Increased NaCl and alkali tolerance of mutant ΔprkA
There is only one annotated PrkA family serine protein kinase in the genome of M. alhagi, denoted PrkA [18]. We have previously found that the expression of prkA was downregulated in high-salt conditions through RNA-Seq validated by RT-qPCR [6]. To determine the function of M. alhagi CCNWXJ12-2 prkA, we constructed a deletion mutant and its tolerance to NaCl and alkali were tested.
The mutant ΔprkA had better salt and alkali resistance than the wild-type strain, while the complemented strain CΔprkA had similar salt and alkali resistance to the wild-type strain (Fig. 1). The expression of prkA in the complementation strain was confirmed using reverse transcription PCR (data not shown).
In Mycobacterium tuberculosis, the pknE deletion mutant showed higher tolerance to acidic stress, SDS, and kanamycin than the wild-type, which means pknE is involved in stress adaptation [13]. Although the protein sequence similarity of Mesorhizobium alhagi PrkA and Mycobacterium tuberculosis PknE is only 12.9 %, the function of pknE in M. tuberculosis was to some degree similar to that of prkA in M. alhagi in stress adaption. However, our results showed that wild-type M. alhagi had the same resistance to antibiotics (kanamycin, gentamicin, ampicillin, tetracycline, streptomycin, and rifampicin; 50 ug/ml for all), SDS (0.01 %), and acid (pH 6) as did the mutant ΔprkA (data not shown).
In E. coli, the deletion mutant of yeaG showed no difference in salt tolerance compared with the wild-type [12], although the similarity in protein sequence between E. coli YeaG and M. alhagi PrkA is relatively high (65.5 %). Despite the similar genomic background of prkA in R. etli and Mesorhizobium alhagi (data not shown) and the high similarity in protein sequences (71.49 %), the prkA deletion mutant of R. etli showed no phenotype changes compared with the wild-type strain [7], in contrast to M. alhagi in the present work.
The functions of these genes in the different bacteria are obviously different. These results show that serine protein kinases can have very different functions in different bacteria.
Measurement of total cellular Na+ content
Bacteria can efflux the extra Na+ from the cells by Na+/H+ antiporters using energy of proton motive force [19]. We have previously found that the expression of a Na+/H+ antiporter gene, nhaA, was upregulated in high-salt conditions [6]. Here we measured the total cellular Na+ content of the wild-type, ΔprkA, and CΔprkA strains in salt-free and high-salt (0.4 M NaCl) conditions to find out whether PrkA influences the total cellular Na+ content. However, our results showed no significant difference among these three strains (p ≥ 0.05) when grown in the same conditions (Fig. 2). The Na+ content of the three strains grown on 0.4 M NaCl TY agar plates was almost 20-fold higher than that in the controls grown on salt-free medium. The similar Na+ content of the three strains implied that prkA does not depress the strain growth in high-salt conditions by adjusting the cellular Na+ content. Therefore, we speculate that the mechanism of PrkA depressing the growth of Mesorhizobium alhagi under salt stress is complex, which involves many components of metabolism.
Increased antioxidative capacity of mutant ΔprkA
Stressful conditions such as heat, acid and high-salt concentrations, can lead to secondary oxidative stress in bacteria [20]. Previous study has shown the intracellular level of reactive oxygen species (ROS) was increased significantly when cells were stressed by high salinity [21, 22]. Because high salinity can trigger a high level of intracellular ROS, we tested the oxidative resistance of the M. alhagi prkA deletion mutant to identify any antioxidant function of PrkA. The survival rates of the wild-type, ΔprkA and CΔprkA strains treated with 10 mM H2O2 for 30 min showed a significant difference (Fig. 3); survival of ΔprkA treated with H2O2 was significantly (p ≤ 0.05) higher than that of the wild-type and complemented strains. These results suggest that PrkA depresses the antioxidative capacity of M. alhagi.
Although H2O2 can be damaging for rhizobium, it appears that H2O2 is required for successful infection. The overexpression of the housekeeping catalase in Sinorhizobium meliloti RmkatB++ results in a delay of symbiosis formation and has negative effects on the development of infection threads [23].
Unfortunately, the symbiosis formation of M. alhagi and Alhagi sparsifolia is very unstable. Great effort has been made to conduct the plant experiments, but the results are always unreliable and unauthentic (data not shown). Therefore, we can only hypothesize that PrkA has positive effects on symbiosis formation.
Antioxidant enzyme activity determination
Catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) are major antioxidases in bacteria, which can eliminate the intracellular ROS [24]. Therefore, we measured the CAT, SOD, and POD activities of M. alhagi in different conditions. Figure 4 shows the enzyme activity in the three strains (without treatment, H2O2 treated, or 0.4 M NaCl treated). The CAT and SOD activities could be detected in all strains in each condition, while the POD activity was not detectable. We checked the RNA-Seq data and found that the genes coding CATs and SODs are highly expressed in M. alhagi, while PODs are only expressed at a low level [6]; thus POD activity in cells may have been below the detection limit of the kit used in our experiments.
In control cells (normal conditions), the CAT activities of the three strains were almost the same, while the SOD activity of ΔprkA was significantly higher (p ≤ 0.05) than that in the other two strains. Salt stress and H2O2 treatment triggered increased CAT and SOD activities in all three strains (Fig. 4). The CAT and SOD activities under high-salt stress were markedly higher than those in the H2O2 treatment group, which could also suggest that high-salt stresses trigger oxidative stress. The lower CAT and SOD activities in the H2O2 treatment group were possibly caused by the low H2O2 concentration or the short treatment time.
The CAT and SOD activities of ΔprkA under salt stress and H2O2 treatment were extremely significantly higher (p ≤ 0.001) than those of the wild-type and CΔprkA (Fig. 4). The increase in CAT activity units of ΔprkA compared to the other two strains was much smaller than that in SOD activity units. Therefore, we hypothesize that PrkA influences the antioxidative capacity of M. alhagi mainly by affecting SOD activity. However, in favorable conditions (e.g., as in the control group), the high expression of SOD genes in ΔprkA could waste energy. PrkA may play a role in control of SOD gene expression. The adjustment of SOD gene expression may need the high expression of prkA in salt-free conditions.