University of Bucharest Collage of Engineering

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University of Bucharest Collage of Engineering Faculty of Biology Bucharest - Romania Peking University Collage of Engineering Beijing - China Genotypic and phenotypic alkane-degradation features and the phylogeny of two Rhodococcus strains isolated from oil-polluted soil Iulia CHICIUDEAN1,2, Yong NIE2, Ana-Maria TANASE1,Ileana STOICA1, Xiao-Lei WU2* Department of Genetics, Faculty of Biology, University of Bucharest, Bucharest, Romania Department of Energy and Resources Engineering, College of Engineering, Peking University, Beijing 100871, PR China * corresponding author: Xiao-Lei Wu: xiaolei_wu@pku.edu.cn INTRODUCTION DRAFT GENOMES information Rhodococcus species, previously described as “masters of catabolic versatility”, by their constant presence in oil-contaminated areas and by their abilities to degrade hydrocarbons, are important candidates for bioremediation processes. In this study we investigated the phylogeny and genotypic and phenotypic alkane-degradation features of two Rhodococcus strains (designated MH15 and FH8) isolated from oil-polluted soil of Romania. Genome features of Rhodococcus sp. Feature Value strain MH15 strain FH8 Genome size (bp) 6.849.809 6.910.321 GC content 62.50% 62.40% Total number of contigs 88 84 Total genes 6.385 6.458 rRNA genes 3 5 5S rRNA 1 2 16S rRNA 23S rRNA tRNA genes 54 53 Protein-coding genes (CDSs) 6.179 6.259 CDSs with predicted function (by COG) 5.087 5.137 MiSeq system (Illumina) (250-bp GL; fold coverage - MH15:127; FH8:119) de novo assembled using SOAPdenovo 2.04 automatically annotation by PGAAP (Prokaryotic Genomes Automatic Annotation Pipeline, NCBI) and COG (Clusters of Orthologous Groups of proteins database) MOLECULAR PHYLOGENY GENOTYPIC alkane-degradation features 99,2% (16S rRNA) R. erythropolis PR4 R. baikonurensis A1-22 Rhodococcus sp. MH15 R. qingshengii djl-6 99,7% (16S rRNA) 99 % (16S rRNA) 100 % (16S rRNA) 96,2% (alkB) 96,4% (alkB) 96,1% (alkB) 96,6% (alkB) 96,7% (alkB) 99,8% (alkB) 99,9% (16S rRNA) FH8 95,9% (alkB) 99,7% (alkB) Comparisons of 16S rRNA and alkB2 nucleotide sequence homologies between closely related species of the genus Rhodococcus. (Tancsics et al., 2015) Maximum-Likelihood tree based on 16S rRNA gene sequences showing the phylogenetic relationships between Rhodococcus sp. strain MH15 and FH8 and other alkane-degrading Actinobacteria members. Bootstrap values (%) of at least 50% are shown at the branches (scale bar= 0.01). Nucleotide accession numbers are given in parentheses. Rhodococcus sp. strain MH15 - 4 alkane monooxygenase genes homologous alkB1-like gene cluster alkB2-like gene cluster alkB3-like gene alkB-like gene The alkB-like gene and alk gene clusters organizations: homologous alkane monooxygenase genes (alkB-like) ( ) rubredoxin ( ) rubredoxin reductase ( ) transcriptional regulatory proteins ( ). Rhodococcus sp. strain FH8 - 5 alkane monooxygenase genes homologous alkB1-like gene cluster alkB2-like gene cluster alkB3-like gene alkB4-like gene alkB-like gene Additionally, strain MH15 and FH8 have 3 and, respectively, 2 CYP153 genes (P450 cytochrome alkane monooxygenase system for C5–C16 n-alkanes initial oxidation). PHENOTYPIC alkane-degradation features C12 C16 C24 C32 CONCLUSIONS 16S rRNA gene phylogenetic analysis indicated that both strains belong to genus Rhodococcus, with 100% 16S rRNA gene similarity with R. qingshengii djl-6T (DSM 44587T). Alkane-degradation phenotype: Investigated strains (MH15 and FH8) showed the ability to grow on and to degrade medium and long-chain alkanes. Alkane-degradation genotype: both organisms contained 4/5 alkane monooxygenase gene homologs alkB1 and alkB2 homologs were part of alk gene clusters, each encoding rubredoxins, a putative TetR transcriptional regulatory protein, and, in the alkB1 cluster, a rubredoxin reductase multiple alkane hydroxylases systems – 3/2 CYP153 genes This study strengthen the validity of using alkB2 genes as a phylogenetic marker in Rhodococcus inter-species level differentiation. This is the first genome-sequenced based, alkane-degrading- genetic-feature characterization of Rhodococcus strains strongly related with R. qingshengii species. Those alkane-degradation results and genome analyses offer the bases for further metabolic and genetic studies and underline oil- spill bioremediation potential of MH15, FH8 Rhodococcus strains. Reference: Whyte L. G, Smits T. H. M., Labbe D., Witholt B., Greer C. W., van Beilen J. B., (2002) Gene Cloning and Characterization of Multiple Alkane Hydroxylase Systems in Rhodococcus Strains Q15 and NRRL B-16531, Applied and Environmental Microbiology., vol. 68, no. 12: 5933–5942. Táncsicsa A., Benedeka T., Szoboszlayb S., Veresb P. G., Farkasb M., Máthéc I., Márialigetid K., Kukolyae J., Lányic S., Krisztba B., (2015) The detection and phylogenetic analysis of the alkane1-monooxygenase gene of members of the genus Rhodococcus, Systematic and Applied Microbiology, 38:1-7.