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.     

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.     

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。     

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。     

广西蚕沙细菌组成多样性解析和VBNC菌群的发掘

【目的】解析广西蚕沙中细菌群落组成与多样性,为蚕沙中菌种资源发掘和蚕沙的综合利用提供科学依据。【方法】通过高通量测序技术研究细菌群落组成特征,同时利用常规稀释涂布平板法和基于复苏促进因子(Rpf)的MPN培养系统解析并筛选蚕沙中可培养和活的非可培养(VBNC)状态的优势菌群,并经16SrRNA基因测序对筛选得到的菌株作初步分类鉴定。【结果】高通量测序表明,广西蚕沙样品中细菌归属于10个门、18个纲、27个目、57个科、96个属,其中4个属的丰度达1%以上,优势菌群为变形菌门(Proteobacteria)肠杆菌属(Enterobacter);通过稀释涂布平板法共获得14个属的33株可培养细菌,其中4个属(Citrobacter、Weissella、Chitinophaga、Pseudoclavibacter)在高通量测序中未被检测到;而在MPN培养系统中,基于复苏促进因子的处理组细菌总数最大检出丰度提高了100倍,并从中共检出21株对Rpf敏感的VBNC菌株,其中6个属(Paenibacillus、Caulobacter、Roseomonas、Pantoea、Erwinia、Acine...     

细菌有活力但不可培养状态及其机制研究进展

有活力但不可培养(viable but non-culturable,VBNC)状态是细菌遭遇逆境时进入的一种特殊状态,该状态下的菌体在条件适宜时可复苏并恢复其致病性,被认为是细菌躲避不良环境的一种生存策略。VBNC状态菌体对人类医学和工农业生产具有巨大的潜在威胁,开展关于VBNC状态的检测及诱导、复苏及其机制研究可为减少或避免该状态细菌的危害提供理论基础。本文简要综述了细菌VBNC状态在诱导、复苏及致病性等方面的研究进展,并结合本实验室及国内外相关团队近年来在植物病原细菌VBNC状态研究中的结果,详细总结了VBNC状态细菌的形成和复苏机制,对植物病原细菌在环境胁迫下的存活机制、病害田间初侵染来源分析及VBNC状态菌体在病害循环中的作用等相关研究具有重要参考意义。     

氧化胁迫诱导苍白杆菌JP1形成VBNC状态及其特征

石油降解菌在各种有害环境因素作用下会进入活的非可培养(viable but non-culturable, VBNC)状态，从而影响其生长及石油降解率。为了研究有害环境因素对石油降解菌生长及石油降解率的影响，采用分光光度法、荧光染色-激光共聚焦显微镜观测H₂O₂胁迫下苍白杆菌(Ochrobactrum sp.)JP1细胞的生长及VBNC状态形成情况。结果表明，不同浓度H₂O₂对其生长有一定抑制作用，当培养液中H₂O₂浓度为75.0 mmol/L时，可有效抑制苍白杆菌JP1生长，处理12 h后苍白杆菌JP1进入VBNC状态。VBNC状态的苍白杆菌JP1细胞缩小变成球体，周质间隙增大；在适宜条件下，VBNC状态苍白杆菌JP1能够复苏为可培养状态，添加丙酮酸钠能够促进VBNC状态细菌细胞的复苏。复苏后的苍白杆菌RJP1具有良好的环境适应性和石油降解能力，为石油污染生物修复的菌种筛选及应用提供了新的策略。     

Rpf蛋白：一种激活休眠状态放线菌的复苏促进因子

放线菌能产生多样性丰富的小分子化合物,但大多数放线菌因为处于“活的尚未培养”状态而无法分离培养。造成“活的尚未培养”状态的原因之一可能是由于一些环境因素,例如有机物、重金属、抗生素等胁迫使细胞处于休眠保护状态,直到遇到适宜的条件才能继续复苏生长。复苏促进因子（Resuscitation-Promoting Factor,Rpf）是由某些放线菌分泌的一类蛋白质,首次在藤黄微球菌（Micrococcus luteus）中发现,之后人们对Rpf蛋白的功能和分布给予了更多的关注。Rpf蛋白能促进一些休眠的革兰氏阳性细菌复苏,这为“活的尚未培养”放线菌的分离培养提供了可能性。同时,针对一些致病放线菌物种,开发Rpf蛋白抑制物为相关疾病的治疗也提供了一条新的途径。基于此,本文对Rpf蛋白的结构组成、特征功能、作用机制及应用前景进行了简要的概述。     

空肠弯曲菌活而不可培养状态的产毒素能力及毒素基因表达的检测

目的 通过诱导空肠弯曲菌进入活的不可培养(VBNC)状态建立细胞学模型,用VeroE6细胞和HeLa细胞检测空肠弯曲菌产细胞膨胀性肠毒素(cytotoxic distendingtoxin,CDT)能力的改变,并在RNA水平上进一步验证毒素的表达。方法 采用4℃冷藏及-70℃冷冻的方法诱导空肠弯曲菌进入VBNC状态,利用细胞总数计算、活细胞染色计数及可培养细胞计数间接确证VBNC状态细菌的存在,细胞对于VBNC状态下的细菌仍然敏感,并用反转录-聚合酶链反应(RT-PCR)证实空肠弯曲菌毒素基因的表达。结果 3种不同状态的细胞计数结果表明存活细胞进入了VBNC状态,细菌经4℃冷藏及-70℃冷冻后数量减少约1个数量级,在冷冻前,细菌对细胞毒性较强,72h时70%以上细胞凋亡,冷冻后的VBNC状态细菌对VeroE6和HeLa细胞的生长仍有影响,72h时约有40%细胞进入凋亡状态。结论 低温、贫养条件下空肠弯曲菌可进入VBNC状态,用常规传统的羊血平板培养基仅能使不到10%的空肠弯曲菌复苏生长,从而可以检测,在VBNC状态时,空肠弯曲菌产毒素能力改变较大,远远低于正常状态,但仍具备产毒素的能力,也从RNA水平实验得到证实。     

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.     

Evaluation of Rpf protein of Micrococcus luteus for cultivation of soil actinobacteria

Rpf protein, a kind of resuscitation promoting factor, was first found in the culture supernatant of Micrococcus luteus. It can resuscitate the growth of M. luteus in “viable but non-culture, VBNC” state and promote the growth of Gram-positive bacteria with high G + C content. This paper investigates the resuscitating activity of M. luteus ACCC 41016^T Rpf protein, which was heterologously expressed in E. coli, to cells of M. luteus ACCC 41016^T and Rhodococcus marinonascens HBUM200062 in VBNC state, and examines the effect on the cultivation of actinobacteria in soil. The results showed that the recombinant Rpf protein had resuscitation effect on M. luteus ACCC 41016^T and R. marinonascens HBUM200062 in VBNC state. 83 strains of actinobacteria, which were distributed in 9 families and 12 genera, were isolated from the experimental group with recombinant Rpf protein in the culture medium. A total of 41 strains of bacteria, which were distributed in 8 families and 9 genera, were isolated from the control group without Rpf protein. The experimental group showed richer species diversity than the control group. Two rare actinobacteria, namely HBUM206391^T and HBUM206404^T, were obtained in the experimental group supplemented with Rpf protein. Both may be potential new species of Actinomadura and Actinokineospora, indicating that the recombinant expression of M. luteus ACCC 41016^T Rpf protein can effectively promote the isolation and culture of actinobacteria in soil.     

Revealing potential functions of VBNC bacteria in polycyclic aromatic hydrocarbons biodegradation

The bioremediation of polycyclic aromatic hydrocarbon (PAH)‐contaminated sites is not running smoothly, because of the lower activity of PAH‐degrading bacteria in actual bioremediation applications. The phenomenon of “viable but nonculturable” (VBNC) state may be a main limiting factor for their poor biodegradation capabilities of PAHs. Due to their abilities of entering into the VBNC state, most of bacterial populations with PAH‐degradation potential remain unculturable. Resuscitation of VBNC bacteria will enhance the degradation capability of indigenous bacteria which will eventually obtain their better capabilities in environmental bioremediation. Although evidences have been presented indicating that resuscitation of VBNC bacteria in polychlorinated biphenyl (PCB)‐contaminated environments not only significantly enhanced PCB degradation, but also obtained novel highly efficient PCB‐degrading bacteria, scanty information is available on the VBNC bacteria in PAH‐contaminated sites. VBNC bacteria, as a vast majority of potential microbial resource could be the repository of novel highly efficient PAH‐biodegraders. Therefore, studies need to be done on resuscitation of VBNC bacteria to overcome key bottlenecks in bioremediation of PAH‐contaminated sites. This mini‐review provides a new insight into the potential functions of VBNC bacteria in PAHs biodegradation.

Significance and Impact of the Study

As the vast majority microbial resource, viable but nonculturable (VBNC) bacteria, which showed their potential functions in polycyclic aromatic hydrocarbons (PAHs) biodegradation, can be of great significance in environmental bioremediation. It is therefore important to resuscitate VBNC bacteria for their better capabilities. Meanwhile, preventing the indigenous functional community from entering into the VBNC state will also maintain the high activity of PAH‐degrading bacteria in actual bioremediation applications. Undoubtedly, much more work needs to be done to reveal indigenous micro‐organisms in the VBNC state from the perspective of environmental functions.     

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.     

Resuscitation rate in different enterococcal species in the viable but non-culturable state

AIMS: The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stress. When in this state bacteria are no longer culturable on conventional growth media, but cells display metabolic activity and maintain pathogenicity factors/genes and, in some cases, resuscitation from the VBNC state has been shown. This state has been described for both human pathogens and faecal pollution indicators. In this study, we present evidence for entry of different enterococcal species into the VBNC state in an oligotrophic microcosm. METHODS AND RESULTS: The duration of the viability of the cells in the VBNC state was measured either by detecting the presence of pbp5 mRNA or by quantifying their resuscitation capability. Enterococci showed different behaviours. Enterococcus faecalis and Enterococcus hirae entered into the VBNC state within 2 weeks and remained in that state for 3 months. In the experiments described the resuscitation rate was 1:10 000 cells as soon as the cells entered the VBNC state and decreased gradually to undetectable levels over the following 3 months. Enterococcus faecium, however, remained culturable up to 4 weeks. After this time period, when the population was totally unculturable, the cells were far less resuscitable than other enterococci and only over a narrow time interval (2 weeks). CONCLUSIONS: These results suggest that Ent. faecalis and Ent. hirae enter the VBNC state but that Ent. faecium, in an oligotrophic laboratory environment, tends to die instead of entering the VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when enterococci are released by humans and animals in natural environments.