Biology subjects

Mark Welch, J. L.

Publications and source records attributed to Mark Welch, J. L..

9 recordsLinked to original sources

bioRxiv2022

Phages are important unrecognized players in the ecology of the oral pathogen Porphyromonas gingivalis

BackgroundPorphyromonas gingivalis (hereafter "Pg") is an oral pathogen that can act as a keystone driver of inflammation and periodontal disease. Although Pg is most readily recovered from individuals with actively progressing periodontal disease, healthy individuals and those with stable non-progressing disease are also colonized by Pg. Insights into the factors shaping the striking strain-level variation in Pg, and its variable associations with disease, are needed to achieve a more mechanistic understanding of periodontal disease and its progression. A key force shaping strain level diversity in all microbial communities is infection of bacteria by their viral (phage) predators and symbionts. Surprisingly, although Pg has been the subject of study for over 40 years, essentially nothing is known of its phages, and the prevailing paradigm is that phages are not important in the ecology of Pg. ResultsHere we systematically addressed the question of whether Pg are infected by phages - and we found that they are. We found that prophages are common in Pg, they are genomically diverse, and they encode genes that have the potential to alter Pg physiology and interactions. We found that phages represent unrecognized targets of the prevalent CRISPR-Cas defense systems in Pg, and that Pg strains encode numerous additional mechanistically diverse candidate anti-phage defense systems. We also found that phages and candidate anti-phage defense system elements together are major contributors to strain level diversity and the species pangenome of this oral pathogen. Finally, we demonstrate that prophages harbored by a model Pg strain are active in culture, producing extracellular viral particles in broth cultures. DiscussionThis work definitively establishes that phages are a major unrecognized force shaping the ecology and intraspecies strain-level diversity of the well-studied oral pathogen Pg. The foundational phage sequence datasets and model systems that we establish here add to the rich context of all that is already known about Pg, and point to numerous avenues of future inquiry that promise to shed new light on fundamental features of phage impacts on human health and disease broadly.

microbiology↗

bioRxiv2021

Site-specificity of streptococci in the oral microbiome

Patterns of microbial distribution are determined by as-yet poorly understood rules governing where microbes can grow and thrive. Therefore, a detailed understanding of where bacteria localize is necessary to advance microbial ecology and microbiome-based therapeutics. The site-specialist hypothesis predicts that most microbes in the human oral cavity have a primary habitat within the mouth where they are most abundant. We asked whether this hypothesis accurately describes the distribution of the members of the genus Streptococcus, a clinically relevant taxon that dominates most oral sites. Prior analysis of 16S rRNA gene sequencing data indicated that some oral Streptococcus clades are site-specialists while others may be generalists. However, within complex microbial populations composed of numerous closely-related species and strains, such as the oral streptococci, genome-scale analysis is necessary to provide the resolution to discriminate closely related taxa with distinct functional roles. Here we assess whether individual species within this genus are generalists using publicly available genomic sequence data that provides species-level resolution. We chose a set of high-quality representative genomes for Streptococcus species from the human oral microbiome. Onto these genomes, we mapped short-read metagenomic sequences from supragingival plaque, tongue dorsum, and other sites in the oral cavity. We found that every reliably detectable Streptococcus species in the human oral cavity was a site-specialist and that even closely related species such as S. mitis, S. oralis, and S. infantis specialized in different sites. These findings indicate that closely related bacteria can have distinct habitat distributions in the absence of dispersal limitation and under similar environmental conditions and immune regimes. These three species also share substantially the same species-specific core genes indicating that neither taxonomy nor gene content are clear predictors of site-specialization. Site-specificity may instead be influenced by subtle characteristics such as nucleotide-level divergences within conserved genes.

microbiology↗

bioRxiv2020

Mechanisms underlying proximity between oral commensal bacteria

Complex polymicrobial biofilm communities are abundant in nature particularly in the human oral cavity where their composition and fitness can affect health. While the study of these communities during disease is essential and prevalent, little is known about interactions within the healthy plaque community. Here we describe interactions between two of the most abundant species in this healthy microbiome, Haemophilus parainfluenzae and Streptococcus mitis. We discovered that H. parainfluenzae typically exists adjacent to Mitis group streptococci in vivo with which it also positively correlated based on microbiome data. By comparing in vitro coculture data to ex vivo microscopy we revealed that this co-occurrence is density dependent and further influenced by H2O2 production. We discovered that H. parainfluenzae has a more redundant, multifactorial response to H2O2 than related organisms and that the integrity of this system enhances streptococcal fitness. We also show that Mitis group streptococci can act as an in vivo source of NAD for H. parainfluenzae and that streptococci in vitro evoke patterns of carbon utilization from H. parainfluenzae that are similar to those observed in vivo. Our findings describe mechanistic interactions between two of the most abundant and prevalent members of healthy supragingival plaque that contribute to their survival in vivo.

microbiology↗

bioRxiv2020

Metapangenomics of the oral microbiome provides insights into habitat adaptation and cultivar diversity

BackgroundThe increasing availability of microbial genomes and environmental shotgun metagenomes provides unprecedented access to the genomic differences within related bacteria. The human oral microbiome with its diverse habitats and abundant, relatively well-characterized microbial inhabitants presents an opportunity to investigate bacterial population structures at an ecosystem scale. ResultsHere, we employ a metapangenomic approach that combines public genomes with Human Microbiome Project (HMP) metagenomes to study the diversity of microbial residents of three oral habitats: tongue dorsum, buccal mucosa, and supragingival plaque. For two exemplar taxa, Haemophilus parainfluenzae and the genus Rothia, metapangenomes revealed distinct genomic groups based on shared genome content. H. parainfluenzae genomes separated into three distinct subgroups with differential abundance between oral habitats. Functional enrichment analyses identified an operon encoding oxaloacetate decarboxylase as diagnostic for the tongue-abundant subgroup. For the genus Rothia, grouping by shared genome content recapitulated species-level taxonomy and habitat preferences. However, while most R. mucilaginosa were restricted to the tongue as expected, two genomes represented a cryptic population of R. mucilaginosa in many buccal mucosa samples. For both H. parainfluenzae and the genus Rothia, we identified not only limitations in the ability of cultivated organisms to represent populations in their native environment, but also specifically which cultivar gene sequences were absent or ubiquitous. ConclusionsOur findings provide insights into population structure and biogeography in the mouth and form specific hypotheses about habitat adaptation. These results illustrate the power of combining metagenomes and pangenomes to investigate the ecology and evolution of bacteria across analytical scales.

microbiology↗

bioRxiv2020

Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome

Microbial residents of the human oral cavity have long been a major focus of microbiology due to their influence on host health and their intriguing patterns of site specificity amidst the lack of dispersal limitation. Yet, the determinants of niche partitioning in this habitat are yet to be fully understood, especially among the taxa that belong to recently discovered branches of microbial life. Here we assembled metagenomes from daily tongue and dental plaque samples from multiple individuals and reconstructed 790 non-redundant genomes, 43 of which resolved to TM7 that formed six monophyletic clades distinctly associated either with plaque or with tongue. Both pangenomic and phylogenomic analyses grouped tongue-specific TM7 clades with other host-associated TM7 genomes. In contrast, plaque-specific TM7 grouped together with environmental TM7 genomes. Besides offering deeper insights into the ecology, evolution, and the mobilome of the cryptic members of the oral microbiome, our study reveals an intriguing resemblance between dental plaque and non-host environments indicated by the TM7 evolution, suggesting that plaque may have served as a stepping stone for environmental microbes to adapt to host environments for some clades of human associated microbes. Additionally, we report that prophages are widespread amongst oral-associated TM7, while absent from environmental TM7, suggesting that prophages may have played a role in adaptation of TM7 to the host environment.

microbiology↗

bioRxiv2020

Spatial organization of the kelp microbiome at micron scales

Macroalgae are colonized by complex and diverse microbial communities that are distinct from those on inert substrates, suggesting intimate symbioses that likely play key roles in both macroalgal and bacterial biology. Canopy-forming kelp fix teragrams of carbon per year in coastal kelp forest ecosystems, yet little is known about the structure and development of their associated microbial communities. We characterized the spatial organization of bacterial communities on blades of the canopy-forming kelp Nereocystis luetkeana using fluorescence in situ hybridization and spectral imaging with a probe set combining phylum, class and genus-level probes to target >90% of the microbial community. We show that kelp blades host a dense microbial biofilm, generally less than 20 m thick, in which disparate microbial taxa live in close contact with one another. The biofilm is spatially differentiated, with tightly clustered cells of the dominant symbiont Granulosicoccus sp. (Gammaproteobacteria) close to the kelp surface and filamentous Bacteroidetes and Alphaproteobacteria relatively more abundant near the biofilm-seawater interface. Further, a community rich in Bacteroidetes colonized the interior of kelp tissues. Microbial community structure and cell density increased along the length of the kelp blade, from sparse microbial colonization of newly produced tissues at the meristematic base of the blade to an abundant microbial biofilm on older tissues at the blade tip. Finally, kelp from a declining population hosted fewer microbial cells compared to kelp from a stable population, indicating that biofilms are characteristic of health and that biofilm loss may be related to the condition of the host. ImportanceThe microbial community coating the surfaces of macroalgae may play a key but underexplored role both in the biology of the macroalgal host and in the biogeochemistry of the coastal ocean. We show that photosynthetic blades of the canopy-forming kelp Nereocystis luetkeana host a complex microbial biofilm that is both dense and spatially differentiated. Microbes of different taxa are in intimate cell-to-cell contact with one another; microbial cells invade the interior of kelp cells as well as cover their external surfaces; and a subset of the surface microbiota projects into the water column. These results highlight the potential for metabolic interactions between key members of the kelp microbiome as well as between microbes and their host. The dense layer of microbes coating the surface of the kelp blade is well-positioned to mediate interactions between the host and surrounding organisms and to modulate the chemistry of the surrounding water column.

microbiology↗

bioRxiv2019

Semi-blind sparse affine spectral unmixing of autofluorescence-contaminated micrographs

Spectral unmixing methods attempt to determine the concentrations of different fluorophores present at each pixel location in an image by analyzing a set of measured emission spectra. Unmixing algorithms have shown great promise for applications where samples contain many fluorescent labels; however, existing methods perform poorly when confronted with autofluorescence-contaminated images. We propose an unmixing algorithm designed to separate fluorophores with overlapping emission spectra from contamination by autofluorescence and background fluorescence. First, we formally define a generalization of the linear mixing model, called the affine mixture model (AMM), that specifically accounts for background fluorescence. Second, we use the AMM to derive an affine nonnegative matrix factorization method for estimating endmember spectra from reference images. Lastly, we propose a semi-blind sparse affine spectral unmixing (SSASU) algorithm that uses knowledge of the estimated endmembers to learn the autofluorescence and background fluorescence spectra on a per-image basis. When unmixing real-world spectral images contaminated by autofluorescence, SSASU was shown to have a similar reconstruction error but greatly improved proportion indeterminacy as compared to existing methods. The source code used for this paper was written in Julia and is available with the test data at https://github.com/brossetti/ssasu.

bioinformatics↗

bioRxiv2018

A simple microbiome in esophagus and gills of the European common cuttlefish, Sepia officinalis

The European common cuttlefish, Sepia officinalis, is used extensively in biological and biomedical research yet its microbiome remains poorly characterized. We analyzed the microbiota of the digestive tract, gills, and skin in mariculture-raised S. officinalis using a combination of 16s rRNA amplicon sequencing and fluorescence spectral imaging. Sequencing revealed a highly simplified microbiota consisting largely of two single bacterial amplicon sequence variants (ASVs) of Vibrionaceae and Piscirickettsiaceae. The esophagus was dominated by a single ASV of the genus Vibrio. Imaging revealed a striking organization of bacteria distributed in a discrete layer that lines the esophagus. Imaging with specific probes confirmed the identity of these bacteria as Vibrionaceae. This Vibrio was also abundant in the microbiota of the stomach, cecum, and intestine, but occurred at lower density and in the lumen rather than in a discrete layer; it was present in only trace proportions in tank water and in the microbiome of shrimp that were used as feed for the cuttlefish. These Vibrio were resilient to treatment of animals with the commonly-used antibiotic, enrofloxacin. The gills were colonized by a single ASV in the family Piscirickettsiaceae, which imaging visualized as small clusters of cells. We conclude that bacteria belonging to the Gammaproteobacteria, especially Vibrionaceae, are the major symbionts of the cuttlefish Sepia officinalis cultured from eggs in captivity, and that the esophagus and gills are major colonization sites.\n\nIMPORTANCEMicrobes can play critical roles in the physiology of their animal hosts, as evidenced in cephalopods by the role of Vibrio (Aliivibrio) fischeri in the light organ of the bobtail squid and the role of Alpha- and Gammaproteobacteria in the reproductive system and egg defense in a variety of cephalopods. We sampled the cuttlefish microbiome throughout the digestive tract, gills, and skin and found dense colonization of an unexpected site, the esophagus, by a microbe of the genus Vibrio, as well as colonization of gills by Piscirickettsiaceae. We found these associations to be resilient to the treatment of animals with a common antibiotic, enrofloxacin. This finding expands the range of organisms and body sites known to be associated with Vibrio and is of potential significance for understanding host-symbiont associations as well as for understanding and maintaining the health of cephalopods in mariculture.

microbiology↗

bioRxiv2017

Preservation of three-dimensional spatial structure in the gut microbiome

Preservation of three-dimensional structure in the gut is necessary in order to analyze the spatial organization of the gut microbiota and gut luminal contents. In this study, we evaluated preparation methods for mouse gut with the goal of preserving micron-scale spatial structure while performing fluorescence imaging assays. Our evaluation of embedding methods showed that commonly used media such as Tissue-Tek Optimal Cutting Temperature (OCT) compound, paraffin, and polyester waxes resulted in redistribution of luminal contents. By contrast, a hydrophilic methacrylate resin, Technovit H8100, preserved three-dimensional organization. Our mouse intestinal preparation protocol optimized using the Technovit H8100 embedding method was compatible with microbial fluorescence in situ hybridization (FISH) and other labeling techniques, including immunostaining and staining with both wheat germ agglutinin (WGA) and 4,6-diamidino-2-phenylindole (DAPI). Mucus labeling patterns of the samples fixed with paraformaldehyde (PFA) and Carnoys fixative were comparable. The protocol optimized in this study enabled simultaneous visualization of micron-scale spatial patterns formed by microbial cells in the mouse intestines along with biogeographical landmarks such as host-derived mucus and food particles.

microbiology↗