The genome analysis of Oleiphilus messinensis ME102 (DSM 13489T) ANGOR UNIVERSITY reveals backgrounds of its obligate alkane-devouring marine lifestyle Toshchakov, Stepan V.; Korzhenkov, Alexei A.; Chernikova, Tatyana; Ferrer, Manuel; Golyshina, Olga; Yakimov, Michail M.; Golyshin, Peter Marine Genomics DOI: 10.1016/j.margen.2017.07.005 PRIFYSGOL BANGOR / B Published: 01/12/2017 Peer reviewed version Cyswllt i'r cyhoeddiad / Link to publication Dyfyniad o'r fersiwn a gyhoeddwyd / Citation for published version (APA): Toshchakov, S. V., Korzhenkov, A. A., Chernikova, T., Ferrer, M., Golyshina, O., Yakimov, M. M., & Golyshin, P. (2017). The genome analysis of Oleiphilus messinensis ME102 (DSM 13489T) reveals backgrounds of its obligate alkane-devouring marine lifestyle. 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Sep. 2021 1 2 The genome analysis of Oleiphilus messinensis ME102 (DSM 13489T) reveals backgrounds of 3 its obligate alkane-devouring marine lifestyle 4 5 6 Stepan V. Toshchakov1, Alexei A. Korzhenkov1, Tatyana N. Chernikova2, Manuel Ferrer3, 7 Olga V. Golyshina2, Michail M. Yakimov1,4 and Peter N. Golyshin2 8 1 Immanuel Kant Baltic Federal University, 236040 Kaliningrad, Russia 9 2 School of Biological Sciences, Bangor University, LL57 2UW Bangor, Gwynedd, UK 10 3 Institute of Catalysis CSIC, Campus Cantoblanco, 28049 Madrid, Spain 11 4 Institute for Coastal Marine Environment, CNR, 98122 Messina, Italy 12 13 14 15 16 17 Keywords: 18 Oceanospirillales, marine alkane-degrading bacteria, hydrocarbonoclastic, Oleiphilus 19 messinensis 20 21 1 22 Abstract 23 Marine bacterium Oleiphilus messinensis ME102 (DSM 13489T) isolated from the sediments of 24 the harbor of Messina (Italy) is a member of the order Oceanospirillales, class 25 Gammaproteobacteria, representing the physiological group of marine obligate 26 hydrocarbonoclastic bacteria (OHCB) alongside the members of the genera Alcanivorax, 27 Oleispira, Thalassolituus, Cycloclasticus and Neptunomonas. These organisms play a crucial 28 role in the natural environmental cleanup in marine systems. Despite having the largest 29 genome (6.379.281 bp) among OHCB, O. messinensis exhibits a very narrow substrate profile. 30 The alkane metabolism is pre-determined by three loci encoding for two P450 family 31 monooxygenases, one of which formed a cassette with ferredoxin and alcohol dehydrogenase 32 encoding genes and alkane monoxygenase (AlkB) gene clustered with two genes for 33 rubredoxins and NAD+-dependent rubredoxin reductase. Its genome contains the largest 34 numbers of genomic islands (15) and mobile genetic elements (140), as compared with more 35 streamlined genomes of its OHCB counterparts. Among hydrocarbon-degrading 36 Oceanospirillales, O. messinensis encodes the largest array of proteins involved in the signal 37 transduction for sensing, and responding to the, environmental stimuli (345 vs 170 in Oleispira 38 antarctica, the bacterium with the second highest number). This must be an important trait 39 to adapt to the conditions in marine sediments with a high physico-chemical patchiness and 40 heterogeneity as compared to those in the water column. 41 42 2 43 1. Introduction 44 Oleiphilus messinensis is one of the organisms that represent the functional group of so- 45 called obligate marine hydrocarbonoclastic bacteria (OHCB), which plays a pivotal role in 46 the degradation of petroleum constituents in the sea (Yakimov et al., 2007). The strain O. 47 messinensis ME102 (DSM 13489T) was isolated from the sediments of the Messina harbor, 48 which is severely affected by the ferry traffic, after the enrichment of the sample with 49 tetradecane in the artificial seawater medium ONR7a. O. messinensis was described to 50 represent a new species within the new genus Oleiphilus, within the novel family 51 Oleiphilacea of Oceanosprillales (Gammaproteobacteria) (Golyshin et al., 2002). These 52 Gram-negative aerobic bacteria have a very restricted substrate range, consistent with 53 their OHCB designation, preferring aliphatic hydrocarbons, fatty acids and alcohols, as 54 carbon and energy sources over sugars and amino acids (Golyshin et al., 2002). Figure 1 55 depicts the placement of O. messinensis pointing at its relatively distant placement on the 56 phylogenetic tree with other Oceanospirillales. Worth attention, the genus Oleiphilus is 57 currently only represented by a single isolate (type strain) with another one to share 98% 58 SSU rRNA sequence identity (GenBank Acc Nr FJ845394), pointing at a rather endemic 59 nature of this particular species. This is in the stark contrast with other OHCBs that include 60 very ubiquitous Alcanivorax spp. (Yakimov et al., 1998, Schneiker et al., 2006), Oleispira 61 spp. important in polar and deep (cold) marine environments (Yakimov et al., 2003; Kube 62 et al., 2013), Thalassolituus spp., an oil-degrader occupying various marine niches, 63 including estuarine waters (Yakimov et al., 2004; McKew et al., 2007; Golyshin et al., 2013) 64 and PAH-degrading specialists from the genus Cycloclasticus (Dyksterhouse et al., 1995; 65 Geiselbrecht et al., 1998; Lai et al., 2012; Messina et al., 2016). Here, we report on the 66 genome-based analysis of obligate marine hydrocarbonoclastic bacterium, Oleiphilus 67 messinensis ME102. Genome analysis revealed exceptional genome plasticity of 3 68 ME102, showing an unprecedented abundance of mobile elements for a member of the 69 Oceanospirillales, which could potentially play an important role in the of genome 70 regulatory circuits. Halomonas Cobetia 100 Halomonas Halomonas Halomonas muralis LMG 20969 NR 025486.1 Salinicola peritrichatus DY22 NR 109731.1 95 Salinicola Larsenimonas salina M1-18 NR 134755.1 Chromohalobacter 75 Kushneria 92 97 Halotalea alkalilenta AW-7 NR 043806.1 Carnimonas nigrificans CTCBS 1 NR 029342.1 Zymobacter 100 Terasakiispira papahanaumokuakeensis PH27A NR 137414.1 Marinospirillum 65 100 100 Salicola Halovibrio denitrificans HGD 3 NR 043524.1 100 Litoricola Alcanivorax 100 Pseudospirillum 100 Gynuella sunshinyii YC6258 NR 126269.1 93 Hahella 100 Zooshikella Salinispirillum marinum GCWy1 NR 134169.1 Pleionea mediterranea MOLA115 NR 135696.1 Oleiphilus messinensis ME102 NR 025432.1 Aliikangiella marina GYP-15 NR 144575.1 92 Kangiella 88 94 Endozoicomonas Kistimonas 100 Motiliproteus sediminis HS6 NR 134770.1 74 Saccharospirillum 55 Reinekea 97 59 Oceanospirillum Balneatrix alpica 4-87 NR 026465.1 79 Litoribrevibacter albus Y32 NR 134757.1 Litoribacillus peritrichatus JYr12 NR 117511.1 Oleispira 100 Oleibacter marinus NBRC 105760 NR 114287.1 84 Oceanobacter kriegii IFO 15467 NR 113758.1 Thalassolituus 88 Marinobacterium 93 Bacterioplanes sanyensis GYP-2 NR 126264.1 100 Marinomonas 62 Nitrincola lacisaponensis 4CA NR 042984.1 Marinobacterium rhizophilum CL-YJ9 NR 044157.1 Marinobacterium nitratireducens CN44 NR 044528.1 100 Marinobacterium jannaschii Corallomonas stylophorae KTSW-6 NR 108836.1 92 Amphritea 95 Neptunibacter Bermanella marisrubri RED65 NR 042750.1 93 Oceaniserpentilla haliotis DSM 19503 NR 042641.1 89 Spongiispira norvegica Gp 4 7.1 NR 042453.1 98 Marinobacterium Profundimonas piezophila YC-1 NR 117943.1 Neptunomonas 71 0.01 4 72 Fig. 1. Phylogenetic position of O. messinensis ME102 and other OHCB (marked in blue) among 250 species of 73 Oceanospirillales, as revealed by SSU rRNA gene sequence analysis. The evolutionary relationships were 74 inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch 75 length = 3.41994391 is shown. The percentage of replicate trees in which the associated taxa clustered 76 together in the bootstrap test > 50% (1000 replicates) are shown next to the branches (Felsenstein, 1985). 77 The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used 78 to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method 79 (Jukes and Cantor, 1969) and are in the units of the number of base substitutions per site. The rate variation 80 among sites was modeled with a gamma distribution (shape parameter = 1). The analysis involved 251 81 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 82 464 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). 83 84 2.1 General features of O. messinensis genome. 85 The genome of O. messinensis was sequenced using hybrid approach using Roche 454 and 86 Illumina sequencing technologies. Assembly was performed with Newbler and 87 Phred/Phrap/Consed de novo assemblers resulting in 6.38 Mb circular chromosome 88 sequenced with