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Time as a microbial resource
Environmental Microbiology Reports ( IF 3.3 ) Pub Date : 2020-10-04 , DOI: 10.1111/1758-2229.12892
Karen G Lloyd 1
Affiliation  

Microbes need resources for energy and cellular building material. They also need access to clement conditions with liquid water and a cellular damage rate that is lower than repair. When deprived of resources and clement conditions, microbes often enter some form of dormancy (e.g., by ceasing cell division, slowing metabolic rate, or forming an endospore) until they can grow again (Lennon and Jones, 2011). For example, at night, phototrophs wait for the sun to return. In winter, soil microbes wait for warmer temperatures. Microbes that cause diseases like tuberculosis can stay dormant for years, waiting for the cessation of antibiotic or immune system bombardment (Alnimr, 2015). But what about longer timescales? Unlike multicellular life, microbes survive in an extremely broad range of conditions and can access an amazing variety of resources to maintain cellular functions in the absence of cell division (Finkel and Kolter, 1999). This means that they have the potential to be dormant for much longer than a few months or years. There is no theoretical reason microbes cannot survive on maintenance energy for hundreds or thousands of years, or longer, with little to no cell proliferation (Hoehler and Jørgensen, 2013; Lever et al., 2015). Given this lack of theoretical constraints on the length of microbial dormancy intervals, two questions arise, (i) is there evidence for the existence of organisms experiencing very long dormancies? And (ii) what could be the advantages of such long wait times?

The only way to be certain about a microbe's physiology is to isolate it from its natural environment and grow it in a laboratory (Overmann et al., 2017). Many dormant populations have individual cells that act as ‘scouts’, coming out of dormancy at random times to periodically sample for the return of conditions conducive to growth (Epstein, 2009; Buerger et al., 2012). Some of these scouts can then be given growth factors and cultured. However, there are two problems with relying on vegetative growth to study microbes capable of long‐term dormancy. The first is that direct sequencing of DNA from many environments shows that most microbial cells are phylogenetically divergent from those that have ever been cultured by anyone (Lloyd et al., 2018; Steen et al., 2019a). This may be the driver behind ‘the great plate count anomaly’, which posits that <1% of cells in many samples can be cultured easily (Staley and Konopka, 1985). It is important to note that not all environments are subject to the great plate count anomaly; environments that undergo rapid environmental change or have copious nutrients, such as the human gut (Lloyd et al., 2018) and lakes recently inundated with volcanic ash (Staley and Konopka, 1985) are often dominated by easily cultured cells. However, for many environments under stable conditions, not‐yet‐cultured microbial clades dominate total cell abundance and the great plate count anomaly applies. There is good evidence to support the notion that the reason these groups have not yet yielded to culture is that they are obligate slow growers that are not easily sped up to grow on normal laboratory timescales. It has long been recognized that many more cultures can be obtained from natural samples if they are incubated for many months (Davis et al., 2005). In fact, the biggest recent advances in culturing culture‐resistant clades have been in very slow‐growing organisms. Highly abundant seawater microbes like Nitrosopumilus sp., Pelagibacter ubiquitans, and Prochlorococcus sp. have doubling times of a day or longer meaning that it takes them up to a month or longer to reach stationary phase (Partensky et al., 1999; Rappe et al., 2002; Könneke et al., 2014). Marine sediment microbial cultures and enrichments operate over even longer timescales, with Atribacteria doubling over 5 days, Lokiarchaeota doubling over 14–25 days, and uncultured members of the Methanosarcinales, called ANME‐2, doubling over 7 months (Nauhaus et al., 2007; Imachi et al., 2020; Katayama et al., 2019). These cultures grow so slowly that detailed physiological assessments and genetic manipulations are nearly impossible on human timescales, even though they have technically been cultured. The second problem with relying on cultures to study extremely long‐lived microbes is that physiologies in the vegetative state may differ greatly than when a microbe is subsisting at low metabolic activity over thousands of years. Therefore, while culturing is essential to be sure about an organism's physiology, it cannot be used to study every type of ultra‐slow growing microbe in every type of condition.

For this reason, culturing efforts must be coupled to direct study of ultra‐slow organisms in natural samples. However, identifying microbes that are dormant for many years while they wait for infrequent events is challenging in natural samples. To observers on the human timescale, such ultra‐slow organisms would appear to be doing nothing. As an analogy, the California coastline is a constantly churning mass of rocks over the geological timescales, but to humans, it is stable enough to build houses on. These houses must be sound enough to withstand the occasional earthquake, but they will not survive the reorientations of land as they are spun, submerged, and exhumed over the course of a few million years. Luckily, modern marine sediments offer a natural laboratory in which to study long‐term dormant microbes without either speeding them up in laboratory cultures or waiting thousands of years for changes to occur. Subtle geochemical changes can be quantified over long timescales by comparing changes in concentration of nutrients with the rate of sediment deposition (Berner, 1980). The resulting reaction‐transport models show that the total rate of energy delivery to marine sediment microbial communities is often many orders of magnitude lower than that required to support laboratory cultures (Hoehler and Jørgensen, 2013; Larowe and Amend, 2015; Bradley et al., 2020). This means that these microbial communities do not have enough energy to maintain a steady rate of cell division. Further evidence that microbial communities buried over many meters in marine sediments are largely in a non‐growing state is the fact that very little genetic novelty appears even after timescales over which mutations or ecological competition would arise if the populations were growing normally (Walsh et al., 2016; Starnawski et al., 2017). Turnover times for these subsisting microbial communities have been calculated to be a few tens of years (Braun et al., 2016). This does not necessarily mean that cells undergo traditional replication and cell division once every 30 years. A turnover of biomass can occur by gradually replacing all the cellular material, lipid by lipid, nucleotide by nucleotide, such that over roughly half a century, all the molecules have been replaced. Actual cell division events are likely to take much longer, or possibly never occur until resources return, which could take hundreds to millions of years.

Are these microbial cells that do not replicate for multiple decades, or possibly even hundreds, thousands, or millions of years doing so because they are waiting for an event that occurs over these timescales? An alternative option would be that these microbes are not adapted to ultra‐long dormancy, but instead just happen to find themselves in some sort of suspended animation for millions of years before they are eventually subducted under a continent and crushed or scalded to death in a subduction zone. While accidental subsistence in multi‐thousand‐year stationary phase is an option that must remain on the table, there is some evidence that the organisms who find themselves in this predicament are evolutionarily poised to do so. With increasing depth in estuarine sediments, microbes express enzymes with a higher specificity for the substrates that are available in the subsurface, suggesting that they are adapted for subsurface dormancy with some amount of metabolic activity (Steen et al., 2019b). Subsurface microbes also have physiological adaptations for ultra‐slow metabolisms and cell divisions (Bird et al., 2019). Additionally, the microbial clades found in the subsurface are not just leftovers of pelagic communities that persist rather than perish as they are buried; instead they are distinct from those found in seawater (Teske and Sørensen, 2008; Durbin and Teske, 2012).

Therefore, it is likely that the organisms found in marine sediments are adapted to live in marine sediments, despite not really being able to grow there. But, even if they are well‐adjusted and ‘happy’ during this long period of dormancy, they must grow somewhere – these cells cannot have been dormant since Earth began 4.5 billion years ago. We then must ask question two: What are they waiting for? If we encounter a dormant microbe in soil in winter, we can presume that it aspires to become vegetative in summer. What is the equivalent for a deeply buried marine sediment organism that is dormant for thousands to millions of years? What is their version of summer?

To determine what events cause long‐term dormant organisms to regain their vegetative state, we must assume an evolutionary framework where long‐term dormancy is an adaptation that has an eventual evolutionary pay‐off. The pay‐off would be the dormant microbe someday ‘wakes up’ and produces progeny that receives a survival benefit from having been one of the first to access the resources when they become available. Evidence for this model comes from laboratory cultures that have been studied in stationary phase for many years. When E. coli cultures kept in stationary phase for months or years are competed against freshly grown E. coli cultures under starvation conditions, the pre‐adapted cultures outcompete the freshly grown cultures, a trait that has been named growth advantage in stationary phase (GASP) (Finkel, 2006). If the same is true for microbes living in marine sediments, then they would have an advantage over fresher organisms if they got the chance to compete for meagre resources, like a yogi accustomed to deprivation competing with a glutton during a famine.

Adaptation to long‐term dormancy is likely driven by growth resources that vary with periodicities of equivalently long timescales. Since marine sediment microbes are dormant over hundreds to millions of years, they are likely ‘waiting’ for events that occur over these timescales. Geological processes occur on a range of timescales long enough to suffice. On the short end of the timescale, microbes could be adapted to multiyear flood, drought, or storm cycles, much like cicadas that undergo a 17‐year diapause. But geological events over longer timescale events could also drive dormancy. Dormant microbes in marine sediments could re‐enter a vegetative state upon return to the seafloor where nutrients are fresh. Sediments over the upper meter can be exhumed and redeposited on the seafloor with bioturbation, small gravity flows, or extreme storm events (if the water is shallow enough). More deeply buried sediments could be exhumed over much longer timescales and by much larger events. Whole submarine cliffsides can be redistributed by submarine landslides, slumping, or turbidite flows. Over even longer timescales, microbes that have managed to survive burial many hundreds of meters deep into marine sediments could be exhumed when oceanic plates strike other oceanic or continental plates in subduction zones. Here, accretionary prisms or mud volcano eruptions offer potential opportunities for bringing a few of the deeply buried microbes out of dormancy (Hoshino et al., 2017). Other environments such as ancient permafrost (Gilichinsky et al., 2007; MacKelprang et al., 2017; Liang et al., 2019) also likely have long‐term dormant organisms. The evolutionary pay‐off for such organisms could be the end of glaciated periods following Milankovitch cycles, although it is important to note that modern day permafrost is thawing faster than expected due to climate change (Crowther et al., 2019).

Just as microbes are not dependent on oxygen, they are also not dependent on achieving a certain growth rate. It is well known that the ability to respire anaerobically increases microbes' environmental range, preventing their imprisonment in oxic environments. Likewise, the ability of microbes to survive long, perhaps extraordinarily long, periods of deprivation enables their habitat expansion into a greater range of timescales. Time itself becomes a resource that microbes can exploit to access new habitats. They can wait for resource‐replenishment events that are beyond the temporal reach of organisms that are constrained to faster growth rates. Such ultra‐slow microbes could be viewed as K strategists within the classic ecological framework of r vs. K strategies, which have slower reproduction rates, longer lifespans and maintain steady‐state populations to maximize use of the carrying capacity of an environment (Pianka, 1970). The caveat, of course, is that ecological paradigms such as this one are designed around multicellular eukaryotes and include predictions about offspring rearing and body size that do not perfectly translate to microbes surviving over geological timescales. The novelty of viewing time as a microbial resource does not, therefore, signify a new ecological paradigm, but a new ecological niche. By actively focusing on how microbes exploit a vast range of timescales, perhaps longer than previously recognized as possible, we can open up new understandings for how microbes and Earth systems interact.



中文翻译:

时间作为微生物资源

微生物需要能源和细胞建筑材料的资源。他们还需要获得液态水和低于修复率的细胞损伤率的温和条件。当资源和条件被剥夺时,微生物通常会进入某种形式的休眠(例如,通过停止细胞分裂、减慢代谢率或形成内生孢子),直到它们可以再次生长(Lennon 和 Jones,2011)。例如,在晚上,光养生物等待太阳回来。在冬天,土壤微生物等待温度升高。导致结核病等疾病的微生物可以休眠多年,等待抗生素或免疫系统轰击停止(Alnimr,2015 年))。但是更长的时间尺度呢?与多细胞生命不同,微生物可以在极其广泛的条件下生存,并且可以在没有细胞分裂的情况下获得多种惊人的资源来维持细胞功能(Finkel 和 Kolter,1999 年)。这意味着它们有可能休眠超过几个月或几年的时间。没有任何理论上的理由微生物不能依靠维持能量存活数百年或数千年或更长时间,几乎没有细胞增殖(Hoehler 和 Jørgensen,2013 年;Lever等人2015 年))。鉴于缺乏对微生物休眠间隔长度的理论限制,出现了两个问题:(i)是否有证据表明存在经历很长时间休眠的生物?(ii) 这么长的等待时间有什么好处?

确定微生物生理学的唯一方法是将其与自然环境隔离并在实验室中培养(Overmann等人2017 年)。许多休眠种群都有充当“侦察兵”的单个细胞,它们会在随机时间从休眠状态中出来,定期采样以恢复有利于生长的条件(Epstein,2009 年;Buerger2012 年))。然后可以给这些侦察员中的一些人提供生长因子并进行培养。然而,依靠营养生长来研究能够长期休眠的微生物存在两个问题。首先,对来自许多环境的 DNA 进行直接测序表明,大多数微生物细胞在系统发育上与任何人培养过的细胞不同(Lloyd2018;Steen2019a)。这可能是“大板计数异常”背后的驱动因素,它假定许多样品中 <1% 的细胞可以轻松培养(Staley 和 Konopka,1985)。需要注意的是,并非所有环境都受到板块计数异常的影响。经历快速环境变化或具有丰富营养的环境,例如人类肠道(Lloyd等人2018 年)和最近被火山灰淹没的湖泊(Staley 和 Konopka,1985 年)) 通常以容易培养的细胞为主。然而,对于稳定条件下的许多环境,尚未培养的微生物进化枝在总细胞丰度中占主导地位,并且大板块计数异常适用。有充分的证据支持这样一种观点,即这些群体尚未屈服于文化的原因是他们是专性的缓慢生长者,在正常的实验室时间尺度上不容易加速生长。人们早就认识到,如果将天然样品培养数月,可以从天然样品中获得更多的培养物(Davis2005)。事实上,最近在培养抗培养进化枝方面取得的最大进展是在生长非常缓慢的生物体中。高度丰富的海水微生物,如Nitrosopumilus sp.,Pelagibacter ubiquitansProchlorococcus sp。具有一天或更长时间的倍增时间,这意味着它们需要长达一个月或更长时间才能达到稳定阶段(Partensky等人1999 年;Rappe等人2002 年;Könneke等人2014 年)。海洋沉积物微生物培养物和富集工作在甚至更长的时间尺度,与Atribacteria超过5天倍增,Lokiarchaeota倍增超过14-25天,未培养的成员Methanosarcinales,称为ANME-2,超过7个月(倍增Nauhaus等人。,2007年;今町2020 年;Katayama等人2019 年)。这些培养物生长得如此缓慢,以至于在人类时间尺度上进行详细的生理评估和基因操作几乎是不可能的,即使它们在技术上是经过培养的。依靠培养来研究寿命极长的微生物的第二个问题是,植物人的生理机能可能与微生物在数千年的低代谢活动下生存时的生理机能有很大不同。因此,虽然培养对于确定生物体的生理机能至关重要,但它不能用于研究在各种条件下的每种类型的超慢生长微生物。

因此,培养工作必须与对天然样品中超慢生物的直接研究相结合。然而,在自然样本中识别休眠多年而等待偶发事件的微生物具有挑战性。对于人类时间尺度上的观察者来说,这种超慢生物似乎什么都不做。打个比方,加利福尼亚的海岸线在地质时间尺度上是一块不断搅动的岩石,但对人类来说,它足够稳定,可以建造房屋。这些房屋必须足够坚固以承受偶尔发生的地震,但它们在经过数百万年的旋转、淹没和挖掘过程中无法经受住土地的重新定位。幸运的是,现代海洋沉积物提供了一个天然实验室,可以在其中研究长期休眠的微生物,而无需在实验室培养中加速它们或等待数千年的变化发生。通过将养分浓度的变化与沉积物沉积速率进行比较,可以量化长期内细微的地球化学变化(Berner,1980 年)。由此产生的反应传输模型表明,海洋沉积物微生物群落的总能量传输速率通常比支持实验室培养所需的能量低许多数量级(Hoehler 和 Jørgensen,2013 年;Larowe 和 Amend,2015 年;Bradley等人。 ,2020 年)。这意味着这些微生物群落没有足够的能量来维持稳定的细胞分裂速度。海洋沉积物中埋藏数米的微生物群落大部分处于非生长状态的进一步证据是,即使在种群正常生长时会出现突变或生态竞争的时间尺度之后,也很少出现遗传新奇性(Walsh2016 年;Starnawski等人2017 年)。据计算,这些现存微生物群落的周转时间为几十年(Braun等人2016 年)。这并不一定意味着细胞每 30 年进行一次传统的复制和细胞分裂。生物质的周转可以通过逐渐替换所有细胞材料、脂质一个脂质、一个核苷酸一个核苷酸来发生,这样大约半个世纪以来,所有分子都被替换了。实际的细胞分裂事件可能需要更长的时间,或者在资源返回之前可能永远不会发生,这可能需要数百到数百万年。

这些微生物细胞在数十年甚至数百年、数千年或数百万年内都不会复制,是因为它们在等待发生在这些时间尺度上的事件吗?另一种选择是,这些微生物不适应超长休眠,而是碰巧发现自己处于某种假死状态数百万年,然后最终被淹没在一块大陆之下并被压碎或烫死。俯冲带。虽然在数千年的静止期意外生存是一种必须保留在桌面上的选择,但有一些证据表明,发现自己处于这种困境的生物在进化上已经做好了这样做的准备。随着河口沉积物深度的增加,2019b)。地下微生物也对超慢代谢和细胞分裂具有生理适应性(Bird2019)。此外,在地下发现的微生物进化枝不仅仅是远洋群落的残余物,它们在被掩埋时会持续存在而不是灭亡;相反,它们不同于在海水中发现的那些(Teske 和 Sørensen,2008 年;Durbin 和 Teske,2012 年)。

因此,在海洋沉积物中发现的生物很可能适合生活在海洋沉积物中,尽管它们并不能真正在那里生长。但是,即使它们在这段漫长的休眠期间得到了很好的调整和“快乐”,它们也必须在某个地方生长——这些细胞自 45 亿年前地球诞生以来就不可能处于休眠状态。那么我们必须问第二个问题:他们在等什么?如果我们在冬天在土壤中遇到一种休眠的微生物,我们可以推测它渴望在夏天变成植物人。对于一个沉睡了数千到数百万年的深埋海洋沉积物生物来说,相当于什么?他们的夏天是什么版本?

为了确定哪些事件导致长期休眠的生物体重新获得植物人状态,我们必须假设一个进化框架,其中长期休眠是一种具有最终进化回报的适应。回报将是休眠的微生物有一天“醒来”并产生后代,这些后代在资源可用时成为第一个获得资源的人,从而获得生存利益。该模型的证据来自在固定相研究多年的实验室培养物。当大肠杆菌培养物保持在静止期数月或数年时与新鲜生长的大肠杆菌竞争饥饿条件下的培养物,预先适应的培养物胜过新鲜培养的培养物,这种特性被称为静止期生长优势 (GASP) (Finkel, 2006 )。如果生活在海洋沉积物中的微生物也是如此,那么如果它们有机会争夺微薄的资源,那么它们就会比新鲜的生物更有优势,就像一个习惯于在饥荒中与贪食者竞争的瑜伽修行者。

对长期休眠的适应可能是由增长资源驱动的,这些资源随着相当长的时间尺度的周期性而变化。由于海洋沉积物微生物休眠了数百到数百万年,它们很可能“等待”在这些时间尺度内发生的事件。地质过程发生在足够长的时间范围内。在时间尺度的短端,微生物可以适应多年的洪水、干旱或风暴周期,就像经历 17 年滞育的蝉一样。但较长时间尺度事件中的地质事件也可能导致休眠。海洋沉积物中的休眠微生物在返回到营养新鲜的海底后可能会重新进入营养状态。在生物扰动、小重力流、或极端风暴事件(如果水足够浅)。更深埋的沉积物可以在更长的时间尺度和更大的事件中被挖掘出来。整个海底悬崖边可以通过海底滑坡、坍塌或浊流重新分布。在更长的时间尺度上,当海洋板块撞击俯冲带的其他海洋或大陆板块时,能够在数百米深的海洋沉积物中存活下来的微生物可能会被挖掘出来。在这里,增生棱柱或泥火山喷发提供了潜在的机会,使一些深埋的微生物摆脱休眠状态(星野 塌陷或浊流。在更长的时间尺度上,当海洋板块撞击俯冲带的其他海洋或大陆板块时,能够在数百米深的海洋沉积物中存活下来的微生物可能会被挖掘出来。在这里,增生棱柱或泥火山喷发提供了潜在的机会,使一些深埋的微生物摆脱休眠状态(星野 塌陷或浊流。在更长的时间尺度上,当海洋板块撞击俯冲带的其他海洋或大陆板块时,能够在数百米深的海洋沉积物中存活下来的微生物可能会被挖掘出来。在这里,增生棱柱或泥火山喷发提供了潜在的机会,使一些深埋的微生物摆脱休眠状态(星野等人2017 年)。其他环境,如古代永久冻土(Gilichinsky et al ., 2007 ; MacKelprang et al ., 2017 ; Liang et al ., 2019)也可能有长期休眠的生物。这种生物的进化回报可能是米兰科维奇循环之后冰川期的结束,尽管重要的是要注意,由于气候变化,现代永久冻土的融化速度比预期的要快(Crowther2019 年)。

正如微生物不依赖氧气一样,它们也不依赖于达到一定的生长速度。众所周知,厌氧呼吸的能力增加了微生物的环境范围,防止它们被困在有氧环境中。同样,微生物能够在长期,也许是非常长的剥夺期中存活下来,使它们的栖息地扩展到更大的时间范围。时间本身成为微生物可以利用以进入新栖息地的资源。他们可以等待资源补充事件,这些事件超出了受限制于更快增长速度的生物体的时间范围。这种超慢微生物可以被视为 r vs. K 策略的经典生态框架内的 K 策略师,它们的繁殖速度较慢,1970 年)。当然,需要注意的是,像这样的生态范式是围绕多细胞真核生物设计的,包括对后代抚养和体型的预测,这些预测不能完美地转化为微生物在地质时间尺度上的生存。因此,将时间视为微生物资源的新颖性并不意味着一种新的生态范式,而是一种新的生态位。通过积极关注微生物如何利用广泛的时间尺度,可能比以前认为的时间更长,我们可以对微生物和地球系统如何相互作用开辟新的认识。

更新日期:2020-10-15
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