Control of the acetamidase gene of Mycobacterium smegmatis by multiple regulators

The acetamidase of Mycobacterium smegmatis is an inducible enzyme which enables the organism to utilise several amides as sole carbon sources. The acetamidase structural gene (amiE) is located downstream of four other genes, of which three form a probable operon with amiE; the fourth (amiC) is divergently transcribed. We constructed deletion mutants in two of these genes in order to determine their role in acetamidase expression. Both AmiC and AmiD were shown to be positive regulators of acetamidase expression required for induction. Combinations of regulatory gene deletions were made which revealed that AmiC interacts with the previously characterised negative regulator AmiA, whereas AmiD does not.     

Identification of strong promoter elements of Mycobacterium smegmatis and their utility for foreign gene expression in mycobacteria

The isolation of elements driving high-level expression of foreign genes in mycobacteria would significantly aid characterization of mycobacterial antigens and recombinant vaccine development. Mycobacterium smegmatis is a widely employed host for recombinant mycobacterial gene expression. This report describes the identification of strong promoter elements of M. smegmatis. Fluorescence-activated cell sorting was employed to isolate DNA fragments permitting high-level expression of the Aequorea victoria green fluorescent protein within recombinant M. smegmatis. Ten postulated M. smegmatis promoters were identified which showed activity two to six times that of the strong beta-lactamase promoter of Mycobacterium fortuitum. The utility of one of these promoters for the over-expression of foreign genes in mycobacteria was demonstrated by the efficient purification of the Mycobacterium leprae 35-kDa antigen from recombinant M. smegmatis.     

耻垢分枝杆菌中反式翻译报告体系的构建

细菌在翻译过程中,mRNA受到损伤(如缺失终止密码子)时会使翻译提前终止,导致核糖体熄火,细菌自身会启动核糖体拯救途径。由tmRNA-SmpB介导的反式翻译系统是结核分枝杆菌中的核糖体拯救途径,对结核分枝杆菌的生长繁殖有重大影响。为探究分枝杆菌中反式翻译途径的启动及其功能特点,本研究选取耻垢分枝杆菌为实验菌株,分别以mCherry和egfp作为报告基因,通过在报告基因3′端添加大肠埃希菌终止子序列,构建能在菌体中反映反式翻译表达的报告体系,并初步探究该体系中报告基因的动态表达特点。结果显示,相比正常表达mCherry的对照菌株,实验菌株中表达的错误mCherry蛋白很快被水解,菌体颜色均明显浅于前者,增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)定量检测数据也显示错误EGFP水平显著低于正常表达的EGFP水平,表明两种反式翻译报告体系均构建成功。报告基因的动态表达数据显示,蛋白出现翻译异常时,耻垢分枝杆菌可在蛋白翻译过程中快速启动反式翻译途径,并于40～45h将不成熟错误蛋白完全水解。本研究构建的反式翻译报告体系可为后续开展分枝杆菌反式翻译途径的功能研究及抗结核药物筛选提供帮助。     

异烟肼影响重组耻垢分支杆菌Rv1776c基因表达的实验研究

目的 研究经典抗结核药物异烟肼对重组耻垢分支杆菌生长增殖及其Rv1776c基因表达的影响。方法 将重组菌MS-Rv1776c接种于LB培养基培养作为对照组,实验组给予异烟肼药物处理,不同时间点取菌液测量A600值,根据所测A值绘制增殖曲线;提取菌液DNA以RT-PCR方法检测异烟肼对Rv1776c基因表达的影响;免疫印迹法SDS-PAGE及Western blot检测异烟肼对Rv1776c蛋白表达的影响。结果 异烟肼对两组重组耻垢分枝杆菌增殖无显著影响（P>0.05）;异烟肼可抑制Rv1776c基因的表达（P<0.05）;SDS-PAGE及Western blot检测发现异烟肼可显著降低ERv1776c蛋白的表达（P<0.05）。结论 异烟肼对重组耻垢分支杆菌的增殖无影响,但可抑制结核分枝杆菌Rv1776c基因及其表达的蛋白。该结果对研究结核菌从休眠菌到复苏初期及活跃期的药物预防提供了实验依据。     

Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies

Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over‐express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non‐pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.     

构建CRISPR-Cas9介导的耻垢分枝杆菌基因组高效删除系统

【背景】耻垢分枝杆菌具有生长迅速和非致病性的特点,可作为结核分枝杆菌致病机理研究替代菌株和类固醇激素生产的工程菌,但目前耻垢分枝杆菌中缺乏高效率的基因组敲除方法。【目的】基于CRISPR-Cas9介导的定点、高效的DNA切割能力,构建耻垢分枝杆菌染色体DNA片段无痕敲除系统。【方法】构建了包含四环素诱导型启动子驱动的密码子优化的cas9基础载体pCas9101,在双侧同源臂长度约为1 kb条件下选用合适的gRNA表达模块,分别测试了对耻垢分枝杆菌mc2155染色体上的3β-羟基类固醇脱氢酶基因(MSMEG_5228,1 071 bp)和胆固醇降解基因簇(MSMEG_5990-MSMEG_6043,约48kb)敲除效率,使用相同大小的同源臂以经典p2NIL-pGOAL方法进行对照,并计算效率。【结果】使用CRISPR-Cas9方法对耻垢分枝杆菌mc2155的3β-羟基类固醇脱氢酶基因敲除效率为22%,胆固醇降解基因簇敲除效率也达到18%,两者连续敲除效率为4%。但对照p2NIL-pGOAL方法未能获得目标DNA片段敲除的菌株。【结论】本文建立的基于CRISPR-Cas9的耻垢分枝杆菌基因组无痕敲除系统显示出较高的敲除效率,该方法可为耻垢分枝杆菌后续研究提供快速高效的基因组操作方法。     

Molecular and biochemical characterisation of Mycobacterium smegmatis alcohol dehydrogenase C

The gene encoding of an alcohol dehydrogenase C (ADHC) from Mycobacterium smegmatis was cloned and sequenced. The protein encoded by this gene has 78% identity with Mycobacterium tuberculosis and Mycobacterium bovis BCG ADHC. The M. smegmatis ADHC was purified from M. smegmatis and the kinetic parameters of this enzyme showed that using NADPH as electron donor it has a strong preference for aliphatic and aromatic aldehyde substrates. Like the M. bovis BCG ADHC, this enzyme is more likely to act as an aldehyde reductase than as an alcohol dehydrogenase. The discovery of such an ADHC in a fast-growing, and easily engineered mycobacterial species opens the way to the utilisation of this M. smegmatis enzyme as a convenient model for the study of the physiological role of this alcohol dehydrogenase in mycobacteria.     

A Histidine Aspartate Ionic Lock Gates the Iron Passage in Miniferritins from Mycobacterium smegmatis

Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8–2.2 Å for the various mutants to compare structural alterations vis à vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release.     

Identification of novel metronidazole-inducible genes in Mycobacterium smegmatis using a customized amplification library

The incidence of antibiotic resistance in pathogenic bacteria is rising. Bacterial resistance may be a natural defense of organisms, or it may result from spontaneous mutations or the acquisition of exogenous resistance genes. We grew spontaneous metronidazole-resistant Mycobacterium smegmatis mutants on solid medium cultures and employed differential expression using a customized amplification library to analyze the global gene profiles of metronidazole-resistant mutants under hypoxic conditions. In total, 66 genes involved in metronidazole resistance were identified and functionally characterized using the gene role category of M. smegmatis. Overall, genes associated with cell wall synthesis, such as methyltransferase and glycosyltransferase, and genes encoding drug transporters were highly expressed. The genes may be involved in the natural drug resistance of mycobacteria by increasing mycobacterial cell wall permeability and the efflux pumps of active drugs. In addition, the genes may play a role in dormancy. The genes identified in this study may lead to a better understanding of the mechanisms of metronidazole resistance during dormancy.     

结核杆菌活动期高表达基因Rv1776c的克隆及其在耻垢分枝杆菌中的表达

目的 构建表达结核分枝杆菌Rv1776c基因的重组耻垢分支杆菌,并鉴定该基因在重组耻垢分支杆菌中的活性。方法 采用PCR技术克隆结核分枝杆菌Rv1776c基因,构建大肠埃希菌‒分支杆菌穿梭表达质粒pMV-Rv1776c,通过酶切和测序鉴定其正确性,用电穿孔法将重组质粒转染到耻垢分支杆菌mc2155中。以SDS-PAGE及Western blot检测证实Rv1776c蛋白在重组耻垢分支杆菌内的表达。结果 重组耻垢分支杆菌构建成功,生长曲线说明重组质粒不会影响耻垢分支杆菌的体外生长;SDS-PAGE及Western blot检测证实Rv1776c在耻垢分枝杆菌内表达出相对分子量约56 kD的Rv1776c蛋白。结论 成功构建了Rv1776c基因的穿梭质粒pMV-Rv1776c,且该质粒在耻垢分枝杆菌内具有生物活性,为进一步研究其表达产物的功能提供基础。     

An efficient system for high-level expression and easy purification of authentic recombinant proteins

Expression of recombinant proteins as fusions to the eukaryotic protein ubiquitin has been found to significantly increase the yield of unstable or poorly expressed proteins. The benefit of this technique is further enhanced by the availability of naturally occurring deubiquitylating enzymes, which remove ubiquitin from the fusion product. However, the versatility of the system has been constrained due to the lack of a robust, easily purified deubiquitylating enzyme. Here we report the development of an efficient expression system, utilizing the ubiquitin fusion technique, which allows convenient high yield and easy purification of authentic protein. An Escherichia coli vector (pHUE) was constructed for the expression of proteins as histidine-tagged ubiquitin fusions, and a histidine-tagged deubiquitylating enzyme to cleave these fusions was expressed and purified. The expression system was tested using several proteins varying in size and complexity. These results indicate that this procedure will be suitable for the expression and rapid purification of a broad range of proteins and peptides, and should be amenable to high-throughput applications.     

流式细胞术检测胞内重组耻垢分枝杆菌的活力研究

目的建立一种快速检测胞内分枝杆菌活力的方法。方法将一定量培养至对数生长期的含pMV-eis的重组耻垢分枝杆菌感染U937巨噬细胞,以含空质粒的耻垢分枝杆菌为对照,吞噬作用2 h后洗去胞外细菌,再分别培养4、12、24和48 h后收集细胞并裂解之。获得的胞内细菌用FDA荧光染料染色后用流式细胞仪检测死亡率,并与平板菌落计数法进行比较。结果流式细胞仪检测出感染12 h后重组耻垢分枝杆菌胞内死亡率较对照组均有显著下降（P〈0.05）,流式细胞仪检测法与平板菌落计数法相比差异无统计学意义（P〉0.05）。结论流式细胞术与传统的平板计数法相比具有快速、敏感、方便的特点,可用于分枝杆菌活菌快速检测。     

Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa3

Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multidrug-resistant tuberculosis and eliminate pathogen in the dormant state. One family of drugs currently under development, the imidazo[1,2-a]pyridine derivatives, is believed to target the pathogen''s homolog of the mitochondrial bc₁ complex. This complex, denoted cytochrome bcc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, making it a good target for drug development. There is no soluble cytochrome c in mycobacteria to transport electrons from the bcc complex to cytochrome oxidase. Instead, the bcc complex exists in a “supercomplex” with a cytochrome aa₃-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa₃ supercomplex using a strain of M. smegmatis that has been engineered to express the M. tuberculosis cytochrome bcc. The resulting hybrid supercomplex is stable during extraction and purification in the presence of dodecyl maltoside detergent. It is hoped that this purification procedure will potentiate functional studies of the complex as well as crystallographic studies of drug binding and provide structural insight into a third class of the bc complex superfamily.     

绿茶粗提物对耻垢分枝杆菌的生长抑制研究

采用乙酸乙酯在中性条件下萃取绿茶,得到含有表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)的粗提物。通过纸片琼脂扩散法和细菌生长曲线来评价绿茶粗提物对耻垢分枝杆菌的抑菌效果,利用透射电子显微镜(Transmission electron microscopy,TEM)观察其对耻垢分枝杆菌细胞壁结构的影响。结果显示,绿茶粗提物对耻垢分枝杆菌生长产生明显抑制作用,且抑菌作用随着浓度的升高逐渐加强;经绿茶粗提物处理的耻垢分枝杆菌细胞壁呈现膨出、变形等形态学变化。因此,绿茶粗提物具有抑制耻垢分枝杆菌生长的功能,其作用机制可能与其主要成分EGCG影响细胞壁肽聚糖的生物合成有关。     

Isolation and characterization of carboxymycobactins as the second extracellular siderophores in Mycobacterium smegmatis

The structure of a chloroform-extractable siderophore from the supernatant of a nonpathogenic mycobacteria, Mycobacterium smegmatis, has been determined and it closely resembles the structure of mycobactin, the intracellular siderophore found in all mycobacteria. The difference in structures is that the extracellular siderophore has a family of short carboxylic chains attached to the mycobactin nucleus instead of a long alkyl chain and hence the name 'carboxymycobactin' is proposed to distinguish it from mycobactin itself as well as from the major siderophore that is produced - exochelin MS. © Rapid Science 1998.     

Expression,purification and functional characterization of AmiA of acetamidase operon of Mycobacterium smegmatis

Regulation of gene expression is one of the mechanisms of virulence in pathogenic organisms. In this context, we would like to understand the gene regulation of acetamidase enzyme of Mycobacterium smegmatis, which is the first reported inducible enzyme in mycobacteria. The acetamidase is highly inducible and the expression of this enzyme is increased 100-fold when the substrate acetamide is added. The acetamidase structural gene (amiE) is found immediately downstream of three predicted open reading frames (ORFs). Three of these genes along with a divergently expressed ORF are predicted to form an operon and involved in the regulation of acetamidase enzyme. Here we report expression, purification and functional characterization of AmiA which is one of these predicted ORFs. Electrophoretic mobility shift assays showed that AmiA binds to the region between the amiA and amiD near the predicted promoter (P2). Over-expression of AmiA significantly lowered the expression of acetamidase compared to the wild type as demonstrated by qRT-PCR and SDS-PAGE. We conclude that AmiA binds near P2 promoter and acts as a repressor in the regulation of acetamidase operon. The described work is a further step forward toward broadening the knowledge on understanding of the complex gene regulatory mechanism of Mycobacterium sp.