Rhizobacter gummiphilus NS21 has two rubber oxygenases (RoxA and RoxB) acting synergistically in rubber utilisation

Biodegradation of poly(cis-1,4-isoprene) (rubber) by Gram-negative bacteria has been investigated on the enzymatic level only in Steroidobacter cummioxidans 35Y (previously Xanthomonas sp. 35Y). This species produces two kinds of rubber oxygenases, RoxA_35Y and RoxB_35Y, one of which (RoxB_35Y) cleaves polyisoprene to a mixture of C₂₀- and higher oligoisoprenoids while the other (RoxA_35Y) cleaves polyisoprene and RoxB_35Y-derived oligoisoprenoids to the C₁₅-oligoisoprenoid 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). ODTD can be taken up by S. cummioxidans and used as a carbon source. Gram-positive rubber-degrading bacteria employ another type of rubber oxygenase, latex clearing protein (Lcp), for the initial oxidative attack of the polyisoprene molecule. In this contribution, we examined which type of rubber oxygenase is present in the only other well-documented Gram-negative rubber-degrading species, Rhizobacter gummiphilus NS21. No homologue for an Lcp protein but homologues for a putative RoxA and a RoxB protein (the latter identical to a previously postulated LatA-denominated rubber cleaving enzyme) were identified in the genome of strain NS21. The roxA_NS21 and roxB_NS21 genes were separately expressed in a ∆roxA_35Y/∆roxB_35Y background of S. cummioxidans 35Y and restored the ability of the mutant to produce oligoisoprenoids. The RoxA_NS21 and RoxB_NS21 proteins were each purified and biochemically characterised. The results—in combination with in silico analysis of databases—indicate that Gram-negative rubber-degrading bacteria generally utilise two synergistically acting rubber oxygenases (RoxA/RoxB) for efficient cleavage of polyisoprene to ODTD.

     

Phe317 Is Essential for Rubber Oxygenase RoxA Activity

RoxA is an extracellular c-type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during growth on rubber. RoxA cleaves poly(cis-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (≈5 Å) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a Xanthomonas sp. ΔroxA strain. Residual clearing zone formation on opaque latex agar was found for Xanthomonas sp. strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced to ≈3% and 10%, respectively. UV-visible spectroscopy of RoxA muteins confirmed that both heme groups were present in an oxidized form, but spectral responses to the addition of low-molecular-weight (inhibitory) ligand molecules such as imidazole and pyridine were different from those of wild-type RoxA. Our results show that residue 317 is involved in interaction with substrates. This is the first report on structure-function analysis of a polyisoprene-cleaving enzyme and on the identification of an amino acid that is essential for polyisoprene cleavage activity.     

Heme-Dependent Rubber Oxygenase RoxA of Xanthomonas sp. Cleaves the Carbon Backbone of Poly(cis-1,4-Isoprene) by a Dioxygenase Mechanism

Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of ¹⁶O₂, ¹⁸O₂, H₂¹⁶O, and H₂¹⁸O. 12-Oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of ¹⁸O-compounds. Incorporation of one ¹⁸O atom in ODTD was found if the cleavage reaction was performed in the presence of ¹⁸O₂ and H₂¹⁶O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H₂¹⁸O in the presence of ¹⁶O₂ indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of ¹⁸O₂, H₂¹⁶O, and alcohol dehydrogenase/NADH, incorporation of two atoms of ¹⁸O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.     

Development of a homologous expression system for rubber oxygenase RoxA from Xanthomonas sp.

Aims: Natural rubber (poly‐[cis‐1,4‐isoprene]) can be cleaved into 12‐oxo‐4,8‐dimethyltrideca‐4,8‐diene‐1‐al by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA is a novel type of dihaem dioxygenase with unknown cleavage mechanism of the rubber carbon backbone. Analysis of mutant RoxA after mutagenesis could be a way to investigate the function of selected amino acids of RoxA during catalysis. Unfortunately, expression of functional RoxA in recombinant Escherichia coli or in recombinant γ‐Proteobacteria such as Pseudomonas putida was not possible in our hands. Therefore, expression of recombinant RoxA in the homologous host, Xanthomonas, was performed. Methods and Results: A transformation system via electroporation was established, and a conjugation system was optimized for Xanthomonas sp. Inactivation of the chromosomal roxA gene by insertional mutagenesis resulted in inability of Xanthomonas sp. to produce active RoxA and to utilize rubber as a sole source of carbon and energy. When an intact copy of roxA was cloned under control of a rhamnose‐inducible promoter in a broad host range vector and was transferred to Xanthomonas sp., high expression levels of functional RoxA in the presence of rhamnose were obtained. Conclusions and Significance and Impact of the Study: Purification of recombinantly expressed RoxA was simplified because of drastically shortened fermentation times and because separation of RoxA from remaining rubber latex particles was not necessary with rhamnose‐induced cultures. About 6 mg purified RoxA were obtained from 1 l of cell‐free culture fluid. Purified recombinant RoxA was highly active and revealed comparable spectral properties as RoxA purified from the wild type. The results of our study are the methodical basis for molecular biological manipulation in Xanthomonas sp. and will simplify investigation into the biochemical mechanisms by which rubber can be biodegraded in the environment by this novel extracellular dihaem dioxygenase RoxA.     

Heme-dependent rubber oxygenase RoxA of Xanthomonas sp. cleaves the carbon backbone of poly(cis-1,4-Isoprene) by a dioxygenase mechanism

Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of (16)O(2), (18)O(2), H(2)(16)O, and H(2)(18)O. 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of (18)O-compounds. Incorporation of one (18)O atom in ODTD was found if the cleavage reaction was performed in the presence of (18)O(2) and H(2)(16)O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H(2)(18)O in the presence of (16)O(2) indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of (18)O(2), H(2)(16)O, and alcohol dehydrogenase/NADH, incorporation of two atoms of (18)O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.     

Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b-Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties

Specific polyisoprene-cleaving activities of 1.5 U/mg and 4.6 U/mg were determined for purified Strep-tagged latex clearing protein (Lcp) of Streptomyces sp. strain K30 at 23°C and 37°C, respectively. Metal analysis revealed the presence of approximately one atom of iron per Lcp molecule. Copper, which had been identified in Lcp1_VH2 of Gordonia polyisoprenivorans previously, was below the detection limit in Lcp_K30. Heme was identified as a cofactor in purified Lcp_K30 by (i) detection of characteristic α-, β-, and γ (Soret)-bands at 562 nm, 532 nm, and 430 nm in the visible spectrum after chemical reduction, (ii) detection of an acetone-extractable porphyrin molecule, (iii) determination of a heme b-type-specific absorption maximum (556 nm) after chemical conversion of the heme group to a bipyridyl-heme complex, and (iv) detection of a b-heme-specific m/z value of 616.2 via mass spectrometry. Spectroscopic analysis showed that purified Lcp as isolated contains an oxidized heme-Fe³⁺ that is free of bound dioxygen. This is in contrast to the rubber oxygenase RoxA, a c-type heme-containing polyisoprene-cleaving enzyme present in Gram-negative rubber degraders, in which the covalently bound heme firmly binds a dioxygen molecule. Lcp_K30 also differed from RoxA in the lengths of the rubber degradation cleavage products and in having a higher melting point of 61.5°C (RoxA, 54.3°C). In summary, RoxA and Lcp both are equipped with a heme cofactor and catalyze an oxidative C-C cleavage reaction but differ in the heme subgroup type and in several biochemical and biophysical properties. These findings suggest differences in the catalytic reaction mechanisms.     

Novel Type of Heme-Dependent Oxygenase Catalyzes Oxidative Cleavage of Rubber (Poly-cis-1,4-Isoprene)

An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH₂— and —CH₂-COCH₃. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40°C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA [rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.     

Non-contiguous finished genome sequence and description of Oceanobacillus massiliensis sp. nov.

Oceanobacillus massiliensis strain N’Diop^T sp. nov. is the type strain of O. massiliensis sp. nov., a new species within the genus Oceanobacillus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. O. massiliensis is an aerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,532,675 bp long genome contains 3,519 protein-coding genes and 72 RNA genes, including between 6 and 8 rRNA operons.     

Non-contiguous finished genome sequence and description of Brevibacillus massiliensis sp. nov.

Brevibacillus massiliensis strain phR^T sp. nov. is the type strain of B. massiliensis sp. nov., a new species within the genus Brevibacillus. This strain was isolated from the fecal flora of a woman suffering from morbid obesity. B. massiliensis is a Gram-positive aerobic rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,051,018 bp long genome (1 chromosome but no plasmid) contains 5,051 protein-coding and 84 RNA genes, and exhibits a G+C content of 53.1%.     

Non contiguous-finished genome sequence and description of Enorma timonensis sp. nov.

Enorma timonensis strain GD5^T sp. nov., is the type strain of E. timonensis sp. nov., a new member of the genus Enorma within the family Coriobacteriaceae. This strain, whose genome is described here, was isolated from the fecal flora of a 53-year-old woman hospitalized for 3 months in an intensive care unit. E. timonensis is an obligate anaerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,365,123 bp long genome (1 chromosome but no plasmid) contains 2,060 protein-coding and 52 RNA genes, including 4 rRNA genes.     

Non-contiguous finished genome sequence and description of Anaerococcus provenciensis sp. nov.

Anaerococcus provenciensis strain 9402080^T sp. nov. is the type strain of A. provenciensis sp. nov., a new species within the genus Anaerococcus. This strain was isolated from a cervical abscess sample. A. provenciensis is a Gram-positive anaerobic cocci. Here, we describe the features of this organism, together with the complete genome sequence and annotation. The 2.26 Mbp long genome contains 2099 protein-coding and 57 RNA genes including 8 rRNA genes and exhibits a G+C content of 33.48%.     

Historical and recent achievements in the field of microbial degradation of natural and synthetic rubber

This review intends to provide an overview of historical and recent achievements in studies of microbial degradation of natural and synthetic rubber. The main scientific focus is on the key enzymes latex-clearing protein (Lcp) from the Gram-positive Streptomyces sp. strain K30 and rubber oxygenase A (RoxA) from the Gram-negative Xanthomonas sp. strain 35Y, which has been hitherto the only known rubber-degrading bacterium that does not belong to the actinomycetes. We also emphasize the importance of knowledge of biodegradation in industrial and environmental biotechnology for waste natural rubber disposal.     

Non contiguous-finished genome sequence and description of Peptoniphilus senegalensis sp. nov.

Peptoniphilus senegalensis strain JC140^T sp. nov., is the type strain of P. senegalensis sp. nov., a new species within the genus Peptoniphilus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. P. senegalensis strain JC140^T is an obligate Gram-positive anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,840,641 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 32.2% and contains 1,744 protein-coding and 23 RNA genes, including 3 rRNA genes.Key words: Peptoniphilus senegalensis, genome     

Non-contiguous finished genome sequence and description of Fenollaria massiliensis gen. nov., sp. nov., a new genus of anaerobic bacterium

Fenollaria massiliensis strain 9401234^T, is the type strain of Fenollaria massiliensis gen. nov., sp. nov., a new species within a new genus Fenollaria. This strain, whose genome is described here, was isolated from an osteoarticular sample. F. massiliensis strain 9401234^T is an obligate anaerobic Gram-negative bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1.71 Mbp long genome exhibits a G+C content of 34.46% and contains 1,667 protein-coding and 30 RNA genes, including 3 rRNA genes.     

The evolution of menopause in cetaceans and humans: the role of demography

Human females stop reproducing long before they die. Among other mammals, only pilot and killer whales exhibit a comparable period of post-reproductive life. The grandmother hypothesis suggests that kin selection can favour post-reproductive survival when older females help their relatives to reproduce. But although there is an evidence that grandmothers can provide such assistance, it is puzzling why menopause should have evolved only among the great apes and toothed whales. We have previously suggested (Cant & Johnstone 2008 Proc. Natl Acad. Sci. USA 105, 5332–5336 (doi:10.1073/pnas.0711911105)) that relatedness asymmetries owing to female-biased dispersal in ancestral humans would have favoured younger females in reproductive competition with older females, predisposing our species to the evolution of menopause. But this argument appears inapplicable to menopausal cetaceans, which exhibit philopatry of both sexes combined with extra-group mating. Here, we derive general formulae for ‘kinship dynamics’, the age-related changes in local relatedness that occur in long-lived social organisms as a consequence of dispersal and mortality. We show that the very different social structures of great apes and menopausal whales both give rise to an increase in local relatedness with female age, favouring late-life helping. Our analysis can therefore help to explain why, of all long-lived, social mammals, it is specifically among the great apes and toothed whales that menopause and post-reproductive helping have evolved.     

Non-contiguous finished genome sequence and description of Paucisalibacillus algeriensis sp. nov.

Paucisalibacillus algeriensis strain EB02^T is the type strain of Paucisalibacillus algeriensis sp. nov., a new species within the genus Paucisalibacillus. This strain, whose genome is described here, was isolated from soil sample from the hypersaline lake Ezzemoul Sabkha in northeastern Algeria. Paucisalibacillus algeriensis is a Gram-positive and strictly aerobic bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,006,766 bp long genome (1 chromosome but no plasmid) exhibits a low G+C content of 36% and contains 3,956 protein-coding and 82 RNA genes, including 9 rRNA genes.     

Non-contiguous finished genome sequence and description of Paenibacillus senegalensis sp. nov.

Paenibacillus senegalensis strain JC66^T, is the type strain of Paenibacillus senegalensis sp. nov., a new species within the genus Paenibacillus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. P. senegalensis strain JC66^T is a facultative Gram-negative anaerobic rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,581,254 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 48.2% and contains 5,008 protein-coding and 51 RNA genes, including 9 rRNA genes.     

Genome sequence of the lupin-nodulating Bradyrhizobium sp. strain WSM1417

Bradyrhizobium sp. strain WSM1417 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen (N₂) fixing root nodule of Lupinus sp. collected in Papudo, Chile, in 1995. However, this microsymbiont is a poorly effective N₂ fixer with the legume host Lupinus angustifolius L.; a lupin species of considerable economic importance in both Chile and Australia. The symbiosis formed with L. angustifolius produces less than half of the dry matter achieved by the symbioses with commercial inoculant strains such as Bradyrhizobium sp. strain WSM471. Therefore, WSM1417 is an important candidate strain with which to investigate the genetics of effective N₂ fixation in the lupin-bradyrhizobia symbioses. Here we describe the features of Bradyrhizobium sp. strain WSM1417, together with genome sequence information and annotation. The 8,048,963 bp high-quality-draft genome is arranged in a single scaffold of 2 contigs, contains 7,695 protein-coding genes and 77 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.