Diversity of the ectoines biosynthesis genes in the salt tolerant Streptomyces and evidence for inductive effect of ectoines on their accumulation

Abstract

Streptomyces commonly produce ectoines as compatible solutes to prevent osmotic stresses. Fine structure of the genes producing ectoine (ectC) and hydroxyectoine (ectD) enzymes in Streptomyces rimosus C-2012 as a slightly halophilic bacterium is reported in this study. Deduced amino acid sequences of ectC and ectD genes from strain C-2012 and some other related species were compared and 72-90% and 13-81% identities were detected for ectC and ectD, respectively. High similarity of ectC between closely or distantly related Streptomyces to the strain C-2012 may indicate horizontal transfer of this gene. However, phylogenetic relationships of ectD were correlated with phylogenetic affiliation of the strains. It suggests that the ability of Streptomyces to produce hydroxyectoine has been the result of a vertical transfer event. HPLC analysis showed that strain C-2012 was able to produce ectoine and hydroxyectoine both in the presence and absence of external salinity (up to 0.45 M NaCl). Accordingly, reverse transcription quantitative PCR (RT-qPCR) showed that ectABCD operon in this strain is positively affected by salt. Also, inductive effect of the salt was increased when it was applied with 1 mM of ectoines. Transcription level of ectC was increased 2.7- and 2.9-fold in the medium supplied with salt and ectoine and salt and hydroxyectoine, respectively. The effect of salinity with or without ectoines was more on ectD transcription level than that of ectC. In S. rimosus under salt stress, ectoine and hydroxyectoine biosynthesis primarily depends on the stimulation of ectABCD operon transcription. However, drastic accumulation of ectoine and hydroxyectoine without increase in ectC and ectD transcripts was observed in the medium supplied with salt and ectoines and that suggest there might be additional posttranscriptional level of control. Increases in ratio of some intracellular free amino acids in salt stressed to unstressed conditions were observed in cells grown with ectoines. Our results suggest the possibility of a supplementary role of ectoines to improve structure and function of the cells in stressful environments as well as their important role as osmoprotectants.

Biogeography and Adaptive evolution of Streptomyces Strains from saline environments

“…Mg 2+ was a chaotropic agent and a limiting factor in the diversity of microbes in the saline environment27. In addition, salinity of sediments was important regulators of some extremozymes in Streptomyces genus, for example, xylanases, alpha amylases, thermoalklotolerant β -1,3-glucanases and cellulases17, which is also consistent with our study in that Streptomyces strains from Xinjiang salt lakes have higher abilities of regulating expression of ectA - D genes (especially, ectC  > 20 fold changes; ectD  > 30 fold changes, Fig. 4) and biosynthetic potential of larger quantities of (>10 fold changes, Fig.…”

“…salinity) (Table 3 and Fig. S3; p value < = 0.05), which leads to the catalytic regulation function of their biosynthesis of hydroxyectoine17.…”

“…Genomic DNA of the obtained strains was extracted and 16S rRNA genes were PCR amplified16. PCR amplification of rpoB , recA , atpD and ectA - D genes was performed according to the methods described previously1617. The amplified PCR products were purified using a TaKaRa DNA fragment purification kit (Ver.…”

“…The amplified PCR products were purified using a TaKaRa DNA fragment purification kit (Ver. 2.0) and were sequenced using an ABI 3100 automated sequencer with primers of four genes (16S rRNA, 27f and 1525r; rpoB , rpoBPF and rpoBPR ; recA , recAF and recAR ; atpD , atpDF and atpDR ; ectA , ectAF and ectAR ; ectB , ectBF and ectBR ; ectC , ectCF and ectCR ; ectD , ectDF and ectDR ) at Shanghai Sangon Biotech (Shanghai, China)1617. The 16S rRNA gene sequences obtained from the candidate strains were compared with reference taxa via the EzTaxon-e database.…”

“…Previous studies have shown that 16S rRNA gene together with rpoB , recA and atpD genes could provide better phylogenetic resolution than one single gene16. Furthermore, the expressional variation of salty-tolerence related genes ( ectA-ectD ) could show adaptive evolution of Streptomyces strains in saline environments17. Here, we applied a multilocus phylogenetic analysis of four house-keeping genes (16S rRNA, rpoB , recA and atpD ) and salty-tolerence related genes ( ectA-ectD ) to investigate the biogeographic patterns of Streptomyces strains retrieved from the saline environments in Xinjiang and Yunnan Provinces of western China, and assessed relative effects of spatial distance and environmental factors on biogeography and salty-tolerence mechanism of Streptomyces strains from different salty-habitats (salt mines vs lakes) by Q-PCR analyses.…”