Complete Genome Sequence of Anoxybacillus flavithermus TNO-09.006, a Thermophilic Sporeformer Associated with a Dairy-Processing Environment

Spores of thermophilic spore-forming bacteria are a common cause of contamination in dairy products. We isolated the thermophilic strain Anoxybacillus flavithermus TNO-09.006 from a milk-processing plant, and we report the complete genome of this isolate consisting of a single chromosome of 2.65 Mb.

centrates is contamination by heat-resistant spores from the biofilms of thermophilic bacteria of the genera Anoxybacillus and Geobacillus. We isolated 20 thermophilic species from fouling samples of two dairy-production plants and screened for their ability to form biofilms. We identified three strains with relatively high biofilm-forming capacities and sequenced their genomes: Geobacillus thermoglucosidans TNO-09.020 (1), Anoxybacillus flavithermus TNO-09.006 (this work), and Geobacillus stearothermophilus TNO-09.008 (unpublished data).
The isolated strain A. flavithermus TNO-09.006 showed significant planktonic growth and biofilm-forming capacity at high temperatures (43 to 62°C; optimum at 57°C) under lab conditions (data not shown). This finding allows studies on the development of biofilm formation with the purpose of preventing the contamination of spores in end products.
The present view on genomic diversity, metabolic diversity, and adaptation of A. flavithermus to a dairy environment is limited, as only the genome of the strain A. flavithermus WK1 has been reported to date. This strain has been isolated from a wastewater drain at the Wairakei geothermal power station in New Zealand (2).
Genome sequencing was performed by GATC Biotech (Konstanz, Germany) using 454 Life Sciences GS20 pyrosequencing (1 M titanium; 454-Life Sciences, Roche). This sequence produced 181,707 reads totaling 72 Mb (average read length, 396 bp). In addition, Illumina mate pair sequencing was conducted (5,819,367 paired reads of 50 bp per read, 582 Mb). Assembly into contigs was performed using a whole-genome shotgun (WGS) assembler and SSPACE (3), which is a stand-alone program for scaffolding preassembled contigs using paired-end read data. A pseudoassembly was created by mapping the scaffolds to the most-similar complete genome sequence, that of A. flavithermus WK1 (GenBank accession no. CP000922.1) (2). A total of 82 gaps were filled using the consensus reads from PacBio RS sequencing (GATC Biotech) (5,402 reads; 7,857,232 bp; average read length, 1,456 bp). In short, the reads were mapped against the pseudoassembly using BLAST (PMID2231712) and Muscle (15034147), followed by manual inspection.
Automatic open reading frame (ORF) calling and annotation were performed using Integrated Services for Genomic Analysis (ISGA). Manual curation of ORFs was conducted using Artemis (4) and Artemis Comparison Tool (ACT) (5) by comparison with the reference A. flavithermus WK1 genome. Improved manual annotation was performed using Pfam (6), InterProScan (7), and NCBI BLAST. The genome of A. flavithermus TNO-09.006 consists of a single chromosome (2.65 Mb; 42% GϩC content) in 1 scaffold containing 68 contigs; no plasmids were found. The chromosome contains approximately 2,761 protein-encoding genes, 53 tRNA-encoding genes, and 7 rRNA-encoding operons (usually at contig boundaries). As the genome of A. flavithermus TNO-09.006 is our second reported genome of a thermophilic isolate from a dairy-processing environment, a more detailed analysis of this genome and a comparative analysis with other thermophilic isolates will provide further insight into the specific properties related to the adaptation of these strains to the dairy-processing environment.
Nucleotide sequence accession number. The complete genome of A. flavithermus TNO-09.006 has been deposited in Gen-Bank under the accession no. AMCM00000000.

ACKNOWLEDGMENTS
We thank Bernadet Renckens for help with sequence data analysis. We gratefully acknowledge Roy Moezelaar and Masja Nierop Groot for coordination of the project.
The project was funded by the Top Institute Food & Nutrition, Wageningen, The Netherlands.