Salt resistance genes revealed by functional metagenomics from brines and moderate-salinity rhizosphere within a hypersaline environment

Abstract

Hypersaline environments are considered one of the most extreme habitats on earth and microorganisms have developed diverse molecular mechanisms of adaptation to withstand these conditions. The present study was aimed at identifying novel genes from the microbial communities of a moderate-salinity rhizosphere and brine from the Es Trenc saltern (Mallorca, Spain), which could confer increased salt resistance to Escherichia coli. The microbial diversity assessed by pyrosequencing of 16S rRNA gene libraries revealed the presence of communities that are typical in such environments and the remarkable presence of three bacterial groups never revealed as major components of salt brines. Metagenomic libraries from brine and rhizosphere samples, were transferred to the osmosensitive strain E. coli MKH13, and screened for salt resistance. Eleven genes that conferred salt resistance were identified, some encoding for well-known proteins previously related to osmoadaptation such as a glycerol transporter and a proton pump, whereas others encoded proteins not previously related to this function in microorganisms such as DNA/RNA helicases, an endonuclease III (Nth) and hypothetical proteins of unknown function. Furthermore, four of the retrieved genes were cloned and expressed in Bacillus subtilis and they also conferred salt resistance to this bacterium, broadening the spectrum of bacterial species in which these genes can function. This is the first report of salt resistance genes recovered from metagenomes of a hypersaline environment.

Keywords: DNA repair; brine; functional metagenomics; hypersaline; rhizosphere; salt resistance genes; saltern; stress response.

FIGURE 1

16S rRNA phylogenetic reconstruction for bacterial (A) and archaeal (B) sequences. The presence of OPUs with abundances >0.5% in each sample type (B = brines; R = rhizosphere) is indicated with a dot. Each OPU results from the phylogenetic inference resulting from the parsimony insertion of representatives of each sequence cluster at 99% identity, each representing independent OTUs.

FIGURE 2

Growth curves of Escherichia coli MKH13 cells carrying plasmids with salt resistance genes (pSR1-pSR8) and MKH13-pSKII⁺ in LB broth and LB broth supplemented with 3% NaCl. Clones pSR1, pSR2, pSR3, pSR4, and MKH13-pSKII⁺ in LB broth (A) and LB broth supplemented with 3% NaCl (B). Clones pSR5, pSR6, pSR7, pSR8, and MKH13-pSKII⁺ in LB broth (C) and LB broth supplemented with 3% NaCl (D).

FIGURE 3

Schematic organization of the ORFs identified in the pSR1-pSR8 plasmids. Arrows denote the location and the transcriptional orientation of the ORFs in the different plasmids. ORFs involved in NaCl resistance are indicated by gray arrows and those whose phenotype was not resistant are shown in white arrows. The presence of predicted transmembrane helices is represented by arrows shaded with vertical bars. Asterisks indicate incomplete ORFs. HP, hypothetical protein.

FIGURE 4

Growth curve of E. coli MKH13 cells carrying pSR4, the E. coli nth gene, and MKH13-pSKII⁺ in LB broth (A) and LB broth supplemented with 3% NaCl (B).

FIGURE 5

Growth curve of E. coli MKH13 cells carrying pSR7, the E. coli rhlE gene and MKH13-pSKII⁺ in LB broth (A) and LB broth supplemented with 3% NaCl (B).

FIGURE 6

Growth of Bacillus subtilis clones in NaCl. B. subtilis clones pSR1-orf2 (A), pSR4-orf1 (B), pSR6-orf2 (C) and pSR7-orf1 (D) were grown in LB broth supplemented with 6% NaCl in the presence and in the absence of 1mM IPTG. B. subtilis strain PY79 with the empty plasmid pdr111inserted in the chromosome was used as negative control.

FIGURE 7

Test for cellular content of Na⁺ ion in E. coli clones pSR1 to pSR8 and MKH13-pSKII⁺ after 1 h of growth with 6% NaCl. Values are the averages of two independent ICP-MS measurements. Error bars indicate standard deviation. An asterisk indicates significantly different from control cells as determined by one-way ANOVA followed by Tukey’s test (p < 0.05).

FIGURE 8

Test for cellular content of Na⁺ ion in E. coli clones pSR6, pSR6-orf2, pSR6-orf3, and MKH13-pSKII⁺ after 1 h of growth with 6% NaCl. Values are the averages of two independent ICP-MS measurements. Error bars indicate standard deviation. An asterisk indicates significantly different from pSR6, pSR6-orf3 and control cells as determined by one-way ANOVA followed by Tukey’s test (p < 0.05).