Development and application of polymerase chain reaction primers based on fhcD for environmental detection of methanopterin-linked C1-metabolism in bacteria

In this work we describe development and testing of a novel pair of environmental primers targeting fhcD, a conserved gene in the H4MTP-linked C1-transfer pathway, and demonstrate that these primers enable confident detection of a broad variety of fhcD genes originating from phylogenetically diverse bacteria. The new primer pair was employed to analyse fhcD diversity in Lake Washington sediment, uncovering the presence of 40 fhcD phylotypes. Based on phylogenetic analyses, the phylotypes identified were affiliated with alpha-, beta- and gamma-proteobacteria, and Planctomycetes, while a number of sequences formed deep branches suggesting the presence of unknown groups of microorganisms. To assess the physiological potential and the possible substrate repertoire of the fhcD-containing species in Lake Washington, we conducted enrichments of natural populations on a variety of C1 substrates, and observed specific shifts in community structure in response to different C1 substrates. A specific shift in community structure was also observed in the presence of humic acids suggesting that C1 transfer metabolism linked to H4MPT may be part of the degradation pathway for this natural polymer, possibly involving formaldehyde production. Overall, our data suggest that C1 oxidation reactions linked to H4MPT are much more widespread in natural environments than previously thought.     

Analysis of fae and fhcD Genes in Mono Lake, California

Genes for two enzymes of the tetrahydromethanopterin-linked C₁ transfer pathway (fae and fhcD) were detected in hypersaline, hyperalkaline Mono Lake (California), via PCR amplification and analysis. Low diversity for fae and fhcD was noted, in contrast to the diversity previously detected in a freshwater lake, Lake Washington (Washington).     

Estimation of methanotroph abundance in a freshwater lake sediment

The numbers of methane-oxidizing bacteria (methanotrophs) in the sediments of Lake Washington were estimated using three culture-independent methods. Quantitative slot-blot hybridizations were performed with type I and type II methanotroph-specific probes. These data were compared to data from quantitative hybridizations using a pmoA-specific probe and a eubacterial probe. From the combined hybridization data, the methanotroph population in Lake Washington was estimated to be 3.6 x 10(8)-7.4 x 10(8) cells/g dry weight. Methanotroph community structure and number were also investigated using polar lipid fatty acid (PLFA) analysis. Analysis of biomarker PLFAs characteristic of both type I (16:1 omega 8) and type II (18:1 omega 8) methanotrophs was used to estimate the abundance of these bacteria in Lake Washington sediments. From the PLFA data, the methanotroph population in Lake Washington was estimated to be 7.1 x 10(8)-9.4 x 10(9) cells/g dry weight. As a third method of quantitation, we calculated the methanotroph population using the total methane oxidation rate for whole cells in Lake Washington sediment to be 1.3 x 10(8)-1.2 x 10(9) cells/g dry weight. The three independent estimates of the number of methanotrophs in Lake Washington sediment agree within a two- to fourfold range. These data suggest that the three techniques used in this study detect the functionally significant population of methanotrophs in Lake Washington. Furthermore, these techniques will be useful for obtaining estimates of methanotroph abundance in additional environments.     

Methanotrophic communities of Saanich Inlet: a microcosm perspective

Saanich Inlet (British Columbia, Canada) is a seasonally anoxic fjord characterized by high rates of both methane production and consumption. In this study, the diversity of microbial populations residing in intermediate waters, characterized by having a high methane content, was assessed using CH(4)-microcosm experiments coupled with PCR surveys of phylogenetic (16S rRNA gene) and functional gene markers (pmoA and fhcD genes). The experiments revealed that bacteria represented by sequences affiliated with Methylomicrobium within the Methylococcales, Methylophaga and Cycloclasticus within the Thiotrichales, and uncultured Planctomycetes were enriched in response to CH(4) addition.     

Sulfur-oxidizing bacteria in Soap Lake (Washington State), a meromictic, haloalkaline lake with an unprecedented high sulfide content

Culture-dependent and -independent techniques were used to study the diversity of chemolithoautotrophic sulfur-oxidizing bacteria in Soap Lake (Washington State), a meromictic, haloalkaline lake containing an unprecedentedly high sulfide concentration in the anoxic monimolimnion. Both approaches revealed the dominance of bacteria belonging to the genus Thioalkalimicrobium, which are common inhabitants of soda lakes. A dense population of Thioalkalimicrobium (up to 10(7) cells/ml) was found at the chemocline, which is characterized by a steep oxygen-sulfide gradient. Twelve Thioalkalimicrobium strains exhibiting three different phenotypes were isolated in pure culture from various locations in Soap Lake. The isolates fell into two groups according to 16S rRNA gene sequence analysis. One of the groups was closely related to T. cyclicum, which was isolated from Mono Lake (California), a transiently meromictic, haloalkaline lake. The second group, consisting of four isolates, was phylogenetically and phenotypically distinct from known Thioalkalimicrobium species and unique to Soap Lake. It represented a new species, for which we suggest the name Thioalkalimicrobium microaerophilum sp. nov.     

Morphological and genetic differentiation of the African clupeid Limnothrissa miodon 34 years after its introduction to Lake Kivu

The evolutionary consequences of an artificial introduction of the clupeid Limnothrissa miodon from Lake Tanganyika into Lake Kivu, East Africa were examined. In 1959, 57 400 fry (mixture of Limnothrissa and the related clupeid, Stolothrissa tanganicae ), were released into Lake Kivu to boost fisheries production. Comparisons were made between respective source and transplant populations 34 years later (1993) using morphometrics ('truss' method), allozymes (29 enzyme-coding loci) and mitochondrial (mt) DNA variation (RFLP analysis of PCR-amplified ND5/6 genes). Significant morphological and genetic differentiation between source and transplant samples was detected, with a distinct clustering of Kivu Limnothrissa on respective dendrograms, especially at the morphometric and mtDNA levels. Differentiation within Lake Tanganyika was, however, consistently higher than that between lakes. Allozymic diversity was similar in samples from both lakes (Lake Tanganyika: heterozygosity = 0.0658, mean number of alleles=1.44; Lake Kivu: heterozygosity = 0.0655; mean number of alleles = 1.48), however, a significantly lower mtDNA haplotype diversity was detected in Lake Kivu (Lake Tanganyika: 0.905; Lake Kivu: 0.755). Data suggest that high post-introduction mortality and various demographic factors reduced the effective population size of the introduced population to tens rather than thousands of individuals, resulting in a reduction in genetic diversity and founder effect.     

西湖水域外来生物福寿螺的种类鉴定及种群遗传多样性研究

为了解杭州西湖景区外来生物福寿螺的分布现状和群体遗传变异特征,研究详细调研了西湖景区福寿螺的分布现状,并针对连续六年采集的福寿螺样本开展了分子鉴定及种群遗传多样性研究.调查结果表明,西湖福寿螺集中发生在苏堤以西,其中西北部的西里湖、茅家埠、杭州花圃及岳湖水域的发生密度高,在西南部的乌龟潭、浴鹄湾等水域发生密度较低.在6...     

基于环境DNA宏条形码的洱海鱼类多样性研究

研究使用环境DNA宏条形码(eDNA metabarcoding)检测洱海鱼类多样性,探索适用于洱海鱼类多样性监测和保护的新方法。通过水样采集、过滤、eDNA提取、遗传标记扩增、测序与生物信息分析的环境DNA宏条形码标准化分析流程,从洱海16个采样点中获得可检测的9个采样点数据,共检测出17种鱼类,其中土著种5种、外来种12种;鲫(Carassius auratus)、鳙(Hypophthalmichthys nobilis)、麦穗鱼(Pseudorasbora parva)、泥鳅(Misgurnus anguillicaudatus)和食蚊鱼(Gambusia affinis)为优势种。研究结果表明虽然环境DNA宏条形码无法完全替代传统的鱼类监测方法,但作为一种新兴的生物多样性监测手段,其可用于快速检测洱海鱼类多样性及其空间分布。     

A reintroduced lake sturgeon population comes of age: A genetic evaluation of stocking success in the St. Louis River

Lake sturgeon (Acipenser fulvescens) have experienced significant declines throughout the Great Lakes, leading to stocking efforts at some locations to reestablish extirpated populations. Given the late sexual maturity of the species, assessment of stocking program success is often delayed. Lake sturgeon were extirpated from the St. Louis River in western Lake Superior. Stocking began in 1983 and continued until 1994, using the Wolf River (Lake Winnebago) in the Lake Michigan basin as the source. Stocking resumed from 1998 to 2000, using the Sturgeon River in Lake Superior as the source. Our objectives were to (a) determine the movement patterns of stocked Wolf River sturgeon, (b) determine whether stocked individuals are migrating into natural spawning populations during the spawning season, (c) identify the origins of individuals captured in the St. Louis River, and (d) assess the genetic diversity of the reintroduced St. Louis River population. All collected samples were analyzed using 12 microsatellite loci, followed by genetic assignment testing to achieve the first three objectives. Genetic diversity was compared to natural spawning populations in Lake Superior. The highest proportion of stocked Wolf River lake sturgeon was detected in western Lake Superior, close to the stocking site. However, individuals were detected throughout Lake Superior. Wolf River individuals were detected in most of the spawning populations in Lake Superior, with the greatest number in the Sturgeon River and the Goulais River. The majority of individuals captured in the St. Louis River were of stocked origin (88.1%), with 73.5% from the Wolf River and 14.6% from the Sturgeon River. These observed proportions differed from the expected proportions based on the number of sturgeon released from each source (χ² = 55.37, p < 0.00001), with a higher representation of Sturgeon River individuals. Evidence of natural recolonization in the St. Louis River was detected from Lake Huron and Goulais River individuals. Genetic diversity of the St. Louis River population was comparable to levels observed in the remnant natural populations in Lake Superior. However, the effective population size of the St. Louis River was small (N_e = 38.1) and average relatedness among individuals was relatively high (r = 0.151). Monitoring of movement of stocked Wolf River sturgeon throughout Lake Superior should continue, with careful attention to the potential for outbreeding depression in remnant populations. Genetic diversity of the St. Louis River population should also continue to be monitored to see if it improves with increased natural reproduction.     

The abundance and biological activity of manganese-oxidizing bacteria and Metallogenium-like morphotypes in Lake Washington, USA

The distribution of bacteria, ATP, Metallogenium morphotypes and manganese-oxidizing activities were studied in Lake Washington, Seattle, WA, U.S.A. In accordance with earlier studies, we have found that Metallogenium morphotypes show a stable seasonal distribution in Lake Washington. We have used ⁵⁴Mn(II) tracer studies coupled with poisoned and no-oxygen controls to demonstrate that biological manganese oxidation was not linked to the numbers of Metallogenium morphotypes. The data suggest that these morphotypes do not contribute significantly to the biological oxidation of manganese in Lake Washington.     

不同环境样本类型对蚌类环境DNA监测的差异研究

为了阐明在使用环境DNA宏条形码技术时不同环境样本类型如何影响蚌类物种的可检测性,于2021年冬季和春季在鄱阳湖分别采集表层水、底层水和沉积物进行环境DNA宏条形码分析,并结合传统方法采集验证。基于环境DNA宏条形码技术共检测到鄱阳湖蚌类33种,传统方法共采集蚌类18种,环境DNA宏条形码技术能检测出传统方法采集到的所有蚌类物种。表层水和底层水注释到的蚌类物种数均分别高于沉积物的,且表层水和底层水注释到的蚌类物种分别完全覆盖沉积物的。基于环境DNA宏条形码技术的蚌类α多样性水平季节差异不显著,但蚌类β多样性水平季节差异显著。表层水和底层水的蚌类多样性均显著高于沉积物样本的, Beta多样性分析也显示水体样本(表层水和底层水分别)和沉积物样本存在显著性差异。但表层水和底层水的蚌类多样性和群落结构均无显著差异。鄱阳湖蚌类群落结构与环境因子的关联分析表明水深(WD)、透明度(SD)、水温(WT)和总氮(TN)显著影响蚌类群落结构。环境DNA宏条形码技术在蚌类的多样性监测中可行,且采水样比采沉积物效果好,表层水和底层水无显著差异。     

Methylotrophy in a lake: from metagenomics to single-organism physiology

This review provides a brief summary of ongoing studies in Lake Washington (Seattle, WA) directed at an understanding of the content and activities of microbial communities involved in methylotrophy. One of the findings from culture-independent approaches, including functional metagenomics, is the prominent presence of Methylotenera species in the site and their inferred activity in C(1) metabolism, highlighting the local environmental importance of this group. Comparative analyses of individual genomes of Methylophilaceae from Lake Washington provide insights into their genomic divergence and suggest significant metabolic flexibility.     

Sulfur-Oxidizing Bacteria in Soap Lake (Washington State), a Meromictic, Haloalkaline Lake with an Unprecedented High Sulfide Content

Culture-dependent and -independent techniques were used to study the diversity of chemolithoautotrophic sulfur-oxidizing bacteria in Soap Lake (Washington State), a meromictic, haloalkaline lake containing an unprecedentedly high sulfide concentration in the anoxic monimolimnion. Both approaches revealed the dominance of bacteria belonging to the genus Thioalkalimicrobium, which are common inhabitants of soda lakes. A dense population of Thioalkalimicrobium (up to 10⁷ cells/ml) was found at the chemocline, which is characterized by a steep oxygen-sulfide gradient. Twelve Thioalkalimicrobium strains exhibiting three different phenotypes were isolated in pure culture from various locations in Soap Lake. The isolates fell into two groups according to 16S rRNA gene sequence analysis. One of the groups was closely related to T. cyclicum, which was isolated from Mono Lake (California), a transiently meromictic, haloalkaline lake. The second group, consisting of four isolates, was phylogenetically and phenotypically distinct from known Thioalkalimicrobium species and unique to Soap Lake. It represented a new species, for which we suggest the name Thioalkalimicrobium microaerophilum sp. nov.     

湖泊富营养化对轮虫群落结构及物种多样性的影响

2005年7月至2006年6月, 我们对安徽省芜湖市境内5个湖泊中轮虫的群落结构及部分水质特征参数进行了调查, 用相关加权综合营养状态指数(TLIc)评价了各湖泊的营养水平, 分析了其中的轮虫群落结构、多样性指数及其与TLI_C之间的关系。经鉴定, 5个湖泊中共采集到轮虫79种, 隶属18科31属; 不同营养水平湖泊中出现的轮虫种类数有明显的差异(P<0.01)。除龙窝湖外, 其余4个湖泊中的轮虫种类数与TLI_C均呈显著负相关关系(r＝–0.984, P<0.05)。湖泊水体的富营养化进程显著地提高了轮虫总密度和第一优势种的优势度, 降低了轮虫群落的均匀度和物种多样性指数。     

Spatial and temporal distribution of Caulobacter spp. in two mesotrophic lakes

The seasonal distribution of Caulobacter spp. has been determined in the water column of two mesotrophic lakes using most probable number (MPN) viable counting techniques from April, 1972 to March, 1973. Concentrations in Lake Washington, a monomictic lake, peaked at 1000–3300 per ml in the epilimnion during the late spring and summer and reached lows in October of less than 2 per ml prior to fall turnover. The reason for the decrease in viable numbers is not known. Caulobacter spp. began to increase gradually in December, during the winter mixing period well before the spring bloom. Concentrations in Hall Lake, a small kettle lake, were somewhat higher than in Lake Washington, ranging from 0.27 per ml in October to over 5000 per ml during May. In contrast to Lake Washington, the highest populations were found in the metalimnion and upper hypolimnion of Hall Lake. Caulobacter spp. were also found in the anaerobic depths of the hypolimnion. However, these did not grow anaerobically, suggesting that they had settled into the hypolimnion and survived in the cold anaerobic zone. The populations of Caulobacter spp. reached maximum numbers at the approximate times and depths at which algal biomass would be expected to be greatest.     

Diversity of extremophilic purple phototrophic bacteria in Soap Lake, a Central Washington (USA) Soda Lake

Culture-based and culture-independent methods were used to explore the diversity of phototrophic purple bacteria in Soap Lake, a small meromictic soda lake in the western USA. Among soda lakes, Soap Lake is unusual because it consists of distinct upper and lower water bodies of vastly different salinities, and its deep waters contain up to 175 mM sulfide. From Soap Lake water new alkaliphilic purple sulfur bacteria of the families Chromatiaceae and Ectothiorhodospiraceae were cultured, and one purple non-sulfur bacterium was isolated. Comparative sequence analysis of pufM, a gene that encodes a key photosynthetic reaction centre protein universally found in purple bacteria, was used to measure the diversity of purple bacteria in Soap Lake. Denaturing gradient gel electrophoresis and subsequent phylogenetic analyses of pufMs amplified from Soap Lake water revealed that a significant diversity of purple bacteria inhabit this soda lake. Although close relatives of several of the pufM phylotypes obtained from cultured species could also be detected in Soap Lake water, several other more divergent pufM phylotypes were also detected. It is possible that Soap Lake purple bacteria are major contributors of organic matter into the ecosystem of this lake, especially in its extensive anoxic and sulfidic deep waters.     

Analysis of sMMO-containing type I methanotrophs in Lake Washington sediment

Methane-oxidizing bacteria (methanotrophs) containing soluble methane monooxygenase (sMMO) are of interest in natural environments due to the high co-metabolic activity of this enzyme with contaminants such as trichloroethylene. We have analysed sMMO-containing methanotrophs in sediment from a freshwater lake. Environmental clone banks for a gene encoding a diagnostic sMMO subunit (mmoX) were generated using DNA extracted from Lake Washington sediment and subjected to RFLP analysis. Representatives from the six RFLP groups were cloned and sequenced, and all were found to group with Type I Methylomonas mmoX, although a majority were divergent from known Methylomonas mmoX sequences. Direct hybridization of Lake Washington sediment DNA was carried out using a series of sMMO- and Methylomonas-specific probes to assess the significance of these sMMO-containing Methylomonas-like strains in the sediment. The total sMMO-containing population and the sMMO-containing Methylomonas-like population were estimated to be similar to previous estimates for total methanotrophs and Type I methanotrophs. These results suggest that the major methanotrophic population in Lake Washington sediment consists of sMMO-containing Methylomonas-like (Type I) methanotrophs. The whole-cell TCE degradation kinetics of such a strain, LW15, isolated from this environment, were determined and found to be similar to values reported for other sMMO-containing methanotrophs. The numerical significance of sMMO-containing Methylomonas-like methanotrophs in a mesotrophic lake environment suggests that these methanotrophs may play an important role in methanotroph-mediated transformations, including co-metabolism of halogenated solvents, in natural environments.     

Biodiversity of methanogenic and other archaea in the permanently frozen Lake Fryxell, Antarctica

Archaea were detected in molecular diversity studies of the permanently frozen Lake Fryxell, Antarctica. Two clusters of methanogens were detected in the sediments, and another cluster of possibly methanotrophic Euryarchaeota was detected in the anoxic water column just above the sediments. One crenarchaeote was detected in water just below the oxycline. The Archaea present in Lake Fryxell are likely involved in the major biogeochemical cycles that occur there.