Revealing the Saline Adaptation Strategies of the Halophilic Bacterium Halomonas beimenensis through High-throughput Omics and Transposon Mutagenesis Approaches

Abstract

Studies on the halotolerance of bacteria are attractive to the fermentation industry. However, a lack of sufficient genomic information has precluded an investigation of the halotolerance of Halomonas beimenensis. Here, we describe the molecular mechanisms of saline adaptation in H. beimenensis based on high-throughput omics and Tn5 transposon mutagenesis. The H. beimenensis genome is 4.05 Mbp and contains 3,807 genes, which were sequenced using short and long reads obtained via deep sequencing. Sixteen Tn5 mutants with a loss of halotolerance were identified. Orthologs of the mutated genes, such as nqrA, trkA, atpC, nadA, and gdhB, have significant biological functions in sodium efflux, potassium uptake, hydrogen ion transport for energy conversion, and compatible solute synthesis, which are known to control halotolerance. Other genes, such as spoT, prkA, mtnN, rsbV, lon, smpB, rfbC, rfbP, tatB, acrR1, and lacA, function in cellular signaling, quorum sensing, transcription/translation, and cell motility also shown critical functions for promoting a halotolerance. In addition, KCl application increased halotolerance and potassium-dependent cell motility in a high-salinity environment. Our results demonstrated that a combination of omics and mutagenesis could be used to facilitate the mechanistic exploitation of saline adaptation in H. beimenensis, which can be applied for biotechnological purposes.

Figure 1

Phylogenetic position and growth conditions of Halomonas beimenensis. (A) Growth curve of H. beimenensis in medium containing various concentrations of NaCl, including 0%, 5%, 10%, 15%, 20%, and 25% (w/v) NaCl. (B) The slope of growth curve in the 5%, 10%, 15%, and 20% NaCl conditions between 3 and 12 h represents the growth rate of H. beimenensis under different NaCl conditions. (C) The length of the lag phase of H. beimenensis under different NaCl conditions. (D) The concentration of H. beimenensis (OD₆₀₀) under different NaCl conditions at 48 h. (E) A phylogenetic tree based on 16S rRNA sequences was constructed by the neighbor-joining method with the Juke-Cantor correction. Bootstrap values were calculated from 1,000 samplings, and values > 50% are shown. Bar, 0.02 substitutions per nucleotide position.

Figure 2

Genome map and gene comparison of Halomonas beimenensis. (A) Genome map of H. beimenensis. Rings from the outside are as follows: 1^st circle: scale marks (unit, kb); 2^nd circle: predicted genes on the + strand; 3^rd circle: predicted genes on the − strand; 4^th circle: read counts mapping to predicted genes on the + strand in 5% NaCl; 5^th circle: read counts mapping to predicted genes on the + strand in 20% NaCl; 6^th circle: read counts mapping to predicted genes on the - strand in 5% NaCl; 7^th circle: read counts mapping to predicted genes on the − strand in 20% NaCl; 8^th circle: significantly differentially expressed genes determined based on log₂ fold-changes (log₂FC) in FPKM values on the + strand that were greater than 2 (up-regulated genes, red lines) or smaller than −2 (down-regulated genes, blue lines); 9^th circle: significantly differentially expressed genes determined based on log₂FC in FPKM values on the - strand greater than 2 (up-regulated gene, red lines) or smaller than -2 (down-regulated genes, blue lines); 10^th circle: GC content, 11^th circle: GC skew (above-average values in pink; below-average values in purple). (B) Venn diagram of gain and loss of genes between H. beimenensis and four Halomonas spp.: H. campaniensis, H. chromatireducens, H. elongata, and H. huangheensis.

Figure 3

Gene features of Halomonas beimenensis. (A) The average TPM (transcripts per million reads) of CDSs and the intergenic regions in the 5% and 20% NaCl conditions. (B) The differentially expressed genes (DEGs) of H. beimenensis between the 5% NaCl and 20% NaCl conditions. The pink columns represent the up-regulated DEGs, for which the log₂FC of FPKM was higher than 2; the blue columns represent the down-regulated DEGs, for which log₂FC in FPKM was lower than −2; the gray columns represent genes that did not significantly change (the absolute value of the log₂FC of FPKM did not exceed 2). Numbers above the column represent the numbers of genes. (C) The top 5 clusters of orthologous groups (COG) categories indicated for the DEGs of H. beimenensis. (D) The top 8 clusters of Gene Ontology (GO) categories indicated for the DEGs of H. beimenensis. Up-regulated DEGs (log₂FC of FPKM > 2) are represented by pink bars and numbers; the down-regulated DEGs (log₂FC of FPKM < −2) are represented by blue bars and numbers; gray bars indicate genes with no significant change (−2 < log₂FC of FPKM < 2). (E) The transcriptome profile of differentially expressed flagella-related genes in 5% and 20% NaCl.

Figure 4

Growth and gene expression levels of 16 halotolerance-related genes in the wild-type and Tn5 mutants of Halomonas beimenensis under different salt conditions. (A) The growth of wild-type (WT) H. beimenensis and 16 Tn5 mutant lines under 5% and 15% NaCl conditions. (B) The expression of 16 halotolerance-related genes in WT H. beimenensis was determined by the FPKM values from the transcriptome data and qRT-PCR analysis of bacteria grown in 5% and 20% NaCl conditions. (C) The highlighted gene expression of the 16 genes in the WT bacteria and the 16 Tn5 mutants of H. beimenensis under 5% and 15% NaCl conditions analyzed by qRT-PCR. Each data point was compared with the gene expression in WT bacteria grown in 5% NaCl.

Figure 5

The network of 16 halotolerance-related genes of Halomonas beimenensis. (A) The putative network in the 5% NaCl condition. (B) The putative network in the 15% NaCl condition. The symbols for each gene suggest involved functions, e.g., (K) potassium; (B) betaine; (E) ectoine; (P) proline; (T) trehalose; (M) cell motility; and (M^K) potassium-dependent cell motility.

Figure 6

Chemical complementation of wild-type and Tn5-mutated Halomonas beimenensis in 15% NaCl. (A) The complementation analysis of the wild-type bacteria with a variety of compatible solutes. (B) The growth of Tn5 mutant lines in which the mutant phenotype was slightly or fully rescued by a variety of compatible solutes. (C) The growth of Tn5 mutant lines that were inhibited by a variety of compatible solutes. (D) The growth of Tn5 mutant lines in which the mutant phenotype was slightly or fully rescued by 200 mM KCl.

Figure 7

Motility and auto-aggregation phenotype of wild-type and 16 Tn5 mutants of Halomonas beimenensis. (A) Motility phenotype assay of the wild-type (WT) and 16 Tn5 mutants. (i) to (vi) were assayed on 3% agar plates containing 5% NaCl for 24 h. (v) to (viii) were assayed on 3% agar plates containing 15% NaCl for 48 h. (B) Motility phenotype assay of WT bacteria and 16 Tn5 mutants with 200 mM KCl. (i) to (vi) were assayed on 3% agar plates containing 5% NaCl for 24 h. (v)-(viii) were assayed on 3% agar plates containing 15% NaCl for 48 h. (C) Auto-aggregation phenotype of the rfbP and rfbC mutants under different salt conditions.