南海深海沉积物放线菌多样性分析

【目的】免培养和纯培养相结合分析南海深海沉积物放线菌多样性。【方法】免培养方法通过提取沉积物宏基因组DNA,利用放线菌门特异性引物扩增放线菌16S r RNA基因序列,构建放线菌16S r RNA基因克隆文库,文库经RFLP(Restriction fragment length polymorphism)分析后挑选代表序列测序并进行多样性指数分析和系统发育分析。可培养方法利用8种培养基进行菌株分离,对排重后的菌株进行16S r RNA基因序列多样性分析。【结果】构建的两个深海位点的16S r RNA基因克隆文库在放线菌门的放线菌纲(Actinobacteria)、酸微菌纲(Acidimicrobiia)、腈基降解菌纲(Nitriliruptoria)和嗜热油菌纲(Thermoleophilia)4个纲中均有分布;两个位点中的种群结构有差异,N40-4位点的优势种群是放线菌纲的链霉菌目(Streptomycetales);N63-4位点的优势种群是腈基降解菌纲的腈基降解菌目(Nitriliruptorales)。8种培养基共分离出41株放线菌,根据形态特征排重后得到的19株菌分布于10个不同的属,12个不同的种,其中稀有放线菌属比例较高,菌株OAct400为潜在的微杆菌属(Microbacterium)新种。【结论】南海深海沉积物蕴含着丰富的放线菌物种资源及大量未知种群,具有进一步研究的价值。     

Key roles for freshwater Actinobacteria revealed by deep metagenomic sequencing

Freshwater ecosystems are critical but fragile environments directly affecting society and its welfare. However, our understanding of genuinely freshwater microbial communities, constrained by our capacity to manipulate its prokaryotic participants in axenic cultures, remains very rudimentary. Even the most abundant components, freshwater Actinobacteria, remain largely unknown. Here, applying deep metagenomic sequencing to the microbial community of a freshwater reservoir, we were able to circumvent this traditional bottleneck and reconstruct de novo seven distinct streamlined actinobacterial genomes. These genomes represent three new groups of photoheterotrophic, planktonic Actinobacteria. We describe for the first time genomes of two novel clades, acMicro (Micrococcineae, related to Luna2,) and acAMD (Actinomycetales, related to acTH1). Besides, an aggregate of contigs belonged to a new branch of the Acidimicrobiales. All are estimated to have small genomes (approximately 1.2 Mb), and their GC content varied from 40 to 61%. One of the Micrococcineae genomes encodes a proteorhodopsin, a rhodopsin type reported for the first time in Actinobacteria. The remarkable potential capacity of some of these genomes to transform recalcitrant plant detrital material, particularly lignin‐derived compounds, suggests close linkages between the terrestrial and aquatic realms. Moreover, abundances of Actinobacteria correlate inversely to those of Cyanobacteria that are responsible for prolonged and frequently irretrievable damage to freshwater ecosystems. This suggests that they might serve as sentinels of impending ecological catastrophes.     

Actinobacterial community dynamics in long term managed grasslands

Palace Leas, a long-term experiment at Cockle Park Farm, Northumberland, UK was established in winter 1896–1897 since when the 13 plots have received regular and virtually unchanged mineral fertiliser and farm yard manure inputs. Fertilisers have had a profound impact on soil pH with the organically fertilised plots showing a significantly higher pH than those receiving mineral fertiliser where ammonium sulphate has led to soil acidification. Here, we investigate the impact of organic and mineral fertilisers on the actinobacterial community structure of these soils using terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene analysis. To differentiate fertiliser effects from seasonal variation, soils were sampled three times over one growing season between May and September 2004 and January 2005. Community profiles obtained using T-RFLP were analysed using multivariate statistics to investigate the relationship between community structure, seasonality and fertiliser management. Soil pH was shown to be the most significant edaphic factor influencing actinobacterial communities. Canonical correspondence analysis, used to investigate the relationship between the 16S rRNA gene community profiles and the environmental parameters, showed that actinobacterial communities also responded to soil water content with major changes evident over the summer months between May and September. Quantitative PCR of the actinobacterial and fungal 16S and 18S rRNA genes, respectively suggested that fungal rRNA gene copy numbers were negatively correlated (P = 0.0131) with increasing actinobacterial signals. A similar relationship (P = 0.000365) was also evident when fatty acid methyl esters indicative of actinobacterial biomass (10-methyloctadecanoic acid) were compared with the amounts of fungal octadecadienoic acid (18:2ω9,12). These results show clearly that soil pH is a major driver of change in actinobacterial communities and that genera such as Arthrobacter and Micrococcus are particularly abundant in soils receiving organic inputs whilst others such as Streptomyces, Acidimicrobium and Actinospica are more prevalent in acid soils. The importance of these findings in terms of fungal abundance and potential disease suppression are discussed.     

Dispersal limitation and the assembly of soil Actinobacteria communities in a long-term chronosequence

It is uncertain whether the same ecological forces that structure plant and animal communities also shape microbial communities, especially those residing in soil. We sought to uncover the relative importance of present-day environmental characteristics, climatic variation, and historical contingencies in shaping soil actinobacterial communities in a long-term chronosequence. Actinobacteria communities were characterized in surface soil samples from four replicate forest stands with nearly identical edaphic and ecological properties, which range from 9500 to 14,000 years following glacial retreat in Michigan. Terminal restriction fragment length polymorphism (TRFLP) profiles and clone libraries of the actinobacterial 16S rRNA gene were constructed in each site for phenetic and phylogenetic analysis to determine whether dispersal limitation occurred following glacial retreat, or if community composition was determined by environmental heterogeneity. At every level of examination, actinobacterial community composition most closely correlated with distance, a surrogate for time, than with biogeochemical, plant community, or climatic characteristics. Despite correlation with leaf litter C:N and annual temperature, the significant and consistent relationship of biological communities with time since glacial retreat provides evidence that dispersal limitation is an ecological force structuring actinobacterial communities in soil over long periods of time.     

13C-Isotopomer-based metabolomics of microbial groups isolated from two forest soils

Soil microorganisms are the primary mediators of organic matter decomposition and humification processes in soil, which represent a critical C flux in the global C cycle. Little is known about how soil microbes regulate carbon cycling including the contribution of their own biomass to stable soil organic matter. A comprehensive understanding of microbial composition is a first step to unraveling microbial regulation of soil humification processes. For this purpose, we isolated 23 microbial strains representing four major groups (Gram (+) bacteria, Gram (−) bacteria, Actinobacteria, and Fungi) from a temperate and a tropical forest soil. The microbial isolates were cultured with uniformly ¹³C-labeled glucose as the C source such that all biochemical components synthesized from glucose were ¹³C labeled. This approach enabled field mesocosm experiments on tracking microbial decomposition, while facilitating solution- and solid-state NMR analysis of microbial composition. Polar and lipid extracts of labeled biomass of the four microbial groups from the two forest sites were profiled by 2D NMR methods, including high-resolution heteronuclear single quantum coherence spectroscopy and HCCH-total correlation spectroscopy. This ¹³C labeling approach also enabled the analysis of intact biomass by 2D solid-state ¹³C–¹³C correlation spectroscopy. Distinction between microbial groups and sites was observed in the polar and lipophilic metabolite profiles. Dominant differences could also be related to the capacity for lipid β-oxidation or adaptation to desiccation. Solid-state NMR further revealed differential synthetic capacity for glycolipids among groups. This technology coupled with ¹³C metabolite profiling should facilitate future functional annotation of indigenous microbial genomes.     

The microbial signature of aerosols produced during the thermophilic phase of composting

Aims:  The microbial diversity of bioaerosols released during operational activities at composting plants is poorly understood. Identification of bacteria and fungi present in such aerosols is the prerequisite for the definition of microbial indicators that could be used in dispersal and exposure studies.
Methods and Results:  A culture-independent analysis of composting bioaerosols collected at five different industrial open sites during the turning of composting piles in fermentation was performed by building 16S rDNA and 18S rDNA libraries. More than 800 sequences were analysed. Although differences in the phylotypes distribution were observed from one composting site to another, similarities in the structure of microbial diversity were remarkable. The same phyla dominated in the five bioaerosols: Ascomycota among fungi, Firmicutes and Actinobacteria among bacteria. For each phylum, some dominant phylotypes were common to at least four bioaerosols. These common phylotypes belonged to Thermomyces , Aspergillus , Penicillium , Geobacillus, Planifilum , Thermoactinomyces , Saccharopolyspora , Thermobifida and Saccharomonospora .
Conclusions:  The microbial signature of aerosols produced during the thermophilic phase of composting was determined. The similarities observed may be explained by the selection of thermophilic and sporulating species.
Significance and Impact of the Study:  Several bacteria and fungi identified in this study may represent potential indicators of composting bioaerosols in air.     

Culturable bacteria from Zn‐ and Cd‐accumulating Salix caprea with differential effects on plant growth and heavy metal availability

Aims: To characterize bacteria associated with Zn/Cd‐accumulating Salix caprea regarding their potential to support heavy metal phytoextraction. Methods and Results: Three different media allowed the isolation of 44 rhizosphere strains and 44 endophytes, resistant to Zn/Cd and mostly affiliated with Proteobacteria, Actinobacteria and Bacteroidetes/Chlorobi. 1‐Aminocyclopropane‐1‐carboxylic acid deaminase (ACCD), indole acetic acid and siderophore production were detected in 41, 23 and 50% of the rhizosphere isolates and in 9, 55 and 2% of the endophytes, respectively. Fifteen rhizosphere bacteria and five endophytes were further tested for the production of metal‐mobilizing metabolites by extracting contaminated soil with filtrates from liquid cultures. Four Actinobacteria mobilized Zn and/or Cd. The other strains immobilized Cd or both metals. An ACCD‐ and siderophore‐producing, Zn/Cd‐immobilizing rhizosphere isolate (Burkholderia sp.) and a Zn/Cd‐mobilizing Actinobacterium endophyte were inoculated onto S. caprea. The rhizosphere isolate reduced metal uptake in roots, whereas the endophyte enhanced metal accumulation in leaves. Plant growth was not promoted. Conclusions: Metal mobilization experiments predicted bacterial effects on S. caprea more reliably than standard tests for plant growth‐promoting activities. Significance and Impact of the Study: Bacteria, particularly Actinobacteria, associated with heavy metal‐accumulating Salix have the potential to increase metal uptake, which can be predicted by mobilization experiments and may be applicable in phytoremediation.     

Production and Purification of a Bioactive Substance Inhibiting Multiple Drug Resistant Bacteria and Human Leukemia Cells from a Salt-Tolerant Marine Actinobacterium sp. Isolated from the Bay of Bengal

Four marine actinobacteria tolerant to 200 g NaCl l⁻¹ were screened for antibacterial activity against eight patient-derived multiple drug resistant (MDR) bacteria. The active compound (MW 300.2, predicted molecular formula C₂₀H₂₈O₂) from an actinobacterium, was inhibitory to three Gram-positive and three Gram-negative MDR bacteria, seven non-clinical Gram-positive, four Gram-negative bacteria and five fungi (MIC: 3.5–4.0 µg ml⁻¹). Also, 54% of human leukemia (HL-60) cells were killed by the compound at 0.05 µg ml⁻¹. Bioreactor production demonstrated unusual primary metabolite kinetics. Molecular phylogenetic analysis showed this typical intertidal inhabitant to be a member of the Streptomyces genus and distinct from other salt-tolerant actinobacteria. As no compound was found to match the properties in several electronic databases, our screening strategy should increase the possibility of discovering bioactive molecules from rare actinobacteria.