Associated bacterial communities, confrontation studies, and comparative genomics reveal important interactions between Morchella with Pseudomonas spp.

Members of the fungal genus Morchella are widely known for their important ecological roles and significant economic value. In this study, we used amplicon and genome sequencing to characterize bacterial communities associated with sexual fruiting bodies from wild specimens, as well as vegetative mycelium and sclerotia obtained from Morchella isolates grown in vitro. These investigations included diverse representatives from both Elata and Esculenta Morchella clades. Unique bacterial community compositions were observed across the various structures examined, both within and across individual Morchella isolates or specimens. However, specific bacterial taxa were frequently detected in association with certain structures, providing support for an associated core bacterial community. Bacteria from the genus Pseudomonas and Ralstonia constituted the core bacterial associates of Morchella mycelia and sclerotia, while other genera (e.g., Pedobacter spp., Deviosa spp., and Bradyrhizobium spp.) constituted the core bacterial community of fruiting bodies. Furthermore, the importance of Pseudomonas as a key member of the bacteriome was supported by the isolation of several Pseudomonas strains from mycelia during in vitro cultivation. Four of the six mycelial-derived Pseudomonas isolates shared 16S rDNA sequence identity with amplicon sequences recovered directly from the examined fungal structures. Distinct interaction phenotypes (antagonistic or neutral) were observed in confrontation assays between these bacteria and various Morchella isolates. Genome sequences obtained from these Pseudomonas isolates revealed intriguing differences in gene content and annotated functions, specifically with respect to toxin-antitoxin systems, cell adhesion, chitinases, and insecticidal toxins. These genetic differences correlated with the interaction phenotypes. This study provides evidence that Pseudomonas spp. are frequently associated with Morchella and these associations may greatly impact fungal physiology.

Ecophysiology and interactions of a taurine-respiring bacterium in the mouse gut.

Taurine-respiring gut bacteria produce H2S with ambivalent impact on host health. We report the isolation and ecophysiological characterization of a taurine-respiring mouse gut bacterium. Taurinivorans muris strain LT0009 represents a new widespread species that differs from the human gut sulfidogen Bilophila wadsworthia in its sulfur metabolism pathways and host distribution. T. muris specializes in taurine respiration in vivo, seemingly unaffected by mouse diet and genotype, but is dependent on other bacteria for release of taurine from bile acids. Colonization of T. muris in gnotobiotic mice increased deconjugation of taurine-conjugated bile acids and transcriptional activity of a sulfur metabolism gene-encoding prophage in other commensals, and slightly decreased the abundance of Salmonella enterica, which showed reduced expression of galactonate catabolism genes. Re-analysis of metagenome data from a previous study further suggested that T. muris can contribute to protection against pathogens by the commensal mouse gut microbiota. Together, we show the realized physiological niche of a key murine gut sulfidogen and its interactions with selected gut microbiota members.

The endohyphal microbiome: current progress and challenges for scaling down integrative multi-omic microbiome research.

As microbiome research has progressed, it has become clear that most, if not all, eukaryotic organisms are hosts to microbiomes composed of prokaryotes, other eukaryotes, and viruses. Fungi have only recently been considered holobionts with their own microbiomes, as filamentous fungi have been found to harbor bacteria (including cyanobacteria), mycoviruses, other fungi, and whole algal cells within their hyphae. Constituents of this complex endohyphal microbiome have been interrogated using multi-omic approaches. However, a lack of tools, techniques, and standardization for integrative multi-omics for small-scale microbiomes (e.g., intracellular microbiomes) has limited progress towards investigating and understanding the total diversity of the endohyphal microbiome and its functional impacts on fungal hosts. Understanding microbiome impacts on fungal hosts will advance explorations of how "microbiomes within microbiomes" affect broader microbial community dynamics and ecological functions. Progress to date as well as ongoing challenges of performing integrative multi-omics on the endohyphal microbiome is discussed herein. Addressing the challenges associated with the sample extraction, sample preparation, multi-omic data generation, and multi-omic data analysis and integration will help advance current knowledge of the endohyphal microbiome and provide a road map for shrinking microbiome investigations to smaller scales. Video Abstract.

Bacteria of eleven different species isolated from biofilms in a meat processing environment have diverse biofilm forming abilities.

Biofilms are formed by microorganisms protected by a self-produced matrix, most often attached to a surface. In the food processing environments biofilms endanger the product safety by the transmission of spoilage and pathogenic bacteria. In this study, we characterised the biofilm formation of the following eleven strains isolated from biofilms in a meat-processing environment: Acinetobacter harbinensis BF1, Arthrobacter sp. BF1, Brochothrix thermosphacta BF1, Carnobacterium maltaromaticum BF1, Kocuria salsicia BF1, Lactococcus piscium BF1, Microbacterium sp. BF1, Pseudomonas fragi BF1, Psychrobacter sp. BF1, Rhodococcus erythropolis BF1, Stenotrophomonas sp. BF1. We applied whole- genome sequencing and subsequent genome analysis to elucidate genetic features associated with the biofilm lifestyle. We furthermore determined the motility and studied biofilm formation on stainless steel using a static mono-species biofilm model mimicking the meat processing environment. The biomass and the EPS components carbohydrates, proteins and extracellular DNA (eDNA) of the biofilms were investigated after seven days at 10 °C. Whole-genome analysis of the isolates revealed that all strains except the Kocuria salsicia BF1 isolate, harboured biofilm associated genes, including genes for matrix production and motility. Genes involved in cellulose metabolism (present in 82% of the eleven strains) and twitching motility (present in 45%) were most frequently found. The capacity for twitching was confirmed using plate assays for all strains except Lactococcus piscium BF1, which showed the lowest motility behaviour. Differences in biofilm forming abilities could be demonstrated. The bacterial load ranged from 5.4 log CFU/cm2 (Psychrobacter sp. isolate) to 8.7 log CFU/cm2 (Microbacterium sp. isolate). The amount of the matrix components varied between isolates. In the biofilm of six strains we detected all three matrix components at different levels (carbohydrates, proteins and eDNA), in two only carbohydrates and eDNA, and in three only carbohydrates. Carbohydrates were detected in biofilms of all strains ranging from 0.5 to 4.3 µg glucose equivalents/cm2. Overall, the Microbacterium sp. strain showed the highest biofilm forming ability with high bacterial load (8.7 log CFU/cm2) and high amounts of carbohydrates (2.2 µg glucose equivalents/cm2), proteins (present in all experiments) and eDNA (549 ng/cm2). In contrast, Brochothrix thermosphacta was a weak biofilm former, showing low bacterial load and low levels of carbohydrates in the matrix (6.2 log CFU/cm2 and 0.5 µg glucose equivalents/cm2). This study contributes to our understanding of the biofilm forming ability of bacteria highly abundant in the meat processing environment, which is crucial to develop strategies to prevent and reduce biofilm formation in the food producing environment.

Sulfoquinovose is a select nutrient of prominent bacteria and a source of hydrogen sulfide in the human gut.

Responses of the microbiota to diet are highly personalized but mechanistically not well understood because many metabolic capabilities and interactions of human gut microorganisms are unknown. Here we show that sulfoquinovose (SQ), a sulfonated monosaccharide omnipresent in green vegetables, is a selective yet relevant substrate for few but ubiquitous bacteria in the human gut. In human feces and in defined co-culture, Eubacterium rectale and Bilophila wadsworthia used recently identified pathways to cooperatively catabolize SQ with 2,3-dihydroxypropane-1-sulfonate as a transient intermediate to hydrogen sulfide (H2S), a key intestinal metabolite with disparate effects on host health. SQ-degradation capability is encoded in almost half of E. rectale genomes but otherwise sparsely distributed among microbial species in the human intestine. However, re-analysis of fecal metatranscriptome datasets of four human cohorts showed that SQ degradation (mostly from E. rectale and Faecalibacterium prausnitzii) and H2S production (mostly from B. wadsworthia) pathways were expressed abundantly across various health states, demonstrating that these microbial functions are core attributes of the human gut. The discovery of green-diet-derived SQ as an exclusive microbial nutrient and an additional source of H2S in the human gut highlights the role of individual dietary compounds and organosulfur metabolism on microbial activity and has implications for precision editing of the gut microbiota by dietary and prebiotic interventions.

Environmental and Intestinal Phylum Firmicutes Bacteria Metabolize the Plant Sugar Sulfoquinovose via a 6-Deoxy-6-sulfofructose Transaldolase Pathway.

Bacterial degradation of the sugar sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) produced by plants, algae, and cyanobacteria, is an important component of the biogeochemical carbon and sulfur cycles. Here, we reveal a third biochemical pathway for primary SQ degradation in an aerobic Bacillus aryabhattai strain. An isomerase converts SQ to 6-deoxy-6-sulfofructose (SF). A novel transaldolase enzyme cleaves the SF to 3-sulfolactaldehyde (SLA), while the non-sulfonated C3-(glycerone)-moiety is transferred to an acceptor molecule, glyceraldehyde phosphate (GAP), yielding fructose-6-phosphate (F6P). Intestinal anaerobic bacteria such as Enterococcus gilvus, Clostridium symbiosum, and Eubacterium rectale strains also express transaldolase pathway gene clusters during fermentative growth with SQ. The now three known biochemical strategies for SQ catabolism reflect adaptations to the aerobic or anaerobic lifestyle of the different bacteria. The occurrence of these pathways in intestinal (family) Enterobacteriaceae and (phylum) Firmicutes strains further highlights a potential importance of metabolism of green-diet SQ by gut microbial communities to, ultimately, hydrogen sulfide.

Community dynamics and functional characteristics of naphthalene-degrading populations in contaminated surface sediments and hypoxic/anoxic groundwater.

Earlier research on the biogeochemical factors affecting natural attenuation in coal-tar contaminated groundwater, at South Glens Falls, NY, revealed the importance of anaerobic metabolism and trophic interactions between degrader and bacterivore populations. Field-based characterizations of both phenomena have proven challenging, but advances in stable isotope probing (SIP), single-cell imaging and shotgun metagenomics now provide cultivation-independent tools for their study. We tracked carbon from 13 C-labelled naphthalene through microbial populations in contaminated surface sediments over 6 days using respiration assays, secondary ion mass spectrometry imaging and shotgun metagenomics to disentangle the contaminant-based trophic web. Contaminant-exposed communities in hypoxic/anoxic groundwater were contrasted with those from oxic surface sediments to identify putative features of anaerobic catabolism of naphthalene. In total, six bacteria were responsible for naphthalene degradation. Cupriavidus, Ralstonia and Sphingomonas predominated at the earliest stages of SIP incubations and were succeeded in later stages by Stenotrophomonas and Rhodococcus. Metagenome-assembled genomes provided evidence for the ecological and functional characteristics underlying these temporal shifts. Identical species of Stenotrophomonas and Rhodococcus were abundant in the most contaminated, anoxic groundwater. Apparent increases in bacterivorous protozoa were observed following exposure to naphthalene, though insignificant amounts of carbon were transferred between bacterial degraders and populations of secondary feeders.

Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells.

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.

In situ expression of nitrite-dependent anaerobic methane oxidation proteins by Candidatus Methylomirabilis oxyfera co-occurring with expressed anammox proteins in a contaminated aquifer.

Deciphering the in situ activities of microorganisms is essential for understanding the biogeochemical processes occurring in complex environments. Here, we used environmental metaproteomics to obtain information about the identity of subsurface microbial populations in coal tar-contaminated groundwater and the metabolic processes they catalyze. Metaproteomic libraries (two shotgun and seven slices from one SDS-PAGE gel) were generated from replicate samples of microbial biomass. Peptide fragment analysis using nano-liquid chromatography (LC)-mass spectrometry (MS)/MS of the three protein pools generated a total of 95,725 mass spectra. When analyzed using mascot v.2.3.02 and searched against the NCBInr bacterial database [confidence interval 99% (P < 0.01)], a total of 1,270 proteins had at least two peptide matches. Replication of identified proteins across the three libraries was low (3.3%); however, in each library, the most frequently identified protein host was Candidatus Methylomirabilis oxyfera (15, 12 and 62 proteins for each shotgun and the gel-slice library respectively). Remarkably, eight of the nine proteins in the nitrite-dependent anaerobic methane oxidation pathway were found. Additionally, 39 proteins were matched to known anammox bacteria including hydroxylamine and hydrazine oxidase. Metaproteomics thus revealed a microbial population, closely related to Ca. Methylomirabilis oxyfera, actively engaged in nitrite-dependent anaerobic methane oxidation and likely competing for nitrite with anammox bacteria.

Metaproteomic survey of six aquatic habitats: discovering the identities of microbial populations active in biogeochemical cycling.

Our goal is to strengthen the foundations of metaproteomics as a microbial community analysis tool that links the functional identity of actively expressed gene products with host phylogeny. We used shotgun metaproteomics to survey waters in six disparate aquatic habitats (Cayuga Lake, NY; Oneida Lake, NY; Gulf of Maine; Chesapeake Bay, MD; Gulf of Mexico; and the South Pacific). Peptide pools prepared from filter-gathered microbial biomass, analyzed by nano-liquid chromatography-mass spectrometry (MS/MS) generating 9,693 ± 1,073 mass spectra identified 326 ± 107 bacterial proteins per sample. Distribution of proteobacterial (Alpha and Beta) and cyanobacterial (Prochlorococcus and Synechococcus spp.) protein hosts across all six samples was consistent with the previously published biogeography for these microorganisms. Marine samples were enriched in transport proteins (TRAP-type for dicarboxylates and ATP binding cassette (ABC)-type for amino acids and carbohydrates) compared with the freshwater samples. We were able to match in situ expression of many key proteins catalyzing C-, N-, and S-cycle processes with their bacterial hosts across all six habitats. Pelagibacter was identified as the host of ABC-type sugar-, organic polyanion-, and glycine betaine-transport proteins; this extends previously published studies of Pelagibacter's in situ biogeochemical role in marine C- and N-metabolism. Proteins matched to Ruegeria confirmed these organism's role in marine waters oxidizing both carbon monoxide and sulfide. By documenting both processes expressed in situ and the identity of host cells, metaproteomics tested several existing hypotheses about ecophysiological processes and provided fodder for new ones.

Role of nitrogen fixation in the autecology of Polaromonas naphthalenivorans in contaminated sediments.

Polaromonas naphthalenivorans strain CJ2 is a Gram-negative betaproteobacterium that was identified, using stable isotope probing in 2003, as a dominant in situ degrader of naphthalene in coal tar-contaminated sediments. The sequenced genome of strain CJ2 revealed several genes conferring nitrogen fixation within a 65.6 kb region of strain CJ2's chromosome that is absent in the genome of its closest sequenced relative Polaromonas sp. strain JS666. Laboratory growth and nitrogenase assays verified that these genes are functional, providing an alternative source of nitrogen in N-free media when using naphthalene or pyruvate as carbon sources. Knowing this, we investigated if nitrogen-fixation activity could be detected in microcosms containing sediments from the field site where strain CJ2 was isolated. Inducing nitrogen limitation with the addition of glucose or naphthalene stimulated nitrogenase activity in amended sediments, as detected using the acetylene reduction assay. With the use of fluorescence microscopy, we screened the microcosm sediments for the presence of active strain CJ2 cells using a dual-labelling approach. When we examined the carbon-amended microcosm sediments stained with both a strain CJ2-specific fluorescent in situ hybridization probe and a polyclonal fluorescently tagged antibody, we were able to detect dual-labelled active cells. In contrast, in sediments that received no carbon addition (showing no nitrogenase activity), no dual-labelled cells were detected. Furthermore, the naphthalene amendment enhanced the proportion of active strain CJ2 cells in the sediment relative to a glucose amendment. Field experiments performed in sediments where strain CJ2 was isolated showed nitrogenase activity in response to dosing with naphthalene. Dual-label fluorescence staining of these sediments showed a fivefold increase in active strain CJ2 in the sediments dosed with naphthalene over those dosed with deionized water. These experiments show that nitrogen fixation may play an important role in naphthalene biodegradation by strain CJ2 and contribute to its ecological success.

Dynamic secondary ion mass spectrometry imaging of microbial populations utilizing C-labelled substrates in pure culture and in soil.

We demonstrate that dynamic secondary ion mass spectrometry (SIMS)-based ion microscopy can provide a means of measuring (13)C assimilation into individual bacterial cells grown on (13)C-labelled organic compounds in the laboratory and in field soil. We grew pure cultures of Pseudomonas putida NCIB 9816-4 in minimal media with known mixtures of (12)C- and (13)C-glucose and analysed individual cells via SIMS imaging. Individual cells yielded signals of masses 12, 13, 24, 25, 26 and 27 as negative secondary ions indicating the presence of (12)C(-), (13)C(-), (24)((12)C(2))(-), (25)((12)C(13)C)(-), (26)((12)C(14)N)(-) and (27)((13)C(14)N)(-) ions respectively. We verified that ratios of signals taken from the same cells only changed minimally during a approximately 4.5 min period of primary O(2)(+) beam sputtering by the dynamic SIMS instrument in microscope detection mode. There was a clear relationship between mass 27 and mass 26 signals in Pseudomonas putida cells grown in media containing varying proportions of (12)C- to (13)C-glucose: a standard curve was generated to predict (13)C-enrichment in unknown samples. We then used two strains of Pseudomonas putida able to grow on either all or only a part of a mixture of (13)C-labelled and unlabelled carbon sources to verify that differential (13)C signals measured by SIMS were due to (13)C assimilation into cell biomass. Finally, we made three key observations after applying SIMS ion microscopy to soil samples from a field experiment receiving (12)C- or (13)C-phenol: (i) cells enriched in (13)C were heterogeneously distributed among soil populations; (ii) (13)C-labelled cells were detected in soil that was dosed a single time with (13)C-phenol; and (iii) in soil that received 12 doses of (13)C-phenol, 27% of the cells in the total community were more than 90% (13)C-labelled.