Characterization of candidate genes involved in halotolerance using high-throughput omics in the halotolerant bacterium Virgibacillus chiguensis

Abstract

We previously used whole-genome sequencing and Tn5 transposon mutagenesis to identify 16 critical genes involved in the halotolerance of Halomonas beimenensis, a species in the phylum Proteobacteria. In this present study, we sought to determine if orthologous genes in another phylum are also critical for halotolerance. Virgibacillus spp. are halotolerant species that can survive in high-saline environments. Some Virgibacillus species are used in different aspects of food processing, compatible solute synthesis, proteinase production, and wastewater treatment. However, genomic information on Virgibacillus chiguensis is incomplete. We assembled a draft V. chiguensis strain NTU-102 genome based on high-throughput next-generation sequencing (NGS) and used transcriptomic profiling to examine the high-saline response in V. chiguensis. The V. chiguensis draft genome is approximately 4.09 Mbp long and contains 4,166 genes. The expression profiles of bacteria grown in 5% and 20% NaCl conditions and the corresponding Gene Ontology (GO) and clusters of orthologous groups (COG) categories were also analyzed in this study. We compared the expression levels of these 16 orthologs of halotolerance-related genes in V. chiguensis and H. beimenensis. Interestingly, the expression of 7 of the 16 genes, including trkA2, smpB, nadA, mtnN2, rfbP, lon, and atpC, was consistent with that in H. beimenensis, suggesting that these genes have conserved functions in different phyla. The omics data were helpful in exploring the mechanism of saline adaptation in V. chiguensis, and our results indicate that these 7 orthologs may serve as biomarkers for future screening of halotolerant species in the future.

Fig 1. Growth conditions for Virgibacillus chiguensis.

(A) Growth curves of V. chiguensis in 0%, 5%, 10%, 15%, 20%, and 25% (w/v) NaCl medium. (B) The slope of the growth curve of V. chiguensis between 9 and 19 h in the various NaCl concentrations. (C) The length of the lag phase for V. chiguensis growth under different NaCl conditions. (D) The OD₆₀₀ concentration of V. chiguensis under various NaCl conditions at 48 h.

Fig 2. Phylogenetic tree for Virgibacillus spp.

The 16S rRNA phylogenetic tree for Virgibacillus spp. was generated by the neighbor-joining method with Juke-Cantor correction. Bootstrap = 1,000. Bar, 0.01 substitutions per nucleotide position.

Fig 3. Contig length and gene number distributions of Virgibacillus chiguensis.

(A) The contig length distribution of Virgibacillus chiguensis. The x-axis represents the number of contigs. The y-axis represents the contig length range. Bp, base pair. (B) The gene number distribution of Virgibacillus chiguensis. The x-axis represents the number of genes. The y-axis represents the contig length range. Bp, base pair.

Fig 4. Gene comparison of Virgibacillus chiguensis with other species.

(A) Venn diagram showing the gain and loss of genes between V. chiguensis and four Virgibacillus spp.: V. halodenitrificans, V. necropolis, Virgibacillus sp. SK37, and Virgibacillus sp. LM2416. (B) Venn diagram showing the gain and loss of genes between V. chiguensis and four other species, including Halomonas beimenensis (CP021435), Escherichia coli (NC_000913), Pseudomonas aeruginosa (NC_002516), and Bacillus subtilis (CP021889). The distributions of the 9 orthologous halotolerant-related genes in V. chiguensis and H. beimenensis are indicated on the Venn diagrams.

Fig 5. Gene features of Virgibacillus chiguensis for gene ontology (GO) and clusters of orthologous groups of proteins (COG).

(A) The top 5 GO sub-categories for DEGs of V. chiguensis. The pink bars represent up-regulated DEGs (log₂FC of FPKM > 2); the blue bars represent down-regulated DEGs (log₂FC of FPKM < −2); the gray bars represent DEGs that did not significantly change. (B) The top 5 clusters of COG categories for the DEGs of V. chiguensis.

Fig 6. Differentially expressed genes (DEGs) of Virgibacillus chiguensis among various NaCl conditions.

The up-regulated DEGs are represented by pink columns showing log₂FC of FPKM was > 2; the down-regulated DEGs are represented by blue columns showing log₂FC of FPKM was < -2; the not significantly changed genes are represented by gray columns showing log₂FC of FPKM did not exceed 2. The numbers indicate the number of genes.

Fig 7. Gene expression of halotolerance-related genes in Halomonas beimenensis and Virgibacillus chiguensis based on the transcriptome profiles and qRT-PCR validation.

Genes related to halotolerance included the potassium transporter gene (trkA2), tmRNA-binding protein (smpB), quinolinate synthetase gene (nadA), 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase gene (mtnN2), undecaprenyl-phosphate galactosephosphotransferase gene (rfbP), (p)ppGpp synthetase/guanosine-3',5'-bis(diphosphate) 3'-diphosphatase gene (spoT), ATP-dependent protease gene (lon), PrkA family serine protein kinase gene (prkA), and ATP synthase gene (atpC).