微生物VBNC状态形成及复苏机制

99%以上的微生物因处于活的但非可培养(viable but non-culturable,VBNC)状态而无法分离培养。复苏促进因子(resuscitation-promoting factors,Rpfs)是培养获取VBNC菌的最重要突破。结合课题组近十余年从环境功能视角利用Rpf复苏培养VBNC菌的研究,本文在阐述微生物VBNC状态的形成及复苏进展的基础上,从VBNC菌形成及复苏过程出发,探究"探索因子"与群体感应的内在关系。并总结了课题组利用Rpf所复苏培养的具有潜在环境功能的VBNC菌种。本论文将为揭示微生物VBNC状态的形成及复苏机制提供新的思路,并为认识和重新评价Rpf法复苏培养VBNC菌在污染环境微生物修复中的作用提供理论依据。     

藤黄微球菌Rpf活性蛋白的制取及其对红球菌VBNC菌体的复苏作用

【目的】克隆藤黄微球菌Micrococcus luteus IAM 14879(=NCIMB 13267)的复苏促进因子Rpf(resuscitation promoting factor)的基因,在大肠杆菌中表达获取基因重组蛋白,考察对近缘高GC革兰氏阳性菌红球菌Rhodococcus sp.DS471活的非可培养VBNC(viable but non-culturable)菌体的复苏促进生长能力。【方法】抽提制备藤黄微球菌的DNA,确定rpf基因引物进行PCR扩增,利用pET15b质粒载体并转化大肠杆菌DE3表达,以SDS-PAGE检验获取纯化重组蛋白;在培养基中添加Rpf,以MPN(most probable number)法计数、评价对VBNC状态菌体的复苏促进生长效果。【结果】基因测序证实获得藤黄微球菌的rpf基因并在大肠杆菌中表达;SDS-PAGE分析表明获得rpf基因的重组蛋白;该蛋白对处于VBNC状态的红球菌具有近100倍的复苏促进生长能力。【结论】成功克隆了藤黄微球菌的rpf基因,在大肠杆菌中获得了表达,表明了Rpf蛋白对处于VBNC状态的红球菌具有复苏促进生长效果。     

细菌复苏促进因子--Rpf

细菌复苏促进因子Rpf是第一个被发现的原核生物自分泌生长因子,该因子在放线菌门细菌中广泛存在,具有促进细菌休眠体复苏的作用.Rpf在细菌生长代谢过程中所发挥的重要调节作用,使其具有潜在的应用价值.     

活的但非可培养功能菌群对多氯联苯降解的探索进展

活的但非可培养(“viable but non-culturable”,简称VBNC)状态是微生物应对各种环境压力的一种生存机制.面对日益严重的异生质污染问题,研究污染胁迫下VBNC状态菌的潜在环境功能具有重要意义.该文阐述了VBNC状态菌的研究现状,针对多氯联苯微生物降解存在的问题,重点介绍环境中潜在VBNC功能菌群的复苏培养.提出利用藤黄球菌(Micrococcus luteus)复苏促进因子(resuscitation-promoting factor,简称Rpf)探索具有潜在多氯联苯降解功能的VBNC状态菌群,为多氯联苯高效降解菌群的筛选提供新思路.同时,结合VBNC状态菌絮凝、硝化除臭等方面的探索研究,对VBNC状态菌的潜在环境功能进行了展望.     

活的但非可培养(VBNC) 状态菌的研究进展

VBNC(viable but non-culturable)是指处于"活的但非可培养"状态的微生物,此微生物体的细胞仍有代谢活性,但用常规方法无法分离培养.本文阐述了VBNC状态菌的形成机理、转变与种类、复苏、研究意义及其应用展望.并报道了我们在十余年间针对生态环境中处于VBNC状态菌的复苏、可培养化、系统进化关系及潜在功能等方面的一些研究成果,拟为微生物资源的开发与应用提供新的科学依据.     

南极乔治王岛冰锥洞微生物培养探索

【目的】南极洲具备独特的环境和相对的生物地理隔离,南极洲各类生境中蕴藏了大量尚未培养和难培养的微生物,也是新颖微生物物种的重要来源之一。本研究以南极冰锥洞这类特殊生境为研究对象,通过培养条件的多样化提升南极微生物的培养率和多样性,揭示南极冰锥洞可培养微生物类群多样性,为该环境可培养微生物功能研究奠定基础,也为南极极端环境未培养微生物的培养方法提供借鉴。【方法】通过采用不同培养基添加复苏促进因子(resuscitation promoting factor, Rpf)的方式,提高南极柯林斯冰盖冰锥洞生境中微生物的可培养率,探究该生境中微生物的多样性。采用4种不同营养水平的培养基,平行添加Rpf进行菌株培养,经分离纯化与16S rRNA基因鉴定,分析冰锥洞可培养微生物的多样性及培养条件对多样性的影响。【结果】本研究共分离培养细菌407株,涵盖5个门、18个科、29个属,其中：放线菌门(Actinomycetota)为优势门,占72.73%;微杆菌科(Microbacteriaceae)为优势科,占69.78%;Lacisediminihabitans属为优势属,占45.70%。从培养基效果...     

来源于Micrococcus luteus的复苏促进因子异源表达及其对活性污泥中休眠细菌的促生长作用

【背景】复苏促进因子(Resuscitation promoting factor,RPF)是由某些放线菌分泌的一类活性蛋白质,最初在Micrococcusluteus中被发现。它不但能够复苏休眠细菌恢复生长,而且对正常的细菌也有促生长效果。然而关于RPF对于环境样品中除原始宿主以外细菌的复苏促进作用研究还鲜有报道。【目的】以寻找新的菌种资源、开发环境功能菌角度研究了RPF对环境样品中潜在休眠菌群的复苏促进作用。【方法】首先对来源于Micrococcus luteus IAM 14879的RPF(Ml RPF)蛋白序列进行生物信息学分析和同源模建;随后克隆了Ml RPF编码基因并在大肠杆菌中异源表达;最后利用重组Ml RPF对活性污泥样品中潜在的休眠细菌开展了复苏促生长研究。【结果】Ml RPF在进化树上处于单独分支,远离其它RPF家族成员;模建结构分析表明Ml RPF具有与C型溶菌酶相似的催化结构域,这有助于解释RPF家族蛋白具有溶解酶活性;重组Ml RPF在大肠杆菌中获得了可溶性表达,它能够促进休眠态M. luteus IAM 14879生长,保留了其生理活性;利用重组Ml RPF从某污水厂活性污泥样品中分离获得了Dietzia、Paracoccus、Rhodococcus和Brevundimonas菌株。【结论】为环境样品中微生物多样性研究及特殊微生物资源的发掘提供新的方法,也为基于Ml RPF的废水生物强化处理技术研发提供理论依据。     

自然界中处于VBNC状态微生物的研究进展

自然界中大约有90％以上的微生物尚未被分离鉴定。80年代初提出VBNC状态微生物的存在,所谓的VBNC是指微生物在自然界中生存的一种状态,本综述了在实验室条件下VBNC状态的形成条件、培养性的恢复及处于VBNC状态微生物的检测方法等方面的研究进展,并介绍了第一个微生物生长因子Rpf.     

结核分枝杆菌RPF样蛋白家族及其功能

上世纪末,Mukamolova等从藤黄微球菌（Micrococcus luteus）发现一种分泌蛋白-复苏促进因子（Rpf）.该因子在皮摩尔浓度下能促进休眠期的藤黄微球菌及其他数种高G＋C含量的革兰氏阳性菌复苏和生长.结核分枝杆菌全基因组发现和该因子同源性较高的5个基因,预测产物都是分泌蛋白,一级结构都含有一个转糖基域.5个基因有一个共同的功能未知的域,3级结构预测该保守结构域和溶菌酶同源.重组表达的Rpf对不同种均有活性.5个rpf在不同生长时期均有表达,但调控有差异.     

自然界中处于VBNC状态微生物的研究进展*

自然界中大约有 90 %以上的微生物尚未被分离鉴定。 80年代初提出VBNC状态微生物的存在 ,所谓的VBNC是指微生物在自然界中生存的一种状态 ,本文综述了在实验室条件下VBNC状态的形成条件、培养性的恢复及处于VBNC状态微生物的检测方法等方面的研究进展 ,并介绍了第一个微生物生长因子Rpf。     

Structural Changes and Cellular Localization of Resuscitation-Promoting Factor in Environmental Isolates of Micrococcus luteus

Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in “spent culture medium.” Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.     

Rpf proteins are the factors of reactivation of the dormant forms of actinobacteria

As the response to unfavorable growth conditions, nonsporulating mycobacteria transform into the dormant state with the concomitant formation of the specialized dormant forms characterized by low metabolic activity and resistance to antibiotics. Such dormant cells can be reactivated under the influence of several factors including proteins of Rpf (Resuscitation promoting factor) family, which possess peptidoglycan hydrolase activity and were considered to belong to the group of the autocrine growth factors of the bacteria. Remarkable interest toward Rpf family is determined by its par-ticipation in resuscitation of the dormant forms of Mycobacterium tuberculosis, what in turn is the key element in resuscitation of the latent tuberculosis – an infectious disease that affects one third of the World’s population. Experiments with Rpf mutant forms and with strains deleted in these proteins revealed a relationship between the enzymatic activity of this protein and its ability to resuscitate mycobacteria both in vitro and in vivo. This review discusses possible mechanisms of Rpf action including those related to possible participation of the products of mycobacterial Rpf-mediated cell wall hydrolysis (muropeptides) as signaling molecules. The unique ability of Rpf proteins to resuscitate the dormant forms of mycobacteria and to stimulate their proliferation would allow these proteins to occupy their niche in medicine–in diagnostics and in creation of antituberculosis subunit vaccines.     

Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

Background

Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs.

Methodology/Principal Findings

Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC₅₀ 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC₅₀. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations.

Conclusions/Significance

NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.     

Resuscitation Promoting Factors: a Family of Microbial Proteins in Survival and Resuscitation of Dormant Mycobacteria

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an extraordinarily successful pathogen of humankind. It has been estimated that up to one-third of the world’s population is infected with M. tuberculosis, and this population is an important reservoir for disease reactivation. Resuscitation promoting factor (Rpf) is a secretory protein, which was first reported in Micrococcus luteus. There are five functionally redundant Rpf-like proteins found in M. tuberculosis. Rpf promotes the resuscitation of dormant bacilli to yield normal, viable colony forming bacteria. All Rpfs share a conserved domain of about 70 amino acids and possess a lysozyme-like activity. The structural studies of the conserved domain suggest that Rpfs could be considered as a c-type lysozyme and lytic transglycosylases. Recently a novel class of nitrophenylthiocyanates (NPT) inhibitors of the muralytic activity of Rpf were reported which opens a new approach in the study of cell-wall hydrolyzing enzymes. This review describes molecular and structural studies conducted on Rpf proteins, their role in the resuscitation of dormant bacteria, in the reactivation of latent infection and identification of low molecular weight inhibitors of resuscitation promoting factors.     

Resuscitation of viable but non‐culturable bacteria to enhance the cellulose‐degrading capability of bacterial community in composting

Nowadays, much of what we know regarding the isolated cellulolytic bacteria comes from the conventional plate separation techniques. However, the culturability of many bacterial species is controlled by resuscitation‐promoting factors (Rpfs) due to entering a viable but non‐culturable (VBNC) state. Therefore, in this study, Rpf from Micrococcus luteus was added in the culture medium to evaluate its role in bacterial isolation and enhanced effects on cellulose‐degrading capability of bacterial community in the compost. It was found that Proteobacteria and Actinobacteria were two main phyla in the compost sample. The introduction of Rpf could isolate some unique bacterial species. The cellulase activity of enrichment cultures with and without Rpf treatment revealed that Rpf treatment significantly enhanced cellulase activity. Ten isolates unique in Rpf addition displayed carboxymethyl‐cellulase (CMCase) activity, while six isolates possessed filter paper cellulase (FPCase) activity. This study provides new insights into broader cellulose degraders, which could be utilized for enhancing cellulosic waste treatment.     

Evidence against dormacy in the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida

Results indicate that A. salmonicida does not enter an unculturable dormant state. The resuscitation of dormant cells by nutrient broth described by previous workers appears to be due to the presence of small numbers of viable, culturable cells too few to be detected by the sampling protocol employed.     

Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.     

Exploring the potential environmental functions of viable but non-culturable bacteria

A conventional plate count is the most commonly employed method to estimate the number of living bacteria in environmental samples. In fact, judging the level of viable culture by plate count is limited, because it is often several orders of magnitude less than the number of living bacteria actually present. Most of the bacteria are in “viable but non-culturable” (VBNC) state, whose cells are intact and alive and can resuscitate when surrounding conditions are more favorable. The most exciting recent development in resuscitating VBNC bacteria is a bacterial cytokine, namely, the resuscitation-promoting factor (Rpf), secreted by Micrococcus luteus, which promotes the resuscitation and growth of high G+C Gram-positive organisms, including some species of the genus Mycobacterium. However, most of studies deal with VBNC bacteria only from the point of view of medicine and epidemiology. It is therefore of great significance to research whether these VBNC state bacteria also possess some useful environmental capabilities, such as degradation, flocculation, etc. Further studies are needed to elucidate the possible environmental role of the VBNC bacteria, rather than only considering their role as potential pathogens from the point view of epidemiology and public health. We have studied the resuscitation of these VBNC bacteria in polluted environments by adding culture supernatant containing Rpf from M. luteus, and it was found that, as a huge microbial resource, VBNC bacteria could provide important answers to dealing with existing problems of environmental pollution. This mini-review will provide new insight for considering the potentially environmental functions of VBNC bacteria.