Presence of mycobacteriophage I3-like DNA sequences in the genome of its host Mycobacterium smegmatis

The genomes of phage I3 and its host Mycobacterium smegmatis have been compared. From thermal melting studies the GC contents of DNA from mycobacteriophage I3 and its host M. smegmatis were found to be 66%. A new method, based only on the initial rates of reassociation, has been developed for calculating the DNA homology. Analysis of DNA reassociation kinetics suggested the presence of one equivalent of the phage I3 genome within the M. smegmatis genome. Southern analysis revealed the presence of almost all of the phage I3 specific sequences within the host genome.     

Metabolism of cyclic AMP in non-pathogenic Mycobacterium smegmatis

The intracellular concentration of cyclic AMP reached a maximum in 3.5-day old cultures of Mycobacterium smegmatis grown in the presence of glycerol as the main source of carbon. Glucose-grown cells exhibited decreased cyclic AMP levels at all stages of growth. When M. smegmatis cells were incubated with various metabolites, pyruvate increased whereas glucose, citric acid, succinic acid and lactic acid decreased intracellular cyclic AMP levels. No cyclic AMP was detected in the incubation medium. The presence of a cyclic AMP-binding protein was demonstrated in cellfree extracts of M. smegmatis.     

Turnover of lipids in Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354

The rates of breakdown and renewal of individual lipids in cultures of Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354 were investigated by means of a pulse labelling technique using palmitate-1-¹⁴C. The results indicated that in growing cultures of both strains phospholipids were broken down, and cardiolipin had a very rapid turnover. In chase experiments, almost 45% and 40% of the radioactivity of this component were lost respectively from M. smegmatis and M. phlei during one generation time of the cell. The other two major components, phosphatidyl ethanolamine and phosphatidylinositol mannosides showed relatively low turnover. The loss of radioactivity from phosphatidylinositol mannosides was greater in M. phlei than in M. smegmatis but the loss of radioactivity from phosphatidyl ethanolamine was higher in M. smegmatis. The pattern of loss of radioactivity from lipids was almost the same in both strains, the difference being only in the extent of loss. The differences in the cellular localization of the phospholipids indicate their different roles within the cell. Results obtained with the glyceride fraction indicated a very rapid turnover of triglycerides in both strains.Abbreviations CL Cardiolipin - PE Phosphatidyl ethanolamine - PIMx phosphatidylinositol mannosides - PIM₂A phosphatidylinositol dimannoside tetra acylated - PIM₂B phosphatidylinositol dimannoside tri acylated - PIM₅ phosphatidylinositol pentamannoside tetra acylated     

Inhibition of DNA gyrase activity by Mycobacterium smegmatis MurI

Glutamate racemase (MurI) catalyzes the interconversion of l-glutamate to d-glutamate, one of the essential amino acids present in the peptidoglycan. In addition to this essential enzymatic function, MurI from Escherichia coli, Bacillus subtilis and Mycobacterium tuberculosis inhibit DNA gyrase activity. A single gene for murI found in the Mycobacterium smegmatis genome was cloned and overexpressed in a homologous expression system to obtain a highly soluble enzyme. In addition to the racemization activity, M. smegmatis MurI inhibits DNA gyrase activity by preventing DNA binding of gyrase. The sequestration of the gyrase by MurI results in inhibition of all reactions catalyzed by DNA gyrase. More importantly, MurI overexpression in vivo in mycobacterial cells provides protection against the action of ciprofloxacin. The DNA gyrase-inhibitory property thus appears to be a typical characteristic of MurI and would have probably evolved to either modulate the function of the essential housekeeping enzyme or to provide protection to gyrase against gyrase inhibitors, which cause double-strand breaks in the genome.     

Growth ofMycobacterium smegmatis in minimal and complete media

The growth patterns ofMycobacterium smegmatis SN2 in a minimal medium and in nutrient broth have been compared. The growth was monitored by absorbancy (Klett readings), colony forming units, wet weight and content of DNA, RNA and protein. During the early part of the growth cycle, the bacteria had higher wet weight and macromolecular content in nutrient broth than in minimal media. During the latter half of the growth cycle however, biosynthesis stopped much earlier in nutrient broth and the bacteria had a much lower content of macromolecules than in the minimal medium. In both the media, a general pattern of completing biosynthesis rapidly in the initial phase and a certain amount of cell division at a later time involving the distribution of preformed macromolecules was seen. The possible adaptive significance of this observation has been discussed.     

Evaluation of the role of sigma B in Mycobacterium smegmatis

The alternate sigma factor, sigB, is known to play a crucial role in maintaining the stationary phase in mycobacteria. In this communication, we have studied the proteomics of Mycobacterium smegmatis mc(2)155 and its two derivatives, one of which has a disrupted sigB gene and the other, PMVSigB, which contains a multicopy plasmid containing sigB. We have identified by two-dimensional gel analyses, several proteins that are over-expressed in PMVSigB compared to mc(2)155. These proteins are either stress proteins or participate actively in different metabolic pathways of the organisms. On the other hand, when sigB deleted mycobacteria were grown until the stationary phase and its two-dimensional protein profile was compared to that of mc(2)155, few DNA binding proteins were found to be up-regulated. We have shown recently that upon over-expressing sigB, the cell surface glycopeptidolipids of M. smegmatis are hyperglycosylated, a situation similar to what was observed for nutritionally starved bacteria. Gene expression profile through quantitative PCR presented here identified a Rhamnosyltransferase responsible for this hyperglycosylation.     

Development of a method to investigate the hydrolysis of xenobiotic esters by a Mycobacterium smegmatis homogenate

One of the main problems in combating tuberculosis is caused by a poor penetration of drugs into the mycobacterial cells. A prodrug approach via activation inside mycobacterial cells is a possible strategy to overcome this hurdle and achieve efficient drug uptake. Esters are attractive candidates for such a strategy and we and others communicated previously the activity of esters of weak organic acids against mycobacteria. However very little is known about ester hydrolysis by mycobacteria and no biological model is available to study the activation of prodrugs by these microorganisms. To begin filling this gap, we have embarked in a project to develop an in vitro method to study prodrug activation by mycobacteria using Mycobacterium smegmatis homogenates. Model ester substrates were ethyl nicotinate and ethyl benzoate whose hydrolysis was monitored and characterized kinetically. Our studies showed that in M. smegmatis most esterase activity is associated with the soluble fraction (cytosol) and is preserved by storage at 5 °C or at room temperature for one hour, or by storage at − 80 °C up to one year. In the range of homogenate concentrations studied (5-80% in buffer), k_obs varied linearly with homogenate concentration for both substrates. We also found that the homogenates showed Michaelis-Menten kinetics behavior with both prodrugs. Since ethyl benzoate is a good substrate for the mycobacterial esterases, this compound can be used to standardize the esterasic activity of homogenates, allowing results of incubations of prodrugs with homogenates from different batches to be readily compared.     

Identification of small RNAs in Mycobacterium smegmatis using heterologous Hfq

Gene regulation by small RNAs (sRNAs) has been extensively studied in various bacteria. However, the presence and roles of sRNAs in mycobacteria remain largely unclear. Immunoprecipitation of RNA chaperone Hfq to enrich for sRNAs is one of the effective methods to isolate sRNAs. However, the lack of an identified mycobacterial hfq restricts the feasibility of this approach. We developed a novel method that takes advantage of the conserved inherent sRNAs-binding capability of heterologous Hfq from Escherichia coli to enrich sRNAs from Mycobacterium smegmatis, a model organism for studying Mycobacterium tuberculosis. We validated 12 trans-encoded and 12 cis-encoded novel sRNAs in M. smegmatis. Many of these sRNAs are differentially expressed at exponential phase compared with stationary phase, suggesting that sRNAs are involved in the growth of mycobacteria. Intriguingly, five of the cis-encoded novel sRNAs target known transposases. Phylogenetic conservation analysis shows that these sRNAs are pathogenicity dependent. We believe that our findings will serve as an important reference for future analysis of sRNAs regulation in mycobacteria and will contribute significantly to the development of sRNAs prediction programs. Moreover, this novel method of using heterologous Hfq for sRNAs enrichment can be of general use for the discovery of bacterial sRNAs in which no endogenous Hfq is identified.     

Transfection of Mycobacterium smegmatis SN2 with mycobacteriophage I3 DNA

Mycobacterium smegmatis SN2 does not exhibit natural competence for the uptake of phage I3 DNA. Competence can artificially be induced by treatment with glycine or CaCl₂, and the combination of both is even more effective. The efficiency of transfection can be improved by inclusion of protamine sulphate and heterologous RNA in the system. From ³²P DNA uptake studies the major barrier for the entry of DNA has been found to be the complex cell wall. The efficiency of transfection calculated on the basis of fraction of DNA which has entered the cell is comparable to that of other bacterial systems. The phage development takes a longer time (7 h for one cycle) after transfection, as compared to infection (4 h).     

Bacterioferritin from Mycobacterium smegmatis contains zinc in its di-nuclear site

Bacterioferritins, also known as cytochrome b (1), are oligomeric iron-storage proteins consisting of 24 identical amino acid chains, which form spherical particles consisting of 24 subunits and exhibiting 432 point-group symmetry. They contain one haem b molecule at the interface between two subunits and a di-nuclear metal binding center. The X-ray structure of bacterioferritin from Mycobacterium smegmatis (Ms-Bfr) was determined to a resolution of 2.7 A in the monoclinic space group C2. The asymmetric unit of the crystals contains 12 protein molecules: five dimers and two half-dimers located along the crystallographic twofold axis. Unexpectedly, the di-nuclear metal binding center contains zinc ions instead of the typically observed iron ions in other bacterioferritins.     

结核分枝杆菌耐药相关单碱基突变的计算方法比较

[目的] 耐药结核分枝杆菌（drug-resistant Mycobacterium tuberculosis）的产生给结核病（tuberculosis）的治疗带来巨大困难。[方法] 使用基于全基因组测序的关联分析探究耐药强相关的单核苷酸多态性（single nucleotide polymorphism,SNP）突变,主要有GEMMA、phyc、plink。为了阐明其中最优的耐药相关SNP计算方法,本研究下载NCBI上已有的1504株结核分枝杆菌数据,并获取它们对于3种常见的一线抗结核治疗药物（isoniazid、rifampicin、ethambutol）的耐药性检验结果。并使用这3种耐药相关SNP计算方法计算与结核分枝杆菌耐药相关的SNP;并评估计算得到的耐药相关SNP在预测耐药表型的敏感性和特异性。[结果] 发现通过phyc可以预测到最多的已知耐药相关SNP和最少的耐药无关SNP,而且phyc预测的耐药相关SNP的敏感性和特异性恒定大于52.49%。[结论] phyc在预测结核分枝杆菌耐药相关SNP中结果最准确,但考虑到运行时间和表型数据的更新,GEMMA和plink的结果也应作为参考。     

副干酪乳杆菌PC-01全基因组测序及不同副干酪乳杆菌菌株比较基因组学分析

【背景】副干酪乳杆菌(Lactobacillus paracasei)作为乳酸菌中重要菌种之一,常被认为是优良益生菌开发的潜在资源。【目的】以L.paracasei PC-01和L.paracasei Zhang为例,分析不同L.paracasei的基因组差异和遗传背景,为菌株的鉴定和开发奠定基础。【方法】采用PacBioSMRT三代测序技术对L.paracasei PC-01进行全基因组测序,结合2株L.paracasei模式菌株和公开的36株全基因组数据,通过比较基因组学方法揭示39株L.paracasei菌株之间的差异。【结果】L.paracasei PC-01基因组不包含质粒,染色体大小为2 829 251 bp,GC含量为46.64%;L.paracasei Zhang包含一个质粒基因组大小为2 898 456 bp,GC含量为46.51%;不同L.paracasei菌株基因组大小、质粒数及GC含量均存在一定差异。L.paracasei群体为开放式基因组,基因组具有高度多样性。基于核心基因构建系统发育树对于L.paracasei种内区分效果最好,L.paracasei PC-...     

Biosynthesis of d-arabinose in Mycobacterium smegmatis: specific labeling from d-glucose

d-Arabinose is a major sugar in the cell wall polysaccharides of Mycobacterium tuberculosis and other mycobacterial species. The reactions involved in the biosynthesis and activation of d-arabinose represent excellent potential sites for drug intervention since d-arabinose is not found in mammalian cells, and the cell wall arabinomannan and/or arabinogalactan appear to be essential for cell survival. Since the pathway involved in conversion of d-glucose to d-arabinose is unknown, we incubated cells of Mycobacterium smegmatis individually with [1-(14)C]glucose, [3,4-(14)C]glucose, and [6-(14)C]glucose and compared the specific activities of the cell wall-bound arabinose. Although the specific activity of the arabinose was about 25% lower with [6-(14)C]glucose than with other labels, there did not appear to be selective loss of either carbon 1 or carbon 6, suggesting that arabinose was not formed by loss of carbon 1 of glucose via the oxidative step of the pentose phosphate pathway, or by loss of carbon 6 in the uronic acid pathway. Similar labeling patterns were observed with ribose isolated from the nucleic acid fraction. Since these results suggested an unusual pathway of pentose formation, labeling studies were also done with [1-(13)C]glucose, [2-(13)C]glucose, and [6-(13)C]glucose and the cell wall arabinose was examined by NMR analysis. This method allows one to determine the relative (13)C content in each carbon of the arabinose. The labeling patterns suggested that the most likely pathway was condensation of carbons 1 and 2 of fructose 6-phosphate produced by the transaldolase reaction with carbons 4, 5, and 6 (i.e., glyceraldehyde 3-phosphate) formed by fructose-1,6 bisphosphate aldolase. Cell-free enzyme extracts of M. smegmatis were incubated with ribose 5-phosphate, xylulose 5-phosphate, and d-arabinose 5-phosphate under a variety of experimental conditions. Although the ribose 5-phosphate and xylulose 5-phosphate were converted to other pentoses and hexoses, no arabinose 5-phosphate (or free arabinose) was detected in any of these reactions. In addition, these enzyme extracts did not convert arabinose 5-phosphate to any other pentose or hexose. In addition, incubation of [(14)C]glucose 6-phosphate and various nucleoside triphosphates (ATP, CTP, GTP, TTP, and UTP) with cytosolic or membrane fractions from the mycobacterial cells did not result in formation of a nucleotide form of arabinose, although other radioactive sugars including rhamnose and galactose were found in the nucleotide fraction. Furthermore, no radioactive arabinose was found in the nucleotide fraction isolated from M. smegmatis cells grown in [(3)H]glucose, nor was arabinose detected in a large-scale extraction of the sugar nucleotide fraction from 300 g of cells. The logical conclusion from these studies is that d-arabinose is probably produced from d-ribose by epimerization of carbon 2 of the ribose moiety of polyprenylphosphate-ribose to form polyprenylphosphate-arabinose, which is then used as the precursor for formation of arabinosyl polymers.     

The animal in the genome: comparative genomics and evolution

Comparisons between completely sequenced metazoan genomes have generally emphasized how similar their encoded protein content is, even when the comparison is between phyla. Given the manifest differences between phyla and, in particular, intuitive notions that some animals are more complex than others, this creates something of a paradox. Simplistic explanations have included arguments such as increased numbers of genes; greater numbers of protein products produced through alternative splicing; increased numbers of regulatory non-coding RNAs and increased complexity of the cis-regulatory code. An obvious value of complete genome sequences lies in their ability to provide us with inventories of such components. I examine progress being made in linking genome content to the pattern of animal evolution, and argue that the gap between genomic and phenotypic complexity can only be understood through the totality of interacting components.     

Superoxide dismutase from Mycobacterium species,strain Takeo

Superoxide dismutase from Mycobacterium species, strain Takeo, has been purified to homogeneity as judged by disc gel electrophoresis and ultracentrifugation. The enzyme was found to have a molecular weight of approximately 61 500 by sedimentation equilibrium and to contain manganese by atomic absorption and electron spin resonance spectra. The amino acid composition was also determined. The enzyme was considerably stable to the treatment with sodium dodecyl sulfate; unless incubating at 80°C for 2 min, it was not completely dissociated into the subunits. The molecular weight of the subunit was found to be approximately 21 000. Antibodies against the superoxide dismutase were produced by immunization of rabbits with the enzyme, and the -globulin fraction was purified. Superoxide dismutase preparations obtained from various species of mycobacteria and nocardia cross-reacted to different degrees with these antibodies on the Ouchterlony double diffusion plates. Comparative immunological studies indicated that strain Takeo might be most closely related to Myobacterium smegmatis among species of mycobacteria and nocardia tested. The antibodies against superoxide dismutase may be used as a valuable tool for the classification of mycobacteria.     

五株克雷伯氏菌噬菌体的生物学特性及比较基因组学研究

【目的】耐药性克雷伯氏菌属(Klebsiella)的细菌作为人类感染的重要病原,是临床治疗重要的挑战。本研究对多株克雷伯氏菌裂解性噬菌体的生物学特性和基因组特征进行比较分析,为其应用提供更多科学数据。【方法】使用双层平板法从人类和动物新鲜粪便、污水中分离纯化裂解性克雷伯氏菌噬菌体;通过磷钨酸染色和透射电镜观察其形态;采用双层平板噬菌斑法确定其宿主范围,测定温度和pH稳定性、一步生长曲线和体外抑菌效果等生物学特性;基于全基因组测序对分离株进行比较基因组学分析;通过体内抑菌试验评估噬菌体对多重耐药变栖克雷伯氏菌(Klebsiella variicola)BS375-3感染的大蜡螟(Galleria mellonella)幼虫的保护作用。【结果】5株噬菌体分别属于Schitoviridae(pKP-BM327-1.2)、Autographiviridae(pKP-M186-2.1、pKP-M186-2.2和pKV-BS375-3.1)、Drexlerviridae(pKP-BS317-1.1)家族;噬菌体pKV-BS375-3.1可裂解受试菌中的8株,pKP-BM327-1.2可裂解受试菌中的3株,pKP-M186-2.1、pKP-M186-2.2和pKP-BS317-1.1则分别裂解受试菌中的1株;5株噬菌体感染10–20 min后即进入指数增长期,在–20–37℃、pH 6–10环境下均能够保持稳定活性;感染变栖克雷伯氏菌BS375-3后经噬菌体pKV-BS375-3.1处理[感染复数(multiplicity of infection,MOI)=100]的大蜡螟幼虫96 h内存活率达到80%(8/10);5株噬菌体基因组长度在42–77 kb之间,未携带抗生素抗性基因和毒力基因,基于内溶素(endolysin)的溯源分析显示该蛋白在克雷伯氏菌噬菌体中呈现多样性,属内呈保守性。【结论】5株克雷伯氏菌噬菌体均具有较好的体外抑菌活性,生物学特性稳定,endolysin在噬菌体属内呈现保守性。宿主谱宽、潜伏期短的噬菌体pKV-BS375-3.1在治疗Klebsiella pneumoniae和K.variicola临床感染方面具有潜在应用前景。     

Snapshots of RecA protein involving movement of the C-domain and different conformations of the DNA-binding loops: crystallographic and comparative analysis of 11 structures of Mycobacterium smegmatis RecA

Mycobacterium smegmatis RecA and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals reported here and five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P6(1). They include one obtained by reducing relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 6(1) screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported for Escherichia coli RecA, the variation in the pitch among the three forms correlates well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue, which is believed to provide the trigger for transmitting the effect of nucleotide binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue similar to that caused by nucleotide binding. The ordering of the DNA-binding loops, which present ensembles of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide binding, presumably on account of the movement of the switch residue that forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications for intermolecular communications within the RecA filament. The structures resulting from different orientations of the C domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA at different phases of activity, and provide insights into the mechanism of action of RecA.     

Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer

The genus Mycobacterium comprises significant pathogenic species that infect both humans and animals. One species within this genus, Mycobacterium tuberculosis, is the primary killer of humans resulting from bacterial infections. Five mycobacterial genomes belonging to four different species (M. tuberculosis, Mycobacterium bovis, Mycobacterium leprae and Mycobacterium avium ssp. paratuberculosis) have been sequenced to date and another 14 mycobacterial genomes are at various stages of completion. A comparative analysis of the gene products of key metabolic pathways revealed that the major differences among these species are in the gene products constituting the cell wall and the gene families encoding the acidic glycine-rich (PE/PPE/PGRS) proteins. Mycobacterium leprae has evolved by retaining a minimal gene set for most of the gene families, whereas M. avium ssp. paratuberculosis has acquired some of the virulence factors by lateral gene transfer.     

Functional Analysis of a c-di-AMP-specific Phosphodiesterase MsPDE from Mycobacterium smegmatis

Cyclic di‑AMP (c-di-AMP) is a second signaling molecule involved in the regulation of bacterial physiological processes and interaction between pathogen and host. However, the regulatory network mediated by c-di-AMP in Mycobacterium remains obscure. In M. smegmatis, a diadenylate cyclase (DAC) was reported recently, but there is still no investigation on c-di-AMP phosphodiesterase (PDE). Here, we provide a systematic study on signaling mechanism of c-di-AMP PDE in M. smegmatis. Based on our enzymatic analysis, MsPDE (MSMEG_2630), which contained a DHH-DHHA1 domain, displayed a 200-fold higher hydrolytic efficiency (k_cat/K_m) to c-di-AMP than to c-di-GMP. MsPDE was capable of converting c-di-AMP to pApA and AMP, and hydrolyzing pApA to AMP. Site-directed mutations in DHH and DHHA1 revealed that DHH domain was critical for the phosphodiesterase activity. To explore the regulatory role of c-di-AMP in vivo, we constructed the mspde mutant (Δmspde) and found that deficiency of MsPDE significantly enhanced intracellular C₁₂-C₂₀ fatty acid accumulation. Deficiency of DAC in many bacteria results in cell death. However, we acquired the M. smegmatis strain with DAC gene disrupted (ΔmsdisA) by homologous recombination approach. Deletion of msdisA reduced bacterial C₁₂-C₂₀ fatty acids production but scarcely affected bacterial survival. We also provided evidences that superfluous c-di-AMP in M. smegmatis could lead to abnormal colonial morphology. Collectively, our results indicate that MsPDE is a functional c-di-AMP-specific phosphodiesterase both in vitro and in vivo. Our study also expands the regulatory network mediated by c-di-AMP in M. smegmatis.