yhcZ基因在苏云金芽胞杆菌生长中的功能研究

The functions of yhcZ gene during Bacillus thuringiensis growth

在枯草芽胞杆菌和蜡样芽胞杆菌中,yhcZ基因和yhc Y基因组成双组分系统调控细菌生长,但yhcZ基因在苏云金芽胞杆菌中发挥的生物学功能尚未明确。本研究通过基因功能注释、上下游基因排列分析和氨基酸序列比对,证实苏云金芽胞杆菌库斯塔克亚种HD73中HD73_5824基因为yhcZ基因,推测其与HD73_5825基因(yhc Y基因)共同组成双组份系统调控细菌生长。利用同源重组技术敲除HD73菌株中的yhcZ基因获得缺失突变体HD(ΔyhcZ),其在LB和SSM培养基中生长均慢于野生型HD73,而互补菌株HD(ΔyhcZ::yhcZ)菌株则能够部分恢复生长,表明yhcZ基因的缺失影响了该菌株细胞的生长。在以0.4%葡萄糖为唯一碳源的M9培养基中,HD(ΔyhcZ)生长速度快于HD73,表明yhcZ基因在该菌株吸收利用葡萄糖的过程中发挥重要作用。Biolog实验显示HD(ΔyhcZ)的单孔颜色变化率低于HD73,且对D/L-丝氨酸、甲酸、D-葡糖酸、L-组胺,D-乳酸甲酯以及柠檬酸等的吸收利用能力低于HD73,表明yhcZ基因能显著影响HD73菌株对碳源的利用。同时,HD(ΔyhcZ)对8%Na Cl的耐受能力弱于HD73,表明该基因可能参与细菌细胞应力响应相关基因的表达与调控。以上结果表明yhcZ基因在HD73菌株生长过程中对葡萄糖及其他碳源的利用具有重要的促进作用。本研究结果为解析yhcZ基因调控葡萄糖及碳源利用的分子机制奠定基础,且为进一步研究细菌生长及发酵提供参考。

yhcZ and yhcY genes constitute a two-component system in Bacillus subtilis and B. cereus that regulates bacterial growth. However, the exact biological function of yhcZ gene in B. thuringiensis has not been fully elucidated. In this study, we proved that HD73_5824 is an yhcZ gene in B. thuringiensis subsp. kurstaki HD73 strain by combining gene functional annotation, analysis of upstream and downstream genes arrangement, and amino acid sequence alignment. This yhcZ gene may co-regulate bacterial growth with HD73_5825 gene（yhcY gene） by constituting a two-component system. Homologous recombination technology was employed to knock out yhcZ gene of HD73, resulting in a mutant strain HD（ΔyhcZ）. The HD（ΔyhcZ） strain grew slower than wild-type strain HD73 in both LB and SSM medium. Re-introduction of yhcZ gene in HD（ΔyhcZ） strain can partially restore the growth, indicating that the deletion of yhcZ gene impacts the cell growth of HD73 strain. HD（ΔyhcZ） strain grew faster than HD73 strain in M9 medium with 0.4% glucose as the sole carbon source, implying that the yhcZ gene plays an important role in glucose utilization by HD73 strain. The results of biolog assay showed that HD（ΔyhcZ） exhibits a lower average well color development compared to HD73. HD（ΔyhcZ） cells also demonstrated a decreased capacity for absorbing and utilizing D/L-serine, formic acid, D-gluconic acid, L-histamine, D-methyl lactate, and citric acid, indicating that yhcZ gene could dramatically influence carbon source utilization of HD73 strains. Additionally, HD（ΔyhcZ） was less resistant to 8% Na Cl, suggesting that yhcZ gene may be involved in the expression and regulation of genes related to high-salt stress response in bacterial cells. The results above show that the yhcZ gene significantly promotes glucose and other carbon sources utilization of HD73 strain during growth. Our findings will lay a foundation not only for analyzing the regulatory mechanisms of glucose and carbon sources utilization by yhcZ gene, but also providing a reference for the further research on bacterial growth and fermentation.