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欧阳夷
, 彭华松, 王威, 胡洪波, 张雪洪
上海交通大学 生命科学技术学院, 微生物代谢国家重点实验室, 上海 200240
OUYANG Yi, PENG Hua-song, WANG Wei, HU Hong-bo, ZHANG Xue-hong
文献标识码: 1671-9964(2016)01-0005-06
文章编号: 1671-9964(2016)01-0005-06
收稿日期: 2015-03-25
网络出版日期: 2016-01-20
版权声明: 2016 上海交通大学期刊中心 版权所有
基金资助:
作者简介:
作者简介: 欧阳夷(1990-), 女, 研究方向:微生物资源与代谢, E-mail:oyy1028@163.com;
通讯作者: 彭华松(1974-), 男, 副教授, 研究方向:微生物资源与代谢, E-mail:hspeng@sjtu.edu.cn
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摘要
绿针假单胞菌(Pseudomonas chlororaphis)HT66是一株分泌高水平吩嗪-1-甲酰胺(phenazine-1-carboxamide, 简称PCN)的植物根际促生细菌。通过全基因组测序与分析, 发现phz基因簇上游存在着PhzI-PhzR双元调控系统。显色实验表明, 野生株信号分子抽提物不能使指示菌紫色杆菌(Chromobacterium violaceum)CV026显紫色, 但能使指示菌根癌农杆菌(Agrobacterium tumefaciens)NTL4显蓝色。研究采用基因无痕敲除方法构建了突变株ΔphzⅠ、ΔphzR;与野生株相比, ΔphzⅠ突变株丧失了PCN合成能力, 对终极腐霉的抑制作用明显下降;而且突变株ΔphzⅠ的信号分子抽提物也不能使指示菌NTL4菌显色。由此可见, 菌株HT66以高丝氨酸内酯作为群体感应的信号分子, 且吩嗪的生物合成受到了PhzI-PhzR的严格调控。进一步形态观察表明, 突变株ΔphzⅠ的菌落颜色变为乳白色, 鞭毛泳动性与野生株相比大大降低, 但其群体从动性变化不显著。
关键词：
Abstract
Pseudomonas chlororaphis HT66 is a significant growth-promoting rhizobacteriaum in plants.It is reportedly the highest phenazine-1-carboxamide(PCN)producing wild type microbe studied thus far. Based on the whole-genome sequence analysis, we find that two genes phzⅠ and phzR are located just upstream of the phenazine biosynthesis gene cluster.Plate assays with Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTL4 were used to analyze the signals of strain HT66.With the addition of the fermentation broth extracts, strain NTL4 produced blue pigment, but the CV026 did not produce purple pigment.Then, using a gene knock-out method, we constructed ΔphzⅠ and ΔphzR HT66 mutants.The results show that both the ΔphzⅠ and ΔphzR mutants are unable to produce PCN.And the plate assay of ΔphzⅠ culture extracts shows no blue pigment is produced by strain NTL4.It suggests that phenazine biosynthesis is regulated by the quorum sensing system and AHL signals in strain HT66.Additionally, the ΔphzⅠ and ΔphzR mutants have some changes in the morphology of colonies and antifungal activity in vitro.Moreover, compared with wild type, the swimming motility of ΔphzⅠ mutant decreases greatly, while the swarming motility has no significant change.
Keywords：
群体感应(quorum sensing, QS)是细菌根据细胞密度变化进行基因表达调控的一种生理行为。革兰氏阴性菌中的QS系统主要由高丝氨酸内酯(N-acyl homoserine lactones, AHLs)类信号分子及其受体蛋白组成。QS系统中, AHL类信号分子在菌体内的合成途径一般是在LuxI或LuxM、AinS等几类蛋白酶催化下合成[1], AHL在细胞中合成后, 能在细胞内外自由地扩散, 其浓度随着细菌密度的增加而增加。当群体密度不断增加, 信号分子浓度超过一定的阈值时, 便能与转录调控蛋白LuxR形成复合物, 而后与上游的lux-box结合, 从而调控相关基因的转录。QS系统控制或改变着细菌的各种生命活动, 比如生物发光[2]、共生现象[3]和抗生素的形成[4]等等。
假单胞菌(Pseudomonas spp.)作为一类重要的生防菌, 能够产生多种抗生素, 主要有2, 4-二乙酰藤黄(2, 4-diacetylphloroglucinol, DAPG或Phl)、氯化苯基吡咯类广谱抗生素硝吡咯菌素(pyrrolnitrin, Prn)、芳香族聚酮类抗生素藤黄绿菌素(pyoluteorin, Plt)和吩嗪类(phenazines)化合物[5]。由假单胞菌产生的吩嗪衍生物有很多种, 如常见的吩嗪-1-羧酸(phenazine-1-carboxylic acid, PCA), 吩嗪-1-甲酰胺(phenazine-1-carboxamide, PCN)和绿脓菌素(pyocyanin, PYO)等。吩嗪类化合物具有广谱抗菌活性, 可以有效抑制多种植物病原菌的生长, 有着重要的生物防治作用[6]。
绿针假单胞菌(Pseudomonas chlororaphis)HT66是一株从水稻根际分离得到的植物根际促生菌株(plant growth-promoting rhizobacteria, PGPR), 它能分泌高水平的吩嗪类化合物PCN, 是目前文献报导的吩嗪类化合物产量最高的野生菌[7], 具有良好的生防应用前景。通过全基因组测序并分析, 发现HT66的phz基因簇上游存在着PhzI-PhzR双元调控系统。本文通过无痕敲除构建phzⅠ、phzR基因缺失菌株, 并对突变菌株的吩嗪产物、菌落形态、运动性等后续变化展开研究。
1.1.1 菌种与质粒
绿针假单胞菌Pseudomonas chlororaphis HT66, 检测AHLs信号分子的报告菌株Agrobac-terium tumefaciens NTL4, Chromobacterium violaceum CV026, 大肠杆菌E.coli DH5α和基因敲除所用克隆载体pK18mobsacB质粒均由本实验室保存。
1.1.2 酶及主要试剂
Taq DNA聚合酶、T4 DNA连接酶、各种限制性内切酶、DNA marker均购于TaKaRa公司;质粒抽提试剂盒、DNA纯化试剂盒、基因组提取试剂盒购于北京全式金生物技术有限公司;引物合成与测序由桑尼公司完成;常规化学试剂均为国产分析纯。
1.1.3 培养基和菌株生长条件
PDA培养基:去皮马铃薯200 g, 切碎后加水煮25 min, 双层纱布过滤, 取滤液添加葡萄糖20 g, 琼脂20 g, 加水至1 L。鞭毛泳动性(swimming)培养基:每升含0.3%的琼脂糖, 10 g胰蛋白胨以及5 g氯化钠;群体从动性(swarming)培养基:每升含0.5%的琼脂, 5 g酵母提取物, 10 g胰蛋白胨以及5 g葡萄糖。
1.2.1 培养液中的AHLs信号分子萃取
取1 mL培养过夜的HT66菌液, 接种至100 mL KB液体培养基中, 28 ℃培养24 h后, 8 000 r/min离心10 min去除菌体。上清液用等体积的乙酸乙酯萃取2次, 并合并有机相, 旋转浓缩后蒸发干燥, 回溶于2 mL乙酸乙酯中, 进一步浓缩干燥, 用100 μL乙腈溶解并放置于-20 ℃保存, 每10 μL相当于10 mL发酵液中信号分子的量。
1.2.2 信号分子的报告菌株检测
挑选活化过的NTL4单菌落至庆大霉素30 μg/mL的LB液体培养基中28 ℃培养过夜。另取100 mL的ABm琼脂固体培养基融化后, 保温在50 ℃, 加入庆大霉素至终浓度为30 μg/mL、加入X-gal至终浓度为60 μg/mL, 然后按1∶9的比例缓缓加入过夜活化的NTL4菌液, 混合均匀并倒平板, 凝固后形成厚度约为8 mm左右的菌液混合凝胶层, 在无菌状态下打孔, 在孔内滴加10 μL提取的信号分子。在28 ℃下培养24 h后, 观察孔周围菌落的显色反应。报告菌株CV026的平板检测方法按照参考文献[8]进行。
1.2.3 无痕敲除目标基因
菌株HT66已经过全基因组测序, 测序结果在NCBⅠ上的序列号为ATBG00000000。根据其全基因组测序的结果设计引物, 引物序列如表1所示。敲除方法以phzⅠ基因为例, 设计phzⅠ的2对上下游引物, 以HT66基因组为模板, 分别扩增上下游同源臂;第2轮PCR以上下游同源臂为模板, 扩增融合PCR产物, 该PCR片段不包含phzⅠ基因;而后将融合片段与pK18mobsacB质粒分别用限制性内切酶EcoRⅠ与HindⅢ进行双酶切, 而后用T4连接酶连接, 构建敲除质粒。敲除质粒先转化DH5a感受态细胞, 在添加卡那霉素(50 μg/mL)、IPTG(20 μg/mL)和X-gal(40 μg/mL)的LB平板上涂板, 37 ℃过夜培养后, 利用蓝白斑筛选, 挑取白色克隆, 进一步PCR验证。阳性克隆PCR产物送测序, 测序验证后将阳性质粒电转入HT66感受态细胞, 涂布卡那抗性LB平板。28 ℃过夜培养后, 挑取阳性克隆涂布LB平板(添加10%蔗糖), 再进一步通过PCR验证筛选出敲除成功的菌株。
其中基因组提取、质粒抽提、酶切反应、DNA片段回收、感受态细胞制备等操作均按相关试剂盒步骤说明进行。
表1 引物名称及序列
Tab.1 List of primers and sequences
| 引物名称 Primer name | 引物序列(5’-3’) Primer sequence(5’-3’) |
|---|---|
| phzⅠ-F1 | CCGGAATTCCGGACTGAAGGTTGCTGAGAG |
| phzⅠ-R1 | TTACTATCTCCGAGTCGACCATCGAAGGCGACA GTTT |
| phzⅠ-F2 | GGTCGACTCGGAGATAGTAAATGCCCCTC |
| phzⅠ-R2 | CCCAAGCTTCGGTTTGATTTCTTTGCCTACGG |
| phzR-F1 | CCGGAATTCATGGAAGAGCACACACTGAG |
| phzR-R1 | TGTCACATTGAGGGTCTTGCATTTACTATCTCC GAGT |
| phzR-F2 | GCAAGACCCTCAATGTGACAGCCGTAAA |
| phzR-R2 | CCCAAGCTTTTGGCGAAGTTCAAGATGATCATT |
1.2.4 PCN产量测定
挑取KB平板上活化的HT66单菌落, 接种KB小瓶过夜活化, 以1%的接种量接种到100 mL KB液体培养基中, 28 ℃、180 r/min发酵24 h, 取样。以HPLC检测PCN浓度, 流动相采用乙腈(A)与5 mmol/L乙酸铵溶液(B), 色谱柱为WondaSil C18-WR反相柱(5 μm;4.6 mm×250 mm, Shimadzu, Japan), 检测波长254 nm, 检测条件:0~2 min, 8%A与92%B;2~20 min, A相浓度从8%升至60%, B相浓度从92%下降至40%;20~21 min, 继续回到8%A相与92%B相。柱温维持在30 ℃, 流速为1.0 mL/min。
1.2.5 鞭毛泳动性、群体从动性和菌落形态检测
培养基高压灭菌后冷却倒数块平板, 使用前在室温下干燥数小时, 并在平板中央放置一块灭菌的小滤纸片。鞭毛泳动性与群体从动性使用待检测的菌株过夜小瓶活化, 用PBS缓冲液将菌液OD值稀释至0.1, 吸取2 μL滴在检测平板的滤纸片上, 28 ℃培养过夜并观察。野生株以及突变株的菌落形态实验主要根据Friedman与Kolter的方法进行[9]。
1.2.6 抑菌活性检测
抑菌活性检测采用实验室保存的终极腐霉病原菌[10], 先在PDA培养基平板上充分活化病原菌, 同时在KB平板上活化绿针假单胞菌HT66, 接种于KB液体小瓶活化过夜。然后取一块新鲜的PDA培养基, 在培养基左侧接入活化的终极腐霉菌丝块, 在培养皿对称的右侧放置一块灭菌的小滤纸片, 滤纸片上接入HT66菌液10 μL, 待检测菌落均设置3块平行平板。另取一块PDA培养基, 仅在左侧接种病原菌, 作为对照。置于28 ℃培养箱中培养1周, 观察菌株对真菌的抑菌能力。
大量研究表明, 许多革兰氏阴性菌都应用群体感应系统, 通过AHLs作为交流的信号分子, 从而调控许多基因的表达。在产吩嗪衍生物的多种假单胞菌中, 多由PhzI-PhzR双元调控蛋白组成群体感应系统, phzⅠ能合成AHLs, AHLs能与PhzR蛋白形成复合物, 如P.chlororaphis PCL1391、Pseudomonas aureofaciens 30-84等, 而不同假单胞菌所产生的AHLs在碳链长度、取代基上有所不同。
绿针假单胞菌HT66的全基因测序的结果已经在NCBI上公布, 通过NCBI的BLAST比较发现, 在菌株HT66中phzⅠ/phzR在基因组上的位置与其他已公布的假单胞菌的排列次序一致, phzⅠ位于phzR上游, 而phzR下游则是吩嗪基因簇。通过BLAST比对, 发现其phzⅠ序列与菌株PCL1391的phzⅠ序列同源性达到99%、与Pseudomonas aureofaciens 30-84同源性为95%。基于菌株HT66全基因组测序的结果, 成功构建phzⅠ、phzR基因的敲除质粒, 转入菌株HT66后, 经过一系列筛选以及PCR验证, 成功获得突变株ΔphzⅠ、ΔphzR。phzⅠ基因全长591 bp, phzR基因长度为552 bp, 突变株的PCR验证结果如图1所示:a图中, 1为突变株ΔphzⅠ中phzⅠ上下游的扩增片段, 2为野生型HT66中phzⅠ上下游的扩增片段, 野生型因为有phzⅠ基因存在所以扩增片段更大;b图中, 1与2均为突变株ΔphzR中phzR上下游的扩增片段, 3为野生型HT66中phzR上下游的扩增片段。
图1 敲除株ΔphzⅠ、ΔphzR的PCR验证电泳图谱注: a图, M为DNA marker, 1为突变株ΔphzⅠ, 2为野生型HT66;b图, M为DNA marker, 1与2均为突变株ΔphzR, 3为野生型HT66
Fig.1 Confirmation of ΔphzⅠ and ΔphzR mutants by PCRNote:(a)M:DNA marker;1:ΔphzI mutant;2:strain HT66;(b)M:DNA marker;1 and 2:ΔphzR mutant;3:strain HT66
由图2可知, 菌株HT66所产生的PCN浓度高达445 mg/L;而突变株ΔphzⅠ、ΔphzR, 在24、48 h均检测不到PCN的产生, 表明其丧失了合成PCN的能力, 说明phzⅠ、phzR对HT66的PCN的合成起着决定性的作用。
我们采用QS系统信号分子报告菌紫色杆菌CV026与根癌农杆菌NTL4来检测样品中是否含有高丝氨酸内酯AHLs, 指示菌CV026与NTL4所能感应的AHLs种类有所不同。显色实验表明, HT66野生菌株的信号分子提取物不能使指示菌CV026显色紫色, 但能使指示菌NTL4显蓝色, 说明HT66菌株所产生的信号分子可能是在3位碳原子上存在羰基或者羟基取代的AHLs[11]。
为了进一步研究phzⅠ对于菌株HT66中信号
图2 假单胞菌HT66中野生株和敲除株ΔphzⅠ、ΔphzR的PCN产量
Fig.2 PCN production by strain HT66 and ΔphzⅠ, ΔphzR mutants
分子合成的影响, 分别提取了HT66野生株及phzⅠ敲除株发酵液中的信号分子。样品用乙腈溶解, 每孔添加的量相当于10 mL发酵液中的信号分子, 10 μL乙腈作为阴性对照, 报告菌株NTL4平板检测。实验结果如图3所示。由图3可知, 从HT66野生株发酵液中所提取的信号分子使NTL4培养基孔周围出现蓝色, 而phzⅠ敲除株发酵液和阴性对照的周围均未能变蓝, 说明绿针假单胞菌HT66野生株发酵液中存在高丝氨酸内酯, 而phzⅠ突变使得其不产生AHLs信号。
图3 假单胞菌HT66中信号分子的NTL4平板检测注: 1: HT66;2:ΔphzⅠ 突变抹;3:乙腈
Fig.3 Detection of AHL signals by plate assaywith Agrobacterium tumefaciens NTL4Note: 1: HT66;2:ΔphzⅠ mutant;3:acetonitrile
图4显示HT66野生株周围有圆环出现, 而ΔphzⅠ菌株没有相应圆环, 菌体只在接种处原处生长, 说明敲除phzⅠ会导致鞭毛泳动能力大大降低。与野生株HT66相比, ΔphzⅠ突变株的群体从动性并无显著变化, 两者生长轨迹大致相同, 说明敲除phzⅠ对该菌的群体从动性影响不大。
图4 鞭毛泳动性与群体从动性实验
Fig.4 Swimming and swarming motility of strain HT66 and ΔphzI mutant
phzⅠ、phzR突变使得HT66的菌落形态发生了变化, 如图5所示。在7 d的培养周期里, 第1~2天, 野生株与突变株并无太大差异;从第3天开始, 野生株HT66呈现出淡黄色, 其中可以看到形成了少量PCN的绿色结晶, 然后从第6、7天开始菌落呈现出衰退的迹象, 略有变褶皱的趋势。而ΔphzⅠ与ΔphzR突变株由于失去了吩嗪物质的产生, 菌落发白, 呈乳白色, 即使到第7天也未出现褶皱。
图5 假单胞菌HT66野生株与ΔphzⅠ、ΔphzR菌落形态的比较
Fig.5 Colonial morphology change among strain HT66, ΔphzⅠ and ΔphzR
phzⅠ、phzR突变对HT66抑菌活性的影响结果如图5所示。经过1周的培养, 空白对照组的病原菌菌丝长满了整块平板;在右侧接种了HT66菌株的平板, 病原菌的生长受到了抑制;而ΔphzⅠ与ΔphzR突变菌株对病原菌的抑制没有野生株HT66的抑制效果显著, 这表明PhzⅠ-PhzR系统对HT66的抑菌活性有着很大的影响。
绿针假单胞菌Pseudomonas chlororaphis HT66是本实验室从水稻根际分离得到的一株具有生防功能的植物根际促生菌, 它产生的吩嗪衍生物PCN, 不仅具有广谱抗菌活性, 还使它在周围微生物生长环境中具有更强的竞争优势。经过本实验室对HT66的全基因组测序与分析, 发现了处于phz操纵子phzA、B、C、D、E、F、G、H 8个基因上游的phzⅠ、phzR双元调控基因, 并通过无痕敲除的方法成功构建了ΔphzⅠ与ΔphzR突变株。
图6 假单胞菌HT66的野生株与ΔphzⅠ、ΔphzR突变株的抑菌活性
Fig.6 Effects of antifungal activity of HT66 and its mutants on the growth of Pythium ultimum, Pythium ultimum (left);HT66 and its mutants(right)
ΔphzⅠ与ΔphzR突变株完全不能产生PCN, 这表明HT66菌株的PhzⅠ-PhzR系统直接参与了PCN生物合成的调控。利用报告菌株NTL4对野生株与ΔphzⅠ发酵液所提取的信号分子样品进行检测, 发现ΔphzⅠ突变株信号分子的缺失, 这可能与PCN的合成有着直接的联系。这与PCL1391中的情况相似[12], 在PCL1391中同样存在着PhzI/PhzR双元调控系统, 其中phzⅠ能合成主要2种信号分子——C4、C6-HSL(homoserine lactone ring), 通过与PhzR蛋白结合形成复合物进而启动下游phz基因簇的表达, 这表明HT66中吩嗪的代谢调控是相对保守的。同样在Pseudomonas aureofaciens 30-84中也有着相似的调控机制, 它的phzⅠ基因合成的信号分子包括C6-HSL与3-OH-C6, C7, C8, C10-HSL[1]。在PCL1391中, phzⅠ基因合成的2种信号分子C4-HSL与C6-HSL, 均能使CV026变色, 而菌株HT66的信号分子提取物并不能使指示菌CV026变色, 却能与NTL4作用产生蓝色反应, 表明菌株HT66的phzⅠ基因尽管与PCL1391的相似度高达99%, 但其产生的信号分子并不相同。
对于植物根际促生菌来说, 鞭毛对于获取营养物质、躲避有害物质以及向环境扩散传播都起着重要作用, 尤其在生物膜形成的早期, 鞭毛的泳动起着关键的作用[13]。调控鞭毛合成与功能的基因有很多, 在本研究中phzⅠ敲除株鞭毛泳动能力大大降低, 表明phzⅠ参与了鞭毛的相关调控。同样在绿针假单胞菌PA23中, PA23-6863(携带AHLs降解质粒pME6863)的运动性对比野生株PA23大幅下降, 而PA23phzR与PA23相比并无显著差异[14]。在HT66菌株中, 对比野生株与ΔphzⅠ、ΔphzR突变株, 菌落形态以及抑菌活性均有较大改变, 吩嗪的缺失使得其抑菌活性有所降低, 而菌落颜色也产生了变化, 表明PhzⅠ-PhzR调控系统对于HT66菌株发挥其生防功能有着重要影响, 但它是如何参与PCN生物合成以及其它相关生防活性的调控, 仍有待进一步的实验。
The authors have declared that no competing interests exist.
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ABSTRACT Bacterial cell-to-cell signalling has emerged as a new area in microbiology. Individual bacterial cells communicate with each other and co-ordinate group activities. Although a lot of detail is known about the mechanisms of a few well-characterized bacterial communication systems, other systems have been discovered only recently. Bacterial intercellular communication has become a target for the development of new anti-virulence drugs.
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The quorum-sensing (QS) signal autoinducer-2 (AI-2) has been proposed to promote interspecies signaling in a broad range of bacterial species. AI-2 is spontaneously derived from 4,5-dihydroxy-2,3-pentanedione that, along with homocysteine, is produced by cleavage of S-adenosylhomocysteine (SAH) and S-ribosylhomocysteine by the Pfs and LuxS enzymes. Numerous phenotypes have been attributed to AI-2 QS signaling using luxS mutants. We have previously reported that the luxS mutation also affects the synthesis of the AI-3 autoinducer that activates enterohemorrhagic Escherichia coli virulence genes. Here we show that several species of bacteria synthesize AI-3, suggesting a possible role in interspecies bacterial communication. The luxS mutation leaves the cell with only one pathway, involving oxaloacetate and l-glutamate, for de novo synthesis of homocysteine. The exclusive use of this pathway for homocysteine production appears to alter metabolism in the luxS mutant, leading to decreased levels of AI-3. The addition of aspartate and expression of an aromatic amino acid transporter, as well as a tyrosine-specific transporter, restored AI-3-dependent phenotypes in an luxS mutant. The defect in AI-3 production, but not in AI-2 production, in the luxS mutant was restored by expressing the Pseudomonas aeruginosa S-adenosylhomocysteine hydrolase that synthesizes homocysteine directly from SAH. Furthermore, phenotype microarrays revealed that the luxS mutation caused numerous metabolic deficiencies, while AI-3 signaling had little effect on metabolism. This study examines how AI-3 production is affected by the luxS mutation and explores the roles of the LuxS/AI-2 system in metabolism and QS.
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N-acylhomoserine-lactone mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms [J].DOI:10.1046/j.1432-1327.1999.00963.x URL PMID: 11739757 [本文引用: 1] 摘要
Pseudomonas aeruginosa and Burkholderia cepacia are capable of forming mixed biofilms in the lungs of cystic fibrosis patients. Both bacteria employ quorum-sensing systems, which rely on N-acylhomoserine lactone (AHL) signal molecules, to co-ordinate expression of virulence factors with the formation of biofilms. As both bacteria utilize the same class of signal molecules the authors investigated whether communication between the species occurs. To address this issue,novel Gfp-based biosensors for non-destructive, in situ detection AHLs were construvcted and characterized. These sensors were used to visualize AHL-mediated communication in mixed biofilms, which were cultivated either in artificial flow chambers or in alginate beads in mouse lung tissue. In both model systems B. cepacia was capable of perceiving the AHL signals produced by P.aeruginosa, while the latter strain did not respond to the molecules produced by B.cepacia. Measurements of extracellular proteolytic activities of difined quorum-sensing mutants grown in media complemented with AHL extracts prepared from culture supernatants of various wild-type and mutant strains supported the view of unidirectional signalling between the two strains.
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The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa [J].DOI:10.1089/dna.1993.12.455 URL PMID: 8508773 [本文引用: 1] 摘要
Erwinia carotovora and Pseudomonas aeruginosa secrete exoenzymes that contribute to the pathogenesis of plant and mammalian infections respectively. E.carotovora mutants defective in synthesis of the pectinase, cellulase and protease exoenzymes were isolated and classified into two groups. Group 2 mutants were found to be defective in the production of a small freely diffusible molecule, N-3-(oxohexanoyl)-L-homoserine, lactone (HSL), and were avirulent. Addition of exogenous HSL to these group 2 mutants restores synthesis of the exoenzymes and virulence in planta. Of the exoenzymes of P.aeruginosa the metalloprotease, elastase, is an established virulence determinant. Mutants of P.aeruginosa that are defective in elastase production have been isolated and were again found to fall into two groups. Analogous to the group 2 mutants of E.carotovora, group 2 mutants of P. aeruginosa are defective in the synthesis of HSL and exogenous HSL restores elastase production. HSL has now been linked to the control of bioluminescence in Vibrio fischeri, carbapenem antibiotic production of E.carotovora and the above exoenzyme virulence determinants. This information significantly enhances our understanding of the extent and nature of pheromone mediated gene expression control in prokaryotes.
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Recent insights into the diversity, frequency and ecological roles of phenazines in fluorescent Pseudomonas spp [J].DOI:10.1111/j.1462-2920.2012.02846.x Magsci [本文引用: 1] 摘要
Phenazine compounds represent a large class of bacterial metabolites that are produced by some fluorescent Pseudomonas spp. and a few other bacterial genera. Phenazines were first noted in the scientific literature over 100 years ago, but for a long time were considered to be pigments of uncertain function. Following evidence that phenazines act as virulence factors in the opportunistic human and animal pathogen Pseudomonas aeruginosa and are actively involved in the suppression of plant pathogens, interest in these compounds has broadened to include investigations of their genetics, biosynthesis, activity as electron shuttles, and contribution to the ecology and physiology of the cells that produce them. This minireview highlights some recent and exciting insights into the diversity, frequency and ecological roles of phenazines produced by fluorescent Pseudomonas spp.
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产量低是限制生物农药吩嗪-1-甲酰胺(Phenaizne- 1-carboximade,PCN)推广应用的主要因素.本文综合使用诱变育种和基因工程改造来提高绿针假单胞菌的PCN产量.诱变育种使用亚硝基胍 (NTG)和紫外线(UV)处理绿针假单胞菌HT66野生型菌株,以平板菌落表面的绿色PCN晶体量为指标建立高通量诱变筛选方法,通过10轮稳定可靠的 诱变处理,获得的诱变高产株P3中PCN产量达到1 697 mg/L,是野生菌株的3.99倍.在固体平板上培养时,P3菌株的菌落更大,PCN晶体更多且出现更早.在诱变育种的基础上进一步进行基因工程改造,敲 除P3株中负调控PCN合成的rpeA基因,获得的P3△rpeA菌株PCN产量达到2 167 mg/L,充分证明了2种育种方法结合使用的高效性.
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Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa [J]. |
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Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli [J].DOI:10.2514/6.2010-8070 URL PMID: 178707 [本文引用: 1] 摘要
The exponential phase of aerobic growth is associated with risk of endogenous oxidative stress in which cells need to cope with an approximately 10-fold increase in the rate of H2O2 generation. We addressed this issue by studying the regulation of the intracellular concentration of H2O2 in aerobically growing Escherichia coli. Intracellular H2O2 was kept at an almost constant steady-state value of approximately 0.2 microM (variation, less than twofold) over a broad range of cell densities in rich medium. This regulation was achieved in part by a transient increase in the OxyR-dependent transcription of the catalase gene katG (monitored by using a katG::lacZ operon fusion) during exponential growth, directly correlated with the increased rate of H2O2 generation. The OxyR-regulated alkyl hydroperoxide reductase encoded by ahpFC did not detectably affect H2O2 or catalase activity levels. Induction of katG, ahpFC, and perhaps other genes prevented the accumulation of oxidatively modified lipids but may not have protected DNA: the spontaneous mutation rate was significantly increased in both wild-type and delta(oxy)R strains during exponential growth compared to that in these strains during lag or stationary phases. Strains lacking oxyR showed throughout growth an 8- to 10-fold-higher frequency of spontaneous mutation than was seen for wild-type bacteria. The ahpdelta5 allele also had a mutator effect half of that of delta(oxy)R in exponential and stationary phases and equal to that of deltaoxyR in lag phase, perhaps by affecting organic peroxide levels. These results show that oxyR-regulated catalase expression is not solely an emergency response of E. coli to environmental oxidative stress, but also that it mediates a homeostatic regulation of the H2O2 produced by normal aerobic metabolism. The activation of the oxyR regulon in this process occurs at much lower levels of H2O2 (approximately 10(-7)M) than those reported for oxyR activation by exogenous H2O2 (approximately 10(-5) M).
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链霉菌P510中吩嗪类抗生物质的分离纯化[D] . |
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Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography [J].DOI:10.1073/pnas.94.12.6036 URL PMID: 9177164 [本文引用: 1] 摘要
Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. With the exception of N-butanoyl-L-homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths tested. The intensity of the response was proportional to the amount of the signal molecule chromatographed. Each of the 3-oxo- and the 3-unsubstituted derivatives migrated with a unique mobility. Using the assay, we showed that some bacteria produce as many as five detectable signal molecules. Structures could be assigned tentatively on the basis of mobility and spot shape. The dominant species produced by Pseudomonas syringae pv. tabaci chromatographed with the properties of N-(3-oxohexanoyl)-L-homoserine lactone, a structure that was confirmed by mass spectrometry. An isolate of Pseudomonas fluorescens produced five detectable species, three of which had novel chromatographic properties. These were identified as the 3-hydroxy- forms of N-hexanoyl-, N-octanoyl-, and N-decanoyl-L-homoserine lactone. The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules.
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Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted into the growth medium [J].DOI:10.1148/radiol.2531082137 URL [本文引用: 1] 摘要
Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp, radicis-lycopersici, The production of phenazine-l-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-L-homoserine lactone (C-6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4HSL) and N-octanoyl-L-homoserine lactone (C-8-HSL), Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C-6-NSL, but not by C-4-HSL, C-8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon dearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C-4-HSL or C-8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.
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Quorum sensing and phenazines are involved in biofilm formation by Pseudomonas chlororaphis(aureofaciens)strain 30-84 [J].DOI:10.1007/s00248-006-9064-6 Magsci [本文引用: 1] 摘要
<a name="Abs1"></a>The biological control bacterium <i>Pseudomonas chlororaphis (aureofaciens)</i> strain 30-84 employs two quorum sensing (QS) systems: PhzR/PhzI regulates the production of the antibiotics phenazine-1-carboxylic acid, 2-hydroxy-phenazine-1-carboxylic acid, and 2-hydroxy-phenazine, whereas CsaR/CsaI regulates currently unknown aspects of the cell surface. Previously characterized derivatives of strain 30-84 with mutations in each QS system and in the phenazine biosynthetic genes were screened for their ability to form surface-attached biofilm populations <i>in vitro</i>, using microtiter plate and flow cell biofilm assays, and on seeds and roots. Results from <i>in vitro</i>, seed, and root studies demonstrated that the PhzR/PhzI and the CsaR/CsaI QS regulatory systems contribute to the establishment of biofilms, with mutations in PhzR/PhzI having a significantly greater effect than mutations in CsaR/CsaI. Interestingly, phenazine antibiotic production was necessary for biofilm formation to the same extent as the PhzR/PhzI QS system, suggesting the loss of phenazines was responsible for the majority of the biofilm defect in these mutants. <i>In vitro</i> analysis indicated that genetic complementation or AHL addition to the growth medium restored the ability of the AHL synthase <i>phzI</i> mutant to form biofilms. However, only phenazine addition or genetic complementation of the phenazine biosynthetic mutation <i>in trans</i> restored biofilm formation by mutants defective in the transcriptional activator <i>phzR</i> or the <i>phzB</i> structural mutant. QS and phenazine production were also involved in the establishment of surface-attached populations on wheat seeds and plant roots, and, as observed <i>in vitro</i>, the addition of AHL extracts restored the ability of <i>phzI</i> mutants, but not <i>phzR</i> mutants, to form surface attached populations on seeds. Similarly, the presence of the wild type in mixtures with the mutants restored the ability of the mutants to colonize wheat roots, demonstrating that AHL and/or phenazine production by the wild-type population could complement the AHL- and phenazine-deficient mutants <i>in situ</i>. Together, these data demonstrate that both QS systems are involved in the formation of surface-attached populations required for biofilm formation by <i>P. chlororaphis</i> strain 30-84, and indicate a new role for phenazine antibiotics in rhizosphere community development beyond inhibition of other plant-associated microorganisms.
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O'toole G A.Microbial biofilms:from ecology to molecular genetics [J].DOI:10.1128/MMBR.64.4.847-867.2000 URL PMID: 11104821 [本文引用: 1] 摘要
Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.
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| [15] |
Fernando W G D, de Kievit T.The PhzI/PhzR quorum-sensing system is required for pyrrolnitrin and phenazine production, and exhibits cross-regulation with RpoS in Pseudomonas chlororaphis PA23 [J].DOI:10.1099/mic.0.054254-0 URL PMID: 22262095 摘要
The aim of the current study was to determine how quorum sensing (QS) affects the production of secondary metabolites in Pseudomonas chlororaphis strain PA23. A phzR mutant (PA23phzR) and an N-acylhomoserine lactone (AHL)-deficient strain (PA23-6863) were generated that no longer inhibited the fungal pathogen Sclerotinia sclerotiorum in vitro. Both strains exhibited reduced pyrrolnitrin (PRN), phenazine (PHZ) and protease production. Moreover, phzA-lacZ and prnA-lacZ transcription was significantly reduced in PA23phzR and PA23-6863. As the majority of secondary metabolites are produced at the onset of stationary phase, we investigated whether cross-regulation occurs between QS and RpoS. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. Finally, we discovered that QS and RpoS have opposing effects on PA23 biofilm formation. While both QS-deficient strains produced little biofilm, the rpoS mutant showed enhanced biofilm production compared with PA23. Collectively, our findings indicate that QS controls diverse aspects of PA23 physiology, including secondary metabolism, RpoS and biofilm formation. As such, QS is expected to play a crucial role in PA23 biocontrol and persistence in the environment.
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