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The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis-1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes, Nocardia farcinica, and Thermomonospora curvata. While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia, strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50°C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearing protein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly(cis-1,4-isoprene), confirming its involvement in initial polymer cleavage.Many bacterial isolates that are able to utilize poly(cis-1,4-isoprene) (rubber) as the sole source of carbon and energy have been described during the last hundred years (for details, see references 14 and 18). These bacteria can be divided into two groups, which follow different strategies to degrade rubber (12). Members of the first group form translucent halos if they are cultivated on solid media containing dispersed latex particles, indicating the excretion of rubber-cleaving enzymes. Mycelium-forming actinomycetes such as Actinoplanes, Micromonospora, and Streptomyces species belong to this group. The second group comprises mycolic acid-containing Actinobacteria belonging to the genera Gordonia, Mycobacterium, and Nocardia. These bacteria are not able to form translucent halos, but they grow adhesively at the surface of rubber particles in liquid culture, and they represent the most potent rubber-degrading bacterial strains (2). All rubber-degrading species described so far are mesophilic species, with only one exception, identified as a Streptomyces sp. closely related to Streptomyces albogriseolus and Streptomyces viridodiastaticus, which was able to grow at temperatures of up to...

Gutta percha, the trans-isomer of polyisoprene, is being used for several technical applications due to its resistance to biological degradation. In the past, several attempts to isolate micro-organisms capable of degrading chemically pure poly(trans-1,4-isoprene) have failed. This is the first report on axenic cultures of bacteria capable of degrading gutta percha. From about 100 different habitats and enrichment cultures, six bacterial strains were isolated which utilize synthetic poly(trans-1,4-isoprene) as sole carbon and energy source for growth. All isolates were assigned to the genus Nocardia based on 16S rRNA gene sequences. Four isolates were identified as strains of Nocardia nova (L1b, SH22a, SEI2b and SEII5a), one isolate was identified as a strain of Nocardia jiangxiensis (SM1) and the other as a strain of Nocardia takedensis (WE30). In addition, the type strain of N. takedensis obtained from a culture collection (DSM 44801 T ) was shown to degrade poly(trans-1,4-isoprene). Degradation of poly(trans-1,4-isoprene) by these seven strains was verified in mineralization experiments by determining the release of CO 2 . All seven strains were also capable of mineralizing poly(cis-1,4-isoprene) and to use this polyisoprenoid as a carbon and energy source for growth. Mineralization of poly(trans-1,4-isoprene) after 80 days varied from 3 % (strain SM1) to 54 % (strain SEI2b) and from 34 % (strain L1b) to 43 % (strain SH22a) for the cis-isomer after 78 days. In contrast, Gordonia polyisoprenivorans strain VH2, which was previously isolated as a potent poly(cis-1,4-isoprene)-degrading bacterium, was unable to degrade poly(trans-1,4-isoprene). Scanning electron microscopy revealed cavities in solid materials prepared from poly(trans-1,4-isoprene) and also from poly(cis-1,4-isoprene) after incubation with N. takedensis strain WE30 or with N. nova strain L1b, whereas solid poly(trans-1,4-isoprene) material remained unaffected if incubated with G. polyisoprenivorans strain VH2 or under sterile conditions.

The latex-clearing protein (Lcp K30 ) from the rubber-degrading bacterium Streptomyces sp. strain K30 is involved in the cleavage of poly(cis-1,4-isoprene), yielding isoprenoid aldehydes and ketones. Lcp homologues have so far been detected in all investigated clearing-zone-forming rubber-degrading bacteria. Internal degenerated oligonucleotides derived from lcp genes of Streptomyces sp. strain K30 (lcp K30 ), Streptomyces coelicolor strain A3(2), and Nocardia farcinica strains IFM10152 and E1 were applied in PCR to investigate whether lcp homologues occur also in the non-clearing-zone-forming rubber-utilizing bacteria Gordonia polyisoprenivorans strains VH2 and Y2K, Gordonia alkanivorans strain 44187, and Gordonia westfalica strain Kb1, which grow adhesively on rubber. The 1,230-and 1,224-bp lcp-homologous genes from G. polyisoprenivorans strain VH2 (lcp VH2 ) and G. westfalica strain Kb1 (lcp Kb1 ) were obtained after screening genomic libraries by degenerated PCR amplification, and their translational products exhibited 50 and 52% amino acid identity, respectively, to Lcp K30 . Recombinant lcp VH2 and lcp Kb1 harboring cells of the non-rubber-degrading Streptomyces lividans strain TK23 were able to form clearing zones and aldehydes on latex overlay-agar plates, thus indicating that lcp VH2 and lcp Kb1 encode functionally active proteins. Analysis by gel permeation chromatography demonstrated lower polymer concentrations and molecular weights of the remaining polyisoprenoid molecules after incubation with these recombinant S. lividans strains. Reverse transcription-PCR analysis demonstrated that lcp VH2 was transcribed in cells of G. polyisoprenivorans strain VH2 cultivated in the presence of poly(cis-1,4-isoprene) but not in the presence of sodium acetate. Anti-Lcp K30 immunoglobulin Gs, which were raised in this study, were rather specific for Lcp K30 and did not cross-react with Lcp VH2 and Lcp Kb1 . A lcp VH2 disruption mutant was still able to grow with poly(cis-1,4-isoprene) as sole carbon source; therefore, lcp VH2 seems not to be essential for rubber degradation in G. polyisoprenivorans.

A gene transfer system for Rhodococcus opacus PD630 based on electroporation was established and optimized employing the Escherichia coli-Rhodococcus shuttle vectors pNC9501 and pNC9503 as well as the E. coli-Corynebacterium glutamicum shuttle vector pJC1 as suitable cloning vectors for R. opacus PD630, resulting in transformation efficiencies up to 1.5 x 10(5) CFUs/microgram plasmid DNA. Applying the optimized electroporation protocol to the pNC9501-derivatives pAK68 and pAK71 harboring the entire PHB synthesis operon from Ralstonia eutropha and the PHA synthase gene phaC1 from Pseudomonas aeruginosa, respectively, recombinant PHA biosynthesis was established in R. opacus PD630 and the TAG-negative mutant ROM34. Plasmid pAK68 enabled synthesis and accumulation of poly(3HB) in R. opacus PD630 and ROM34 during cultivation under storage conditions from 1% (w/v) gluconate, of poly(3HB-co-3HV) from 0.2% (w/v) propionate and of poly(3HV) from 0.1% (w/v) valerate. Under storage conditions, recombinant strains of PD630 and ROM34 harboring pAK71 were able to synthesize and accumulate PHA of the medium chain length hydroxyalkanoic acids 3HHx, 3HO, 3HD and 3HDD from 0.1% (w/v) hexadecane or octadecane and a copolyester composed of 3HHp, 3HN and 3HUD from 0.1% (w/v) pentadecane or heptadecane. In the recombinant strains of PD630 and ROM34, the thiostrepton-induced overexpression of a 20 kDa protein was observed with its N-terminus exhibiting a homology of 60% identical amino acids to TipA from Streptomyces lividans.

About 22,000 1-methyl-3-nitro-1-nitrosoguanidine-and UV-induced mutants of the rubber-degrading bacterium Streptomyces sp. strain K30 were characterized for the ability to produce clear zones on natural rubber latex overlay agar plates. Thirty-five mutants were defective solely in cleavage of rubber and were phenotypically complemented with the wild-type lcp (latex clearing protein) gene. Sixty-nine mutants exhibited a pleiotropic phenotype and were impaired in utilization of rubber and xylan, indicating that the enzymes responsible for the initial cleavage of these polymers are exported by the same secretion pathway (Q. In contrast, oxiB and oxiA, which are located directly downstream of lcp and putatively encode a heteromultimeric aldehyde dehydrogenase oxidizing the primary cleavage products generated by Lcp from poly(cis-1,4-isoprene), were expressed only in the presence of poly(cis-1,4-isoprene). Expression of lcp at a low level is thus required for sensing the polymer in the medium. Rubber degradation products may then induce the transcription of genes coding for enzymes catalyzing the later steps of poly(cis-1,4-isoprene) degradation and the transcription of lcp itself. lcp, oxiB, and oxiA seem to constitute an operon, as a polycistronic mRNA comprising these three genes was detected. The transcriptional start site of lcp was mapped 400 bp upstream of the lcp start codon.KThe microbial degradation of natural and synthetic poly(cis-1,4-isoprene) rubber is currently being intensively investigated (for a review, see reference 24), and two different strategies for degradation of isoprene rubber have been described (18). Members of one group of organisms form clear zones on natural rubber latex agar plates and generally belong to the mycelium-forming actinomycetes, such as Actinoplanes, Streptomyces, and Micromonospora. Members of the second group comprise nocardioform actinomycetes like Gordonia, Mycobacterium, and Nocardia and do not produce translucent halos; instead, they require direct contact with the rubber substrates (37). Xanthomonas sp. strain 35Y is the only known rubberdegrading bacterium that does not belong to the actinomycetes but is a gram-negative bacterium (34); however, in terms of its strategy for rubber degradation, it belongs to the first group and forms halos on rubber-containing agar plates.Whereas no proteins involved in rubber degradation in the nocardioform actinomycetes are known so far, the rubbercleaving dioxygenase RoxA occurring in culture supernatants of Xanthomonas sp. strain 35Y (16, 34) and the lcp gene encoding Lcp (latex clearing protein) in Streptomyces sp. strain K30 have recently been identified and characterized by Braaz et al. (6) and Rose and Steinbüchel (27), respectively. Both of these bacteria belong to the so-called clear-zone-forming group of rubber-degrading bacteria, and obviously, both RoxA and Lcp are secreted into the extracellular medium, leading to the formation of translucent halos on natural rubber latex. Sequence analysis of Lcp and characterization...

Two cis-1,4-polyisoprene (isoprene rubber) degrading bacteria, strains VH2 and Y2K, were identified as strains of the species Gordonia polyisoprenivorans belonging to the Corynebacterineae, a suborder of the order Actinomycetales. Both showed characteristic growth and degradation of isoprene rubber as described previously for the type strain of G. polyisoprenivorans Kd2 (DSM 44302 T ). For strain VH2 the chemotaxonomic properties were investigated, and DNA^DNA hybridization experiments with the type strain revealed the affiliation to the species G. polyisoprenivorans. The comparison of the 16S rDNA sequences, and especially hyper variable regions of these, led to the classification of strain Y2K to the same species. At present, the species G. polyisoprenivorans comprises three different isolates which share the ability to degrade isoprene rubber potently but which were obtained from different geographic regions. ß

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