Biological Functions of Transcription Factor OmpR in Serratia marcescens FZSF02
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摘要:
目的 探索EnvZ/OmpR双组分调控系统的效应蛋白OmpR对粘质沙雷氏菌FZSF02灵菌红素合成及其他生物学特性的影响。 方法 同源重组法构建ompR敲除菌株，琼脂平板产色试验和qPCR检测OmpR对菌株灵菌红素合成的影响，结晶紫染色法和琼脂平板法等研究基因敲除菌株生物被膜形成能力，运动性和对不同环境胁迫因素的耐受性。 结果 序列分析显示OmpR为序列高度保守的蛋白。PCR验证证明ompR基因敲除成功；与野生型菌株相比∆ompR失去灵菌红素合成能力，qPCR试验显示灵菌红素合成基因簇中3个关键基因pigA、pigF和pigN转录水平分别降低为野生型菌株的3.8%，2.0%和2.1%；∆ompR菌株生物被膜生成能力较野生型降低37.5%（37 ℃）和15.1%（27 ℃）；OmpR对该菌的生长能力、运动能力和响应环境胁迫的能力无明显影响。 结论 ompR为一新报道的特异性调控粘质沙雷氏菌灵菌红素合成的基因，且该基因对该菌生物被膜的形成有重要影响。 Abstract:Objective Biological functions of the regulatory protein ompR in the two component EnvZ/OmpR system, including prodigiosin-producing ability and other biological characteristics, of Serratia marcescens FZSF02 were studied. Methods Homologous recombination was used to construct ompR-knockout S. marcescens FZSF02. Effect of OmpR on the prodigiosin-producing ability was examined by LB agar plate incubation and qPCR. Methods of crystal violet staining, agar plate incubation, and others were applied to determine the biofilm-forming, mobility, and stress adaptation abilities of the transcription factor protein under various environmental stresses. Results OmpR was a protein with high conserved amino acid sequences. An ompR- deleted strain, FZSF02 ∆ompR, was successfully obtained by homologous recombination and confirmed by PCR. As a result, FZSF02 ∆ompR lost prodigiosin-producing ability that possessed by the wild strain. The transcriptional levels of pigA, pigF, and pigN of the prodigiosin biosynthesis gene cluster in FZSF02 ∆ompR were respectively 3.8%, 2.0% and 2.1% of the wild type strain. The biofilm formation of FZSF02 ∆ompR declined 37.5% (at 37 ℃) and 15.1% (at 27 ℃) from its wild counterpart. On the other hand, OmpR exhibited no significant effect on the growth, mobility, or response to the environmental stress. Conclusion OmpR was a newly reported gene that specifically regulated the prodigiosin biosynthesis in S. marcescens. It also significantly affected the biofilm formation but not on the growth, mobility, or stress response. -
Key words:
- Serratia marcescens /
- OmpR /
- prodigiosin /
- biological characteristics
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图 1 粘质沙雷氏菌FZSF02菌株OmpR基于氨基酸序列的系统发育分析
注：括号中是对应物种蛋白在GenBank中的序列编号
Figure 1. Phylogenetic analysis on OmpR from S. marcescens FZSF02 with its homologous proteins
Note：Numbers in brackets are GenBank sequence accession for respective proteins.
图 2 粘质沙雷氏菌FZSF02菌株ompR基因敲除验证
注：应用引物OmpRFF和OmpRBR进行PCR。泳道M：DNA分子量标准；泳道1：FZSF02野生型；泳道2：ompR基因Tn5转座子插入突变体FZSF02 ompR:: Tn5；泳道3：ompR基因被敲除120 bp的菌株FZSF02ΔompR。
Figure 2. Agarose gel electrophoresis identification of ompR-knockout S. marcescens FZSF02
Note：PCR were carried out with primers OmpRFF and OmpRBR. Lane M: DNA marker; Lane 1: wild type FZSF02; Lane 2: FZSF02 ompR:: Tn5 with ompR inserted by Tn5 transposon; Lane 3: FZSF02ΔompR with 120 bp ompR-knockout.
图 3 FZSF02野生菌株与ompR敲除菌株FZSF02ΔompR的生长曲线
注：A：野生型菌株WT和基因敲除菌株FZSF02ΔompR在27 ℃条件的生长曲线。B：野生型菌株WT和基因敲除菌株FZSF02ΔompR在37 ℃条件的生长曲线。
Figure 3. Growth curves of WT and ΔompR of FZSF02
Note：A: Growth curves of WT and FZSF02ΔompR at 27 ℃. B: Growth curves of WT and FZSF02ΔompR at 37 ℃.
图 4 OmpR对Serratia marcescens FZSF02产灵菌红素能力及灵菌红素合成基因表达的影响
注：A：野生型菌株FZSF02, ompR 转座子突变菌株和ompR敲除菌株在LB琼脂平板上的生长情况。FZSF02, FZSF02 ompR:: Tn5 and FZSF02ΔompR分别代表野生型菌株，Tn5插入突变菌株和ompR敲除菌株。B：ompR基因敲除对零菌红素合成基因的影响。pigA, pigF和pigN为零菌红素合成基因簇上的3个基因。Log2倍数变化值代表上述三个基因在基因敲除菌株FZSF02ΔompR中相对野生型菌株的表达量变化。
Figure 4. Effects of OmpR on prodigiosin-producing ability and expressions of prodigiosin biosynthesis genes of S. marcescens FZSF02
Note：A: Prodigiosin-producing abilities of FZSF02, ompR mutant strain, and ompR-knockout strain on LB agar at 27 ℃. FZSF02, FZSF02 ompR:: Tn5, and FZSF02ΔompR were WT strain, Tn5 transposon insertion strain, and ompR-knockout strain, respectively. B: Deletion of ompR on expression of prodigiosin biosynthesis genes. pigA, pigF, and pigN were 3 genes in prodigiosin biosynthesis gene cluster. Log 2-fold change values represent expression levels of these genes in FZSF02ΔompR as compared to WT FZSF02.
图 5 OmpR对Serratia marcescens FZSF02生物膜合成能力的影响
注：27 ℃和37 ℃液体培养条件下野生型菌株WT和基因敲除菌株ΔompR的产生物被膜能力检测。
Figure 5. Effect of OmpR on biofilm-producing ability of S. marcescens FZSF02
Note：Biofilm-producing ability of WT andΔompR were assayed after incubation in liquid LB at 27 ℃ and 37 ℃ for 36 h.
图 6 Serratia marcescens FZSF02和FZSF02ΔompR的运动能力
注：在0.3%（w/v）和0.7%（w/v）琼脂的LB固体平板上检测Serratia marcescens FZSF02和基因敲除菌株FZSF02ΔompR运动的菌落直径。
Figure 6. Mobility of S. marcescens FZSF02 and FZSF02ΔompR
Note：Mobility diameters of S. marcescens FZSF02 and FZSF02ΔompR were assayed on solid LB plates containing 0.3% (w/v) and 0.7% (w/v) agar, respectively.
图 7 Serratia marcescens FZSF02和FZSF02ΔompR的产酶能力
注：WT和ΔompR分别代表Serratia marcescens FZSF02野生型菌株和ompR敲除菌株。A：在含1% (w/v)脱脂奶粉的LB固体琼脂培养基上的蛋白酶产生能力。B：在含1%(v/v) tween 80的固体琼脂培养基上的脂肪酶产生能力。C和D：在血琼脂培养基上27 ℃条件下培养24小时和7天观察溶血素产生能力。
Figure 7. Enzyme-producing abilities of S. marcescens FZSF02 and FZSF02ΔompR
Note：WT andΔompR represent WT and ompR-knockout S. marcescens FZSF02, respectively. A: Protease-producing ability on LB agar plates containing 1% (w/v) of skim milk. B: Lipase-producing ability on LB agar plates containing 1% (v/v) of tween 80. C and D: Hemolysin-producing abilities on blood agar base medium after incubation at 27 ℃ for 24 h (C) and 7 days (D), respectively.
表 1 供试引物
Table 1. Primers used in this study
引物 Primer 序列 Sequence（5′-3′） OmpRFF ATGCAAGAGAATCATAAGATCCTG OmpRFR CATCGATGATGGTTGAGAGTCGGCGCCGATTTCCAGCCCCA OmpRBF CTCGATGAGTTTTTCTAAGGCAAATTCAAACTGAACCTCGGC OmpRBR TCATGCCTTGCTGCCGTCCGGTAC KanF TCTCAACCATCATCGATGAATTGT KanR TTAGAAAAACTCATCGAGCATCAA pigAF CGCCATCTTCCACGATTCAA pigAR CATTAGCCGACACTGTTCCA pigFF CACGGTATTCGGCGATGAC pigFR CACGGTGTTGCGAGAAGT pigNF CGGTTACCCTGGTCTATTG pigNR TGTCAGCACGATGTTCAT 16SF CGTTACTCGCAGAAGAAGCA 16SR TCACCGCTACACCTGGAA 
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表 2 胁迫条件下WT与∆ompR的存活率
Table 2. Survival rates of WT and ∆ompR under stress （单位：%）
胁迫条件
Stress factorWT 存活率
Survival rate of WT∆ompR 存活率
Survival rate of ∆ompRpH=3 22.72±3.40 a 28.35±4.10 a 55 ℃ 0.15±0.01 a 0.11±0.05 a 2 mol·L−1 NaCl 92.45±2.60 a 90.65±3.80 a 0.22 mol·L−1 H2O2 22.40±1.30 a 23.84±1.10 a
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