strain K30. only known rubber-degrading bacterium that does not belong to the actinomycetes but is a Gram-negative bacterium (Jendrossek and Reinhardt 2003). A rubber oxygenase RoxA, which is synthesized during growth on NR latex by sp. 35Y, was identified (Jendrossek and Reinhardt 2003; Braaz et al. 2005). This bacterium is strictly aerobic and produces insoluble yellow pigments in the cell. species belong to the phylum Proteobacteria and stain Gram-negative. However, regarding the strategy of rubber degradation, it belongs to the first group and forms halos on rubber-containing agar plates. In a hypothetical pathway supposed for rubber degradation, Bode et al. (2000) postulated a not further characterized oxidation of the degradation product acetonyldiprenylacetoaldehyde to the corresponding acid. This aldehyde compound was previously also identified by Tsuchii and Takeda (1990) after incubation of NR with sp. 35Y and subsequent ether extraction. This oxidation step converting the aldehyde to the corresponding acid could possibly be performed by an enzyme similar to OxiAB whereas Lcp is responsible for the first step in this pathway, the oxidative cleavage of the polyisoprene backbone. These aldehyde and ketones with low molecular weights, which are then possibly further oxidized by OxiAB to the corresponding acids, are activated and metabolized via the -oxidation pathway in sp. K30 (Fig. 1). Open in a separate window Figure 1 Hypothetical pathway of poly(-1,4-isoprene) degradation by Kl sp. strain K30. Rose et al. (2005) identified the gene encoding a latex clearing protein from sp. strain K30. The clear zone forming phenotype was used to identify clones harboring the gene from sp. strain K30 by phenotypic complementation of a clear zone negative mutant. The 1191-bp structural gene was preceded by a putative signal sequence and restored the capability of forming clear zones on NR latex agar plates in the mutant. Like RoxA, also Lcp Coenzyme Q10 (CoQ10) is secreted into the extracellular Coenzyme Q10 (CoQ10) medium leading to the formation of translucent halos on NR latex. However, both proteins share no sequence homologies. The putative translation product of exhibited strong homologies (50% aa identity) to a putative secreted protein from strain A3 (Bagdasarian and Timmis 1982), which is another clear zone forming strain (Rose et al. 2005). Sequence analysis of Lcp and characterization of mutants of sp. strain K30 showed secretion of Lcp via the twin-arginine translocation (Tat) pathway (Yikmis et al. 2008; Thomas et al. 2001). Because expression of functional Lcp in recombinant strains or in recombinant -Proteobacteria such as was not successful, expression of recombinant Lcp in Coenzyme Q10 (CoQ10) other bacteria belonging to the genus sp., was performed. In this study, we show a system optimized for the expression of recombinant Coenzyme Q10 (CoQ10) Lcp and the microbial degradation of rubber by these strains. Three actinomycetes strains, TK23, TK24, and gene to these strains. Furthermore, we have conducted an important experiment to demonstrate Lcp activity using the supernatant of these Lcp-expressing strains in vitro. All three strains obviously secreted a functional Lcp, as indicated by the formation of a halo. We also generated a knock out mutant from sp. strain K30 to characterize the role of Lcp with regard to poly(mutant, we have now confirmed evidence that Lcp is responsible for the initial rubber degradation. Materials and Methods Bacterial strains and culture conditions Bacteria and plasmids used in this study are listed in Table 1. If not otherwise mentioned, cells of sp. were grown in tryptic soy broth (TSB) medium at 30C (Merck, Darmstadt, Germany), whereas cells of were cultivated at 37C in Luria Bertani broth (LB) (Sambrook et al. 1989), mineral salts medium (MSM) (Schlegel et al. 1961), or in standard I (St-I) medium (Merck). Antibiotics were applied according to Sambrook et al. (1989) and as indicated in the text. For growth experiments with natural and synthetic polyisoprene, cells were cultivated in MSM (Schlegel et al. 1961). The following carbon sources were added to liquid MSM: Coenzyme Q10 (CoQ10) 0.5% (v/v) natural latex concentrate (Neotex Latz; Weber & Schaer, Hamburg, Germany) or 0.3% (w/v) synthetic poly(was a gift from Weber & Schaer and was used for the preparation of overlay plates as described previously (Jendrossek et al. 1997). Latex overlay agar plates were used for growth of clear zone forming strains. For this, MSM agar plates were covered with an overlay of MSM agar containing 0.2% (v/v) disperged latex concentrate. Table 1 Bacterial strains, plasmids,.