Complete genome sequence of the moderately thermophilic mineral-sulfide-oxidizing firmicute Sulfobacillus acidophilus type strain (NAL^T)

This organism was selected for sequencing on the basis of its phylogenetic position [38], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [39]. The genome project is deposited in the Genomes OnLine Database [18] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

S. acidophilus strain NAL^T, DSM 10332, was grown in DSMZ medium 709 (Acidomicrobium medium) [40] at 45°C. DNA was isolated from 0.5-1 g of cell paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer with modification st/LALM for cell lysis as described in Wu et al. 2009 [39]. DNA is available through the DNA Bank Network [41].

The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [42]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 104 contigs in three scaffolds was converted into a phrap [43] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (599.7 Mb) were assembled with Velvet [44] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 143.7 Mb of 454 draft data and all of the 454 paired-end data. Newbler parameters were -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package [43] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution (C. Han, unpublished), Dupfinisher [45], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, PCR and Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 640 additional reactions and eight shatter libraries were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI [46]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 219.6 × coverage of the genome. The final assembly contained 612,059 pyrosequence and 16,626,072 Illumina reads.

Genes were identified using Prodigal [47] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [48]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [49].

While the sequencing of the genome described in this paper was underway, Li et al. from the Third Institute of Oceanography, Xiamen, China published the complete genome sequence of strain TPY [7]. The two genomes differ in size by less than 7,000 bp. Here, we take the opportunity to compare the completed genome sequences from these two stains, NAL^T and TPY, both belonging to S. acidophilus. While the biological material for the type stain, NAL^T, is publicly available from the DSMZ open collection for postgenomic analyses, no source of the biological material (MIGS-13 criterion, see Table 2) of strain TPY was provided by Li et al. [7].

To estimate the overall similarity between the genomes of strains NAL^T and TPY (Genbank accession number: CP002901), the GGDC-Genome-to-Genome Distance Calculator [50,51] was used. The system calculates the distances by comparing the genomes to obtain HSPs (high-scoring segment pairs) and interfering distances from three formulae (HSP length / total length; identities / HSP length; identities / total length). The comparison of the genomes of strains NAL^T and TPY revealed that 99.65% of the average of the genome lengths are covered with HSPs. The identity within these HSPs was 99.01%, whereas the identity over the whole genome (counting regions not covered by HSPs as non-identical) was 98.67%. The inferred digital DNA-DNA hybridization values for the two strains are 96.47% (formula 1 in [51]), 86.08% (formula 2 in [51]) and 97.05% (formula 3 in [51]), respectively. These results clearly demonstrate that according to the whole genome sequences of strains NAL^T and TPY, the similarity is very high, supporting the membership of both strains in the same species.

The comparison of the number of genes belonging to the different COG categories revealed few differences between the genomes of strains NAL^T and TPY. Strain NAL^T has 2,740 genes with COGs assigned, while strain TPY has 2,700. We analyzed the differences in COG assignment between the two strains and found that in almost all cases they could be explained by differences in the gene calls or pseudogene assignment, i.e. in one genome two parts of a pseudogene were called as two separate genes, while in the other genome they were combined into one pseudogene. The only clear case of a difference in gene content between the two strains is the presence of a transposable element consisting of two genes (Sulac_1668, Sulac_1669) disrupting a subunit of a potassium transporter (Sulac_1667) in strain NAL^T. There were also cases where a gene in one strain was split into two genes in the other strain. For example, Sulac_2178 corresponds to TPY_1983 and TPY1984, and Sulac_0347 corresponds to TPY_0381 and TPY_0382. In both cases the differences are due to a single base indel.

A dot plot showed that there are large blocks of synteny between the two genomes with some rearrangements (data not shown). The genes found on the plasmid in strain NAL^T are found in two regions of the chromosome in strain TPY. Sulac_3528-3555 corresponds to TPY_0524-0552, while Sulac_3556-3626 corresponds to TPY_2310-2244. This suggests that in strain TPY, the plasmid was inserted into the chromosome and then split into two pieces.

We analyzed CRISPR repeats with the CRISPR Recognition Tool [52] and found major differences between the two strains. They both have two regions of CRISPR repeats, but the strain TPY repeat regions have 8 and 9 repeats while the strain NAL^T repeat regions have 27 and 43 repeats. All of the spacers in the TPY repeat regions are found in NAL^T, but NAL^T has many additional spacers. This agrees with previous results suggesting that CRISPRs evolve quickly, and differences can be found in closely related strains [53].