Draft genome sequence of Bacillus azotoformans MEV2011, a (Co-) denitrifying strain unable to grow with oxygen

Bacillus azotoformans MEV2011, isolated from soil, is a microaerotolerant obligate denitrifier, which can also produce N2 by co-denitrification. Oxygen is consumed but not growth-supportive. The draft genome has a size of 4.7 Mb and contains key genes for both denitrification and dissimilatory nitrate reduction to ammonium. Electronic supplementary material The online version of this article (doi:10.1186/1944-3277-10-4) contains supplementary material, which is available to authorized users.


Introduction
Species of the genus Bacillus are characterized as Grampositive, facultative aerobic bacteria capable of forming endospores [1]. In the absence of oxygen, many Bacillus species can respire with nitrate instead, employing either dissimilatory nitrate reduction to ammonium or denitrification [2,3]. Despite the widespread occurrence of nitratereducing bacilli, their molecular and genetic basis remained poorly investigated [4,5]. Only recently, genome sequencing of two denitrifying type strains, B. azotoformans LMG 9581 T and B. bataviensis LMG 21883 T , has yielded first insights into the genomic inventory of nitrate reduction and denitrification in Gram-positives [6].
Classification and features B. azotoformans MEV2011 (Figure 1) was isolated at 28°C on anoxic King B plates [7] amended with KNO 3 (5 g L −1 ) from a highly diluted top soil sample at Aarhus University, Denmark. Strain MEV2011 resembles the type strain in its chemoorganotrophic growth on shortchain fatty acids, complete denitrification, and absence of fermentation [8]. However, it differs from the type strain by its inability to grow with oxygen, even though it can tolerate and consume oxygen at atmospheric concentrations. Growth by denitrification (verified by 15 N incubations; data not shown) starts at microaerobic conditions (<30 μM O 2 ; Figure 2), yet the initial presence of oxygen in the growth medium leads to longer lag phases and no increase in final density of the culture ( Figure 3); growth without nitrate was never observed. Therefore, we characterize B. azotoformans MEV2011 as microaerotolerant obligate denitrifier. In addition, B. azotoformans MEV2011 is capable of co-denitrification, a co-metabolic process, in which reduced nitrogen compounds like amino acids or hydroxylamine react with NO + formed during denitrification to produce N 2 O or N 2 [9]; co-denitrification was verified by the mass spectrometric detection of 30 N 2 + 29 N 2 in cultures growing on tryptic soy broth (TSB) and 15 NO 3 − , as suggested in [9]. B. azotoformans MEV2011 is available from the BCCM/ LMG Bacteria Collection as strain LMG 28302; its general features are summarized in Table 1.

Genome sequencing and annotation
Genome project history Bacillus azotoformans MEV2011 was selected for whole genome sequencing based on its unusual "obligate" denitrifying phenotype, i.e. its inability to grow under oxic conditions, together with its co-denitrifying capacity. Comparing the genome of strain MEV2011 to that of the oxygen-respiring and conventionally denitrifying type strain [8] may provide insights into the molecular basis of its metabolic features. The draft genome sequence was completed on July 20, 2013. The genome project is deposited in the Genomes OnLine Database (GOLD) as project Gp0043190. Raw sequencing reads have been deposited at the NCBI Sequence Read Archive (SRA) under the experiment numbers SRX527325 (100 bp library) and SRX527326 (400 bp library). This Whole Genome Shotgun project has been deposited at GenBank under the accession number JJRY00000000. The version described in this paper is version 1. Table 2 presents the project information and its association with MIGS version 2.0 compliance [27]. Figure 1 Phylogenetic tree highlighting the position of Bacillus azotoformans MEV2011 (shown in red) relative to closely related (≥95% sequence similarity) type strains within the Bacillaceae. Pre-aligned sequences were retrieved from the Ribosomal Database Project (RDP) [37]. Alignment of the B. azotoformans MEV2011 sequence as well as manual alignment optimization was performed in ARB [38]. The maximum likelihood tree was inferred from 1,478 aligned positions of 16S rRNA gene sequences and calculated based on the General Time Reversible (GTR) model with gamma rate heterogeneity using RAxML 7.4.2 [39]. Type strains with corresponding published genomes are shown in bold face. Open and closed circles indicate nodes with bootstrap support (1,000 replicates ) of 50-80% and >80%, respectively. Escherichia coli ATCC 11577 T (X80725) was used to root the tree (not shown). Scale bar, 0.1 substitutions per nucleotide position.   [12] with the following parameters: job = genome,denovo,accurate; technology = iontor.
In parallel, the reads were also assembled using Newbler 2.6 (Roche) with the following parameters: -mi 96 -ml 50 (i.e. 96% minimum sequence similarity and 50 bp minimum overlap). Contigs shorter than 1,000 bp were removed from both assemblies. All remaining contigs were trimmed by 50 bp from the 5' and the 3' ends using the prinseq-lite.pl script in order to remove errorprone contig ends. The two assemblies were merged and manually inspected using Sequencher 5.0.1 (Genecodes). In cases where the bases of the two assemblies disagreed, the Newbler variant was preferred. Contigs not contained in both assemblies were removed from the data set. The final assembly yielded 56 contigs representing 4.7 Mbp of sequence information.

Genome properties
The  rRNA genes indicates a total of 11 rRNA operons. Most (75.3%) protein-coding genes were assigned to putative functions. The distribution of genes into COG functional categories is presented in Table 4.

Insights from the genome sequence
Overall, the genome of the novel strain MEV2011 appeared highly similar to that of the B. azotoformans type strain LMG 9581 T [8]. In silico DNA-DNA hybridization (DDH) was performed for the assembled MEV2011 genome against the published genome of LMG 9581 T (Acc. number NZ_AJLR00000000); the contigs of B. azotoformans LMG 9581 T were assembled into one FASTA file before uploading to the online genome-to-genome calculator provided by the DSMZ [23]. Using the GGDC 2.0 model, DHH estimates were always >70%, irrespective of the formula used for computing DHH, and with probabilities between 78 and 87%. These results confirm that MEV2011 is a novel strain of the species B. azotoformans. Just as B. azotoformans LMG 9581 T , strain MEV2011 carries multiple copies of key denitrification genes, encodes both membrane-bound and periplasmic nitrate reductases, and the key genes for nitrite reduction to both NO (in denitrification) and ammonium (in DNRA); see (Additional file 1: Table S1) and reference [6] for details. Modularity and redundancy in nitrate reduction pathways has also been observed in other Bacillus species (e.g. B. bataviensis [6], Bacillus sp. strain ZYK [24], Bacillus sp. strain 1NLA3E [25]), and may be a general feature of nitrate-reducing members of this genus.
All genes essential for aerobic respiration were identified, including those for terminal oxidases (see Additional Phylum Firmicutes TAS [29][30][31] Class Bacilli TAS [32,33] Order Bacillales TAS [29,34] Family Bacillaceae TAS [29,35] Genus Bacillus TAS [29,36] Species Evidence codes -IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [10]. file 1: Table S2) and for detoxifying reactive oxygen species (see Additional file 1: Table S3). Therefore, the inability of B. azotoformans MEV2011 to grow with oxygen remains a conundrum and in some way resembles that of various sulfate-reducing bacteria, which also consume oxygen and even produce ATP during oxic respiration but are unable to grow in the presence of oxygen [26].

Conclusion
Based on our whole genome comparison, the microaerotolerant obligate (co-) denitrifying Bacillus sp. MEV2011 (LMG 28302) is a novel strain of Bacillus azotoformans, with similar redundancy in its nitrate reduction pathways, including the potential for DNRA, and a complete set of genes for oxic respiration and oxygen detoxification; its inability to grow with oxygen remains enigmatic.

Additional file
Additional file 1: Table S1. Overview of the genomic inventory for dissimilatory nitrogen transformations in Bacillus azotoformans MEV2011. Table S2. Overview of the genomic inventory for enzymatic reduction of O 2 and ATP synthase in Bacillus azotoformans MEV2011. Table S3.
Overview of the genomic inventory for the detoxification of reactive oxygen species in Bacillus azotoformans MEV2011.

Competing interests
The authors declare that they have no competing interests.  a)The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.