Characterization of the genes responsible for rubber degradation in Actinoplanes sp. strain OR16.

A Gram-positive rubber-degrading bacterium, Actinoplanes sp. strain OR16 (strain NBRC 114529), is able to grow on agar plates containing natural and synthetic rubber as the sole sources of carbon and energy. When this strain was grown on natural rubber latex overlay agar plates, translucent halos around the cells were observed. To identify the natural rubber degradation genes and other features of its metabolism, its complete genome sequence was determined. The genome of OR16 consists of 9,293,892 bp and comprises one circular chromosome (GenBank accession number AP019371.1) with a G + C content of 70.3%. The genome contains 8238 protein-coding and 18 rRNA genes. A homology search of the genome sequence revealed that three genes (lcp1, lcp2, and lcp3) are homologous to an extracellular latex-clearing protein (Lcp) of Streptomyces sp. K30. RT-PCR analysis revealed that lcp1 and lcp2 seem to constitute an operon. Purified lcp gene products have oxygen consumption activity toward natural rubber latex, suggesting that all these genes encode rubber-degrading enzymes in OR16. Quantitative reverse transcription-PCR analysis indicated that the transcription of these genes is induced during the growth of OR16 on natural rubber. The genes located adjacent to lcp1 and lcp3, which code for a TetR/AcrR-type transcriptional regulator, can bind to the promoter regions of these lcp genes. It is suggested that the putative regulators play a role in regulating the transcription of the lcp genes. These results strongly suggested that three lcp genes are required for the utilization of natural rubber in strain OR16. Key Points • The complete genome sequence of Actinoplanes sp. strain OR16 was determined. • Three lcp genes which are involved in the natural rubber degradation in OR16 were identified. • Transcription of these lcp genes is induced during utilization of rubber in OR16. • Two regulators, which bind to the promoter regions of lcp, were determined.

Poly(cis-1,4-isoprene)-cleavage enzymes from natural rubber-utilizing bacteria.

Natural rubber and synthetic poly(cis-1,4-isoprene) are used industrially in the world. Microbial utilization for the isoprene rubbers has been reported in gram-positive and gram-negative bacteria. Poly(cis-1,4-isoprene)-cleavage enzymes that are secreted by rubber-utilizing bacteria cleave the poly(cis-1,4-isoprene) chain to generate low-molecular-weight oligo(cis-1,4-isoprene) derivatives containing aldehyde and ketone groups. The resulting products are converted to the compounds including carboxyl groups, which could then be further catabolized through ß-oxidation pathway. One of poly(cis-1,4-isoprene)-cleavage enzymes is latex-clearing protein (Lcp) that was found in gram-positive rubber degraders including Streptomyces, Gordonia, Rhodococcus, and Nocardia species. The other one is rubber oxygenase A and B (RoxA/RoxB) which have been identified from gram-negative rubber degraders such as Steroidobacter cummioxidans and Rhizobacter gummiphilus. Recently, the transcriptional regulation mechanisms for Lcp-coding genes in gram-positive bacteria have been characterized. Here, the current knowledge of genes and enzymes for the isoprene rubber catabolism were summarized.

Biodegradation of natural rubber and deproteinized natural rubber by enrichment bacterial consortia.

This study examined the biodegradation of natural rubber (NR) and deproteinized natural rubber (DPNR) by bacterial consortia enriched from a rubber-processing factory's waste in Vietnam. The results reveal the degradation in both NR and DPNR, and the DPNR was degraded easier than NR. The highest weight loss of 48.37% was obtained in the fourth enrichment consortium with DPNR, while 35.39% was obtained in the fifth enrichment consortium with NR after 14 days of incubation. Nitrogen content and fatty acid content determined by Kjeldahl method and fourier transform infrared spectroscopy (FTIR), respectively, were decreased significantly after being incubated with the consortia. Structure of degraded rubber film analyzed by nuclear magnetic resonance spectroscopy showed the presence of aldehyde group, a sign of rubber degradation. Bacterial cells tightly adhering and embedding into NR and DPNR films were observed by scanning electron microscopy. There were differences in the bacterial composition of the consortia with NR and DPNR, which were determined by metagenomic analysis using 16S rRNA gene sequencing. The phyla Bacteroidetes and Proteobacteria may play a role in the degradation of non-isoprene compounds such as protein or lipid, while the phylum Actinobacteria plays a crucial role in the degradation of rubber hydrocarbon in all consortia.

Identification of natural rubber degradation gene in Rhizobacter gummiphilus NS21.

A Gram-negative rubber-degrading bacterium, Rhizobacter gummiphilus NS21 grew and produced aldehyde metabolites on a deproteinized natural rubber (DPNR)-overlay agar medium forming a clearing zone. A transposon-insertion mutant, which had lost the ability to degrade DPNR, was isolated to identify the rubber degradation genes. Sequencing analysis indicated that the transposon was inserted into a putative oxygenase gene, latA. The deduced amino acid sequence of latA has 36% identity with that of roxA, which encodes a rubber oxygenase of Xanthomonas sp. strain 35Y. Phylogenetic analysis revealed that LatA constitutes a distinct group from RoxA. Heterologous expression in a Methylibium host and deletion analysis of latA indicated that the latA product is responsible for the depolymerization of DPNR. The quantitative reverse transcription-PCR analysis indicated that the transcription of latA is induced during the growth on DPNR. These results strongly suggest that latA is directly involved in the degradation of rubber in NS21.

Characterization of the catabolic pathway for a phenylcoumaran-type lignin-derived biaryl in Sphingobium sp. strain SYK-6.

Sphingobium sp. strain SYK-6 is capable of degrading various lignin-derived biaryls. We determined the catabolic pathway of a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA) in SYK-6, and identified some of the DCA catabolism genes. In SYK-6 cells, the alcohol group of DCA was oxidized to the carboxyl group, first at the B-ring side chain and then at the A-ring side chain. The resultant metabolite was degraded to 5-formylferulate and vanillin through the decarboxylation and the Cα-Cß cleavage of the A-ring side chain. Based on the DCA catabolic pathway, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) genes are thought to be involved in the conversion of DCA into an aldehyde intermediate (DCA-L) and the conversion of DCA-L into a carboxylic acid intermediate (DCA-C), respectively. SLG_05620 and SLG_24930, which belong to quinohemoprotein ADH and aryl ADH, respectively, were isolated as the genes responsible for the oxidation of DCA. In addition to these genes, multiple genes similar to SLG_05620 and SLG_24930 were found to confer DCA oxidation activities on Escherichia coli cells. In order to identify the DCA-L dehydrogenase genes, the DCA-L oxidation activities of the SYK-6 gene products of putative twenty-one ALDH genes were examined. Significant activities were observed in the four ALDH gene products, including the SLG_27910 product, which showed the highest activity. The disruption of SLG_27910 caused a decreased conversion of DCA-L, suggesting that SLG_27910 plays an important role in the DCA-L oxidation. In conclusion, no specific gene seems to be solely responsible for the conversion of DCA and DCA-L, however, the multiple genes encoding quinohemoprotein ADH and aryl ADH genes, and four ALDH genes are probably involved in the conversion processes.

Complete genome sequence of Sphingobium sp. strain SYK-6, a degrader of lignin-derived biaryls and monoaryls.

Sphingobium sp. strain SYK-6 is able to grow on an extensive variety of lignin-derived biaryls and monoaryls, and the catabolic genes for these compounds are useful for the production of industrially valuable metabolites from lignin. Here we report the complete nucleotide sequence of the SYK-6 genome which consists of the 4,199,332-bp-long chromosome and the 148,801-bp-long plasmid.

Identification and transcriptional analysis of poly(cis-1,4-isoprene) degradation gene in Rhodococcus sp. strain RDE2.

The microbial degradation of synthetic and natural poly(cis-1,4-isoprene) rubber is expected to become an alternative treatment technique for waste from poly(cis-1,4-isoprene) products, such as scrap tires. A gram-positive rubber-degrading bacterium, Rhodococcus sp. strain RDE2, was isolated from the waste of a rubber-processing factory in Vietnam. This strain grew on natural rubber as a sole source of carbon and energy and produced oligo-isoprenoid metabolites containing aldehyde groups from poly(cis-1,4-isoprene). To identify the genes responsible for poly(cis-1,4-isoprene) degradation, the complete genome sequence of this strain was determined. The complete genome sequence consists of a 5,715,406 bp chromosome and 6 plasmids (GenBank accession numbers AP025186.1 to AP025192.1) with an average GC content of 67.9%. The genome contains 5358 protein-coding sequences and 12 and 68 copies of rRNA and tRNA genes, respectively. Based on genome sequence analysis, the lcp gene (RDE2_08,770), responsible for the initial step of poly(cis-1,4-isoprene) degradation, was identified. The gene product obtained from Escherichia coli depolymerizes poly(cis-1,4-isoprene) to low-molecular-weight oligo-isoprenoids. The transcription of this gene is activated during the utilization of poly(cis-1,4-isoprene) in strain RDE2. The lcpR gene (RDE2_08,760), which encodes a putative transcriptional regulator, is located upstream of lcp. The lcpR gene product recognizes the promoter region of lcp. When the lcpR gene is deleted, the constitutive transcription of lcp is observed. Thus, it is inferred that the LcpR negatively regulates lcp transcription. These results strongly suggest that the lcp and lcpR genes are involved in poly(cis-1,4-isoprene) utilization in strain RDE2.

Regulatory system of the protocatechuate 4,5-cleavage pathway genes essential for lignin downstream catabolism.

Sphingobium sp. strain SYK-6 converts various lignin-derived biaryls with guaiacyl (4-hydroxy-3-methoxyphenyl) and syringyl (4-hydroxy-3,5-dimethoxyphenyl) moieties to vanillate and syringate. These compounds are further catabolized through the protocatechuate (PCA) 4,5-cleavage (PCA45) pathway. In this article, the regulatory system of the PCA45 pathway is described. A LysR-type transcriptional regulator (LTTR), LigR, activated the transcription of the ligK-orf1-ligI-lsdA and ligJABC operons in the presence of PCA or gallate (GA), which is an intermediate metabolite of vanillate or syringate, respectively, and repressed transcription of its own gene. LigR bound to the positions -77 to -51 and -80 to -48 of the ligK and ligJ promoters, respectively, and induced DNA bending. In the presence of PCA or GA, DNA bending on both promoters was enhanced. The LigR-binding regions of the ligK and ligJ promoters in the presence of inducer molecules were extended and shortened, respectively. The LTTR consensus sequences (Box-K and Box-J) in the ligK and ligJ promoters were essential for the binding of LigR and transcriptional activation of both operons. In addition, the regions between the LigR binding boxes and the -35 regions were required for the enhancement of DNA bending, although the binding of LigR to the -35 region of the ligJ promoter was not observed in DNase I footprinting experiments. This study shows the binding features of LigR on the ligK and ligJ promoters and explains how the PCA45 pathway genes are expressed during degradation of lignin-derived biaryls by this bacterium.

Regulation of vanillate and syringate catabolism by a MarR-type transcriptional regulator DesR in Sphingobium sp. SYK-6.

Vanillate and syringate are major intermediate metabolites generated during the microbial degradation of lignin. In Sphingobium sp. SYK-6, vanillate is O demethylated to protocatechuate by LigM; protocatechuate is then catabolized via the protocatechuate 4,5-cleavage pathway. Syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and then gallate is subjected to ring cleavage by DesB. Here, we investigated the transcriptional regulation of desA, ligM, and desB involved in vanillate and syringate catabolism. Quantitative reverse transcription-PCR analyses indicated that the transcription of these genes was induced 5.8-37-fold in the presence of vanillate and syringate. A MarR-type transcriptional regulator, SLG_12870 (desR), was identified as the gene whose product bound to the desB promoter region. Analysis of a desR mutant indicated that the transcription of desB, ligM, and desR is negatively regulated by DesR. Purified DesR bound to the upstream regions of desB, ligM, and desR, and the inverted repeat sequences similar to each other in these regions were suggested to be essential for DNA binding of DesR. Vanillate and syringate inhibited DNA binding of DesR, indicating that these compounds are effector molecules of DesR. The transcription of desA was found to be regulated by an as-yet unidentified regulator.

Characterization and functional expression of a rubber degradation gene of a Nocardia degrader from a rubber-processing factory.

A rubber-degrading bacterial consortium named H2DA was obtained from an enrichment culture with natural rubber latex and rubber-processing factory waste in Vietnam. Gel permeation chromatography analysis revealed that only the strain NVL3 degraded synthetic poly(cis-1,4-isoprene) into low-molecular-weight intermediates among the three strains found in the H2DA. The 16S-rRNA gene sequence of NVL3 showed the highest identity with that of Nocardia farcinica DSM 43665T. NVL3 accumulated aldehyde intermediates from synthetic poly(cis-1,4-isoprene) on a rubber-overlay plate as indicated by Schiff's staining. NVL3 also degraded deproteinized natural rubber into low-molecular-weight aldehyde intermediates. A latex-clearing protein (lcp) gene ortholog was identified within the genome sequence of NVL3, and it showed a moderate amino-acid identity (54-75%) with the lcp genes from previously reported rubber degraders. The heterologous expression of the NVL3 lcp in Escherichia coli BL21(DE3) allowed us to purify the 46.8-kDa His-tagged lcp gene product (His-Lcp). His-Lcp degraded synthetic poly(cis-1,4-isoprene) and accumulated aldehyde intermediates from deproteinized natural rubber suggesting the functional expression of the lcp gene from a Nocardia degrader in E. coli. Quantitative reverse transcription PCR analysis indicated the strong transcriptional induction of the lcp gene in NVL3 in the presence of synthetic poly(cis-1,4-isoprene). These results suggest the involvement of the lcp gene in rubber degradation in NVL3.

Rhizobacter gummiphilus sp. nov., a rubber-degrading bacterium isolated from the soil of a botanical garden in Japan.

A rubber-degrading bacterium, the strain NS21, that was isolated from a soil sample in a botanical garden in Japan (Imai et al., 2011) was examined by phenotypic, phylogenetic and chemotaxonomic approaches to determine its taxonomic position. The strain NS21 was motile, possessing a single polar flagellum and a facultatively anaerobic straight rod. Analysis of the 16S rRNA and gyrB gene sequences of NS21 revealed a close relationship to the genus Rhizobacter. The predominant quinone type was Q-8. The G+C content of the NS21 genomic DNA was 70.8 mol%. The major fatty acids were C16:0, C17:0 cyclo, C18:1ω7c and C16:1ω7c and/or iso-C15:0 2-OH. C12:0 2-OH was present. The DNA-DNA hybridization experiments indicated that the DNA relatedness values of the strain NS21 to R. dauci H6(T) and R. fulvus Gsoil 322(T) were lower than 24%. The phenotypic characteristics showed obvious dissimilarities when compared to closely related species. On the basis of these taxonomic properties, a novel species is proposed as Rhizobacter gummiphilus sp. nov., with the strain NS21(T) (NBRC 109400(T), BCC 58006(T)) as the type strain. The emended description of the genus Rhizobacter was also presented.

Isolation and characterization of Streptomyces, Actinoplanes, and Methylibium strains that are involved in degradation of natural rubber and synthetic poly(cis-1,4-isoprene).

Rubber-degrading bacteria were screened for the production of clearing zones around their colonies on latex overlay agar plates. Novel three bacteria, Streptomyces sp. strain LCIC4, Actinoplanes sp. strain OR16, and Methylibium sp. strain NS21, were isolated. To the best of our knowledge, this is the first report on the isolation of a Gram-negative rubber-degrading bacterium other than γ-proteobacteria. Gel permeation chromatography analysis revealed that these strains degraded poly(cis-1,4-isoprene) to low-molecular-weight products. The occurrence of aldehyde groups in the degradation products by NS21 was suggested by staining with Schiff's reagent and ¹H-nuclear magnetic resonance spectroscopy. The lcp gene of LCIC4, which showed 99% amino acid sequence identity with that of Streptomyces sp. strain K30, was cloned, and contained a putative twin-arginine motif at its N terminus. It is located next to oxiB, which is estimated to be responsible for oxidation of degradation intermediate of rubber in K30. Southern hybridization analysis using LCIC4 lcp probe revealed the presence of a lcp-homolog in OR16. These results suggest that the lcp-homologs are involved in rubber degradation in LCIC4 and OR16.

A second 5-carboxyvanillate decarboxylase gene, ligW2, is important for lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6.

A lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), is degraded to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by DDVA O-demethylase (LigX), meta-cleavage oxygenase (LigZ), and meta-cleavage compound hydrolase (LigY) in Sphingomonas paucimobilis SYK-6. 5CVA is then transformed to vanillate by a nonoxidative 5CVA decarboxylase and is further degraded through the protocatechuate 4,5-cleavage pathway. A 5CVA decarboxylase gene, ligW, was isolated from SYK-6 (X. Peng, E. Masai, H. Kitayama, K. Harada, Y, Katayama, and M. Fukuda, Appl. Environ. Microbiol. 68:4407-4415, 2002). However, disruption of ligW slightly affected the 5CVA decarboxylase activity and the growth rate on DDVA of the mutant, suggesting the presence of an alternative 5CVA decarboxylase gene. Here we isolated a second 5CVA decarboxylase gene, ligW2, which consists of a 1,050-bp open reading frame encoding a polypeptide with a molecular mass of 39,379 Da. The deduced amino acid sequence encoded by ligW2 exhibits 37% identity with the sequence encoded by ligW. Based on a gas chromatography-mass spectrometry analysis of the reaction product from 5CVA catalyzed by LigW2 in the presence of deuterium oxide, LigW2 was indicated to be a nonoxidative decarboxylase of 5CVA, like LigW. After disruption of ligW2, both the growth rate on DDVA and the 5CVA decarboxylase activity of the mutant were decreased to approximately 30% of the wild-type levels. The ligW ligW2 double mutant lost both the ability to grow on DDVA and the 5CVA decarboxylase activity. These results indicate that both ligW and ligW2 contribute to 5CVA degradation, although ligW2 plays the more important role in the growth of SYK-6 cells on DDVA.