The Experts below are selected from a list of 2487 Experts worldwide ranked by ideXlab platform
Laurie E Comstock - One of the best experts on this subject based on the ideXlab platform.
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mobile type vi secretion system loci of the gut Bacteroidales display extensive intra ecosystem transfer multi species spread and geographical clustering
PLOS Genetics, 2021Co-Authors: Leonor Garciabayona, Michael J Coyne, Laurie E ComstockAbstract:The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and Type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on Integrative and Conjugative Elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population fixation of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 74 MGE that transferred to multiple Bacteroidales species within individual gut microbiomes. We further show that only three other MGE demonstrate multi-species spread in several human gut microbiomes to the degree demonstrated by the GA1 and GA2 ICE. These data underscore the ubiquity and dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.
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mobile type vi secretion system loci of the gut Bacteroidales display extensive intra ecosystem transfer multi species sweeps and geographical clustering
bioRxiv, 2021Co-Authors: Leonor Garciabayona, Michael J Coyne, Laurie E ComstockAbstract:The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on integrative and conjugative elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population sweeps of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 177 MGE that sweep through multiple Bacteroidales species within individual gut microbiomes. We further show that only eight MGE demonstrate multi-species population sweeps in as many human gut microbiomes as the GA1 and GA2 ICE. These data underscore the ubiquity and rapid dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.
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Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer, multi-species spread and geographical clustering.
'Public Library of Science (PLoS)', 2021Co-Authors: Leonor García-bayona, Michael J Coyne, Laurie E ComstockAbstract:The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and Type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on Integrative and Conjugative Elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population fixation of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 74 MGE that transferred to multiple Bacteroidales species within individual gut microbiomes. We further show that only three other MGE demonstrate multi-species spread in human gut microbiomes to the degree demonstrated by the GA1 and GA2 ICE. These data underscore the ubiquity and dissemination of mobile T6SS loci within Bacteroidales communities and across human populations
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streamlined genetic manipulation of diverse bacteroides and parabacteroides isolates from the human gut microbiota
Mbio, 2019Co-Authors: Leonor Garciabayona, Laurie E ComstockAbstract:Studies of the gut microbiota have dramatically increased in recent years as the importance of this microbial ecosystem to human health and disease is better appreciated. The Bacteroidales are the most abundant order of bacteria in the healthy human gut and induce both health-promoting and disease-promoting effects. There are more than 55 species of gut Bacteroidales with extensive intraspecies genetic diversity, especially in regions involved in the synthesis of molecules that interact with other bacteria, the host, and the diet. This property necessitates the study of diverse species and strains. In recent years, the genetic toolkit to study these bacteria has greatly expanded, but we still lack a facile system for creating deletion mutants and allelic replacements in diverse strains, especially with the rapid increase in resistance to the two antibiotics used for genetic manipulation. Here, we present a new versatile and highly efficient vector suite that allows the creation of allelic deletions and replacements in multiresistant strains of Bacteroides and Parabacteroides using a gain-of-function system based on polysaccharide utilization. These vectors also allow for easy counterselection independent of creating a mutant background strain, using a toxin from a type VI secretion system of Bacteroides fragilis Toxin production during counterselection is induced with one of two different molecules, providing flexibility based on strain phenotypes. This family of vectors greatly facilitates functional genetic analyses and extends the range of gut Bacteroidales strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools.IMPORTANCE We have entered an era when studies of the gut microbiota are transitioning from basic questions of composition and host effects to understanding the microbial molecules that underlie compositional shifts and mediate health and disease processes. The importance of the gut Bacteroidales to human health and disease and their potential as a source of engineered live biotherapeutics make these bacteria of particular interest for in-depth mechanistic study. However, there are still barriers to the genetic analysis of diverse Bacteroidales strains, limiting our ability to study important host and community phenotypes identified in these strains. Here, we have overcome many of these obstacles by constructing a series of vectors that allow easy genetic manipulation in diverse gut Bacteroides and Parabacteroides strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut Bacteroidales, including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.
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Streamlined Genetic Manipulation of Diverse Bacteroides and Parabacteroides Isolates from the Human Gut Microbiota
'American Society for Microbiology', 2019Co-Authors: Leonor García-bayona, Laurie E ComstockAbstract:We have entered an era when studies of the gut microbiota are transitioning from basic questions of composition and host effects to understanding the microbial molecules that underlie compositional shifts and mediate health and disease processes. The importance of the gut Bacteroidales to human health and disease and their potential as a source of engineered live biotherapeutics make these bacteria of particular interest for in-depth mechanistic study. However, there are still barriers to the genetic analysis of diverse Bacteroidales strains, limiting our ability to study important host and community phenotypes identified in these strains. Here, we have overcome many of these obstacles by constructing a series of vectors that allow easy genetic manipulation in diverse gut Bacteroides and Parabacteroides strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut Bacteroidales, including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.Studies of the gut microbiota have dramatically increased in recent years as the importance of this microbial ecosystem to human health and disease is better appreciated. The Bacteroidales are the most abundant order of bacteria in the healthy human gut and induce both health-promoting and disease-promoting effects. There are more than 55 species of gut Bacteroidales with extensive intraspecies genetic diversity, especially in regions involved in the synthesis of molecules that interact with other bacteria, the host, and the diet. This property necessitates the study of diverse species and strains. In recent years, the genetic toolkit to study these bacteria has greatly expanded, but we still lack a facile system for creating deletion mutants and allelic replacements in diverse strains, especially with the rapid increase in resistance to the two antibiotics used for genetic manipulation. Here, we present a new versatile and highly efficient vector suite that allows the creation of allelic deletions and replacements in multiresistant strains of Bacteroides and Parabacteroides using a gain-of-function system based on polysaccharide utilization. These vectors also allow for easy counterselection independent of creating a mutant background strain, using a toxin from a type VI secretion system of Bacteroides fragilis. Toxin production during counterselection is induced with one of two different molecules, providing flexibility based on strain phenotypes. This family of vectors greatly facilitates functional genetic analyses and extends the range of gut Bacteroidales strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools
Michael J Coyne - One of the best experts on this subject based on the ideXlab platform.
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mobile type vi secretion system loci of the gut Bacteroidales display extensive intra ecosystem transfer multi species spread and geographical clustering
PLOS Genetics, 2021Co-Authors: Leonor Garciabayona, Michael J Coyne, Laurie E ComstockAbstract:The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and Type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on Integrative and Conjugative Elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population fixation of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 74 MGE that transferred to multiple Bacteroidales species within individual gut microbiomes. We further show that only three other MGE demonstrate multi-species spread in several human gut microbiomes to the degree demonstrated by the GA1 and GA2 ICE. These data underscore the ubiquity and dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.
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mobile type vi secretion system loci of the gut Bacteroidales display extensive intra ecosystem transfer multi species sweeps and geographical clustering
bioRxiv, 2021Co-Authors: Leonor Garciabayona, Michael J Coyne, Laurie E ComstockAbstract:The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on integrative and conjugative elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population sweeps of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 177 MGE that sweep through multiple Bacteroidales species within individual gut microbiomes. We further show that only eight MGE demonstrate multi-species population sweeps in as many human gut microbiomes as the GA1 and GA2 ICE. These data underscore the ubiquity and rapid dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.
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Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer, multi-species spread and geographical clustering.
'Public Library of Science (PLoS)', 2021Co-Authors: Leonor García-bayona, Michael J Coyne, Laurie E ComstockAbstract:The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and Type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on Integrative and Conjugative Elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population fixation of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 74 MGE that transferred to multiple Bacteroidales species within individual gut microbiomes. We further show that only three other MGE demonstrate multi-species spread in human gut microbiomes to the degree demonstrated by the GA1 and GA2 ICE. These data underscore the ubiquity and dissemination of mobile T6SS loci within Bacteroidales communities and across human populations
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“Cross-glycosylation ” of proteins in Bacteriodales species
2016Co-Authors: Gerald Posch, Laurie E Comstock, Michael J Coyne, Martin Pabst, Laura Neumann, Paul Messner, Christina SchäfferAbstract:D ow nloaded from 2 While it is now evident that the two Bacteroidales species Bacteroides fragilis and Tannerella forsythia both have general O-glycosylation systems and share a common glycosylation sequon, the ability of these organisms to glycosylate a protein native to the other organism has not yet been demonstrated. Here, we report on the glycosylation of heterologous proteins between these two organisms. Using genetic tools previously developed for Bacteroides species, two B. fragilis model glycoproteins were expressed in the fastidious anaerobe T. forsythia and the attachment of the known T. forsythia O-glycan to these proteins was demonstrated by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Likewise, two predominant T. forsythia glycoproteins were expressed in B. fragilis and glycosylation with the B. fragilis O-glycan was confirmed. Purification o
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type vi secretion systems of human gut Bacteroidales segregate into three genetic architectures two of which are contained on mobile genetic elements
BMC Genomics, 2016Co-Authors: Michael J Coyne, Kevin G Roelofs, Laurie E ComstockAbstract:Type VI secretion systems (T6SSs) are contact-dependent antagonistic systems employed by Gram negative bacteria to intoxicate other bacteria or eukaryotic cells. T6SSs were recently discovered in a few Bacteroidetes strains, thereby extending the presence of these systems beyond Proteobacteria. The present study was designed to analyze in a global nature the diversity, abundance, and properties of T6SSs in the Bacteroidales, the most predominant Gram negative bacterial order of the human gut. By performing extensive bioinformatics analyses and creating hidden Markov models for Bacteroidales Tss proteins, we identified 130 T6SS loci in 205 human gut Bacteroidales genomes. Of the 13 core T6SS proteins of Proteobacteria, human gut Bacteroidales T6SS loci encode orthologs of nine, and an additional five other core proteins not present in Proteobacterial T6SSs. The Bacteroidales T6SS loci segregate into three distinct genetic architectures with extensive DNA identity between loci of a given genetic architecture. We found that divergent DNA regions of a genetic architecture encode numerous types of effector and immunity proteins and likely include new classes of these proteins. TheT6SS loci of genetic architecture 1 are contained on highly similar integrative conjugative elements (ICEs), as are the T6SS loci of genetic architecture 2, whereas the T6SS loci of genetic architecture 3 are not and are confined to Bacteroides fragilis. Using collections of co-resident Bacteroidales strains from human subjects, we provide evidence for the transfer of genetic architecture 1 T6SS loci among co-resident Bacteroidales species in the human gut. However, we also found that established ecosystems can harbor strains with distinct T6SS of all genetic architectures. This is the first study to comprehensively analyze of the presence and diversity of T6SS loci within an order of bacteria and to analyze T6SSs of bacteria from a natural community. These studies demonstrate that more than half of our gut Bacteroidales, equivalent to about ¼ of the bacteria of this ecosystem, encode T6SSs. The data reveal several novel properties of these systems and suggest that antagonism between or distributed defense among these abundant intestinal bacteria may be common in established human gut communities.
Leonor Garciabayona - One of the best experts on this subject based on the ideXlab platform.
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mobile type vi secretion system loci of the gut Bacteroidales display extensive intra ecosystem transfer multi species spread and geographical clustering
PLOS Genetics, 2021Co-Authors: Leonor Garciabayona, Michael J Coyne, Laurie E ComstockAbstract:The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and Type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on Integrative and Conjugative Elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population fixation of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 74 MGE that transferred to multiple Bacteroidales species within individual gut microbiomes. We further show that only three other MGE demonstrate multi-species spread in several human gut microbiomes to the degree demonstrated by the GA1 and GA2 ICE. These data underscore the ubiquity and dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.
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mobile type vi secretion system loci of the gut Bacteroidales display extensive intra ecosystem transfer multi species sweeps and geographical clustering
bioRxiv, 2021Co-Authors: Leonor Garciabayona, Michael J Coyne, Laurie E ComstockAbstract:The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on integrative and conjugative elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population sweeps of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 177 MGE that sweep through multiple Bacteroidales species within individual gut microbiomes. We further show that only eight MGE demonstrate multi-species population sweeps in as many human gut microbiomes as the GA1 and GA2 ICE. These data underscore the ubiquity and rapid dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.
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streamlined genetic manipulation of diverse bacteroides and parabacteroides isolates from the human gut microbiota
Mbio, 2019Co-Authors: Leonor Garciabayona, Laurie E ComstockAbstract:Studies of the gut microbiota have dramatically increased in recent years as the importance of this microbial ecosystem to human health and disease is better appreciated. The Bacteroidales are the most abundant order of bacteria in the healthy human gut and induce both health-promoting and disease-promoting effects. There are more than 55 species of gut Bacteroidales with extensive intraspecies genetic diversity, especially in regions involved in the synthesis of molecules that interact with other bacteria, the host, and the diet. This property necessitates the study of diverse species and strains. In recent years, the genetic toolkit to study these bacteria has greatly expanded, but we still lack a facile system for creating deletion mutants and allelic replacements in diverse strains, especially with the rapid increase in resistance to the two antibiotics used for genetic manipulation. Here, we present a new versatile and highly efficient vector suite that allows the creation of allelic deletions and replacements in multiresistant strains of Bacteroides and Parabacteroides using a gain-of-function system based on polysaccharide utilization. These vectors also allow for easy counterselection independent of creating a mutant background strain, using a toxin from a type VI secretion system of Bacteroides fragilis Toxin production during counterselection is induced with one of two different molecules, providing flexibility based on strain phenotypes. This family of vectors greatly facilitates functional genetic analyses and extends the range of gut Bacteroidales strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools.IMPORTANCE We have entered an era when studies of the gut microbiota are transitioning from basic questions of composition and host effects to understanding the microbial molecules that underlie compositional shifts and mediate health and disease processes. The importance of the gut Bacteroidales to human health and disease and their potential as a source of engineered live biotherapeutics make these bacteria of particular interest for in-depth mechanistic study. However, there are still barriers to the genetic analysis of diverse Bacteroidales strains, limiting our ability to study important host and community phenotypes identified in these strains. Here, we have overcome many of these obstacles by constructing a series of vectors that allow easy genetic manipulation in diverse gut Bacteroides and Parabacteroides strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut Bacteroidales, including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.
Stefan Wuertz - One of the best experts on this subject based on the ideXlab platform.
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variably improved microbial source tracking with digital droplet pcr
Water Research, 2019Co-Authors: Stefan Wuertz, Jean Pierre Nshimyimana, Mercedes Cecilia Cruz, Janelle R ThompsonAbstract:Abstract This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85–1.00 with marginal reduction in human stool by ddPCR relative to qPCR (
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Bacteroidales markers for microbial source tracking in southeast asia
Water Research, 2017Co-Authors: Jean Pierre Nshimyimana, Mercedes Cecilia Cruz, Janelle R Thompson, Stefan WuertzAbstract:The island city country of Singapore served as a model to validate the use of host-associated Bacteroidales 16S rRNA gene marker assays for identifying sources of fecal pollution in the urban tropical environment of Southeast Asia. A total of 295 samples were collected from sewage, humans, domesticated animals (cats, dogs, rabbits and chicken), and wild animals (birds, monkeys and wild boars). Samples were analyzed by real time PCR using five human-associated assays (HF183-SYBR Green, HF183, BacHum, BacH and B. thetaiotaomicron α-1-6, mannanase (B. theta), one canine-associated assay (BacCan), and a total Bacteroidales assay (BacUni). The best performing human-associated assay was B. theta with a diagnostic sensitivity of 69% and 100% in human stool and sewage, respectively, and a specificity of 98%. BacHum achieved the second highest sensitivity and specificity for human stool at 65% and 91%, respectively. The canine-associated Bacteroidales assay (BacCan) had a sensitivity and specificity above 80% and was validated for tracking fecal pollution from dogs. BacUni demonstrated a sensitivity and specificity of 100% for mammals, thus BacUni was confirmed for total Bacteroidales detection in the region. We showed for the first time that rabbit fecal samples cross-react with human-associated assays (HF183-SYBR Green, HF183, BacHum and BacH) and with BacCan. Our findings regarding the best performing human-associated assays differ from those reported in Bangladesh and India, which are geographically close to Southeast Asia, and where HF183 and BacHum were the preferred assays, respectively.
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survival of host associated Bacteroidales cells and their relationship with enterococcus spp campylobacter jejuni salmonella enterica serovar typhimurium and adenovirus in freshwater microcosms as measured by propidium monoazide quantitative pcr
Applied and Environmental Microbiology, 2012Co-Authors: Sungwoo Bae, Stefan WuertzAbstract:ABSTRACT The ideal host-associated genetic fecal marker would be capable of predicting the presence of specific pathogens of concern. Flowthrough freshwater microcosms containing mixed feces and inocula of the pathogens Campylobacter jejuni, Salmonella enterica serovar Typhimurium, and adenovirus were placed at ambient temperature in the presence and absence of diurnal sunlight. The total Enterococcus DNA increased during the early periods (23 h) under sunlight exposure, even though cultivable Enterococcus and DNA in intact cells, as measured by propidium monoazide (PMA), decreased with first-order kinetics during the entire period. We found a significant difference in the decay of host-associated Bacteroidales cells between sunlight exposure and dark conditions (P value 0.05). Overall, the ratio of quantitative PCR (qPCR) cycle threshold (CT) values with and without PMA treatment was indicative of the time elapsed since inoculation of the microcosm with (i) fecal material from different animal sources based on host-associated Bacteroidales and (ii) pure cultures of bacterial pathogens. The use of both PMA-qPCR and qPCR may yield more realistic information about recent sources of fecal contamination and result in improved prediction of waterborne pathogens and assessment of health risk.
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estimating true human and animal host source contribution in quantitative microbial source tracking using the monte carlo method
Water Research, 2010Co-Authors: Dan Wang, Sarah S Silkie, Kara L Nelson, Stefan WuertzAbstract:Abstract Cultivation- and library-independent, quantitative PCR-based methods have become the method of choice in microbial source tracking. However, these qPCR assays are not 100% specific and sensitive for the target sequence in their respective hosts’ genome. The factors that can lead to false positive and false negative information in qPCR results are well defined. It is highly desirable to have a way of removing such false information to estimate the true concentration of host-specific genetic markers and help guide the interpretation of environmental monitoring studies. Here we propose a statistical model based on the Law of Total Probability to predict the true concentration of these markers. The distributions of the probabilities of obtaining false information are estimated from representative fecal samples of known origin. Measurement error is derived from the sample precision error of replicated qPCR reactions. Then, the Monte Carlo method is applied to sample from these distributions of probabilities and measurement error. The set of equations given by the Law of Total Probability allows one to calculate the distribution of true concentrations, from which their expected value, confidence interval and other statistical characteristics can be easily evaluated. The output distributions of predicted true concentrations can then be used as input to watershed-wide total maximum daily load determinations, quantitative microbial risk assessment and other environmental models. This model was validated by both statistical simulations and real world samples. It was able to correct the intrinsic false information associated with qPCR assays and output the distribution of true concentrations of Bacteroidales for each animal host group. Model performance was strongly affected by the precision error. It could perform reliably and precisely when the standard deviation of the precision error was small (≤0.1). Further improvement on the precision of sample processing and qPCR reaction would greatly improve the performance of the model. This methodology, built upon Bacteroidales assays, is readily transferable to any other microbial source indicator where a universal assay for fecal sources of that indicator exists.
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rapid decay of host specific fecal Bacteroidales cells in seawater as measured by quantitative pcr with propidium monoazide
Water Research, 2009Co-Authors: Sungwoo Bae, Stefan WuertzAbstract:We investigated the persistence of feces-derived Bacteroidales cells and their DNA in seawater under natural conditions using an optimized chemical method based on co-extraction of nucleic acids with propidium monoazide (PMA), which interferes with PCR amplification of molecular markers from extracellular DNA and dead bacterial cells. The previously validated Bacteroidales assays BacUni-UCD, BacHum-UCD, BacCow-UCD, and BacCan-UCD were utilized to determine concentrations of Bacteroidales genetic markers targeting all warm-blooded animals, humans, cows and dogs, specifically, over a period of 24d. Microcosms containing mixed feces in dialysis tubing were exposed to seawater under flow-through conditions at ambient temperature in the presence and absence of sunlight. Using a two-stage plus linear decay model, the average T(99) (two-log reduction) of host-specific Bacteroidales cells was 28h, whereas that of host-specific Bacteroidales DNA was 177h. Natural sunlight did not affect the survival of uncultivable Bacteroidales cells and their DNA with the exception of the BacCow-UCD marker. Bacteroidales DNA, as measured by quantitative PCR (qPCR) without PMA, persisted for as long as 24d at concentrations close to the limit of detection. Culturable Enterococcus cells were detected for only 70h, whereas Enterococcus cells measured by qPCR with and without PMA persisted for 450h. In conclusion, measuring Bacteroidales DNA without differentiating between intact and dead cells or extracellular DNA may misinform about the extent of recent fecal pollution events, particularly in the case of multiple sources of contamination with variable temporal and spatial scales due to the relatively long persistence of DNA in the environment. In contrast, applying qPCR with and without PMA can provide data on the fate and transport of fecal Bacteroidales in water, and help implement management practices to protect recreational water quality.
Moriya Ohkuma - One of the best experts on this subject based on the ideXlab platform.
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distribution and evolution of nitrogen fixation genes in the phylum bacteroidetes
Microbes and Environments, 2015Co-Authors: Junichi Inoue, Kenshiro Oshima, Wataru Suda, Mitsuo Sakamoto, Takao Iino, Satoko Noda, Yuichi Hongoh, Masahira Hattori, Moriya OhkumaAbstract:Diazotrophs had not previously been identified among bacterial species in the phylum Bacteroidetes until the rapid expansion of bacterial genome sequences, which revealed the presence of nitrogen fixation (nif) genes in this phylum. We herein determined the draft genome sequences of Bacteroides graminisolvens JCM 15093T and Geofilum rubicundum JCM 15548T. In addition to these and previously reported ‘Candidatus Azobacteroides pseudotrichonymphae’ and Paludibacter propionicigenes, an extensive survey of the genome sequences of diverse Bacteroidetes members revealed the presence of a set of nif genes (nifHDKENB) in strains of Dysgonomonas gadei, Dysgonomonas capnocytophagoides, Saccharicrinis fermentans, and Alkaliflexus imshenetskii. These eight species belonged to and were distributed sporadically within the order Bacteroidales. Acetylene reduction activity was detected in the five species examined, strongly suggesting their diazotrophic nature. Phylogenetic analyses showed monophyletic clustering of the six Nif protein sequences in the eight Bacteroidales species, implying that nitrogen fixation is ancestral to Bacteroidales and has been retained in these species, but lost in many other lineages. The identification of nif genes in Bacteroidales facilitates the prediction of the organismal origins of related sequences directly obtained from various environments.
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distribution and evolution of nitrogen fixation genes in the phylum bacteroidetes
Microbes and Environments, 2015Co-Authors: Junichi Inoue, Kenshiro Oshima, Wataru Suda, Mitsuo Sakamoto, Takao Iino, Satoko Noda, Yuichi Hongoh, Masahira Hattori, Moriya OhkumaAbstract:Biological nitrogen fixation, the conversion of atmospheric dinitrogen to ammonia, has a significant impact on nitrogen cycles in ecosystems. Nitrogen-fixing microorganisms (diazotrophs) are widely distributed in diverse prokaryotic phyla, but sparsely within these phyla. This distribution pattern suggests that nitrogen fixing ability is evolutionary ancient and mainly transmitted vertically with the widespread loss of function (12). The recent rapid expansion of microbial genome sequences has revealed the presence of the genes encoding homologous proteins to known nitrogenases, even in prokaryotic species that had not previously been recognized as diazotrophs (9). In some cases, experimental evidence for nitrogen fixation was obtained following identification of the responsible genes in the genome (23, 25). The nitrogenase complex consists of its catalytic components encoded by nifD and nifK and nitrogenase reductase encoded by nifH. In addition to ordinary molybdenum-dependent nitrogenases (Nif), vanadium-dependent (Vnf) and iron-only alternative nitrogenases (Anf) have also been identified, but occur in a limited number of diazotrophs. These Nif, Vnf, and Anf types of nitrogenases are homologous and evolutionarily related, except for an additional subunit, VnfG or AnfG, in the Vnf or Anf type, respectively (39). In addition, the factors involving metal cofactor assembly such as those encoded by nifE, nifN, and nifB are necessary for functional nitrogenase. nifE and nifN are homologous to nifD and nifK, respectively, and may have originated from ancient gene duplications (5, 10). The phylum Bacteroidetes comprises a huge assemblage of diverse bacterial species isolated from various environments and have been classified into four orders (according to the list of prokaryotic names with standing in nomenclature; http://www.bacterio.net/). The order Bacteroidales in this phylum comprises nearly 40 genera and many species in this order are Gram-negative anaerobic rods that are frequently isolated from human and animal gastrointestinal tracts. Although a large number of genome-sequenced strains exist in this order, many are isolates from human specimens. Diazotrophs had not previously been identified among members in the phylum Bacteroidetes; however, a recent genome survey revealed the presence of nitrogenase homologous genes. The first genome of the Bacteroidetes member that encodes nif genes was reported in ‘Candidatus Azobacteroides pseudotrichonymphae’, an abundant endosymbiont Bacteroidales species of a cellulolytic protist in the gut of the termite Coptotermes formosanus (16). In that study, it was hypothesized that nif genes had been acquired via lateral gene transfer from a bacterium co-inhabiting the gut. nif genes were also detected in the genome sequence of another Bacteroidales member, Paludibacter propionicigenes strain WB4T (9). These findings suggested that nif genes are more widespread, but sporadic among Bacteroidetes members than currently recognized. No direct experimental evidence currently exists for the nitrogen fixing abilities of these two species. In the present study, the homologous sequences of nif genes were surveyed in the genomes of diverse Bacteroidetes members. Based on the presence of a set of nif genes and results of the acetylene reduction assay, we identified several Bacteroidales members as new potential diazotrophs. Phylogenetic analyses suggested that the nif genes of the identified species have been vertically transmitted from a common ancestor. The identification of potential diazotrophs in Bacteroidales may have an impact on their ecology and their genes can be useful references for metagenomic studies on microbial communities.
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the motility symbiont of the termite gut flagellate caduceia versatilis is a member of the synergistes group
Applied and Environmental Microbiology, 2007Co-Authors: Yuichi Hongoh, Satoko Noda, Toshiaki Kudo, Tomoyuki Sato, Michael F Dolan, Moriya OhkumaAbstract:The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “Candidatus Tammella caduceiae,” for this unique motility ectosymbiont of Caducaia versatilis.
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endosymbiotic Bacteroidales bacteria of the flagellated protist pseudotrichonympha grassii in the gut of the termite coptotermes formosanus
Applied and Environmental Microbiology, 2005Co-Authors: Satoko Noda, Toshiya Iida, Osamu Kitade, Hideaki Nakajima, Toshiaki Kudo, Moriya OhkumaAbstract:A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.
