Involvement of two latex-clearing proteins during rubber degradation and insights into the subsequent degradation pathway revealed by the genome sequence of Gordonia polyisoprenivorans strain VH2

Abstract

The increasing production of synthetic and natural poly(cis-1,4-isoprene) rubber leads to huge challenges in waste management. Only a few bacteria are known to degrade rubber, and little is known about the mechanism of microbial rubber degradation. The genome of Gordonia polyisoprenivorans strain VH2, which is one of the most effective rubber-degrading bacteria, was sequenced and annotated to elucidate the degradation pathway and other features of this actinomycete. The genome consists of a circular chromosome of 5,669,805 bp and a circular plasmid of 174,494 bp with average GC contents of 67.0% and 65.7%, respectively. It contains 5,110 putative protein-coding sequences, including many candidate genes responsible for rubber degradation and other biotechnically relevant pathways. Furthermore, we detected two homologues of a latex-clearing protein, which is supposed to be a key enzyme in rubber degradation. The deletion of these two genes for the first time revealed clear evidence that latex-clearing protein is essential for the microbial utilization of rubber. Based on the genome sequence, we predict a pathway for the microbial degradation of rubber which is supported by previous and current data on transposon mutagenesis, deletion mutants, applied comparative genomics, and literature search.

Fig 1

Visualization of the genome of G. polyisoprenivorans strain VH2 and its comparison to genomes of other rubber- and non-rubber-degrading bacteria. The chromosome is shown on the left and plasmid p174 on the right. Circle 1 shows positions in the chromosome and plasmid, respectively (in kb); circles 2 and 3 show proteins encoded on leading (red) and lagging (blue) strands of strain VH2; circle 4 shows the proteome of IR-degrading Nocardia farcinica strain IFM 10152; circle 5 shows the proteome of IR-degrading Actinosynnema mirum strain DSM 43827; circle 6 shows the proteome of IR-degrading Streptomyces flavogriseus strain ATCC 33331; circle 7 shows the proteome of non-IR-degrading Rhodococcus jostii strain RHA1; circle 8 shows the proteome of non-IR-degrading Mycobacterium smegmatis strain mc²155. In circles 4 to 8, similarities to proteins of strain VH2 are plotted in different colors (the darker the color, the higher the similarity). Circle 9 is a GC plot; circle 10 (chromosome only) is a GC skew. Comparative genomics was done using BiBag software.

Fig 2

Growth of G. polyisoprenivorans strain VH2 and lcp deletion mutants cultivated in mineral salt medium with 0.2% IR at 30°C (semilogarithmic plot; triple determination of growth). ♦, G. polyisoprenivorans strain VH2 (wild type); ■, G. polyisoprenivorans Δlcp1ΩKm (mutant strain M71); ▲, G. polyisoprenivorans Δlcp2ΩApra (mutant strain M9); ×, G. polyisoprenivorans Δlcp1ΩKm Δlcp2ΩApra (mutant strain C15); , G. polyisoprenivorans Δlcp1ΩKm harboring pMA5096 upstream of lcp2 (mutant strain TH15).

Fig 3

Location of lcp1, lcp2, and adjacent genes. (A) lcp1 and surrounding area (located on chromosome): 1, putative geranylgeranyl pyrophosphate synthase; 2, putative monooxygenase; 3, hypothetical protein; 4, glycosyl transferase family 2; 5, phytoene desaturase; 6, phytoene desaturase; 7, phytoene synthase; 8, isopentenyl-diphosphate delta-isomerase; 9, hypothetical protein; 10, putative protease; 11, putative transcriptional regulator, TetR family; 12, latex-clearing protein 1; 13, CinA domain-containing protein. (B) lcp2 and surrounding area (located on plasmid): 14, MarR family transcriptional regulator; 15, hypothetical protein; 16, latex-clearing protein 2; 17, ApbE family lipoprotein; 18, ferric reductase domain-containing protein; 19, NADH dehydrogenase.

Fig 4

Predicted pathway of poly(cis-1,4-isoprene) rubber degradation by G. polyisoprenivorans strain VH2 based on annotated genome, transposon-induced mutants, comparative genomics, and literature search. The predicted transporter is visualized via a hypothetical arrangement based on that of Casali and Riley (24). CM, cytoplasmatic membrane; CoA and CoASH, coenzyme A.