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Whole-Genome Analysis of Azoarcus Sp. Strain CIB Provides Genetic

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Whole-Genome Analysis of Azoarcus Sp. Strain CIB Provides Genetic ManuscriptCORE Metadata, citation and similar papers at core.ac.uk Provided by Digital.CSIC Whole-genome analysis of Azoarcus sp. strain CIB 1 2 provides genetic insights to its different lifestyles and predicts 3 novel metabolic features 4 5 6 7 Zaira Martín-Moldes a1 , María Teresa Zamarro a1 , Carlos del Cerro a1 , Ana Valencia a, 8 Manuel José Gómez b2 , Aida Arcas b3 , Zulema Udaondo a4 , José Luis García a, Juan 9 a a a* 10 Nogales , Manuel Carmona , and Eduardo Díaz 11 a 12 Centro de Investigaciones Biológicas-CSIC, 28040 Madrid, Spain 13 b Centro de Astrobiología, INTA-CSIC, 28850 Torrejón de Ardoz, Madrid, Spain 14 15 1 16 These authors contributed equally to this work 17 18 19 2 Present address: Centro Nacional de Investigaciones Cardiovasculares, ISCIII, 20 Madrid, Spain 21 3 22 Present address: Instituto de Neurociencias, UMH -CSIC, Alicante, Spain 4 23 Present address: Abengoa Research, Sevilla, Spain 24 25 26 * 27 Corresponding author. Tel.: +34915611800. E-mail address : [email protected] 28 (E.Díaz) 29 30 31 32 33 34 35 36 Abbreviations : ANI, average nucleotide identity; BIMEs, bacterial interspersed mosaic 37 38 elements; IAA, indoleacetic acid; ICE, integrative and conjugative element ; REPs, 39 repeated extragenic palindrome sequences; ROS, reactive oxygen species; TAS, toxin - 40 antitoxin system; TMAO, trimethylamine N-oxide. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 1 63 64 65 1 ABSTRACT 2 3 The genomic features of Azoarcus sp. CIB reflect its most distinguishing phenotypes as 4 5 a diazotroph, facultative anaerobe, capable of degrading either aerobically and/or 6 anaerobically a wide range of aromatic compounds, including some toxic hydrocarbons 7 such as toluene and m-xylene, as well as its endophytic lifestyle. The analyses of its 8 genome have expanded the catabolic potential of strain CIB towards common natural 9 10 compounds, such as certain diterpenes, that were not anticipated as carbon sources. The 11 high number of predicted solvent efflux pumps and heavy metal resistance gene clusters 12 has provided the first evidence for two environmentally-relevant features of this 13 bacterium that remained unknown. Genome mining has revealed several gene clusters 14 likely involved in the en dophytic lifestyle of strain CIB, opening the door to the 15 16 molecular characterization of some plant growth promoting traits . Horizontal gene 17 transfer and mobile genetic elements appear to have played a major role as a mechanism 18 of adaptation of this bacterium to different lifestyles. This work paves the way for a 19 systems biology-based understanding of the abilities of Azoarcus sp. CIB to integrate 20 21 aerobic and anaerobic metabolism of aromatic compounds, tolerate stress conditions, 22 and interact with plants as an endophyte of great potential for phytostimulation and 23 phytoremediation strategies . Comparative genomics provides an Azoarcus pan genome 24 that confirms the global metabolic flexibility of this genus, and suggests that its 25 26 phylogeny should be revisited. 27 28 29 30 31 32 33 34 35 36 37 38 Keywords : Azoarcus , aromatic compounds, endophyte, metals resistance, mobile genetic 39 elements, comparative genomics 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 2 63 64 65 Introduction 1 2 Azoarcus is a genus of betaproteobacteria that belongs to the family 3 Rhodocyclaceae , a physiologically versatile group encompassing bacteria with diverse 4 5 functions [60]. The environmental relevance of Azoarcus strains is supported by their 6 frequent detection in diverse soils, sludges, and wastewaters [39]. The Azoarcus genus, 7 that includes ten recognized species, namely A. indigens (type species of the genus), A. 8 communis, A. tolulyticus, A. toluvorans, A. toluclasticus, A. evansii, A. anaerobius, A. 9 10 buckelii, A. olearius, and A. taiwanensis [2,31,42,49], was shown to comprise bacteria 11 that fit into one of two major phylogenetic and eco-physiological groups [2 2,2 8,3 8,4 9]. 12 One gr oup includes free -living bacteria that usually inhabit waters and soils and 13 participate in the biogeochemical cycling of a large number of organic and inorganic 14 metabolites [3 1,40 ,4 3,4 9]. Many strains of this group have been described and/or 15 16 isolated by th eir ability to degrade aromatic compounds in anoxic conditions, being 17 strain EbN1 (currently "Aromatoleum aromaticum" EbN1) [3 8,3 9] and A. evansii 18 KB740 [2,1 4] the two most studied . The other group includes Azoarcus strains such as 19 A. communis strain SWub3 , A. indigens strain VB32 or the well -characterized Azoarcus 20 sp. strain BH72, that invade roots of Kallar grass and rice, living as endophytic bacteria 21 22 [4 2]. Interestingly, the free -living Azoarcus strains that are anaerobic biodegraders of 23 aromatic compou nds , and whose prototype is the EbN1 strain, have exclusively 24 received particular attention for their degradation and biotransformation abilities 25 [2 2,3 8,3 9]. 26 27 Azoarcus sp. CIB (CECT#5669) was isolated from a DSMZ 12184 culture (not 28 available anymore), which was supposed to be Azoarcus sp. strain M3, isolated from a 29 diesel fuel-contaminated aquifer at Menziken (Switzerland) [20,32]. Azoarcus sp. CIB is 30 a free-living bacterium with the ability to degrade a high number of aromatic 31 compounds under aerobic and/or anaerobic conditions, including some toxic 32 33 hydrocarbons such as toluene and m-xylene [9,25,32,57]. Recently, we have 34 demonstrated that Azoarcus sp. CIB has also the ability to grow in association with 35 plants, colonizing the intercellular spaces of the exodermis of rice roots. In addition, the 36 strain CIB displays plant growth promoting traits such the ability to uptake insoluble 37 38 phosphorous, production of indoleacetic acid (IAA) or nitrogen fixation [1 6]. Thus, 39 Azoarcus sp. CIB may represent the prototype of a subgroup of Azoarcus strains that 40 share the anaerobic biodegradation of aromatic hydrocarbons with a facultative 41 endophytic lifestyle [1 6]. Since Azoarcus sp. CIB presents a robust growth and it is 42 susceptible of genetic manipulation, it became a mode l system to study the complex 43 44 regulatory networks that control the expression of the aerobic and anaerobic aromatic 45 degradation clusters [ 5,9,1 3,5 7,5 8], and some recombinants strains have been 46 engineered for biotechnological prospects [6 3]. 47 In this work, we sequenced the whole genome of Azoarcus sp. CIB and 48 49 accomplished a comparative analysis with the genomes of other strains, such as 50 Azoarcus sp. BH72 and strain EbN1, that are the prototypes of obligate endophytes and 51 free-living strains, respectively. This work provides new insights into the genetic 52 determinants that may account for some of the reported metabolic abilities of the CIB 53 strain, and offers information on genetic characteristics that may be relevant for the 54 55 adoption of a particular lifesytle or that can be of biotechnological interest. 56 57 58 59 60 61 62 3 63 64 65 1 Materials and Methods 2 3 Genome sequence, contigs assembling, and gaps filling 4 5 6 Azoarcus sp. CIB was anaerobically grown at 30 ºC in MC medium [32] 7 containing 3 mM benzoate as sole carbon and energy source and 10 mM nitrate as 8 electron acceptor. Cultures were collected when they reached the early stationary phase 9 10 and genomic DNA was extracted using previously published protocols [32]. 11 The genome sequencing of Azoarcus sp. CIB was carried out using the 454 Life 12 Sciences high-density pyrosequencing methodology in a GSFLX sequencer from Roche 13 at LifeSequencing (Valencia, Spain). FASTQ reads (about 250-nt long) were assembled 14 in contigs by using the Newbler software from Roche. Contigs were ordered in scaffolds 15 16 by performing a long-tag paired-end sequencing according to Roche protocols at 17 LifeSequencing (Valencia, Spain). Gap filling on the scaffolds was performed by 18 manual assembly of FASTQ reads with BioEdit (Ibis Biosciences) and by conventional 19 sequencing methods (ABI Prism 377; Applied Biosystems) of PCR products (purified 20 with Gene Clean Turbo, Q-BIOgene) spanning the regions between flanking contigs. 21 22 23 Gene prediction and genome annotation 24 25 The genome of Azoarcus sp. CIB was annotated by means of a bacterial genome 26 27 annotation pipeline [56], which used tRNAscan-SE to predict tRNA genes, RNAmmer 28 to predict rRNA genes and Glimmer to predict coding sequences. Functional 29 annotations for proteins were generated by comparison against several protein sequence 30 and protein family databases (SwissProt, NCBI protein, COG, Pfam, Smart, Prk) with 31 BLAST and RPS-BLAST [1]. Annotations were summarized in different output 32 33 formats. One of them was used as input for Pathway Tools, for automatic metabolic 34 reconstruction [27]. 35 Transposase and integrase encoding genes were manually annotated with the 36 assistance of the ISFinder database ( http://www-is.biotoul.fr/ ). 37 38 39 Comparative genomics 40 41 The complete genome of Azoarcus sp. CIB was compared with that of all 42 currently sequenced strains: " Aromatoleum aromaticum " strain EbN1 (NC_006513.1 ; 43 44 NC_006823.1 ; NC_006824.1 ); Azoarcus sp. BH72 (NC_008702.1) ; Azoarcus sp. 45 KH32C (NC_020516.1; NC_020548.1); and Azoarcus toluclasticus strain MF63 46 (NZ_ARJX00000000.1). The closely related Thauera sp. strain MZ1T (CP001281.2, 47 CP001282.1) was used as outgroup. Comparative genomic analyses, including Venn 48 49 diagrams, synteny analyses and phylogenetic trees were performed with EDGAR [6]. 50 Average nucleotide identity among the genomes based on MUMmer (ANIm) was 51 calculated with Jspecies [44].
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