铁还原菌Shewanella xiamenensis BC01的有机溶剂应激研究

doi:10.11949/0438-1157.20201845

摘要/Abstract

摘要：

以铁还原菌厦门希瓦氏菌Shewanella xiamenensis BC01（SXM）为研究对象，系统探究了SXM在不同有机溶剂胁迫下的行为变化。生长方面，SXM对有机溶剂的耐受性与其亲水性相关，甲醇、乙醇、丙酮及DMSO的耐受浓度可达5%，对异丙醇、丙醇及叔丁醇的耐受浓度为2%，对丁醇的耐受浓度仅为1%。形态方面，丙醇刺激下的SXM菌体被显著拉长为至少5 μm以上，而丁醇刺激下菌体则缩小至平均不足1 μm，且均为可逆变化；蛋白方面，在丙醇与丁醇刺激下，与铁代谢相关的TonB、IucA及含铁脱氢酶等蛋白表达量均下调，铁还原酶活性也被大幅抑制；基因层面，丙醇刺激下与电子传递相关的mtrC基因表达量提高147.6%。

关键词: 有机溶剂, 希瓦氏菌, 醇类应激, 铁还原酶, 生物工程, 蛋白质, 形态学

Abstract:

The widely distributed genus Shewanella is famous for its capacity of reducing iron. The aim of this study is to decipher the behavioral change on Shewanella xiamenensis BC01 (SXM) under stress of organic solvents. The tolerance of SXM to organic solvents is related to the hydrophilicity of solvents. For methanol, ethanol, acetone and DMSO, the tolerance concentration for SXM is 5%, for n-propanol, isopropanol and tert-butanol, the tolerance concentration is 2%, while for n-butanol, the tolerance concentration is only 1%. The scanning electron microscopy revealed that cells were obviously stretched to at least 5 μm under n-propanol stress, and cells were shrunk to an average of less than 1 μm under n-butanol stress. Protein identification implied that TonB receptor, IucA family protein and iron containing dehydrogenase were down-regulating under propanol and butanol stress, and the enzymatic assay of ferric reductase was obviously dropped down. At the genetic level, the expression of mtrC gene related to electron transport increased by 147.6% under the stimulation of propanol. It would provide practical information on environmental stress and accelerate mechanism of stress in Shewanella geneus.

Key words: organic solvents, Shewanella, alcohol stress, iron reductase, biological engineering, protein, morphology

中图分类号:

Q 81

陈婷婷, 韩恺忻, 陈翠雪, 凌雪萍, 沈亮, 卢英华. 铁还原菌Shewanella xiamenensis BC01的有机溶剂应激研究[J]. 化工学报, 2021, 72(7): 3747-3756.

CHEN Tingting, HAN Kaixin, CHEN Cuixue, LING Xueping, SHEN Liang, LU Yinghua. Study of iron-reducing bacteria Shewanellaxiamenensis BC01 under organic solvents stress[J]. CIESC Journal, 2021, 72(7): 3747-3756.

图/表 9

图1 SXM在不同溶剂应激下的生长情况

Fig.1 Growth curves of SXM in culture with different solvents at 30℃ and 150 r/min in LB medium

表1 有机溶剂的性质与SXM在不同溶剂培养下的比生长速率

Table 1 Physical properties of different organic solvents and specific growth rate for SXM cultured in different solvents

外加溶剂	分子量	lgP	密度/(g/ml)	溶解度/(g/100 H₂O)	SGR^① for 1%/(g/h)	SGR^① for 2%/(g/h)	SGR^① for 5%/ (g/h)
水(对照组)	18.0	—	1.0	无穷大	0.2992^②
甲醇	32.0	-0.5	0.792	无穷大	0.2336	0.2329	0.1836
乙醇	46.1	-0.08	0.790	无穷大	0.2655	0.2328	0.0486
异丙醇	60.1	0.38	0.804	无穷大	0.3144	0.3550	ND
丙醇	60.1	0.59	0.785	无穷大	0.3035	0.1666	ND
叔丁醇	74.1	0.80	0.781	8.8	0.2484	0.1425	ND
丁醇	74.1	1.16	0.809	8.0	0.0244	0.0004	ND
丙酮	78.13	-2.03	1.101	易溶	0.2432	0.2031	0.0115
DMSO	58.08	-0.24	0.788	无穷大	0.2256	0.2047	0.1323

图2 不同有机溶剂应激下SXM的扫描电镜表征

Fig.2 The SEM result of SXM bearing different solvents

表2 有机溶剂应激下SXM的粒径变化

Table 2 Particle size distribution of SXM under cultured in different organic solvents

外加溶剂	测量粒径与原始粒径比值/%	PDI
LB (H₂O)	100.0	0.078
2% 甲醇	109.9	0.025
2% 乙醇	94.3	0.040
2% 丙醇	179.6	0.469
1% 丁醇	69.3	0.159
2% 叔丁醇	129.0	0.229
2% 丙酮	105.7	0.128
2% DMSO	97.5	0.084

图3 不同应激条件下SXM的蛋白电泳分析（Ori：原始条件, P1、P2分别表示1%丙醇与2%丙醇, B1表示1%丁醇；箭头所示蛋白均经MALDI-TOF-TOF进行后续分析）

Fig.3 SDS-PAGE profiles of SXM cultured at normal condition (Ori), 1% n-propanol (P1), 2% n-propanol(P2) and 1% n-butanol (B1) for whole cell and periplasm, respectively(The proteins indicated by the arrows were followed by MALDI-TOF-TOF)

表3 醇类应激下SXM产生的差异蛋白

Table 3 Mascot protein identi?cation of differential proteins produced by SXM under cultured in different alcohols

样品	蛋白及对应菌株	蛋白分数^①	蛋白编号	分子量	等电点
TonB	TonB-dependent receptor Shewanella sp. MR-4	657	gi\|499941499	82239	4.92
IucA	IucA/IucC family protein Shewanella xiamenensis	1079	gi\|659914351	72233	5.89
P1	phosphoglucomutase/phosph-omannomutase alpha/beta/subunit Shewanella xiamenensis	543	gi\|659915240	63150	5.85
P2	iron-containing alcohol dehydrogenase Shewanella xiamenensis	749	gi\|659915731	40394	5.34
OmpA	OmpA family protein Shewanella xiamenensis	328	gi\|659913939	40372	4.72
porin	porin Shewanella xiamenensis	1177	gi\|659913622	39016	4.60
Hsp90	heat shock protein 90 Shewanella oneidensis	336	gi\|499384609	71779	5.25
Ahd	aromatic hydrocarbon degradation membrane protein Shewanella xiamenensis	845	gi\|659914295	45906	4.43

表4 不同溶剂应激时SXM的铁还原酶酶活

Table 4 Fe reductase activities of SXM bearing different solvents

外加溶剂	全细胞酶活/(U/L)
外加溶剂	以铁酸盐为底物	以FeCl₃为底物
LB (H₂O)	286.1±12.0	9.5±0.6
丙醇	74.0±4.1	3.3±0.23
丁醇	72.2±3.2	3.0±0.05

图4 MTR通道基因的标准曲线与不同溶剂应激下基因的表达量

Fig.4 The quantification real time PCR (qRT-PCR) standard curves of genes of MTR pathway (mtrA, mtrB and mrtC) and gene expression level under solvent stress

图5 不同培养条件下MTR通道变化的推测示意图

Fig.5 Proposed mechanism of electrons transfer through the outer membrane associated proteins (MtrA, MtrB and MtrC) under LB medium and 2% n-propanol in LB respectively

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