Genome sequence of Bacillus altitudinis HH-03 isolated from dairy pipeline

TL;DR

This paper reports the genome sequence of a bacterium isolated from a dairy pipeline, which could help improve cleaning processes in the dairy industry.

Contribution

The study provides a new genome sequence for Bacillus altitudinis HH-03 and highlights its potential for improving clean-in-place processes.

Findings

The genome of Bacillus altitudinis HH-03 is 3.85 Mbp with 41.2% GC content.

The genome contains 4,010 predicted protein-coding genes.

The genomic data may aid in developing more effective cleaning processes in dairy pipelines.

Abstract

In this study, Bacillus altitudinis HH-03 was isolated from a dairy pipeline, and its genomic information was obtained by performing sequencing. The genome size of strain HH-03 was 3.85 Mbp with a GC content of 41.2%. Based on the annotation procedure, 4,010 protein-coding genes were identified. The genomic information of strain HH-03 will be helpful for the development of clean-in-place processes.

Figures

The figure represents the distribution of various genomic features, with the following color codes: gray: coding sequences (CDS); orange: GC content; green: positive GC skew (GC Skew+); purple: negative GC skew (GC Skew-); blue: rRNA genes; pink: tRNA genes.

Full text

ANNOUNCEMENT

Dairy products are susceptible to contamination by Bacillus during processing and production since Bacillus can form endospores and biofilms (1), possesses high heat resistance and dispersal abilities, and can grow and multiply in milk pipelines (2). A significantly elevated incidence of Bacillus cereus contamination was reported in unprocessed milk samples (3), and Bacillus altitudinis contamination was detected in pre-pasteurized milk (4).

We isolated heat-tolerant bacteria from the washed water of the milk pipeline in Hubei Province, China, after boiling the sample for 10 min and growing the bacteria on nutrient agar with manganese at 37°C for 2 days. Most colonies were identified as Bacillus altitudinis based on nearly complete 16S rRNA gene sequences obtained by amplification and sequencing. The strain HH-03 was streaked onto nutrient agar with manganese solid medium and incubated at 37°C for 24 h. A distinct single colony was subsequently inoculated into liquid nutrient agar with manganese and cultured at 37°C for 24 h . Then, the 16S rRNA gene sequences were amplified and sequenced to verify culture purity. We selected strain HH-03 for further genome analysis. Genomic DNA from the pure culture of strain HH-03 was extracted using the MagAttract HMW DNA Kit (Qiagen, USA). The sequencing library was constructed using the Transposase Enzyme Linked Long-read Sequencing (TELL-Seq) WGS Library Prep Kit (Universal Sequencing Technology, USA) and the sequencing primers provided by the TELL-Seq Illumina Sequencing Primer Kit (Universal Sequencing Technology, USA), following the manufacturer’s instructions (5). The libraries were sequenced on an Illumina NovaSeq 6000, 150 bp paired-end platform to obtain raw sequencing data. Quality control and assembly of the raw sequencing data of strain HH-03 were conducted using the TELL-Read and TELL-Link processes provided by Universal Sequencing Technology (5).

The 632 M raw reads were assembled de novo using TELL-Read with parameters “-s T507 -g NONE” and TELL-Link with “-p 507.” Contig sequencing depths were quantified using BWA (v0.7.17) (6), with the assembled sequences showing a mean sequencing depth of 112.

The obtained contigs were subjected to BLAST analysis against the NCBI database to identify contaminated sequences. Following the removal of contigs exhibiting inadequate sequencing depth below 80 and contamination, the strain HH-03 yielded 12 contigs, with the largest one being 3.82 Mbp in length.

To gain insight into the genome structure, the coding sequences of the positive and negative strands of the genome, the location of non-coding RNAs, and the spliced genome were plotted in a genome feature circle diagram. The spliced FASTA files were uploaded to CGViewer (7), an online genome feature map website, for online interactive operations, and the genomes were annotated using PGAP (v6.9) (8). The genomic circle diagram of strain HH-03 is shown in Fig. 1. The assembled genome size of the sample was 3,846,532 bp with a GC content of 41.2%. Additionally, the sample contained 3,796 coding sequences (with protein), 11 complete rRNA genes, and 75 tRNA genes.

The figure represents the distribution of various genomic features, with the following color codes: gray: coding sequences (CDS); orange: GC content; green: positive GC skew (GC Skew+); purple: negative GC skew (GC Skew-); blue: rRNA genes; pink: tRNA genes.

Strain HH-03 was subjected to average nucleotide identity (ANI) analysis by using JSpeciesWS (9). The highest ANI value for strain HH-03 genome was 97.85% with Bacillus altitudinis (GCF_001191605.1), indicating that strain HH-03 should be identified as Bacillus altitudinis.

The genomic information of Bacillus altitudinis HH-03 will be helpful for the development of clean-in-place processes.