Effect of major metal ions in sea water on growth and transcription of protease coding gene of 4 Kangiella strains

Abstract:

[Background] Kangiella is gram-negative, obligate marine heterotrophic bacteria. The Kangiella genus forms deep branches within the class Proteobacteria in the phylogenetic analyses based on 16S rRNA gene sequences. The physiology and ecological role of this group of marine bacteria are still unknown. We assume that the extracellular serine proteases play an important role in the growth of Kangiella for recycling of organic nitrogen. [Objective] Analyzed physiological effects of different metal salts (Na salt, Mg salt, Ca salt, K salt) on the growth and the expression of alkaline serine protease genes of 4 Kangiella strains (K. aquimarina DSM 16071, K. geojedonensis YCS-5, K. koreensis DSM 16069 and K. profundi FT102). [Methods] The growth of 4 Kangiella strains in different versions of marine broth 2216E were observed. Using Folin method to test the extracellular protease activities with 2% casein as substrate. Three conserved alkaline serine protease coding sequences (asp1, asp2 and asp3) and phoP, phoQ genes in K. profundi FT102 were amplified by PCR and cloned. Real-time PCR method was used to evaluate the expression level of these genes in the 4?strains. [Results] Based on the growth curve of 4 Kangiella strains in different versions of 2216E, we found that Kangiella strains can’t grow in the absence of NaCl. However, except K. koreensis DSM 16069, the growth curves of other 3 Kangiella strains in different version of marine broth 2216E, in which no supplementation of either magnesium salt, or calcium salt, or potassium salt, were found similar. The extracellular protease activities of 4 strains were found different in logarithmic period, the maximum extracellular protease activity was 11.23 U/mL and lowest was 0.99 U/mL. Moreover, the results of Real time-PCR showed that the variety of the transcription level of 3 conserved genes (asp1, asp2, asp3) in K. aquimarina DSM 16071, K. geojedonensis YCS-5 and K. koreensis DSM 16069 was not obvious. But for K. koreensis DSM 16069, the transcriptional level of these 3 conserved alkaline serine protease coding genes was significantly upregulated, when either magnesium salt or calcium salt was omitted in the marine broth 2216E. [Conclusion] Na salt is necessary for the growth of Kangiella strains, Mg salt and Ca salt have different effects on the transcription of 3 serine protease coding genes asp1, asp2, asp3. The biomass, extracellular protease activity and the transcription level of 3 conserved serine protease genes and phoP, phoQ were significantly elevated in K. koreensis DSM 16069 when Mg salt was not supplemented in the marine broth 2216E.