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Complete genome sequence of the nitrogen-fixing bacterium Azospirillum humicireducens type strain SgZ-5T

Standards in Genomic Sciences volume 13, Article number: 28 (2018) Cite this article

Abstract

The Azospirillum humicireducens strain SgZ-5T, belonging to the Order Rhodospirillales and the Family Rhodospirillaceae, was isolated from a microbial fuel cell inoculated with paddy soil. A previous work has shown that strain SgZ-5T was able to fix atmospheric nitrogen involved in plant growth promotion. Here we present the complete genome of A. humicireducens SgZ-5T, which consists of a circular chromosome and six plasmids with the total genome size of 6,834,379 bp and the average GC content of 67.55%. Genome annotations predicted 5969 protein coding and 85 RNA genes including 14 rRNA and 67 tRNA genes. By genomic analysis, we identified a complete set of genes that is potentially involved in nitrogen fixation and its regulation. This genome also harbors numerous genes that are likely responsible for phytohormones production. We anticipate that the A. humicireducens SgZ-5T genome will contribute insights into plant growth promoting properties of Azospirillum strains.

Introduction

Bacteria that live in the plant rhizosphere and possess a large array of potential mechanisms to enhance plant growth are considered as PGPR [1,2,3]. Azospirillum represents a well characterized genus of PGPR due to its capacity of fixing atmospheric nitrogen [4, 5]. Although the exact contribution of Azospirillum to biological nitrogen fixation in plant growth promotion is debated [2], agricultural applications of the genus Azospirillum have been still developed [6, 7]. Another main characteristic of Azospirillum proposed to explain plant growth promotion has been related to its ability to produce phytohormones [8, 9].

At present, there are 17 species within the genus Azospirillum [10], of which the nitrogen-fixing bacterium A. humicireducens SgZ-5T, the focus species of this study, was initially isolated from the anode biofilm of a MFC. A soil sample collected from paddy field in Guangzhou City, Guangdong Province, China (23.18o N 113.36o E) was used as inoculating source of the MFC. In a previous report [11], the nitrogen-fixing capability of strain SgZ-5T was confirmed by acetylene-reduction assay and identification of a nifH gene. Furthermore, this strain has the ability to grow under anaerobic conditions via the oxidation of various organic compounds coupled to the reduction of humus [11], showing its potential use in plant rhizosphere. Here, we describe the physiological features together with the whole genome sequence of A. humicireducens SgZ-5T.

Organism information

Classification and features

A. humicireducens SgZ-5T is a Gram-negative, facultative anaerobic, motile, spiral, straight to slightly curved rod-shaped bacterium (Fig. 1), belonging to the Order Rhodospirillales and the Family Rhodospirillaceae . The strain grew optimally in the conditions of 30 °C, pH 7.2, and 1% NaCl [11]. On NA, strain SgZ-5T formed cream-colored, round, smooth, convex and non-translucent colonies (Fig. 1). With AQDS as the sole terminal electron acceptor, strain SgZ-5T could utilize pyruvate, glucose and acetate as electron donors under anaerobic conditions [11]. Strain SgZ-5T was able to use a range of carbon substrates including N-Acetyl-glucosamine, citrate, D-ribose, meso-inositol, D-saccharose, D-maltose, L-rhamnose, suberic acid, malonate, acetate, L-serine, salicin, L-lactate, L-alanine, gluconate, 2-keto-gluconate, glycogen, D-mannitol, D-glucose, D-melibiose, L-fucose, D-sorbierite, L-arabinose, L-histidine, 3-hydroxy-butyric acid, 4-hydroxy-benzoic acid, L-proline, capric acid, adipic acid and malic acid [11] (Table 1).

Fig. 1
figure 1

Images of the A. humicireducens SgZ-5T. a Colonies of the strain on NA agar plate, b light microscopy and c transmission electron microscopy of the strain

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Table 1 Classification and general features of A. humicireducens SgZ-5T according to the MIGS recommendations [16]
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A phylogenetic tree was constructed from aligning the 16S rRNA gene sequences of strain SgZ-5T and type strains of the genus Azospirillum by MEGA 5 using the neighbour-joining method [12]. The phylogenetic position of strain SgZ-5T is shown in Fig. 2, where A. humicireducens can be grouped as a Azospirillum species, forms a distinct subclade together with A. lipoferum that are known as a biofertilizer widely used for agricultural production [13, 14]. The 16S rRNA gene of strain SgZ-5T is 98% similar to that of A. lipoferum NCIMB 11861T. Since nifH gene is highly conserved among nitrogen-fixing Proteobacteria [15], a nifH-based phylogenetic tree was constructed to identify the relationship of A. humicireducens to other species within the genus Azospirillum and related genus (Additional file 1). The phylogenetic reconstruction indicated the close relationship of the A. humicireducens SgZ-5T nifH gene with that from Azospirillum sp. B510.

Fig. 2
figure 2

Phylogenetic tree highlighting the position of A. humicireducens SgZ-5T relative to other type strains within the genus Azospirillum . The strains and their corresponding GenBank accession numbers of 16S rRNA genes were indicated in parentheses. The sequences were aligned using Clustal W and the neighbor-joining tree was constructed based on kimura 2-paramenter distance model by using MEGA 5. Bootstrap values above 50 % were obtained from 1000 bootstrap replications. Bar, 0.01 substitutions per nucleotide position. Rhodovulum adriaticum DSM 2781T was used as an outgroup

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Genome sequencing information

Genome project history

A. humicireducens SgZ-5T was selected for genome sequencing on the basis of its biotechnological potential in agricultural applications as a PGPR likely harboring multiple PGPP [11]. The complete genome sequences have been deposited at Gen-Bank/EMBL/DDBJ under the accession numbers CP015285.1, CP028902-CP028907. Project information is available from Genome Online database number Gp0150267 at Joint Genome Institute. In Table 2, we summarize the project information and its association with Minimum Information about a Genome Sequence (MIGS) [16].

Table 2 Genome sequencing project information
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Growth conditions and genomic DNA preparation

A. humicireducens SgZ-5T was routinely cultured in NB medium containing (L− 1) 5 g peptone, 3 g beef extract and 5 g NaCl at 30 °C. For genome sequencing, total genomic DNA was extracted from 10 mL overnight cultures using a DNA extraction kit following the manufacture’s instructions (Aidlab). Quantification and quality control of the genomic DNA were completed by using a Qubit fluorometer (Invitrogen, CA, USA) with Qubit dsDNA BR Assay kit and 0.7% agarose gel electrophoresis with λ-Hind III digest DNA marker.

Genome sequencing and assembly

Complete genome sequencing was performed on an Illumina HiSeq 2500 system by constructing three DNA libraries (a paired-end library with insert size of 491 bp, and two mate pair libraries with insert sizes of 2.5 and 6.9 kb). After filtering low quality and Illumina PCR adapter reads, a total of 1967 Mb clean data were obtained from 2052 Mb raw data. Subsequently, all reads data were denovo assembled into a circular contig with 259 folds of genomic coverage, using SOAPdenovo v.2.04 [17]. Detailed genome sequencing project information is shown in Table 2.

Genome annotation

Gene prediction was carried out by GeneMarkS v.4.6 [18]. Function annotation of predicted ORFs was performed based on a BLASTP search against NCBI nonredundant protein database and COG database. Transfer RNAs, rRNAs and sRNA were predicted using tRNAscan-SE v.1.31 with the bacterial model, RNAmmer v.1.2 and Rfam database v.9.1, respectively [19,20,21]. The CRISPRs were identified by using the CRISPR database [22]. The prediction of genes with signal peptides and transmembrane helices were performed by SignalP server v.4.1 [23] and TMHMM server v.2.0 [24], respectively. The secondary metabolism gene cluster was predicted according to the antiSMASH v.3.0 procedure [25].

Genome properties

The genome of A. humicireducens SgZ-5T comprises a circular chromosome of 3,181,617 bp and six circular plasmids, designated as pYZ1 (715,112 bp), pYZ2 (1,008,603 bp), pYZ3 (252,411 bp), pYZ4 (338,445 bp), pYZ5 (626,509 bp) and pYZ6 (711,682 bp) (Table 3). The total size of the genome is 6,834,379 bp, and the average GC content is 67.55%. The genome contains 6054 genes with the total length of 5,902,731 bp, of which 5969 (98.6%) are protein coding genes. There are 85 RNA genes (1.4%), including 14 rRNA and 67 tRNA genes. A total of 4844 genes (80.0%) have been assigned a predicted function while the rest have been designated as hypothetical proteins. Genome statistics are summarized in Table 4 and a graphical map is represented in Fig. 3. Furthermore, 4550 (75.2%) genes were assigned to 21 COG functional categories. The distribution of genes into different COG functional categories is provided in Table 5. Six Azospirillum species genomes (including A. humicireducens ) of characterized strains are compared in Table 6. Almost all of these Azospirillum genomes consisting of 6–8 replicons have the total size of 6.5–7.6 Mb and the average GC content of 67.5–70.7%, and contain the total genes in the range of 5951 to 6982 [3, 6, 26, 27]. Furthermore, the main features of A. humicireducens SgZ-5T genome are close to those of A. lipoferum 4B genome.

Table 3 Summary of genome: one chromosome and six plasmids
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Table 4 Genome statistics of A. humicireducens SgZ-5T
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Fig. 3
figure 3

Circular map of the chromosome of A. humicireducens SgZ-5T. From center to outside, circle 1 illustrates the GC skew. Circle 2 shows GC content (peaks out/inside the circle indicate values higher or lower than the average G+C content, respectively). Circle 3 denotes ncRNA genes. Circles 4, 5 and 6 indicate the CDSs, colored according to COG, KEGG and GO categories, respectively. Circle 7 demonstrates the predicted protein-coding sequences

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Table 5 Number of genes associated with general COG functional categories
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Table 6 Genome statistics comparison among characterized Azospirillum speciesa
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Insights into the genome sequence

Nitrogen fixation is the major proposed mechanism, by which Azospirillum affects plant growth [2, 4]. A complete set of genes encoding enzymes involved in nitrogen fixation was found in the genomic analysis of A. humicireducens SgZ-5T (Table 7). The main genes involved in this process are nif genes, of which nifDK genes (A6A40_02900 and A6A40_02895) annotated as nitrogenase molybdenum-iron proteins and nifH gene (A6A40_02905) encoding dinitrogenase reductase protein have been identified. In the upstream region of the nifHDK operon, we have found that nifEN genes (A6A40_02875 and A6A40_02870) involved in synthesis of the molybdenum-iron cofactor of nitrogenase are clustered into a single operon together with nifX (A6A40_02865). Furthermore, the genome of A. humicireducens SgZ-5T has nifUSVW genes (A6A40_02235, A6A40_02230, A6A40_02225 and A6A40_02215), which are separated from the structural nifENX operon by about 160 kb.

Table 7 Genes of A. humicireducens SgZ-5T involved in nitrogen fixation
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Organization of the nitrogen fixation gene cluster in A. humicireducens SgZ-5T is presented in Fig. 4. Except for the separately transcribed nifA (A6A40_09040), nifB (A6A40_09050) and nifZ genes (A6A40_09070 and A6A40_09075), all the nif genes have resided in the nitrogen fixation gene cluster of 176.7 kb. Besides, an operon containing fixABCX genes (A6A40_02185, A6A40_02190, A6A40_02195 and A6A40_02220) responsible for electron transfer to nitrogenase is located upstream of this gene cluster. Nevertheless, the fixABCX operon is generally regulated by a transcriptional activator NifA protein for all nitrogen-fixing bacteria in the genus Azospirillum studied so far [5]. Furthermore, draTG genes (A6A40_02920 and A6A40_02925) implicated in posttranslational regulatory process of nitrogenase activity were found in the downstream of and divergently oriented with respect to nifHDK genes. On the whole, the nitrogen fixation gene cluster of A. humicireducens SgZ-5T was in agreement with that in A. brasilense , A. lipoferum and Azospirillum sp. B510 [6, 26, 28, 29], suggesting that nitrogen fixation process demands the systematic action of various genes.

Fig. 4
figure 4

Organization of the nitrogen fixation gene cluster in A. humicireducens SgZ-5T. Arrows represent genes and their respective direction of transcription. Genes are colored as depicted in the lower box

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Since tryptophan is a main precursor for biosynthesis of IAA, a well-known phytohormone [30], the genes in A. humicireducens SgZ-5T related to the production of this amino acid have been analyzed (Additional file 2). The genome harbors three genes trpE, trpG and trpEG (A6A40_04380, A6A40_04655 and A6A40_05775), each encoding the key enzyme anthranilate synthase in tryptophan biosynthesis. Together with trpG, the genes trpD (A6A40_04650) and trpC (A6A40_04645) form a gene cluster of 2.4 kb. Except for anthranilate synthase, this trpGDC gene cluster encodes anthranilate phosphoribosyltransferase and indole-3-glycerol phosphate synthase, which plays a role in synthesis of tryptophan used in multiple biological processes including IAA biosynthesis [31]. The same trpGDC cluster was previously found in A. brasilense [32]. Although the ipdC gene, related to the indole-3-pyruvate pathway for the biosynthesis of IAA [30], was not discovered in the A. humicireducens SgZ-5T genome, alternative pathway might exist in SgZ-5T. In the genome, A6A40_22745 and A6A40_22755 were assigned as candidates for iaaM and iaaH genes, respectively. These two genes were also found in the Azospirillum sp. B510 genome, and are known to be involved in the IAM pathway for IAA biosynthesis by catalyzing the decarboxylation of tryptophan into IAM and the hydrolysis of IAM to produce IAA [6, 30].

The A. humicireducens SgZ-5T genome also contains a terpene gene cluster of 24.0 kb consisting of 23 genes (A6A40_04945, A6A40_04950, A6A40_04955, …, A6A40_05055) (Additional file 3). This gene cluster encodes a series of proteins, which are involved in the biosynthesis of secondary metabolite production of terpenoid. Thereinto, A6A40_05010 was indentified as the crtB gene, encoding phytoene synthase involved in the biosynthesis of carotenoid. Similar genes in this gene cluster were previously observed in the A. lipoferum 4B genome [7, 26]. Furthermore, some phytohormones including gibberellins and abscisic acid with over 120 types found in plants, fungi, and bacteria, are synthesized through the terpenoid pathway [2]. Therefore, A. humicireducens SgZ-5 exhibits an attractive application as a PGPR likely harboring multiple PGPP in agriculture.

Conclusion

We report here an inventory of the genomic features of the nitrogen-fixing bacterium A. humicireducens SgZ-5T. The genome sequence of strain SgZ-5T revealed further genetic elements involved in nitrogen fixation and its regulation, as well as in the production of phytohormones. We anticipate that knowledge of this genome will contribute to new insights into the mechanisms of plant growth stimulation through genomic comparisons among available complete genomes of Azospirillum strains.

Abbreviations

AQDS:

Anthraquinone-2, 6-disulfonate

IAA:

Indole-3-acetic acid

IAM:

Indole-3-actamide

MFC:

Microbial fuel cell

NA:

Nutrient Agar

NB:

Nutrient Broth

PGPP:

Plant growth promoting properties

PGPR:

Plant growth-promoting rhizobacteria

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Acknowledgments

This work was supported by the Guangdong Academy of Sciences Funds for Innovation Driven Development, China (2017GDASCX-0409), the National Natural Science Foundation of China (41501546), the Guangdong Natural Science Foundation, China (2016A030313779), and the Science and Technology Planning Project of Guangdong, China (2017A030303057).

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Authors and Affiliations

  1. Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmental Science & Technology, Guangzhou, 510650, China

    Zhen Yu, Guiqin Yang, Xiaoming Liu & Yueqiang Wang

  2. School of Environment, Guangdong Key Laboratory of Environmental Pollution and Health, Jinan University, Guangzhou, 510632, China

    Li Zhuang

  3. Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Shungui Zhou

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Contributions

LZ and SZ conceived and designed the experiments. GY, YW and XL performed the experiments. ZY assembled and analysed genome. ZY and LZ drafted the manuscript. GY and SZ revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Li Zhuang.

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Additional files

Additional file 1:

Phylogenetic tree based on the partial nifH gene sequences showing the position of A. humicireducens SgZ-5T relative to other species within the genus Azospirillum and related genus. The strains and their corresponding GenBank accession numbers of nifH gene were indicated in parentheses. The sequences were aligned using Clustal W and the neighbor-joining tree was constructed based on kimura 2-paramenter distance model by using MEGA 5. Bootstrap values above 50% were obtained from 1000 bootstrap replications. Bar, 0.01 substitutions per nucleotide position. Leptospirillum ferriphilum YSKT was used as an outgroup. (DOCX 64 kb)

Additional file 2:

Genes of A. humicireducens SgZ-5T involved in biosynthesis of tryptophan. (DOCX 16 kb)

Additional file 3:

Genes of A. humicireducens SgZ-5T located in a terpene gene cluster. (DOCX 16 kb)

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Yu, Z., Yang, G., Liu, X. et al. Complete genome sequence of the nitrogen-fixing bacterium Azospirillum humicireducens type strain SgZ-5T. Stand in Genomic Sci 13, 28 (2018). https://doi.org/10.1186/s40793-018-0322-2

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Keywords

Environmental Microbiome

ISSN: 2524-6372