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1. Kinetics and Identities of Extracellular Peptidases in Subsurface Sediments of the White Oak River Estuary, North Carolina

   https://doi.org/10.1128/aem.00102-19  

   Steen, Andrew D. ; Kevorkian, Richard T. ; Bird, Jordan T. ; Dombrowski, Nina ; Baker, Brett J. ; Hagen, Shane M. ; Mulligan, Katherine H. ; Schmidt, Jenna M. ; Webber, Austen T. ; Royalty, Taylor M. ; et al ( October 2019 , Applied and Environmental Microbiology)

   ABSTRACT Anoxic subsurface sediments contain communities of heterotrophic microorganisms that metabolize organic carbon at extraordinarily low rates. In order to assess the mechanisms by which subsurface microorganisms access detrital sedimentary organic matter, we measured kinetics of a range of extracellular peptidases in anoxic sediments of the White Oak River Estuary, NC. Nine distinct peptidase substrates were enzymatically hydrolyzed at all depths. Potential peptidase activities ( V max ) decreased with increasing sediment depth, although V max expressed on a per-cell basis was approximately the same at all depths. Half-saturation constants ( K m ) decreased with depth, indicating peptidases that functioned more efficiently at low substrate concentrations. Potential activities of extracellular peptidases acting on molecules that are enriched in degraded organic matter ( d -phenylalanine and l -ornithine) increased relative to enzymes that act on l -phenylalanine, further suggesting microbial community adaptation to access degraded organic matter. Nineteen classes of predicted, exported peptidases were identified in genomic data from the same site, of which genes for class C25 (gingipain-like) peptidases represented more than 40% at each depth. Methionine aminopeptidases, zinc carboxypeptidases, and class S24-like peptidases, which are involved in single-stranded-DNA repair, were also abundant. These results suggest a subsurface heterotrophic microbial community that primarily accesses low-quality detrital organic matter via a diverse suite of well-adapted extracellular enzymes. IMPORTANCE Burial of organic carbon in marine and estuarine sediments represents a long-term sink for atmospheric carbon dioxide. Globally, ∼40% of organic carbon burial occurs in anoxic estuaries and deltaic systems. However, the ultimate controls on the amount of organic matter that is buried in sediments, versus oxidized into CO 2 , are poorly constrained. In this study, we used a combination of enzyme assays and metagenomic analysis to identify how subsurface microbial communities catalyze the first step of proteinaceous organic carbon degradation. Our results show that microbial communities in deeper sediments are adapted to access molecules characteristic of degraded organic matter, suggesting that those heterotrophs are adapted to life in the subsurface. 

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2. Predicted functional genes for the biodegradation of xenobiotics in groundwater and sediment at two contaminated Naval Sites

   https://doi.org/10.1007/s00253-021-11756-3  

   Vera, Andrea ; Paes Wilson, Fernanda ; Cupples, Alison M. ( January 2022 , Applied microbiology and biotechnology)

   The goals of this study were to predict the genes associated with the biodegradation of organic contaminants and to examine microbial community structure in samples from two contaminated sites. The approach involved a predictive bioinformatics tool (PICRUSt2) targeting genes from twelve KEGG xenobiotic biodegradation pathways (benzoate, chloroalkane and chloroalkene, chlorocyclohexane and chlorobenzene, toluene, xylene, nitrotoluene, ethylbenzene, styrene, dioxin, naphthalene, polycyclic aromatic hydrocarbons, and metabolism of xenobiotics by cytochrome P450). Further, the predicted phylotypes associated with functional genes early in each pathway were determined. Phylogenetic analysis indicated a greater diversity in the sediment compared to the groundwater samples. The most abundant genera for sediments/microcosms included Pseudomonas, Methylotenera, Rhodococcus, Stenotrophomonas, and Brevundimonas, and the most abundant for the groundwater/microcosms included Pseudomonas, Cupriavidus, Azospira, Rhodococcus, and unclassified Burkholderiaceae. Genes from all twelve of the KEGG pathways were predicted to occur. Seven pathways contained less than twenty-five genes. The predicted genes were lowest for xenobiotics metabolism by cytochrome P450 and ethylbenzene biodegradation and highest for benzoate biodegradation. Notable trends include the occurrence of the first genes for trinitrotoluene and 2,4-dinitrotoluene degradation. Also, the complete path from toluene to benzoyl-CoA was predicted. Twenty-two of the dioxin pathway genes were predicted, including genes within the first steps. The following phylotypes were associated with the greatest number of pathways: unclassified Burkholderiaceae, Burkholderia-Caballeronia-Paraburkholderia, Pseudomonas, Rhodococcus, unclassified Betaproteobacteria, and Polaromonas. This work illustrates the value of PICRUSt2 for predicting biodegradation potential and suggests that a subset of phylotypes could be important for the breakdown of organic contaminants or their metabolites. 

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3. Predominance of Anaerobic, Spore-Forming Bacteria in Metabolically Active Microbial Communities from Ancient Siberian Permafrost

   https://doi.org/10.1128/AEM.00560-19  

   Liang, Renxing ; Lau, Maggie ; Vishnivetskaya, Tatiana ; Lloyd, Karen G. ; Wang, Wei ; Wiggins, Jessica ; Miller, Jennifer ; Pfiffner, Susan ; Rivkina, Elizaveta M. ; Onstott, Tullis C. ; et al ( August 2019 , Applied and Environmental Microbiology)

   ABSTRACT The prevalence of microbial life in permafrost up to several million years (Ma) old has been well documented. However, the long-term survivability, evolution, and metabolic activity of the entombed microbes over this time span remain underexplored. We integrated aspartic acid (Asp) racemization assays with metagenomic sequencing to characterize the microbial activity, phylogenetic diversity, and metabolic functions of indigenous microbial communities across a ∼0.01- to 1.1-Ma chronosequence of continuously frozen permafrost from northeastern Siberia. Although Asp in the older bulk sediments (0.8 to 1.1 Ma) underwent severe racemization relative to that in the youngest sediment (∼0.01 Ma), the much lower d -Asp/ l -Asp ratio (0.05 to 0.14) in the separated cells from all samples suggested that indigenous microbial communities were viable and metabolically active in ancient permafrost up to 1.1 Ma. The microbial community in the youngest sediment was the most diverse and was dominated by the phyla Actinobacteria and Proteobacteria . In contrast, microbial diversity decreased dramatically in the older sediments, and anaerobic, spore-forming bacteria within Firmicutes became overwhelmingly dominant. In addition to the enrichment of sporulation-related genes, functional genes involved in anaerobic metabolic pathways such as fermentation, sulfate reduction, and methanogenesis were more abundant in the older sediments. Taken together, the predominance of spore-forming bacteria and associated anaerobic metabolism in the older sediments suggest that a subset of the original indigenous microbial community entrapped in the permafrost survived burial over geological time. IMPORTANCE Understanding the long-term survivability and associated metabolic traits of microorganisms in ancient permafrost frozen millions of years ago provides a unique window into the burial and preservation processes experienced in general by subsurface microorganisms in sedimentary deposits because of permafrost’s hydrological isolation and exceptional DNA preservation. We employed aspartic acid racemization modeling and metagenomics to determine which microbial communities were metabolically active in the 1.1-Ma permafrost from northeastern Siberia. The simultaneous sequencing of extracellular and intracellular genomic DNA provided insight into the metabolic potential distinguishing extinct from extant microorganisms under frozen conditions over this time interval. This in-depth metagenomic sequencing advances our understanding of the microbial diversity and metabolic functions of extant microbiomes from early Pleistocene permafrost. Therefore, these findings extend our knowledge of the survivability of microbes in permafrost from 33,000 years to 1.1 Ma. 

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4. Landscape characteristics shape surface soil microbiomes in the Chihuahuan Desert

   https://doi.org/10.3389/fmicb.2023.1135800  

   Hansen, Frederick A. ; James, Darren K. ; Anderson, John P. ; Meredith, Christy S. ; Dominguez, Andrew J. ; Pombubpa, Nuttapon ; Stajich, Jason E. ; Romero-Olivares, Adriana L. ; Salley, Shawn W. ; Pietrasiak, Nicole ( June 2023 , Frontiers in Microbiology)

   Introduction Soil microbial communities, including biological soil crust microbiomes, play key roles in water, carbon and nitrogen cycling, biological weathering, and other nutrient releasing processes of desert ecosystems. However, our knowledge of microbial distribution patterns and ecological drivers is still poor, especially so for the Chihuahuan Desert. Methods This project investigated the effects of trampling disturbance on surface soil microbiomes, explored community composition and structure, and related patterns to abiotic and biotic landscape characteristics within the Chihuahuan Desert biome. Composite soil samples were collected in disturbed and undisturbed areas of 15 long-term ecological research plots in the Jornada Basin, New Mexico. Microbial diversity of cross-domain microbial groups (total Bacteria, Cyanobacteria, Archaea, and Fungi) was obtained via DNA amplicon metabarcode sequencing. Sequence data were related to landscape characteristics including vegetation type, landforms, ecological site and state as well as soil properties including gravel content, soil texture, pH, and electrical conductivity. Results Filamentous Cyanobacteria dominated the photoautotrophic community while Proteobacteria and Actinobacteria dominated among the heterotrophic bacteria. Thaumarchaeota were the most abundant Archaea and drought adapted taxa in Dothideomycetes and Agaricomycetes were most abundant fungi in the soil surface microbiomes. Apart from richness within Archaea ( p  = 0.0124), disturbed samples did not differ from undisturbed samples with respect to alpha diversity and community composition ( p  ≥ 0.05), possibly due to a lack of frequent or impactful disturbance. Vegetation type and landform showed differences in richness of Bacteria, Archaea, and Cyanobacteria but not in Fungi. Richness lacked strong relationships with soil variables. Landscape features including parent material, vegetation type, landform type, and ecological sites and states, exhibited stronger influence on relative abundances and microbial community composition than on alpha diversity, especially for Cyanobacteria and Fungi. Soil texture, moisture, pH, electrical conductivity, lichen cover, and perennial plant biomass correlated strongly with microbial community gradients detected in NMDS ordinations. Discussion Our study provides first comprehensive insights into the relationships between landscape characteristics, associated soil properties, and cross-domain soil microbiomes in the Chihuahuan Desert. Our findings will inform land management and restoration efforts and aid in the understanding of processes such as desertification and state transitioning, which represent urgent ecological and economical challenges in drylands around the world. 

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5. Metagenomics as a Public Health Risk Assessment Tool in a Study of Natural Creek Sediments Influenced by Agricultural and Livestock Runoff: Potential and Limitations

   https://doi.org/10.1128/AEM.02525-19  

   Suttner, Brittany ; Johnston, Eric R. ; Orellana, Luis H. ; Rodriguez-R, Luis M. ; Hatt, Janet K. ; Carychao, Diana ; Carter, Michelle Q. ; Cooley, Michael B. ; Konstantinidis, Konstantinos T. ; Ercolini, Danilo ( March 2020 , Applied and Environmental Microbiology)

   ABSTRACT Little is known about the public health risks associated with natural creek sediments that are affected by runoff and fecal pollution from agricultural and livestock practices. For instance, the persistence of foodborne pathogens such as Shiga toxin-producing Escherichia coli (STEC) originating from these practices remains poorly quantified. Towards closing these knowledge gaps, the water-sediment interface of two creeks in the Salinas River Valley of California was sampled over a 9-month period using metagenomics and traditional culture-based tests for STEC. Our results revealed that these sediment communities are extremely diverse and have functional and taxonomic diversity comparable to that observed in soils. With our sequencing effort (∼4 Gbp per library), we were unable to detect any pathogenic E. coli in the metagenomes of 11 samples that had tested positive using culture-based methods, apparently due to relatively low abundance. Furthermore, there were no significant differences in the abundance of human- or cow-specific gut microbiome sequences in the downstream impacted sites compared to that in upstream more pristine (control) sites, indicating natural dilution of anthropogenic inputs. Notably, the high number of metagenomic reads carrying antibiotic resistance genes (ARGs) found in all samples was significantly higher than ARG reads in other available freshwater and soil metagenomes, suggesting that these communities may be natural reservoirs of ARGs. The work presented here should serve as a guide for sampling volumes, amount of sequencing to apply, and what bioinformatics analyses to perform when using metagenomics for public health risk studies of environmental samples such as sediments. IMPORTANCE Current agricultural and livestock practices contribute to fecal contamination in the environment and the spread of food- and waterborne disease and antibiotic resistance genes (ARGs). Traditionally, the level of pollution and risk to public health are assessed by culture-based tests for the intestinal bacterium Escherichia coli . However, the accuracy of these traditional methods (e.g., low accuracy in quantification, and false-positive signal when PCR based) and their suitability for sediments remain unclear. We collected sediments for a time series metagenomics study from one of the most highly productive agricultural regions in the United States in order to assess how agricultural runoff affects the native microbial communities and if the presence of Shiga toxin-producing Escherichia coli (STEC) in sediment samples can be detected directly by sequencing. Our study provided important information on the potential for using metagenomics as a tool for assessment of public health risk in natural environments. 

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