Genetic analysis of phenylacetic acid catabolism in <Emphasis Type="Italic">Arthrobacter oxydans</Emphasis> CECT386

Arthrobacter oxydans CECT386 is a Gram-positive bacterium able to use either phenylacetic acid or phenylacetaldehyde as the sole carbon and energy source for aerobic growth. Genes responsible for the catabolism of these compounds have been located at two chromosomal regions and were organized in one isolated paaN gene and two putative paa operons, one consisting of the paaD, paaF, tetR and prot genes, and one consisting of the paaG, paaH, paaI, paaJ, paaK and paaB genes. The identity of the paaF and paaN genes was supported by functional complementation experiments. A comparison with the paa catabolic genes and/or gene clusters of other bacteria that degrade these aromatic compounds is presented. The results of this study broaden the knowledge regarding the range of metabolic potential of this strain and eventually make it attractive for environmental applications.     

Two P-type ATPases are required for copper delivery in Arabidopsis thaliana chloroplasts

Copper delivery to the thylakoid lumen protein plastocyanin and the stromal enzyme Cu/Zn superoxide dismutase in chloroplasts is required for photosynthesis and oxidative stress protection. The copper delivery system in chloroplasts was characterized by analyzing the function of copper transporter genes in Arabidopsis thaliana. Two mutant alleles were identified of a previously uncharacterized gene, PAA2 (for P-type ATPase of Arabidopsis), which is required for efficient photosynthetic electron transport. PAA2 encodes a copper-transporting P-type ATPase with sequence similarity to PAA1, which functions in copper transport in chloroplasts. Both proteins localized to the chloroplast, as indicated by fusions to green fluorescent protein. The PAA1 fusions were found in the chloroplast periphery, whereas PAA2 fusions were localized in thylakoid membranes. The phenotypes of paa1 and paa2 mutants indicated that the two transporters have distinct functions: whereas both transporters are required for copper delivery to plastocyanin, copper delivery to the stroma is inhibited only in paa1 but not in paa2. The effects of paa1 and paa2 on superoxide dismutase isoform expression levels suggest that stromal copper levels regulate expression of the nuclear genes IRON SUPEROXIDE DISMUTASE1 and COPPER/ZINC SUPEROXIDE DISMUTASE2. A paa1 paa2 double mutant was seedling-lethal, underscoring the importance of copper to photosynthesis. We propose that PAA1 and PAA2 function sequentially in copper transport over the envelope and thylakoid membrane, respectively.     

Aerobic metabolism of phenylacetic acids in Azoarcus evansii

The aerobic metabolism of phenylacetic acid (PA) and 4-hydroxyphenylacetic acid (4-OHPA) was investigated in the beta-proteobacterium Azoarcus evansii. Evidence for the existence of two independent catabolic pathways for PA and 4-OHPA is presented. 4-OHPA metabolism involves the formation of 2,5-dihydroxyphenylacetate (homogentisate) and maleylacetoacetate catalyzed by specifically induced 4-OHPA 1-monooxygenase and homogentisate 1,2-dioxygenase. The metabolism of PA starts by its activation to phenylacetyl-CoA (PA-CoA) via an aerobically induced phenylacetate-coenzyme A ligase. Phenylalanine (Phe) aerobic metabolism in this bacterium proceeds also via PA and PA-CoA. Whole cells of A. evansii transformed [1-(14)C]PA to (14)C-phenylacetyl-CoA and subsequently to a number of unknown labeled products, which were also observed in PA-degrading bacteria from different phylogenetic groups, i.e. Escherichia coli, Rhodopseudomonas palustrisand Bacillus stearothermophilus. A chromosomal region from A. evansiiof 11.5 kb containing a cluster of 11 phenylacetic acid catabolic ( paa) genes ( paaYZGHIKABCDE) was sequenced and characterized. The derived gene products were similar to the characterized putative gene products involved in PA catabolism in E. coli and Pseudomonas putida and to other putative PA catabolic gene products of diverse bacteria. RT-PCR analysis of the paa genes of A. evansiigrowing aerobically with PA showed a probable organization of the paa genes in three operons. The similarity of the PA metabolic products pattern and of gene sequences suggests a common aerobic bacterial PA pathway.     

Cloning and Characterization of Benzoate Catabolic Genes in the Gram-Positive Polychlorinated Biphenyl Degrader Rhodococcus sp. Strain RHA1

Benzoate catabolism is thought to play a key role in aerobic bacterial degradation of biphenyl and polychlorinated biphenyls (PCBs). Benzoate catabolic genes were cloned from a PCB degrader, Rhodococcus sp. strain RHA1, by using PCR amplification and temporal temperature gradient electrophoresis separation. A nucleotide sequence determination revealed that the deduced amino acid sequences encoded by the RHA1 benzoate catabolic genes, benABCDK, exhibit 33 to 65% identity with those of Acinetobacter sp. strain ADP1. The gene organization of the RHA1 benABCDK genes differs from that of ADP1. The RHA1 benABCDK region was localized on the chromosome, in contrast to the biphenyl catabolic genes, which are located on linear plasmids. Escherichia coli cells containing RHA1 benABCD transformed benzoate to catechol via 2-hydro-1,2-dihydroxybenzoate. They transformed neither 2- nor 4-chlorobenzoates but did transform 3-chlorobenzoate. The RHA1 benA gene was inactivated by insertion of a thiostrepton resistance gene. The resultant mutant strain, RBD169, neither grew on benzoate nor transformed benzoate, and it did not transform 3-chlorobenzoate. It did, however, exhibit diminished growth on biphenyl and growth repression in the presence of a high concentration of biphenyl (13 mM). These results indicate that the cloned benABCD genes could play an essential role not only in benzoate catabolism but also in biphenyl catabolism in RHA1. Six rhodococcal benzoate degraders were found to have homologs of RHA1 benABC. In contrast, two rhodococcal strains that cannot transform benzoate were found not to have RHA1 benABC homologs, suggesting that many Rhodococcus strains contain benzoate catabolic genes similar to RHA1 benABC.     

Glyoxylate metabolism is a key feature of the metabolic degradation of 1,4-dioxane by Pseudonocardia dioxanivorans strain CB1190

The groundwater contaminant 1,4-dioxane (dioxane) is transformed by several monooxygenase-expressing microorganisms, but only a few of these, including Pseudonocardia dioxanivorans strain CB1190, can metabolize the compound as a sole carbon and energy source. However, nothing is yet known about the genetic basis of dioxane metabolism. In this study, we used a microarray to study differential expression of genes in strain CB1190 grown on dioxane, glycolate (a previously identified intermediate of dioxane degradation), or pyruvate. Of eight multicomponent monooxygenase gene clusters carried by the strain CB1190 genome, only the monooxygenase gene cluster located on plasmid pPSED02 was upregulated with dioxane relative to pyruvate. Plasmid-borne genes for putative aldehyde dehydrogenases, an aldehyde reductase, and an alcohol oxidoreductase were also induced during growth with dioxane. With both dioxane and glycolate, a chromosomal gene cluster encoding a putative glycolate oxidase was upregulated, as were chromosomal genes related to glyoxylate metabolism through the glyoxylate carboligase pathway. Glyoxylate carboligase activity in cell extracts from cells pregrown with dioxane and in Rhodococcus jostii strain RHA1 cells expressing the putative strain CB1190 glyoxylate carboligase gene further demonstrated the role of glyoxylate metabolism in the degradation of dioxane. Finally, we used (13)C-labeled dioxane amino acid isotopomer analysis to provide additional evidence that metabolites of dioxane enter central metabolism as three-carbon compounds, likely as phosphoglycerate. The routing of dioxane metabolites via the glyoxylate carboligase pathway helps to explain how dioxane is metabolized as a sole carbon and energy source for strain CB1190.     

Adventures in Rhodococcus - from steroids to explosives

Rhodococcus is a genus of mycolic-acid-containing actinomycetes that utilize a remarkable variety of organic compounds as growth substrates. This degradation helps maintain the global carbon cycle and has increasing applications ranging from the biodegradation of pollutants to the biocatalytic production of drugs and hormones. We have been using Rhodococcus jostii RHA1 as a model organism to understand the catabolic versatility of Rhodococcus and related bacteria. Our approach is exemplified by the discovery of a cluster of genes specifying the catabolism of cholesterol. This degradation proceeds via β-oxidative degradation of the side chain and O2-dependent cleavage of steroid ring A in a process similar to bacterial degradation of aromatic compounds. The pathway is widespread in Actinobacteria and is critical to the pathogenesis of Mycobacterium tuberculosis, arguably the world's most successful pathogen. The close similarity of some of these enzymes with biphenyl- and polychlorinated-biphenyl-degrading enzymes that we have characterized is facilitating inhibitor design. Our studies in RHA1 have also provided important insights into a number of novel metalloenzymes and their biosynthesis, such as acetonitrile hydratase (ANHase), a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. Molecular genetic and biochemical studies have identified AnhE as a dimeric metallochaperone that delivers cobalt to ANHase, enabling its maturation in vivo. Other metalloenzymes we are characterizing include N-acetylmuramic acid hydroxylase, which catalyzes an unusual hydroxylation of the rhodococcal and mycobacterial peptidoglycan, and 2 RHA1 dye-decolorizing peroxidases. Using molecular genetic and biochemical approaches, we have demonstrated that one of these enzymes is involved in the degradation of lignin. Overall, our studies are providing fundamental insights into a range of catabolic processes that have a wide variety of applications.     

Roles of ring-hydroxylating dioxygenases in styrene and benzene catabolism in Rhodococcus jostii RHA1

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ΔbphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the β-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta- and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.     

Molecular characterization of a phenanthrene degradation pathway in Mycobacterium vanbaalenii PYR-1

Mycobacterium vanbaalenii PYR-1 is capable of degrading a number of polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites via multiple pathways. Genes for the large and small subunits of a pyrene dioxygenase, nidA and nidB, respectively, were previously identified in M. vanbaalenii PYR-1 [Appl. Environ. Microbiol. 67 (2001) 3577]. A library of the M. vanbaalenii PYR-1 genome was constructed in a fosmid vector to identify additional genes involved in PAH degradation. Twelve fosmid clones containing nidA were identified by Southern hybridization. Sequence analysis of one nidA-positive clone, pFOS608, revealed a number of additional genes involved in PAH degradation. At this locus, one putative operon contained genes involved in phthalate degradation, and another contained genes encoding a putative ABC transporter(s). A number of the genes found in this region are homologous to those involved in phenanthrene degradation via the phthalic acid pathway. The majority of phenanthrene degradation genes were located between putative transposase genes. In Escherichia coli, pFOS608 converted phenanthrene into phenanthrene cis-3,4-dihydrodiol, and converted 1-hydroxy-2-naphthoic acid into 2'-carboxybenzalpyruvate, 2-carboxybenzaldehyde, and phthalic acid. A subclone containing nidA and nidB converted phenanthrene into phenanthrene cis-3,4-dihydrodiol, suggesting that the NidAB dioxygenase is responsible for an initial attack on phenanthrene. This study is the first to identify genes responsible for the degradation of phenanthrene via the phthalic acid pathway in Mycobacterium species.     

Tracing Evolutionary Footprints to Identify Novel Gene Functional Linkages

Systematic determination of gene function is an essential step in fully understanding the precise contribution of each gene for the proper execution of molecular functions in the cell. Gene functional linkage is defined as to describe the relationship of a group of genes with similar functions. With thousands of genomes sequenced, there arises a great opportunity to utilize gene evolutionary information to identify gene functional linkages. To this end, we established a computational method (called TRACE) to trace gene footprints through a gene functional network constructed from 341 prokaryotic genomes. TRACE performance was validated and successfully tested to predict enzyme functions as well as components of pathway. A so far undescribed chromosome partitioning-like protein ro03654 of an oleaginous bacteria Rhodococcus sp. RHA1 (RHA1) was predicted and verified experimentally with its deletion mutant showing growth inhibition compared to RHA1 wild type. In addition, four proteins were predicted to act as prokaryotic SNARE-like proteins, and two of them were shown to be localized at the plasma membrane. Thus, we believe that TRACE is an effective new method to infer prokaryotic gene functional linkages by tracing evolutionary events.     

Catabolism of benzoate and phthalate in Rhodococcus sp. strain RHA1: redundancies and convergence

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (pat), and two branches of the beta-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the beta-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent beta-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.     

Aerobic tryptophan degradation pathway in bacteria: novel kynurenine formamidase

While a variety of chemical transformations related to the aerobic degradation of L-tryptophan (kynurenine pathway), and most of the genes and corresponding enzymes involved therein have been predominantly characterized in eukaryotes, relatively little was known about this pathway in bacteria. Using genome comparative analysis techniques we have predicted the existence of the three-step pathway of aerobic L-tryptophan degradation to anthranilate (anthranilate pathway) in several bacteria. Based on the chromosomal gene clustering analysis, we have identified a previously unknown gene encoding for kynurenine formamidase (EC 3.5.1.19) involved with the second step of the anthranilate pathway. This functional prediction was experimentally verified by cloning, expression and enzymatic characterization of recombinant kynurenine formamidase orthologs from Bacillus cereus, Pseudomonas aeruginosa and Ralstonia metallidurans. Experimental verification of the inferred anthranilate pathway was achieved by functional expression in Escherichia coli of the R. metallidurans putative kynBAU operon encoding three required enzymes: tryptophan 2,3-dioxygenase (gene kynA), kynurenine formamidase (gene kynB), and kynureninase (gene kynU). Our data provide the first experimental evidence of the connection between these genes (only one of which, kynU, was previously characterized) and L-tryptophan aerobic degradation pathway in bacteria.