The genomes of the non-clearing-zone-forming and natural-rubber- degrading species Gordonia polyisoprenivorans and Gordonia westfalica harbor genes expressing Lcp activity in Streptomyces strains

Abstract

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.

FIG. 1.

Formation of aldehydes and clearing zones on latex overlay-agar plates. Recombinant strains of S. lividans strain TK23 harboring plasmid pIJSK::lcp_K30 (A), pIJSK::lcp_VH2 (B), pIJSK::lcp_Kb1 (C), or pIJSK (D) were incubated for 10 days at 30°C on latex overlay-agar plates containing 0.5% (wt/vol) glucose and stained with Schiff's reagent to detect aldehydes; results are shown in the upper part of the figure. Clearing-zone formation was documented for the same recombinant strains of S. lividans strain TK23 in the corresponding panels at the bottom of the figure after 30 days of incubation at 30°C on latex overlay-agar plates containing 0.5% (wt/vol) glucose. Thiostrepton (25 μg/ml) was added to the medium for plasmid maintenance.

FIG. 2.

Changes of molecular masses of poly(cis-1,4-isoprene) after incubation with recombinant strains of S. lividans strain TK23. The diagram represents GPC elution profiles for residual chloroform-soluble polymers after incubation of synthetic poly(cis-1,4-isoprene) with an average molecular mass (M) of 800 kDa (catalog no. 182141; Sigma-Aldrich, Steinheim, Germany) with recombinant strains of S. lividans strain TK23 harboring plasmid pIJSK::lcp_K30, pIJSK::lcp_VH2, pIJSK::lcp_Kb1, or pIJSK for 8 weeks.

FIG. 3.

Transcription analysis of lcp_VH2 in G. polyisoprenivorans strain VH2. Expression of lcp_VH2 was analyzed by RT-PCR of samples containing total RNA isolated from cells of G. polyisoprenivorans strain VH2 in the logarithmic growth phase. The resulting PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide, and a negative image is presented. Cells were grown in MSM with either 0.2% (wt/vol) sodium acetate (lanes 1 and 2) or 0.25% (wt/vol) poly(cis-1,4-isoprene) (lanes 3 and 4) as sole carbon source. Lanes 1 and 3 represent the controls to detect DNA contamination, whereas lanes 2 and 4 represent the RT-PCR assay. A 100-bp DNA ladder (lanes M; MBI Fermentas, Germany) was used for size comparison.

FIG. 4.

Immunological detection of Lcp from Gordonia species. (A) Electropherogram of an SDS-polyacrylamide gel after separation of proteins from crude cell extracts and extracellular protein fractions. Proteins in the gel were stained with Coomassie brilliant blue R250. (B) Western blot employing anti-Lcp_K30 IgG antibodies prepared from an SDS-polyacrylamide gel. Std, molecular mass standard; lanes 1, six-His-tagged Lcp_K30 protein; lanes 2, extracellular protein fraction of S. lividans strain TK23 harboring pIJSK::lcp_K30; lanes 3, crude cell extract of S. lividans strain TK23 harboring pIJSK::lcp_K30; lanes 4, crude cell extract of S. lividans strain TK23 harboring pIJSK; lanes 5, extracellular protein fraction of Streptomyces sp. strain K30 wild type grown on latex; lanes 6, crude cell extract of Streptomyces sp. strain K30 wild type grown on latex; lanes 7, crude cell extract of Streptomyces sp. strain K30 wild type grown on glucose. Streptomyces sp. strain K30 was grown in MSM either with 0.2% (wt/vol) latex or with 0.5% glucose. The recombinant strain of S. lividans strain TK23 containing plasmid pIJSK::lcp_K30 was cultivated in MSM with 0.5% (wt/vol) glucose plus 0.2% (wt/vol) latex as carbon sources. In the Western blot the anti-Lcp_K30 IgG antibodies recognized the approximately 46-kDa six-His-tagged Lcp_K30 protein (a), the approximately 45-kDa Lcp_K30 with signal peptide (b), and the approximately 42-kDa Lcp_K30 after signal peptide cleavage (c). Furthermore, Lcp present in crude cell extracts was detected in dot blot assays. (C) Crude cell extracts of the recombinant strains of S. lividans strain TK23 containing plasmids pIJSK::lcp_VH2 (lane 1), pIJSK::lcp_Kb1 (lane 2), pIJSK::lcp_K30 (lane 3), or pIJSK (lane 4), cultivated for 1 week at 30°C in MSM containing 0.5% (wt/vol) glucose plus 0.2% (wt/vol) latex as carbon sources, were applied to a PVDF membrane, and the immunological analysis was done as described in the manual from the manufacturer (GE Healthcare, Buckinghamshire, United Kingdom).