Investigation of microbial groups involved in the uptake of atmospheric trace gases in upland soils

Atmospheric trace gases play a leading role in the changes occurring in the atmosphere at present, including climate change. A significant part of atmospheric trace gas fluxes occurs at the interface between atmosphere and upland soils. Unfortunately, the microorganisms in charge of these dynamics are not fully understood. This thesis therefore focuses on the investigation of microbial groups in terrestrial environments, responsible for or proposed to be involved in the uptake of atmospheric trace gases (CH4, CO2, H2), namely the potential atmospheric methane oxidizer upland soil cluster α (USCα), the autotrophic ammonia oxidizing archaea (AOA), and the hydrogen oxidizing streptomycetes. Several methods were tested to investigate the incorporation of labeled substrate and to monitor the expression of their functional marker genes, pmoA for the high-affinity particulate methane monooxygenase of USCα, amoA for the ammonia monooxygenase of AOA, and hydB for the high-affinity [NiFe]-hydrogenase of Streptomyces sp. PCB7. Although the upland soil cluster α (USCα) in forest soils is assumed to represent methanotrophic bacteria adapted to the trace level of atmospheric methane and to play an essential part in the removal of this greenhouse gas from the atmosphere, so far it is unclear whether these microorganisms are able to obtain all their energy and carbon solely from CH4 or use additional carbon compounds. Stable isotope probing was applied to investigate incorporation of labeled CH4 and acetate into nucleic acids of USCα. The results of this study indicate that USCα might only use atmospheric CH4 as an additional energy source or survival strategy, but utilizes additional carbon compounds, such as acetate, for growth suggesting the USCα represents rather facultative than obligate methanotrophs. Furthermore, CARD-FISH of pmoA transcripts visualized USCα in situ for the first time. These findings promote the knowledge and understanding of upland soils as a sink for atmospheric methane and the microorganisms proposed to be responsible for this process. While for a long time autotrophic bacteria were believed to be solely responsible for the process of ammonia oxidation, there is now increasing evidence that also Archaea are involved. But to date it remained elusive whether ammonia oxidizing archaea in soil can assimilate CO2 and to what extent they are functionally active. Stable isotope probing of nucleic acids using 13CO2 showed that ammonia oxidizing archaea were actively involved in microbial ammonia oxidation in an agricultural soil and did fix CO2 autotrophically, presumably via the hydroxypropionate-hydroxybutyrate cycle. CARD-FISH further demonstrated the numerical importance of the archaeal ammonia oxidizers to the overall archaeal community in this environment. These results give novel evidence that the contribution of nitrifying Archaea to ammonia oxidation and CO2 fixation in terrestrial environments might be substantial. Although hydrogen is considered to be one of the most important future energy carriers, little is known about the global biogeochemical cycle of this trace gas. Previous findings indicate that microorganisms rather than free soil enzymes are responsible for the uptake of atmospheric H2 in soils. In this thesis, CARD-FISH analyses demonstrated that streptomyces spores instead of the mycelia expressed the high-affinity H2 uptake activity. This suggests that H2-oxidizing streptomycetes, or actinobacteria in general, are essential for the uptake of atmospheric H2 in upland soils

Draft Genome Sequences of Obligate Methylotrophs <i>Methylovorus</i> sp. Strain MM2 and <i>Methylobacillus</i> sp. Strain MM3, Isolated from Grassland Soil

Methylotrophs of the family Methylophilaceae were isolated from grassland soil. Here, we report the draft genome sequences of two obligate methylotrophs, Methylovorus sp. strain MM2 and Methylobacillus sp. strain MM3. These genome sequences provide further insights into the genetic and metabolic diversity of the Methylophilaceae

Assembly of Bacterial Genome Sequences from Metagenomes of Spacecraft Assembly Cleanrooms

Characterizing the microbiome of spacecraft assembly cleanrooms is important for planetary protection. We report two bacterial metagenome-assembled genomes (MAGs) reconstructed from metagenomes produced from cleanroom samples from the Kennedy Space Center’s Payload Hazardous Servicing Facility (KSC-PHSF) during the handling of the Phoenix spacecraft. Characterization of these MAGs will enable identification of the strategies underpinning their survival

Draft Genome Sequence of Methylocella silvestris TVC, a Facultative Methanotroph Isolated from Permafrost

Permafrost environments play a crucial role in global carbon and methane cycling. We report here the draft genome sequence of Methylocella silvestris TVC, a new facultative methanotroph strain, isolated from the Siksik Creek catchment in the continuous permafrost zone of Inuvik (Northwest Territories, Canada)

Insights into toxic prymnesium parvum blooms:The role of sugars and algal viruses

Prymnesium parvum is a toxin-producing microalga that causes harmful algal blooms globally, which often result in large-scale fish kills that have severe ecological and economic implications. Although many toxins have previously been isolated from P. parvum, ambiguity still surrounds the responsible ichthyotoxins in P. parvum blooms and the biotic and abiotic factors that promote bloom toxicity. A major fish kill attributed to P. parvum occurred in Spring 2015 on the Norfolk Broads, a low-lying set of channels and lakes (Broads) found on the East of England. Here, we discuss how water samples taken during this bloom have led to diverse scientific advances ranging from toxin analysis to discovery of a new lytic virus of P. parvum, P. parvum DNA virus (PpDNAV-BW1). Taking recent literature into account, we propose key roles for sialic acids in this type of viral infection. Finally, we discuss recent practical detection and management strategies for controlling these devastating blooms

Targeted metagenomics of active microbial populations with stable-isotope probing

The ability to explore microbial diversity and function has been enhanced by novel experimental and computational tools. The incorporation of stable isotopes into microbial biomass enables the recovery of labeled nucleic acids from active microorganisms, despite their initial abundance and culturability. Combining stable-isotope probing (SIP) with metagenomics provides access to genomes from microorganisms involved in metabolic processes of interest. Studies using metagenomic analysis on DNA obtained from DNA-SIP incubations can be ideal for the recovery of novel enzymes for biotechnology applications, including biodegradation, biotransformation, and biosynthesis. This chapter introduces metagenomic and DNA-SIP methodologies, highlights biotechnology-focused studies that combine these approaches, and provides perspectives on future uses of these methods as analysis tools for applied and environmental microbiology

Assessing the toxicity and mitigating the impact of harmful Prymnesium blooms in eutrophic waters of the Norfolk Broads

Prymnesium parvum is a toxin-producing microalga, which causes harmful algal blooms globally, frequently leading to massive fish kills that have adverse ecological and economic implications for natural waterways and aquaculture alike. The dramatic effects observed on fish are thought to be due to algal polyether toxins, known as the prymnesins, but their lack of environmental detection has resulted in an uncertainty about the true ichthyotoxic agents. Using qPCR, we found elevated levels of P. parvum and its lytic virus, PpDNAV-BW1, in a fish-killing bloom on the Norfolk Broads, United Kingdom, in March 2015. We also detected, for the first time, the B-type prymnesin toxins in Broads waterway samples and gill tissue isolated from a dead fish taken from the study site. Furthermore, Norfolk Broads P. parvum isolates unambiguously produced B-type toxins in laboratory-grown cultures. A 2 year longitudinal study of the Broads study site showed P. parvum blooms to be correlated with increased temperature and that PpDNAV plays a significant role in P. parvum bloom demise. Finally, we used a field trial to show that treatment with low doses of hydrogen peroxide represents an effective strategy to mitigate blooms of P. parvum in enclosed water bodies

Poplar phyllosphere harbors disparate isoprene-degrading bacteria

The climate-active gas isoprene (2-methyl-1,3-butadiene) is released to the atmosphere in huge quantities, almost equaling that of methane, yet we know little about the biological cycling of isoprene in the environment. Although bacteria capable of growth on isoprene as the sole source of carbon and energy have previously been isolated from soils and sediments, no microbiological studies have targeted the major source of isoprene and examined the phyllosphere of isoprene-emitting trees for the presence of degraders of this abundant carbon source. Here, we identified isoprene-degrading bacteria in poplar tree-derived microcosms by DNA stable isotope probing. The genomes of isoprene-degrading taxa were reconstructed, putative isoprene metabolic genes were identified, and isoprene-related gene transcription was analyzed by shotgun metagenomics and metatranscriptomics. Gram-positive bacteria of the genus Rhodococcus proved to be the dominant isoprene degraders, as previously found in soil. However, a wider diversity of isoprene utilizers was also revealed, notably Variovorax, a genus not previously associated with this trait. This finding was confirmed by expression of the isoprene monooxygenase from Variovorax in a heterologous host. A Variovorax strain that could grow on isoprene as the sole carbon and energy source was isolated. Analysis of its genome confirmed that it contained isoprene metabolic genes with an identical layout and high similarity to those identified by DNA-stable isotope probing and metagenomics. This study provides evidence of a wide diversity of isoprene-degrading bacteria in the isoprene-emitting tree phyllosphere and greatly enhances our understanding of the biodegradation of this important metabolite and climate-active gas

Methanethiol-dependent dimethylsulfide production in soil environments

Dimethylsulfide (DMS) is an environmentally important trace gas with roles in sulfur cycling, signalling to higher organisms and in atmospheric chemistry. DMS is believed to be predominantly produced in marine environments via microbial degradation of the osmolyte dimethylsulfoniopropionate (DMSP). However, significant amounts of DMS are also generated from terrestrial environments, for example, peat bogs can emit ~6 μmol DMS m−2 per day, likely via the methylation of methanethiol (MeSH). A methyltransferase enzyme termed ‘MddA’, which catalyses the methylation of MeSH, generating DMS, in a wide range of bacteria and some cyanobacteria, may mediate this process, as the mddA gene is abundant in terrestrial metagenomes. This is the first study investigating the functionality of MeSH-dependent DMS production (Mdd) in a wide range of aerobic environments. All soils and marine sediment samples tested produced DMS when incubated with MeSH. Cultivation-dependent and cultivation-independent methods were used to assess microbial community changes in response to MeSH addition in a grassland soil where 35.9% of the bacteria were predicted to contain mddA. Bacteria of the genus Methylotenera were enriched in the presence of MeSH. Furthermore, many novel Mdd+ bacterial strains were isolated. Despite the abundance of mddA in the grassland soil, the Mdd pathway may not be a significant source of DMS in this environment as MeSH addition was required to detect DMS at only very low conversion rates