Bacterial isolate collection from switchgrass rhizosphere.

We provide a collection of 78 bacterial isolates from the rhizosphere of switchgrass (Panicum virgatum L.) at the Lux Arbor Reserve in Delton, MI, a site of the Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, MI, USA. We include information on isolation conditions and full-length 16S rRNA sequences.

Seasonal activities of the phyllosphere microbiome of perennial crops.

Understanding the interactions between plants and microorganisms can inform microbiome management to enhance crop productivity and resilience to stress. Here, we apply a genome-centric approach to identify ecologically important leaf microbiome members on replicated plots of field-grown switchgrass and miscanthus, and to quantify their activities over two growing seasons for switchgrass. We use metagenome and metatranscriptome sequencing and curate 40 medium- and high-quality metagenome-assembled-genomes (MAGs). We find that classes represented by these MAGs (Actinomycetia, Alpha- and Gamma- Proteobacteria, and Bacteroidota) are active in the late season, and upregulate transcripts for short-chain dehydrogenase, molybdopterin oxidoreductase, and polyketide cyclase. Stress-associated pathways are expressed for most MAGs, suggesting engagement with the host environment. We also detect seasonally activated biosynthetic pathways for terpenes and various non-ribosomal peptide pathways that are poorly annotated. Our findings support that leaf-associated bacterial populations are seasonally dynamic and responsive to host cues.

Do-it-Yourself Mock Community Standard for Multi-Step Assessment of Microbiome Protocols.

Microbiomes provide critical functions that support animals, plants, and ecosystems. High-throughput sequencing (HTS) has become an essential tool for the cultivation-independent study of microbiomes found in diverse environments, but requires effective and meaningful controls. One such critical control is a mock microbial community, which is used as a positive control for nucleic acid extraction, marker gene amplification, and sequencing. While mock community standards can be purchased, they can be costly and often include only medically relevant microbial strains that are not expected to be major players in non-human microbiomes. As an alternative, it is possible to design and construct a do-it-yourself (DIY) mock community, which can then be used as a positive control that is specifically customized to the protocol needs of a particular study system. In this article, we describe protocols to select appropriate microbial strains for the construction of a mock community. We first describe the steps to verify the identity of community members via Sanger sequencing. Then, we provide guidance on assembling and storing the DIY mock community as viable whole cells. This includes steps to create standard growth curves referenced to plate counts for each member, so that the community members can be quantified and later compared in terms of their "expected versus returned" relative contributions after sequencing. We also describe appropriate methods for the cryostorage of the fully assembled mock community as viable whole cells, so that they can be used as a unit in a microbiome analysis, from the lysis and nucleic acid extraction steps onwards. Finally, we provide an example of returned data and interpretation of DIY mock community sequences, discussing how to assess possible contamination and identify protocol biases for particular members. Overall, DIY mock communities serve to determine success and possible bias in a cultivation-independent microbiome analysis. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Strain identification and verification using Sanger sequencing Basic Protocol 2: Creation of glycerol stocks of each mock community strain for long-term cryostorage Basic Protocol 3: Assessment of strain freezer viability without cryoprotectant Basic Protocol 4: Creation of standard curve to determine CFU/ml of a liquid culture as a function of optical density Basic Protocol 5: Full mock community assembly using community concentration calculations and standard curves.

Assembly and seasonality of core phyllosphere microbiota on perennial biofuel crops.

Perennial grasses are promising feedstocks for biofuel production, with potential for leveraging their native microbiomes to increase their productivity and resilience to environmental stress. Here, we characterize the 16S rRNA gene diversity and seasonal assembly of bacterial and archaeal microbiomes of two perennial cellulosic feedstocks, switchgrass (Panicum virgatum L.) and miscanthus (Miscanthus x giganteus). We sample leaves and soil every three weeks from pre-emergence through senescence for two consecutive switchgrass growing seasons and one miscanthus season, and identify core leaf taxa based on occupancy. Virtually all leaf taxa are also detected in soil; source-sink modeling shows non-random, ecological filtering by the leaf, suggesting that soil is an important reservoir of phyllosphere diversity. Core leaf taxa include early, mid, and late season groups that were consistent across years and crops. This consistency in leaf microbiome dynamics and core members is promising for microbiome manipulation or management to support crop production.

Hook length of the bacterial flagellum is optimized for maximal stability of the flagellar bundle.

Most bacteria swim in liquid environments by rotating one or several flagella. The long external filament of the flagellum is connected to a membrane-embedded basal body by a flexible universal joint, the hook, which allows the transmission of motor torque to the filament. The length of the hook is controlled on a nanometer scale by a sophisticated molecular ruler mechanism. However, why its length is stringently controlled has remained elusive. We engineered and studied a diverse set of hook-length variants of Salmonella enterica. Measurements of plate-assay motility, single-cell swimming speed, and directional persistence in quasi-2D and population-averaged swimming speed and body angular velocity in 3D revealed that the motility performance is optimal around the wild-type hook length. We conclude that too-short hooks may be too stiff to function as a junction and too-long hooks may buckle and create instability in the flagellar bundle. Accordingly, peritrichously flagellated bacteria move most efficiently as the distance travelled per body rotation is maximal and body wobbling is minimized. Thus, our results suggest that the molecular ruler mechanism evolved to control flagellar hook growth to the optimal length consistent with efficient bundle formation. The hook-length control mechanism is therefore a prime example of how bacteria evolved elegant but robust mechanisms to maximize their fitness under specific environmental constraints.

Divergent extremes but convergent recovery of bacterial and archaeal soil communities to an ongoing subterranean coal mine fire.

Press disturbances are stressors that are extended or ongoing relative to the generation times of community members, and, due to their longevity, have the potential to alter communities beyond the possibility of recovery. They also provide key opportunities to investigate ecological resilience and to probe biological limits in the face of prolonged stressors. The underground coal mine fire in Centralia, Pennsylvania has been burning since 1962 and severely alters the overlying surface soils by elevating temperatures and depositing coal combustion pollutants. As the fire burns along the coal seams to disturb new soils, previously disturbed soils return to ambient temperatures, resulting in a chronosequence of fire impact. We used 16S rRNA gene sequencing to examine bacterial and archaeal soil community responses along two active fire fronts in Centralia, and investigated the influences of assembly processes (selection, dispersal and drift) on community outcomes. The hottest soils harbored the most variable and divergent communities, despite their reduced diversity. Recovered soils converged toward similar community structures, demonstrating resilience within 10-20 years and exhibiting near-complete return to reference communities. Measured soil properties (selection), local dispersal, and neutral community assembly models could not explain the divergences of communities observed at temperature extremes, yet beta-null modeling suggested that communities at temperature extremes follow niche-based processes rather than null. We hypothesize that priority effects from responsive seed bank transitions may be key in explaining the multiple equilibria observed among communities at extreme temperatures. These results suggest that soils generally have an intrinsic capacity for robustness to varied disturbances, even to press disturbances considered to be 'extreme', compounded, or incongruent with natural conditions.