海水中主要金属盐对4株Kangiella属细菌的生长及其蛋白酶相关基因表达的影响

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

摘要:

【背景】Kangiella是海洋专属的、营异养生长的革兰氏阴性菌。按16S rRNA基因系统发育分析，Kangiella属归属于γ-变形杆菌纲，但此类细菌的生理生态功能未知。推测该类海洋细菌产生的胞外丝氨酸蛋白酶在其利用有机氮源生长的过程中起重要作用。【目的】研究海水盐中主要金属离子Na盐、Mg盐、Ca盐、K盐对海洋来源的4株Kangiella菌株(K. aquimarina DSM 16071、K. geojedonensis YCS-5、K. koreensis DSM 16069和K. profundi FT102)的生长及胞外蛋白酶表达的影响。【方法】测定Kangiella属4个不同种的模式菌株在2216E培养基及不添加Na盐、Mg盐、Ca盐和K盐的2216E培养基中的生长情况。以2%的酪蛋白为底物，采用福林酚法分别检测4株菌株的胞外蛋白酶酶活。克隆K. profundi FT102菌株中的3个丝氨酸蛋白酶编码基因以及phoP和phoQ基因，使用实时荧光定量PCR技术检测Mg盐对这些基因表达的影响。【结果】4株Kangiella菌株在不添加Na盐的2216E培养基中均不能生长；在不添加Mg盐、Ca盐和K盐的2216E培养基中，K. aquimarina DSM 16071、K. geojedonensis YCS-5和K. profundi FT102这3个菌株的生长差异较小；而K. koreensis DSM 16069菌株的生物量在不添加上述3类主要金属盐的2216E培养基中反而增高。各个菌株在对数期时的胞外蛋白酶活差异较大，最高可以达到11.23?U/mL，最低仅为0.99 U/mL。2216E培养基中不添加Mg盐时，K. aquimarina DSM 16071、K. geojedonensis YCS-5和K. profundi FT102这3株菌中的丝氨酸蛋白酶编码基因asp1、asp2和asp3以及phoP和phoQ基因的转录水平无显著差异，而在K. koreensis DSM 16069菌株中这5个基因的表达却显著上调。【结论】Na盐是4株海洋Kangiella菌株生长所必需的金属离子，Mg盐和Ca盐对4株菌株的生长及其中3个保守的丝氨酸蛋白酶基因表达的影响不同。在不添加Mg盐的2216E培养基中，K. koreensis DSM 16069菌株的生物量、胞外蛋白酶活和3个保守的丝氨酸蛋白酶编码基因以及phoP和phoQ基因的转录量都显著上升。

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.