophically with the enzymes of the Calvin cycle for the fixation of CO2, which are specified by the cbbLSXFP gene cluster. Previously, the 5' end of an open reading frame (cbbR), displaying a high sequence similarity to the LysR family of regulatory proteins and transcribed divergently from cbbLSXFP, was identified (W. G. Meijer, A. C. Arnberg, H. G. Enequist, P. Terpstra, M. E. Lidstrom, and L. Dijkhuizen, Mol. Gen. Genet. 225:320-330, 1991). This paper reports the complete nucleotide sequence of cbbR and a functional characterization of the gene. The cbbR gene of X. flavus specifies a 333-amino-acid polypeptide, with a molecular weight of 35,971. Downstream from cbbR, the 3' end of an open reading frame displaying a high similarity to ORF60K from Pseudomonas putida and ORF261 from Bacillus subtilis was identified. ORF60K and ORF261 are located at the replication origin of the bacterial chromosome. Inactivation of cbbR, via the insertion of an antibiotic resistance gene, rendered X. flavus unable to grow autotrophically. This was caused not by an inability to oxidize autotrophic substrates (e.g., formate) but by a complete lack of expression of the cbb genes. The expression of the CbbR protein in Escherichia coli was achieved by placing cbbR behind a strong promoter and optimization of the translational signals of cbbR. CbbR binds specifically to two binding sites in the cbbR-cbbL intergenic region.

Xanthobacter flavus, a gram-negative facultatively autotrophic bacterium, employs the Calvin cycle for the fixation of carbon dioxide. Cells grown under autotrophic growth conditions possess an Fe(2+)-dependent fructosebisphosphate (FBP) aldolase (class II) in addition to a class I FBP aldolase. By nucleotide sequencing and heterologous expression in Escherichia coli, genes encoding transketolase (EC 2.2.1.1.; CbbT) and class II FBP aldolase (EC 4.1.2.13; CbbA) were identified. A partial open reading frame encoding a protein similar to pentose-5-phosphate 3-epimerase was identified downstream from cbbA. A phylogenetic tree of transketolase proteins displays a conventional branching order. However, the class II FBP aldolase protein from X. flavus is only distantly related to that of E. coli. The autotrophic FBP aldolase proteins from X. flavus, Alcaligenes eutrophus, and Rhodobacter sphaeroides form a tight cluster, with the proteins from gram-positive bacteria as the closest relatives.

During autotrophic growth of Xanthobacter flavus, energy derived from the oxidation of hydrogen methanol or formate is used to drive the assimilation of CO2 via the Calvin cycle. The genes encoding the Calvin cycle enzymes are organized in the cbb operon, which is expressed only during autotrophic growth. Although it has been established that the transcriptional activator CbbR is required for the expression of the cbb operon, it is unclear whether CbbR is the only factor contributing to the regulation of the cbb operon. This paper describes the isolation of X. flavus mutants which were affected in the regulation of the cbb operon. One of the mutant strains was subject to an enhanced repression of the cbb operon promoter by the gluconeogenic substrate succinate and in addition failed to grow autotrophically. The rate of growth of the X. flavus mutant on succinate-containing medium was lower than that of the wild-type strain, but rates of growth on medium supplemented with gluconate were identical. A genomic library of X. flavus was constructed and was used to complement the mutant strain. The nucleotide sequence of the DNA fragment required to restore autotrophic growth of the X. flavus mutant was determined. One open reading frame that displayed extensive similarities to phosphoglycerate kinase-encoding genes (pgk) was identified. The X. flavus mutant lacked phosphoglycerate kinase activity following growth on gluconate or succinate. Introduction of the pgk gene into the X. flavus mutant partially restored the activity of phosphoglycerate kinase. Induction of the cbb operon of the X. flavus wild-type strain resulted in a simultaneous and parallel increase in the activities of ribulose-1,5-biphosphate carboxylase and phosphoglycerate kinase, whereas the latter activity remained absent in the X. flavus pgk mutant. It is concluded that X. flavus employees a single phosphoglycerate kinase enzyme and this is not encoded within the cbb operon.

The deduced primary sequence (cbbL and cbbS) of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (rubisco) from Bradyrhizobium japonicum places this enzyme within the Type IC subgroup of red-like rubisco enzymes. In addition, B. japonicum appears to organize most of the structural genes of the Calvin-Benson-Bassham (CBB) pathway in at least one major operon. Functional expression and characterization of the B. japonicum and Xanthobacter flavus enzymes from this group revealed that these molecules exhibit diverse kinetic properties despite their relatively high degree of sequence relatedness. Of prime importance was the fact that these closely related enzymes exhibited CO2 and O2 substrate specificities that varied from relatively low values [tau = (VcKo)/(VoKc) = 45] to values that approximated those obtained for higher plants (tau = 75). These results, combined with the metabolic and genetic versatility of the organisms from which these enzymes were derived, suggest a potential rich resource for future biological selection and structure-function studies aimed at elucidating structural features that govern key enzymological properties of rubisco.