Environmental Microbiology | Spotlight

Response of Methylocystis sp. Strain SC2 to Salt Stress: Physiology, Global Transcriptome, and Amino Acid Profiles

Dongfei Han, Hannes Link, Werner Liesack
Alfons J. M. Stams, Editor
DOI: 10.1128/AEM.00866-17

ABSTRACT

Soil microorganisms have to rapidly respond to salt-induced osmotic stress. Type II methanotrophs of the genus Methylocystis are widely distributed in upland soils but are known to have a low salt tolerance. Here, we tested the ability of Methylocystis sp. strain SC2 to adapt to increased salinity. When exposed to 0.75% NaCl, methane oxidation was completely inhibited for 2.25 h and fully recovered within 6 h. Growth was inhibited for 23.5 h and then fully recovered. Its transcriptome was profiled after 0 min (control), 45 min (early response), and 14 h (late response) of stress exposure. Physiological and transcriptomic stress responses corresponded well. Salt stress induced the differential expression of 301 genes, with sigma factor σ32 being a major controller of the transcriptional stress response. The transcript levels of nearly all the genes involved in oxidizing CH4 to CO2 remained unaffected, while gene expression involved in energy-yielding reactions (nuoA-N) recovered concomitantly with methane oxidation from salt stress shock. Glutamate acted as an osmoprotectant. Its accumulation in late stress response corresponded to increased production of glutamate dehydrogenase 1. Chromosomal genes whose products (stress-induced protein, DNA-binding protein from starved cells, and CsbD family protein) are known to confer stress tolerance showed increased expression. On plasmid pBSC2-1, genes encoding type IV secretion system and single-strand DNA-binding protein were upregulated in late response, suggesting stress-induced activation of the plasmid-borne conjugation machinery. Collectively, our results show that Methylocystis sp. strain SC2 is able to adapt to salt stress, but only within a narrow range of salinities.

IMPORTANCE Besides the oxic interface of methanogenic environments, Methylocystis spp. are widely distributed in upland soils, where they may contribute to the oxidation of atmospheric methane. However, little is known about their ability to cope with changes in soil salinity. Growth and methane oxidation of Methylocystis sp. strain SC2 were not affected by the presence of 0.5% NaCl, while 1% NaCl completely inhibited its activity. This places strain SC2 into the low-salt-tolerance range reported for other Methylocystis species. Our results show that, albeit in a narrow range, strain SC2 is able to respond and adapt to salinity changes. It possesses various stress response mechanisms, which allow resumption of growth within 24 h when exposed to 0.75% NaCl. Presumably, these mechanisms allow Methylocystis spp., such as strain SC2, to thrive in upland soils and to adapt to certain fluctuations in soil salinity.

INTRODUCTION

Aerobic methane-oxidizing bacteria (MOB), or methanotrophs, are able to utilize methane as their sole source of carbon and energy. Methanotrophs play an important role in the global cycling of greenhouse gases, as methane is present in the atmosphere at a mixing ratio of 1.8 ppm by volume (ppmv) and is 20 to 23 times more effective in trapping atmospheric heat than carbon dioxide (1, 2).

All proteobacterial methanotrophs are classified into type I or type II MOB by multiple criteria; however, they are classified mostly according to their pathways of carbon assimilation and phospholipid fatty acid (PLFA) composition (3). While type I MOB (Gammaproteobacteria) are phylogenetically and taxonomically diverse, type II MOB (Alphaproteobacteria) are limited to the traditionally known genera Methylocystis and Methylosinus (3, 4) and the more recently described genera Methylocella (5), Methylocapsa (6), and Methyloferula (7, 8).

Typically, methanotrophs inhabit the aerobic interfaces of methanogenic environments, such as natural wetlands and rice paddies, where there is a high methane supply (3, 9). In these environments, methanotrophic activity is characterized primarily by low apparent affinity [Km(app)] to methane (10, 11). On the contrary, methanotrophs inhabiting upland soils oxidize methane with high apparent affinity. In addition to yet-uncultured MOB (upland soil clusters α and γ), type II MOB of the genera Methylocystis and Methylosinus are abundantly present in upland soils acting as a sink for atmospheric methane (12, 13). While salinity is known to have a major effect on both methane oxidation and MOB community composition in dryland soils (14) and saline alkaline soils (15), no pure-culture study has been performed to date to assess the resilience of methanotrophic bacteria to salt stress.

Here, we investigated how a particular type II MOB, Methylocystis sp. strain SC2, responds to salt stress under short-term and long-term exposure. Strain SC2 is a model organism that possesses functional copies of two different particulate methane monooxygenase (pMMO) isozymes. The low- and high-affinity enzyme systems, pMMO1 and pMMO2, are encoded by pmoCAB1 (two copies) and pmoCAB2 (single copy), respectively (16–18). These two isozymes allow strain SC2 to adapt to a wide range of methane concentrations and thus to changes in its environment (12, 13, 18). High-affinity methane oxidation is assumed to be the relevant methanotrophic activity in upland soils. In such soils, moisture content and osmotic pressure fluctuate seasonally. Desiccation processes and increases in salinity may lead to high-osmolarity microniches (19). This prompted our interest in examining the ability of strain SC2 to cope with osmotic and salt stress. Our research combined measurements of growth rates and methane oxidation activities with global transcriptome analysis and amino acid profiling.

RESULTS AND DISCUSSION

Salt tolerance of strain SC2.The addition of up to 0.5% NaCl had no significant effect on the growth of strain SC2. The optical density at 600 nm (OD600) increased from 0.12 to 0.27 (0.5% NaCl) and 0.29 (control) until day 6. The OD600 of cultures treated with 0.75% NaCl increased from 0.12 (day 3) to 0.18 (day 6) and then reached stationary phase. No increase in optical density was observed after the addition of 1% or 1.5% NaCl (Fig. 1A). Correspondingly, methane oxidation (Fig. 1B) and carbon dioxide production (Fig. 1C) were moderately inhibited at 0.75% NaCl but completely inhibited at 1% and 1.5% NaCl. This placed strain SC2 into the low salt tolerance range of 0.2% to 1% known for Methylocystis spp., with only M. parvus reported to tolerate up to 2% NaCl (20, 21). Consequently, we chose the partially inhibitory concentration of 0.75% NaCl to investigate the global transcriptome response of strain SC2 to salt stress.

FIG 1
FIG 1

Growth, methane consumption, and carbon dioxide production of Methylocystis sp. SC2 as a function of different NaCl concentrations. NaCl was added at the mid-exponential phase, as indicated by arrows.

Changes in the SC2 transcriptome were examined after short-term (45-min) and long-term (14-h) stress exposure. In transcriptome studies, a 45-min stress exposure is commonly applied to examine the salt shock response of bacteria, as has been done for Bacilli (22, 23), Rhodobacter sphaeroides (24), and Desulfovibrio vulgaris (25). In the late stress response, the decision to use 14-h stress exposure was based on preliminary results obtained for the recovery of methane oxidation upon 0.75% NaCl addition (measured as percent [vol/vol] decrease of CH4 h−1 in the headspace): 0.19% within the first 3 h, 0.23% between 3 and 11.5 h, and 0.31% between 11.5 and 23.5 h. The 0.31% rate was comparable to that of the control cultures during exponential growth phase (0.35% [vol/vol] CH4 h−1).

Global transcriptomics.Methylocystis sp. strain SC2 contains a circular chromosome of 3,773,444 bp and two plasmids of 229,614 bp (pBSC2-1) and 143,536 bp (pBSC2-2) (26). The chromosome and the two plasmids are referred to as the SC2 genome, which comprises 4,058 genes. Of these, 301 genes were identified as differentially expressed, with log2 fold changes in reads per kilobase of coding sequence (CDS) (or exon) model per million mapped reads (RPKM) values of less than or equal to −2 or greater than or equal to 2 (Fig. 2A). Their putative functions were categorized using Clusters of Orthologous Groups (COGs) (see Fig. S1 and Table S1 in the supplemental material).

FIG 2
FIG 2

Differential expression of strain SC2 genes in response to salt stress. (A) The histogram indicates the differential expression levels of the complete set of 4,058 genes. (B) Total number of genes up- or downregulated, shown separately for the chromosome and the two plasmids of strain SC2. (C) Number of genes up- or downregulated in early (45-min) and late (14-h) stress responses. (D) Heat map showing a hierarchical cluster analysis of gene expression (RPKM) in Methylocystis sp. SC2 at 0-min (control), 45-min (45 min), and 14-h (14 h) NaCl treatments.

Using the log2 RPKM values of each gene, we calculated the Pearson product-moment correlation coefficient (PCC) (27, 28) between the three independent Illumina RNA sequencing (RNA-Seq) (triplicate) data sets generated for each salt treatment: 0 min (control), 45 min (short term), and 14 h (long term). The PCC value of each triplicate data set was greater than 0.95 (Table S2). Thus, the transcriptome profiles obtained for the three biological replicates of each salt treatment were highly correlated to each other and highly reproducible. In addition, we chose 10 SC2 genes for differential expression analysis by reverse transcription-quantitative PCR (RT-qPCR). Overall, the RT-qPCR results confirmed the reliability of Illumina RNA-Seq for assessing significant changes in global gene expression (see validation of RNA-Seq by RT-qPCR in the supplemental results).

Of the 301 genes that were differentially expressed, 167 and 134 genes were up- and downregulated, respectively. A total of 260 genes was located on the chromosome, while 41 genes were plasmid-borne (Fig. 2B). A set of 53 genes was differentially expressed in both (45-min and 14-h) treatments. Most of these genes were upregulated (46 genes), suggesting that in both early and late responses, their expression products are of importance for the ability of strain SC2 to cope with salt stress. Thirty-five of the 46 upregulated genes were categorized as “function unknown” (24 genes) or “general function prediction only” (11 genes).

Most genes (248), however, were differentially expressed in either the early or late response (Fig. 2C and S2 and Tables S3 to S8). The majority of the genes that showed differential expression only in the early (45-min) response were downregulated (104 of 151 genes). In addition to function unknown (29 genes) or general function prediction only (26 genes), downregulated genes were related to amino acid transport and metabolism (5 genes), cell wall/membrane/envelope biogenesis (6 genes), energy production and conversion (10 genes), inorganic ion transport and metabolism (7 genes), lipid metabolism (3 genes), nucleotide transport and metabolism (5 genes), and translation, ribosomal structure, and biogenesis (3 genes) (Fig. S1). Further discussion on the differential expression of genes encoding cell wall/membrane/envelope proteins, inorganic ion transport, and hypothetical proteins is given in the supplemental results.

In contrast to the early response, the majority of genes that showed differential expression only in late (14-h) response were upregulated (75 of 97 genes). More than half of the upregulated genes were annotated function unknown (36 genes) or general function prediction only (15 genes). Thirty-two of the 75 genes specifically upregulated in the late response were located on pBSC2-1, suggesting a high functional importance of the plasmid for strain SC2. This view is corroborated by plasmid curing attempts that failed (29).

Hierarchical cluster analysis of the transcriptome profiles confirmed that stress exposure time had a significant effect on global gene expression. The control and late-response profiles had greater similarity with each other than either shared with the early response pattern (Fig. 2D). In particular, the number of genes that were downregulated in either early response (105 genes) or late response (22 genes) differed between the 45-min and 14-h NaCl treatments.

Compatible solutes.Two genes involved in the synthesis of amino acids were differentially expressed in either the early (BN69_2878) or late (BN69_0999) response. Gene BN69_2878 encodes ornithine cyclodeaminase, which is involved in proline synthesis (30). Gene BN69_0999 encodes glutamate dehydrogenase 1. This enzyme catalyzes the final step in glutamate synthesis, the conversion of α-ketoglutarate to glutamate (31). Transcripts of additional genes involved in the synthesis of Nδ-acetyl ornithine, proline, glutamate, and glutamine were detected, but their expression levels did not significantly change in response to salt stress (Fig. 3 and S3).

FIG 3
FIG 3

Glutamate synthesis in response to salt stress. Arrows and numbers above indicate for each gene its log2 fold change in RPKM value in 45-min and 14-h treatment, respectively. Up- or downregulation in response to salt stress is indicated by the different direction and color of each arrow. The numbers in parenthesis show the fold changes of intracellular amino acid concentration.

Thus, we suspected that in strain SC2, proline and glutamate are synthesized to act as osmoprotectants in the early and late response, respectively. Metabolomic profiling of amino acids in strain SC2 confirmed glutamate synthesis in the late response but not proline synthesis in the early response (Fig. 3 and S3 and Table S9). Relative to the other amino acids, the intracellular concentration of glutamate was already high in the controls but greatly increased with stress exposure. Its concentration significantly increased (P < 0.05) from 5.17 to 16.85 nmol OD−1 (ml culture)−1 between the control and late response. The glutamate concentration in the early response was 7.15 nmol OD−1 (ml culture)−1 (Table S10). Given these findings, we conclude that glutamate was the major osmoprotectant, and it may have already acted as a compatible solute in the early response. Strain SC2 was cultivated in a synthetic medium with methane as the sole carbon source. No extracellular amino acids were available for stress-induced transport into the cells. Thus, glutamate had to be synthesized via the tricarboxylic acid (TCA) cycle, with α-ketoglutarate as the precursor.

Sigma factors.The SC2 genome contains 16 sigma factor-related genes (Table S11). Of these, three genes encoding sigma factor σ32 and a subunit of extracytoplasmic function (ECF) subfamily sigma factor σ24 were differentially expressed (Table 1). Apparently, sigma factor σ32 is a major controller of the transcriptional stress response in strain SC2. The rpoH gene (BN69_0407) encoding sigma factor σ32 was highly upregulated in both the early and late responses, with log2 fold changes of 5.29 and 3.40, respectively. A second gene (BN69_2909) encoding sigma factor σ32 was significantly upregulated only in the early response, with a log2 fold change of 2.65. In E. coli, sigma factor σ32 controls both heat shock (32) and osmotic stress (33) responses by regulating transcription initiation. The gene product of ftsH is known to be responsible for the degradation of sigma factor σ32. In strain SC2, two ftsH gene copies (BN69_0412 and BN69_0853) were slightly upregulated in both the early and late responses, suggesting the existence of a sigma factor σ32/FtsH regulatory system that is responsive to osmotic stress (see supplemental results for further details on ftsH). The gene (BN69_1388) encoding a subunit of ECF sigma factor σ24 was significantly downregulated only in the late response. ECF sigma factors are involved in sensing and responding to signals that are generated outside the cell or in the cell membrane (34). Depending on the organism, the biological roles of ECF sigma factors may be in envelope stress response, oxidative stress and general stress response, or iron transport (35). One of the best-understood ECF signaling pathways regulates the σ24-mediated expression of periplasmic stress response genes in E. coli. The gene encoding σ24 controls the periplasmic heat shock regulon that is activated by excessive levels of aberrantly folded proteins in the cell envelope caused by heat or other outer membrane stress (35, 36). In particular, σ24 controls the expression of a periplasmic endopeptidase that is essential for bacterial growth under heat stress (37). The genes regulated by σ24 in E. coli also include sigma factor σ32, the housekeeping sigma factor σ70, as well as σ24 itself, with all its regulatory components (36). Thus, it is reasonable to speculate that like in E. coli, sigma factor σ24 in strain SC2 controls the expression of periplasmic stress response genes by cross talk with sigma factors σ32 and σ70.

View this table:
TABLE 1

Chromosomal genes differentially expressed in response to salt stress

Genes conferring stress tolerance.We identified several chromosomal SC2 genes whose products are known to confer stress tolerance in other bacteria. Among these, the stress-induced protein, DNA-binding protein from starved cells, and CsbD family protein were expressed in response to salt stress (Table 1).

The expression of the SC2 gene (BN69_2205) encoding the stress-induced protein was highly upregulated primarily in the early response (Table 1). The production of stress-induced protein is known to be induced by environmental stress, but its functional role remains elusive (38, 39). Homologs of BN69_2205 are widely distributed among alphaproteobacteria, but in particular, in type II methanotrophs, including Methylosinus, Methylocystis, Methylocella, Methylocapsa, and Methyloferula (38, 39). BN69_2205 belongs to a cluster of four genes whose organization is highly conserved among members of the Methylocystis/Methylosinus group (see supplemental results for further details on the PhyR regulon) (39). Homologs of BN69_2205 are not present in any type I methanotroph, as deduced from a BLAST survey against nucleotide collection (nr/nt) and non-redundant protein database of NCBI GenBank (last survey on 27 March 2017). The stress-induced protein encoded by BN69_2205 contains tandem lysine-glycine-glycine (KGG) domains, which belong to the GsiB superfamily. This KGG repeat is found in bacterial proteins which are expressed under conditions of stress (40).

DNA-binding protein from starved cells (Dps) has been studied in various bacteria and archaea. This protein is known to protect bacterial cells against many different types of stressors, including starvation, oxidative stress (41, 42), metal toxicity (43, 44), thermal stress (45), and salt stress (46, 47). Dps has a dual function, with ferroxidase and DNA-binding activities (46). It forms extremely stable complexes with DNA but without specificity to particular sequence motifs (48). In E. coli, dps transcripts were enriched after an incubation period of 60 min with 400 mM NaCl (47). In contrast, in strain SC2 (BN69_2097), the dps transcript level was significantly increased only in the late response (Table 1). This result was validated by RT-qPCR (Table S12). The delayed response may be due to the low metabolic rate of strain SC2 relative to E. coli.

Possibly, the CsbD family protein has the greatest biological significance for the stress response of strain SC2. The expression level of csbD (BN69_2599) was most strongly increased among all the 301 stress-responsive SC2 genes, with a log2 fold change in RPKM value of 6.89 and 7.57 in the early and late responses, respectively (Table 1). CsbD is a general stress response protein, but its exact functional role is not yet known. In addition to involvement in phosphate starvation, heat, acidic, and oxidative stresses, CsbD has been shown to be involved in the salt stress response (49, 50). Depending on the microorganism, its production is under the control of different sigma factors: σB (RpoB) in Bacillus subtilis (49), σS (RpoS) in E. coli (47), and σT (RpoT) in Caulobacter crescentus (51). Given that Methylocystis sp. strain SC2 lacks rpoB, rpoS, and rpoT, it is reasonable to assume that in strain SC2, the production of CsbD is regulated by sigma factor σ32. This view is supported by the fact that the expression levels of both genes, rpoH (σ32) and csbD, are significantly increased in early and late stress responses (Table 1). The differential expression of csbD was validated by RT-qPCR (Table S12). Located directly downstream of csbD, the expression of a hypothetical gene (BN69_2598) was concomitantly upregulated with csbD (Table S3). However, the distribution of BN69_2598 homologs is limited to Methylocystis spp. (Table S13). Two heat shock genes encoding DnaJ domain protein (BN69_2600) and an activator of Hsp90 ATPase 1 family protein (BN69_2601) were located directly upstream of the csbD gene but were not differentially expressed. Thus, a stress response regulon may be located around gene BN69_2599. Interestingly, the SC2 genome also harbors a gene (BN69_1446) that encodes a homolog of the CsbD family protein (56% identity and 83% similarity on nucleotide and amino acid levels, respectively). This gene, however, was not differentially expressed in response to salt stress.

Plasmid-borne genes.Thirty-eight (pBSC2-1) and three (pBSC2-2) salt stress-responsive genes were located on the two plasmids (Fig. 2B). Thus, pBSC2-1 plays a major role in stress response, while pBSC2-2 does not. On pBSC2-1, genes encoding type IV secretion system (virB operon, SC2p1_02210 to SC2p1_02310) and single-strand DNA-binding protein (ssb, SC2p1_01870) were specifically upregulated in the late response (Table 2).

View this table:
TABLE 2

Plasmid-borne genes differentially expressed in response to salt stress

The virB operon consists of 11 genes and encodes a type IV secretion system (T4SS) that can be classified into three subfamilies: conjugation, DNA uptake and release, and effector translocator (52). The structure and function of T4SS were first characterized in Agrobacterium tumefaciens (53) and Campylobacter jejuni (54). virB expression is induced in response to the perception of environmental signals (55–57). Concomitantly with the virB operon and ssb, we observed the increased expression of 15 hypothetical pBSC2-1 genes. Overall, 32 of 38 pBSC2-1 genes were specifically upregulated in the late response, suggesting a functional link between the transcriptional activities of chromosomal and plasmid-borne genes. In B. subtilis, the transfer regions of plasmid pLS20 showed increased expression during early and mid-exponential growth. These regions are mainly characterized by the virB operon (58, 59). Transcriptional profiling by microarray analysis showed that during mid-exponential growth, the expression of more than 5% of the B. subtilis host genes is affected by the presence of the plasmid (59). In addition, the presence of pLS20 has a broad impact on the physiology of its host cells and increases their stress resistance in multiple ways (59). In strain SC2, virB/pBSC2-1 had a response pattern similar to that observed for virB/pLS20 in B. subtilis in response to salt stress. It suggests that in strain SC2, virB functions as a conjugation machinery in the late stress response.

The ssb gene product binds and protects single-stranded DNA (ssDNA) in a sequence-independent manner. ssDNA serves as the template in various cellular functions but is hypersensitive to chemical and nucleolytic attacks that can cause genome damage (60). In both E. coli and B. subtilis, the expression of ssb, regardless of whether chromosome or plasmid borne, was found to be involved in stress response, but in particular in DNA repair (47, 61). Nearly all conjugative plasmids possess ssb genes, but the biological significance of plasmid SSBs is not yet clear (62). To date, there is no evidence that SSBs coat the transferred DNA during its passage to the recipient cell. Possibly, plasmid-carried ssb genes function to prevent the depletion of SSB reserved in conjugating cells, and there may be more stringent requirements for plasmid SSBs under particular stress (63).

Growth recovery from stress-induced inhibition.To elucidate how the stress-induced changes in global transcriptome and amino acid profiles relate to SC2 growth and methane oxidation activities, we examined parameters of growth and methane oxidation activities with high temporal resolution (Fig. 4). SC2 growth was completely inhibited for 23.5 h immediately upon the addition of 0.75% NaCl. Growth was fully recovered after the 23.5-h inhibition period (Fig. 4A). Unlike growth, methane oxidation was inhibited for a shorter period (2.25 h) and gradually recovered within 6 h (Fig. 4B). Apparently, the energy obtained from methane oxidation during the first 23.5 h of salt stress was redirected from growth to stress adaptation. This corresponds well to the tremendous changes observed in the gene expression patterns between the 45-min and 14-h NaCl treatments. While the 45-min transcriptome is linked to the complete inhibition of both growth and methane oxidation, gene expression analysis after 14 h of stress exposure falls well into the recovery and adaptation period of strain SC2. The vast recovery of methane oxidation corresponds well to the fact that only 3 of 36 genes involved in the methane oxidation pathway were downregulated in the early response but none in the late response (see supplemental results and Table S14).

FIG 4
FIG 4

Growth (A and D), methane consumption (B and E), and energy value changes (C) of Methylocystis sp. SC2 shown for control and 0.75% NaCl treatments. Phases 1 to 7 indicate (1) lag phase, (2) early exponential phase prior to NaCl addition, (3) inhibition of growth and methane oxidation activity after NaCl addition to the culture, (4) recovery of methane oxidation, (5) recovery of SC2 growth, (6) early to mid-stationary phase, and (7) mid- to late-stationary phase. Error bars indicate standard deviation calculated from three independent replicates.

Besides the transcriptome research on strain SC2, the only other transcriptome study on a Methylocystis species compared the gene expression of strain SB2 grown on methane or ethanol. On methane, the SB2 genes responsible for converting methane into methanol were expressed at a significantly higher level than those for downstream oxidative transformations. Therefore, the authors concluded that the conversion of methane to methanol may be the rate-limiting step for growth of strain SB2 with methane (64). Overall, we made the same observation for growth of strain SC2 on methane, except for mxa genes responsible for the oxidation of methanol to formaldehyde. The genes mxaF, maxI, and in particular, mxaG, were expressed at a higher level in strain SC2 than in strain SB2. In strain SC2, mxaF, mxaI, and mxaG showed a similar expression level as the pmoCAB genes encoding particulate methane monooxygenase (compare the gene expression levels shown in Table S2 in the study by Vorobev et al. [64] with those indicated in Table S14 of this study).

Energy conservation under salt stress.We calculated the change in energy value that relates to the methane consumed and biomass produced by strain SC2 in different growth phases and compared these values in the control and 0.75% NaCl treatment (Fig. 4C). Prior to the NaCl treatment, there was no significant difference in the energy value change of methane consumption and biomass production between the control and NaCl treatment groups (phases 1 and 2, Fig. 4C). Nearly no change in the energy value of both methane and biomass occurred in the first 2 h after the addition of 0.75% NaCl (phase 3, Fig. 4C). This 2.25-h period is characterized by a shock response that involves a complete inhibition of both methane consumption and growth activity. However, due to the full recovery of methane consumption within 6 h, the energy value in the NaCl treatment group tremendously changed between 2.25 and 23.5 h, while growth and biomass accumulation remained completely inhibited (phase 4, Fig. 4C). A total of 17 genes related to energy production and conversion were differentially expressed in the early and/or late response(s) (Fig. S2 and Table S15). Among them, five genes (nuoEFGHI) belong to a 14-gene cluster (nuoA-N) that encodes NADH-quinone oxidoreductase. These five genes were coordinately downregulated in the early response, but their expression was nearly fully recovered in the late response. Thus, the energy-yielding reactions recovered concomitantly with methane consumption from salt stress shock. The period between 23.5 and 79 h was characterized by resumed growth and thus a significant change in the energy value of SC2 biomass in the NaCl treatment (phase 5, Fig. 4C). Defined by decreasing methane consumption and nearly no biomass production, phases 6 and 7 represent the early and late-stationary phases in both the control and NaCl treatment.

Final remarks.All known Methylocystis spp. have a low range of salt tolerance (20, 21). This prompted us to investigate whether Methylocystis spp. are able to respond to changes in salinity and to adapt to salt stress. Evidently, Methylocystis sp. strain SC2 is able to cope with and acclimatize to 0.75% NaCl, with full recovery of its growth activity after 23.5 h of stress exposure. Methane oxidation activity completely recovered after only 6 h. Correspondingly, stress exposure had no effect on the expression level of the nearly complete set of 36 methane oxidation pathway genes. Overall, transcriptional adaptation of strain SC2 involved the increased expression of genes whose products are well known to control the stress response (e.g., sigma factor σ32) or to confer stress tolerance (e.g., glutamate [osmoprotectant], stress-induced protein, DNA-binding protein from starved cells, and CsbD family protein). These stress-responsive genes are widely, but not consistently, distributed among Methylocystis spp., suggesting that the transcriptional response to salt stress may vary between members of this genus (Table S13).

On the one hand, the SC2 genome contains various genes that we had anticipated to be stress responsive but did not show any differential expression. In other bacteria, the products of these genes had been shown either to be regulators of the transcriptional stress response (e.g., PhyR) or to confer stress tolerance (e.g., the glycine cleavage system [GCV], disulfide bond formation [Dsb] system, and the potassium-transporting system Kdp) (see supplemental results for further details). On the other hand, 44 stress-responsive genes with unknown function were uniquely present in either strain SC2 or Methylocystis spp. (see “hypothetical proteins” in supplemental results and Table S16). Of these, the expression of 12 genes was significantly increased in both the early and late responses (Tables 1 and S16). The presence of these taxon-specific genes with unknown function may suggest that in Methylocystis spp., unique mechanisms have evolved that enhance survival under environmental stress.

MATERIALS AND METHODS

Growth experiment, methane measurement, and NaCl treatment.Methylocystis sp. strain SC2 was inoculated into 120-ml serum bottles containing 40 ml of mineral salts medium with an ionic strength of 25 mM. The medium composition was the same as reported earlier (4), with one modification: KNO3 was used as a nitrogen source instead of NH4Cl.

Inoculated bottles were filled with filter-sterilized 20% (vol/vol) methane in the headspace and incubated on a rotary shaker at 150 rpm and 25°C. Using spectrophotometry (Bio Photometer RS232C; Eppendorf AG, Hamburg, Germany), the growth of strain SC2 was monitored by measuring the optical density at 600 nm (OD600). Methane consumption was analyzed by gas chromatography (SRI Instruments, Earl St. Torrance, CA). Cultures of strain SC2 were grown to the mid-exponential phase, corresponding to an OD600 of 0.12 (third day of incubation), and then treated with different NaCl concentrations of 0, 0.25, 0.5, 0.75, 1.0, and 1.5%. A concentration of 0.75% NaCl was chosen for stress response analysis. Thus, the ionic strength increased to 153 mM after adding 0.75% NaCl. Upon NaCl addition, samples for whole-genome transcriptomics were taken from triplicate cultures after a treatment period of 0 min (control), 45 min (45 min), and 14 h (14 h). The control and NaCl treatments were analyzed by Illumina RNA-Seq.

RNA extraction and mRNA enrichment.SC2 cells were collected into 50-ml conical tubes by centrifuging at 8,000 rpm for 5 min at 4°C. Upon cell harvest, total RNA was extracted using the RiboPure-Bacteria kit (Ambion, Austin, TX, USA), according to the manufacturer's instructions. Purified RNA was treated with RNase-free DNase I (Ambion). mRNA was enriched by subtractive hybridization of rRNA, using the Ribo-Zero magnetic kit for bacteria (Epicentre, Madison, WI, USA). RNA was quantified using the Qubit system (Life Technologies, Carlsbad, CA, USA). RNA quality was assessed by automated capillary electrophoresis using the Experion HighSens microfluidic chip for prokaryotic RNA on an Experion system (Bio-Rad, Hercules, CA, USA).

Library construction and RNA-Seq.Nine cDNA libraries were constructed, with three libraries each from control, 45-min NaCl treatment, and 14-h NaCl treatment group. cDNA libraries were prepared with NEBNext Ultra directional RNA library prep kit for Illumina (New England BioLabs, Ipswich, MA), according to the manufacturer's instructions. Library quality was assessed by an Experion HighSens microfluidic chip for prokaryotic DNA. Illumina single-read (100-bp) RNA-Seq was performed on a HiSeq 2500 instrument at the Max Planck Genome Centre Cologne (Cologne, Germany).

Data analysis.The number of single-end 100-bp reads obtained for each cDNA library varied between 29 and 69 million. Nearly all the reads (>99%) were classified as “good reads,” using the standalone lite version of PRINSEQ and standard filtration criteria: (i) read length, (ii) ambiguous bases, and (iii) sequence complexity (65). Reads with a quality score of >20 were kept for further analysis. Of these, more than 99% were non-rRNA reads (Table S17). Residual rRNA sequences were removed by alignments with the latest releases of the 5S, 16S, and 23S bacterial rRNA databases from SILVA (66), using the standalone version of SortMeRNA (67). The resulting RNA data sets were mapped to the concatenated sequences of the SC2 chromosome and its two plasmids, pBSC2-1 and pBSC2-2. Expression levels were calculated using the CLC Genomics Workbench (CLC bio, Aarhus, Denmark). The mapping efficiency of the non-rRNA reads was between 87 and 90% for each of the nine cDNA libraries (Table S17). Gene expression levels were normalized and reported as RPKM values. RPKM refers to reads per kilobase of CDS (or exon) model per million mapped reads (68). The RPKM mean values of each gene were used for calculating log2 fold changes in RPKM values between control and salt treatments, thereby revealing the differential expression response of each SC2 gene. Log2 fold changes in RPKM values of less than or equal to −2 or greater than or equal to 2 were considered significant (68, 69). The complete linkage method (70) with Euclidean distance measure was used to calculate similarities between global transcriptome profiles.

Real-time quantitative PCR.Ten chromosomal and plasmid-borne SC2 genes were selected for validation of RNA-Seq results. The primers used for RT-qPCR are listed in Table S18. For each sample, 1 μg of total RNA was reverse transcribed in a 2-μl reaction mixture using the SuperScript reverse transcriptase (RT) reagent kit (Invitrogen, Carlsbad, CA, USA). cDNA dilution series were used to quantify mRNA transcript copies. Amplicons (positive-control DNA) of each target gene were cloned into the pGEM-T Easy vector (Promega, Madison, WI). The clones were serially diluted and used to construct calibration curves for real-time PCR (71). The calibration curves ranged from 10 to 108 copies. RT-qPCR was performed using the SYBR green Taq mix (Sigma-Aldrich) on a Bio-Rad CFX96 real-time PCR system. The PCR efficiency was at least 90% (R2 > 0.99). The presence of unspecific products was excluded by melt curve analysis.

Amino acid profiling.Metabolites were measured as described previously (72). Briefly, an Agilent 1290 Infinity II ultrahigh-performance liquid chromatography (UHPLC) system (Agilent Technologies, Waldbronn, Germany) was used for liquid chromatography. The column was an Acquity BEH Amide 30 by 2.1 mm column with 1.7-μm particle size (Waters GmbH, Eschborn, Germany). The temperature of the column oven was 30°C, and the injection volume was 3 μl. LC solvents A and B were as follows: solvent A was water with 10 mM ammonium formate and 0.1% (vol/vol) formic acid, and solvent B was acetonitrile with 0.1% (vol/vol) formic acid. The gradient was 0 min at 90% B, 1.3 min at 40% B, 1.5 min at 40% B, 1.7 min at 90% B, and 2 min at 90% B. The flow rate was 0.4 ml · min−1. An Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies) was used for mass spectrometry. The source gas temperature was set to 200°C, with 14 liters · min−1 drying gas and a nebulizer pressure of 24 lb/in2. The sheath gas temperature was set to 300°C and the flow to 11 liters · min−1. The electrospray nozzle and capillary voltages were set to 500 and 2,500 V, respectively. A 13C-labeled metabolite extract from E. coli was used as an internal standard to obtain absolute quantitative data.

Calculation of energy value change.The growth of strain SC2 and methane consumption in response to salt stress were examined as described above, relative to untreated controls. Both parameters were monitored 41 and 65 h before the addition of NaCl, as well as 2.25, 23.5, 79, 127, and 175 h after the addition of 0.75% NaCl. These time points relate to particular phases in the adaptive response of strain SC2 to salt stress. The energy value changes of cell biomass accumulation and methane consumption for each time point were calculated from the respective mass changes. The grand mean calorific value of dried microorganism (22,538 J · g−1) (73) and calorific value of methane (39,820 J · g−1) (74) were used as the standard values for calculation.

Accession number(s).The raw sequence data have been submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under BioProject number PRJNA369136 and SRA accession number SRP098298.

ACKNOWLEDGMENTS

This study was supported by the Deutsche Forschungsgemeinschaft (DFG) through Collaborative Research Center SFB 987.

FOOTNOTES

    • Received 16 April 2017.
    • Accepted 9 August 2017.
    • Accepted manuscript posted online 11 August 2017.

  • Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00866-17.

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KEYWORDS

methanotrophs
Methylocystis
salt stress
osmotic stress
methane oxidation
transcriptome
differential expression
amino acid profiling
compatible solutes
glutamate

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