Fossil records indicate that life appeared in marine environments ∼3. 5 billion years ago (Gyr) and transitioned to terrestrial ecosystems nearly 2. 5 Gyr. Sequence analysis suggests that “hydrobacteria” and “terrabacteria” might have diverged as early as 3 Gyr. Bacteria of the genus Azospirillum are associated with roots of terrestrial plants; however, virtually all their close relatives are aquatic. We obtained genome sequences of two Azospirillum species and analyzed their gene origins. While most Azospirillum house-keeping genes have orthologs in its close aquatic relatives, this lineage has obtained nearly half of its genome from terrestrial organisms. The majority of genes encoding functions critical for association with plants are among horizontally transferred genes. Our results show that transition of some aquatic bacteria to terrestrial habitats occurred much later than the suggested initial divergence of hydro- and terrabacterial clades. The birth of the genus Azospirillum approximately coincided with the emergence of vascular plants on land.
Fossil records indicate that life appeared in marine environments ∼3. 5–3. 8 billion years ago (Gyr) [1] and transitioned to terrestrial ecosystems ∼2. 6 Gyr [2]. The lack of fossil records for bacteria makes it difficult to assess the timing of their transition to terrestrial environments; however sequence analysis suggests that a large clade of prokaryotic phyla (termed “terrabacteria”) might have evolved on land as early as 3 Gyr, with some lineages later reinvading marine habitats [3]. For example, cyanobacteria belong to the terrabacterial clade, but one of its well-studied representatives, Prochlorococcus, is the dominant primary producer in the oceans [4]. Bacteria of the genus Azospirillum are found primarily in terrestrial habitats, where they colonize roots of important cereals and other grasses and promote plant growth by several mechanisms including nitrogen fixation and phytohormone secretion [5], [6]. Azospirillum belong to proteobacteria, one of the largest groups of “hydrobacteria”, a clade of prokaryotes that originated in marine environments [3]. Nearly all known representatives of its family Rhodospirillaceae are found in aquatic habitats (Figure 1 and Table S1) suggesting that Azospirillum represents a lineage which might have transitioned to terrestrial environments much later than the Precambrian split of “hydrobacteria” and “terrabacteria”. To obtain insight into how bacteria transitioned from marine to terrestrial environments, we sequenced two well studied species, A. brasilense and A. lipoferum, and a third genome of an undefined Azospirillum species became available while we were carrying out this work [7].
Horizontal gene transfer has been long recognized as a major evolutionary force in prokaryotes [12]. Its role in the emergence of new pathogens and adaptation to environmental changes is well documented [32]. While other recent studies indicate that HGT levels in natural environments may reach as much as 20% of a bacterial genome [33], our data suggest that HGT has affected nearly 50% of the Azospirillum genomes, in close association with dramatic changes in lifestyle necessary for transition from aquatic to terrestrial environments and association with plants. Emergence of these globally distributed plant-associated bacteria, which appear to coincide with radiation of land plants and root development, likely has dramatically changed the soil ecosystem.
The genome of Azospirillum lipoferum 4B was sequenced by the whole random shotgun method with a mixture of ∼12X coverage of Sanger reads, obtained from three different libraries, and ∼18X coverage of 454 reads. Two plasmid libraries of 3 kb (A) and 10 kb (B), obtained by mechanical shearing with a Hydroshear device (GeneMachines, San Carlos, California, USA), were constructed at Genoscope (Evry, France) into pcDNA2. 1 (Invitrogen) and into the pCNS home vector (pSU18 modified, Bartolome et al. [34]), respectively. Large inserts (40 kb) (C) were introduced into the PmlI site of pCC1FOS. Sequencing with vector-based primers was carried out using the ABI 3730 Applera Sequencer. A total of 95904 (A), 35520 (B) and 15360 (C) reads were analysed and assembled with 504591 reads obtained with Genome Sequencer FLX (Roche Applied Science). The Arachne “HybridAssemble” version (Broad institute, MA) combining 454 contigs with Sanger reads was used for assembly. To validate the assembly, the Mekano interface (Genoscope), based on visualization of clone links inside and between contigs, was used to check the clones coverage and misassemblies. In addition, the consensus was confirmed using Consed functionalities (www. phrap. org), notably the consensus quality and the high quality discrepancies. The finishing step was achieved by PCR, primer walks and transposon bomb libraries and a total of 5460 sequences (58,602 and 4800 respectively) were needed for gap closure and quality assessment. The genome of strain Azospirillum brasilense Sp245 was sequenced by the whole random shotgun method with a mixture of ∼10X coverage of Sanger reads obtained from three different libraries and ∼25X coverage of 454 reads. A plasmid library of 3 kb, obtained by mechanical shearing with a Hydroshear device (GeneMachines, San Carlos, California, USA), were constructed at Plant Genome Mapping Laboratory (University of Georgia, USA) into pcDNA2. 1 vector (Invitrogen). Large inserts (40 kb) were introduced into the PmlI site of pCC1FOS. Sequencing with vector-based primers was carried out using the ABI 3730 Applera Sequencer. The Arachne “HybridAssemble” version combining 454 contigs with Sanger reads was used for assembly. Contig scaffolds were created using Sequencher (Gene Codes) and validated using clone link inside and between contigs. AMIGene software [35] was used to predict coding sequences (CDSs) that were submitted to automatic functional annotation [36]. The resulting 6233 A. lipoferum 4B CDSs and 7848 A. brasilense Sp245 CDSs were assigned a unique identifier prefixed with “AZOLI” or “AZOBR” according to their respective genomes. Putative orthologs and synteny groups were computed between the sequenced genomes and 650 other complete genomes downloaded from the RefSeq database (NCBI) using the procedure described in Vallenet et al. [36]. Manual validation of the automatic annotation was performed using the MaGe (Magnifying Genomes) interface. IS finder (www-is. biotoul. fr) was used to annotate insertion sequences [37]. The A. lipoferum 4B nucleotide sequence and annotation data have been deposited to EMBL databank under accession numbers: FQ311868 (chromosome), FQ311869 (p1), FQ311870 (p2), FQ311871 (p3), FQ311872 (p4), FQ311873 (p5), FQ311874 (p6). The A. brasilense Sp245 nucleotide sequence and annotation data have been deposited at EMBL databank under accession numbers: HE577327 (chromosome), HE577328 (p1), HE577329 (p2), HE577330 (p3), HE577331 (p4), HE577332 (p5), HE577333 (p6). In addition, all the data (i. e. , syntactic and functional annotations, and results of comparative analysis) were stored in a relational database, called AzospirilluScope [36], which is publicly available at http: //www. genoscope. cns. fr/agc/mage/microscope/about/collabprojects. php? P_id=39. BLAST searches were performed using NCBI toolkit version 2. 2. 24+ [38]. Multiple sequence alignments were built using the L-INS-i algorithm of MAFFT [39] with default parameters. Phylogenetic tree construction was performed using PhyML [40] with default parameters unless otherwise specified. 16S rRNA sequences were retrieved from the Ribosomal Database Project [41]. A concatenated ribosomal protein tree was constructed from sequenced members of alpha-proteobacteria with a 98% 16S rRNA sequence identity cutoff to limit overrepresentation. The following ribosomal proteins were used: L3, L5, L11, L13, L14, S3, S7, S9, S11, and S17. The proteins were identified using corresponding Pfam models and HMMER [42] searches against the genomes of sequenced alpha-proteobacteria selected above. The sequences were aligned and concatenated. GBlocks [43] with default parameters was used to reduce the number of low information columns. The tree was constructed using PhyML with the following options: empirical amino acid frequencies, 4 substitution categories, estimated gamma distribution parameter, and NNI tree topology search. Protein sequences queries from all 3 Azospirillum genomes were used in BLAST searches against the non-redundant microbial genome set constructed by Wuichet and Zhulin [26] supplemented with sequenced members of Rhodospirillales absent in the original set (Acetobacter pasteurianus IFO 3283-01, alpha proteobacterium BAL199, Magnetospirillum gryphiswaldense MSR-1, and Magnetospirillum magnetotacticum MS-1). E-value cutoff of 10∧−4 was used. Only the first occurrence of each species was used in ancestry assignment. Proteins were assigned as being ancestral or horizontally transferred, with varying degrees of confidence, based on the presence of members of Rhodospirillales and Rhodospirillaceae in the top eight BLAST hits. Ancestral assignment was based on the top 8 hits, based on the number of Rhodospirillaceae genomes in the database: 2 Azospirillum, 3 Magnetospirillum, 2 Rhodospirillum, and Nisaea sp. BAL199, excluding the organism on which ancestry assignment is being performed. High confidence ancestral proteins have at least 6 of the top 8 species belonging to Rhodospirillales or all but 1, if the BLAST result had less than 8 species. This rule allows for 1–2 independent events of HGT from Rhodospirillales to other distantly related species. Medium confidence ancestral proteins have at least 4 Rhodospirillaceae in the top 8. Low confidence ancestral proteins have at least 1 Rhodospirillaceae in the top 8, excluding hits to other Azospirillum genomes. High confidence horizontally transferred proteins have 0 hits to Rhodospirillales in the top 10, excluding hits to other Azospirillum genomes. Medium confidence horizontally transferred proteins have 0 hits to Rhodospirillales in the top 5, excluding hits to other Azospirillum genomes. Low confidence horizontally transferred proteins have 0 hits to Rhodospirillaceae in the top 8, excluding hits to other Azospirillum genomes. Unassigned proteins either have no BLAST hits outside Azospirillum, or simultaneously classify as medium confidence horizontally transferred and medium or low confidence ancestral. Bidirectional BLAST was used to identify orthologs of the putative glycoside hydrolase (GH) genes. Phyml package was used to confirm evolutionary relationships and visualize the results. Domain architectures were obtained through Pfam [53] search for each protein. Then information from CAZy [54] and recent analysis [55] was used to assign putative activities of the predicted GHs. Chemotaxis proteins were identified in genomic datasets as previously described [56]. Using CheA sequences from a recent chemotaxis system classification analysis [26], alignments of the P3–P5 regions of CheA were built for each class and for the entire set of CheA sequences. Each alignment was made non-redundant so that no pair of sequences shared more than 80% sequence identity. Hidden Markov Models (HMMs) were built from each non-redundant alignment and used to create library via the HMMER3 software package (version HMMER 3. 0b3) [42] and default parameters. The rhizosphere CheA sequences from a recent study [25] were run against the CheA HMM library. Unclassified sequences (Unc) are those with top hits to the full CheA set HMM rather than a class-specific HMM. The remaining sequences were assigned to the class of the top scoring HMM. Azospirillum strains and control strains (Dickeya dadantii 3937 as a positive control, A. tumefaciens NT1 as a negative control) were cultured for 16 h in liquid AB minimal medium [57] containing 0. 2% malate and 1 mg/L biotin. An aliquot of 107 cells (for Dickeya dadantii 3937) or 2. 107 cells (for all other strains) was deposited on top of AB plates containing 0. 1% carboxymethylcellulose instead of malate. Plates were incubated for 5 days before being stained as previously described [58]. A 211-bp cpaB (AZOBR_p460079) internal fragment was amplified by PCR with primers F6678 (GCGTGGACCTGATCCTGAC) and F6679 (GTGACCGTCTCGCTCTGAC) and subcloned into pGEM-T easy (Promega). White colonies were screened by PCR with primers F6678 and F6679 for correct insertion in pGEM-T easy, resulting in pR3. 37. The insert of plasmid pR3. 37 was digested with NotI and cloned into the NotI site of pKNOCK-Km [59], resulting in pR3. 39 after transfer into chemically-competent cells of E. coli S17. 1 λpir. pR3. 39 was introduced into A. brasilense Sp245 by biparental mating. Transconjugants resulting from a single recombination event of pR3. 39 were selected on AB medium containing 0. 2% malate, ampicillin (100 mg/mL) and kanamycin (40 mg/mL). The correct insertion of pKNOCK into cpaB was confirmed by PCR with primers (F6678 and F5595 TGTCCAGATAGCCCAGTAGC, located on pKNOCK) and sequencing of the PCR amplicon. Sp245 and Sp245cpaB were labelled with pMP2444 [60] allowing the constitutive expression of EGFP. The strains were grown in NFB* (Nitrogen free broth containing 0. 025% of LB) with appropriate antibiotics in glass tubes containing a cover-slide, under a mild lateral agitation for 6 days. After the incubation, the liquid and the cover-slide were removed from the tubes and the biofilm formed at the air/liquid interface was colored by 0. 1% crystal violet. After two washings with distilled water, crystal violet was solubilized by ethanol and quantified by spectrophotometry at 570 nm. The experiment was performed twice in triplicate. In parallel, the colonization of the glass cover-slide was monitored by confocal laser scanning microscopy (510 Meta microscope; Carl Zeiss S. A. S.) equipped with an argon-krypton laser, detectors, and filter sets for green fluorescence (i. e. , 488 nm for excitation and 510 to 531 nm for detection). Series of horizontal (x-y) optical sections with a thickness of 1 µm were taken throughout the full length of the Sp245 and Sp245cpaB biofilms. Three dimensional reconstructions of biofilms were performed using LSM software release 3. 5 (Carl Zeiss S. A. S.).
Genome sequencing and analysis of plant-associated beneficial soil bacteria Azospirillum spp. reveals that these organisms transitioned from aquatic to terrestrial environments significantly later than the suggested major Precambrian divergence of aquatic and terrestrial bacteria. Separation of Azospirillum from their close aquatic relatives coincided with the emergence of vascular plants on land. Nearly half of the Azospirillum genome has been acquired horizontally, from distantly related terrestrial bacteria. The majority of horizontally acquired genes encode functions that are critical for adaptation to the rhizosphere and interaction with host plants.
genome sequencing
genome complexity
genome evolution
microbial evolution
biology
genomics
comparative genomics
microbiology
genetics and genomics
Azospirillum Genomes Reveal Transition of Bacteria from Aquatic to Terrestrial Environments