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In JoVE (1)
Other Publications (11)
- Journal of Bacteriology
- Anaerobe
- Nature
- Molecular Microbiology
- Environmental Microbiology
- Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
- Environmental Microbiology
- The ISME Journal
- PloS One
- Cell
- Environmental Microbiology
Articles by Eric Matson in JoVE
JoVE General
Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes
Department of Environmental Science and Engineering, California Institute of Technology - Caltech
This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.
Other articles by Eric Matson on PubMed
Identification of Genes of VSH-1, a Prophage-like Gene Transfer Agent of Brachyspira Hyodysenteriae
VSH-1 is a mitomycin C-inducible prophage of the anaerobic spirochete Brachyspira hyodysenteriae. Purified VSH-1 virions are noninfectious, contain random 7.5-kb fragments of the bacterial genome, and mediate generalized transduction of B. hyodysenteriae cells. In order to identify and sequence genes of this novel gene transfer agent (GTA), proteins associated either with VSH-1 capsids or with tails were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of 11 proteins were determined. Degenerate PCR primers were designed from the amino acid sequences and used to amplify several VSH-1 genes from B. hyodysenteriae strain B204 DNA. A lambda clone library of B. hyodysenteriae B204 DNA was subsequently screened by Southern hybridization methods and used to identify and sequence overlapping DNA inserts containing additional VSH-1 genes. VSH-1 genes spanned 16.3 kb of the B. hyodysenteriae chromosome and were flanked by bacterial genes. VSH-1 identified genes and unidentified, intervening open reading frames were consecutively organized in head (seven genes), tail (seven genes), and lysis (four genes) clusters in the same transcriptional direction. Putative lysis genes encoding endolysin (Lys) and holin proteins were identified from sequence and structural similarities of their translated protein products with GenBank bacteriophage proteins. Recombinant Lys protein hydrolyzed peptidoglycan purified from B. hyodysenteriae cells. The identified VSH-1 genes exceed the DNA capacity of VSH-1 virions and do not encode traditional bacteriophage early functions involved in DNA replication. These genome properties explain the noninfectious nature of VSH-1 virions and further confirm its resemblance to known prophage-like, GTAs of other bacterial species, such as the GTA from Rhodobacter capsulatus. The identification of VSH-1 genes will enable analysis of the regulation of this GTA and should facilitate investigations of VSH-1-like prophages from other Brachyspira species.
Induction and Transcription of VSH-1, a Prophage-like Gene Transfer Agent of Brachyspira Hyodysenteriae
The anaerobic spirochete Brachyspira hyodysenteriae is host to a bacteriophage-like agent known as VSH-1. VSH-1 is a novel gene transfer mechanism which does not self-propagate and transfers random 7.5kb fragments of host DNA between B. hyodysenteriae cells. In these investigations early events during VSH-1 induction by mitomycin C were examined. Quantitative PCR analysis revealed that VSH-1 hvp38 and hvp53 genes did not detectably increase in copy numbers during induction. Based on Northern blot hybridization assays, transcription of VSH-1 genes hvp38, hvp53, hvp45, hvp101, and lys increased fivefold to tenfold between 2 and 4h after induction whereas mRNA levels for B. hyodysenteriae flaA1 declined over the same time period. Chloramphenicol prevented the mitomycin C-induced increases in VSH-1 gene transcription. Hydrogen peroxide (300muM) substituted for mitomycin C as an inducer of VSH-1 gene transcription and is a possible 'natural' inducer of VSH-1 production in vivo. Northern blot hybridization, RT PCR, and primer extension analyses showed that VSH-1 genes are co-transcribed at an initiation site upstream of the VSH-1 gene operon. Two direct heptanucleotide repeats (ACTTATA) were identified between the putative -35 and -10 positions of the VSH-1 gene operon and are likely to represent a binding site for transcription proteins. These findings indicate VSH-1 virion production does not require genome replication, consistent with the inability of VSH-1 to self-propagate. Early events in VSH-1 induction include de novo synthesis of protein(s) essential for transcription of VSH-1 genes as polycistronic mRNA initiating upstream of the hvp45 gene.
Metagenomic and Functional Analysis of Hindgut Microbiota of a Wood-feeding Higher Termite
From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding 'higher' Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-microl environment can be.
Novel Ultrastructures of Treponema Primitia and Their Implications for Motility
Members of the bacterial phylum Spirochaetes are generally helical cells propelled by periplasmic flagella. The spirochete Treponema primitia is interesting because of its mutualistic role in the termite gut, where it is believed to cooperate with protozoa that break down cellulose and produce H(2) as a by-product. Here we report the ultrastructure of T. primitia as obtained by electron cryotomography of intact, frozen-hydrated cells. Several previously unrecognized external structures were revealed, including bowl-like objects decorating the outer membrane, arcades of hook-shaped proteins winding along the exterior and tufts of fibrils extending from the cell tips. Inside the periplasm, cone-like structures were found at each pole. Instead of the single peptidoglycan layer typical of other Gram-negative bacteria, two distinct periplasmic layers were observed. These layers formed a central open space that contained two flagella situated adjacent to each other. In some areas, the inner membrane formed flattened invaginations that protruded into the cytoplasm. High-speed light microscopic images of swimming T. primitia cells showed that cell bodies remained rigid and moved in a helical rather than planar motion. Together, these findings support the 'rolling cylinder' model for T. primitia motility that posits rotation of the protoplasmic cylinder within the outer sheath.
Selenium Controls Transcription of Paralogous Formate Dehydrogenase Genes in the Termite Gut Acetogen, Treponema Primitia
Summary The termite gut spirochete, Treponema primitia, is a CO(2)-reductive acetogen that is phylogenetically distinct from other distantly related and more extensively studied acetogens such as Moorella thermoacetica. Research on T. primitia has revealed details about the role of spirochetes in CO(2)-reductive acetogenesis, a process important to the mutualism occurring between termites and their gut microbial communities. Here, a locus of the T. primitia genome containing Wood-Ljungdahl pathway genes for CO(2)-reductive acetogenesis was sequenced. This locus contained methyl-branch genes of the pathway (i.e. for the reduction of CO(2) to the level of methyl-tetrahydrofolate) including paralogous genes for cysteine and selenocysteine (Sec) variants of formate dehydrogenase (FDH) and genes for Sec incorporation. The FDH variants affiliated phylogenetically with hydrogenase-linked FDH enzymes, suggesting that T. primitia FDH enzymes utilize electrons derived directly from molecular H(2). Sub-nanomolar concentrations of selenium decreased transcript levels of the cysteine variant FDH gene. Selenium concentration did not markedly influence the level of mRNA upstream of the Sec-codon in the Sec variant FDH; however, the level of transcript extending downstream of the Sec-codon increased incrementally with increasing selenium concentrations. The features and regulation of these FDH genes are an indication that T. primitia may experience dynamic selenium availability in its H(2)-rich gut environment.
"A Window on Tissue" - Using Facial Orientation to Control Endoscopic Views of Tissue Depth
An endoscope is an invaluable tool to interpret conditions within a body. Flexible endoscopes are controlled by a set of rotational knobs requiring a doctor's hands to guide and locate the view. This research uses a combination of a camera, facial recognition techniques and software to create a hands-free gesture recognition application for use by a physician to probe the internals of a human body. The physician will utilize the head movements to move the endoscopic camera freeing their hands to perform a procedure or other functions.
Selenium Controls Transcription of Paralogous Formate Dehydrogenase Genes in the Termite Gut Acetogen, Treponema Primitia
The termite gut spirochete, Treponema primitia, is a CO(2)-reductive acetogen that is phylogenetically distinct from other distantly related and more extensively studied acetogens such as Moorella thermoacetica. Research on T. primitia has revealed details about the role of spirochetes in CO(2)-reductive acetogenesis, a process important to the mutualism occurring between termites and their gut microbial communities. Here, a locus of the T. primitia genome containing Wood-Ljungdahl pathway genes for CO(2)-reductive acetogenesis was sequenced. This locus contained methyl-branch genes of the pathway (i.e. for the reduction of CO(2) to the level of methyl-tetrahydrofolate) including paralogous genes for cysteine and selenocysteine (Sec) variants of formate dehydrogenase (FDH) and genes for Sec incorporation. The FDH variants affiliated phylogenetically with hydrogenase-linked FDH enzymes, suggesting that T. primitia FDH enzymes utilize electrons derived directly from molecular H(2). Sub-nanomolar concentrations of selenium decreased transcript levels of the cysteine variant FDH gene. Selenium concentration did not markedly influence the level of mRNA upstream of the Sec-codon in the Sec variant FDH; however, the level of transcript extending downstream of the Sec-codon increased incrementally with increasing selenium concentrations. The features and regulation of these FDH genes are an indication that T. primitia may experience dynamic selenium availability in its H(2)-rich gut environment.
RNA-seq Reveals Cooperative Metabolic Interactions Between Two Termite-gut Spirochete Species in Co-culture
The hindguts of wood-feeding termites typically contain hundreds of microbial species. Together with their insect host, these gut microbes degrade lignocellulose into usable catabolites. Although past research revealed many facets of the stepwise flow of metabolites in this scheme, not much is known about the breadth of interactions occurring between termite-gut microbes. Most of these microbes are thought to depend on, and to have co-speciated with, their host and each other for millions of years. In this study, we explored the interactions of two spirochetes previously isolated from the very same termite species. As hydrogen (H(2)) is the central free intermediate in termite-gut lignocellulose digestion, we focused on interactions between two closely related termite-gut spirochetes possessing complementary H(2) physiologies: one produces H(2), while the other consumes it. In vitro, these two Treponema species markedly enhanced each other's growth. RNA sequencing resolved the transcriptomes of these two closely related organisms, revealing that co-cultivation causes comprehensive changes in global gene expression. The expression of well over a 100 genes in each species was changed >twofold, with over a dozen changed >10-fold. Several changes implicating synergistic cross-feeding of known metabolites were validated in vitro. Additionally, certain activities beneficial to the host were preferentially expressed during consortial growth. However, the majority of changes in gene expression are not yet understandable, but indicate a broad, comprehensive and mutualistic interaction between these closely related, co-resident gut symbionts. The results suggest that staggeringly intricate networks of metabolic and gene interactions drive lignocellulose degradation and co-evolution of termite gut microbiota.
Anaerobic Carbon Monoxide Dehydrogenase Diversity in the Homoacetogenic Hindgut Microbial Communities of Lower Termites and the Wood Roach
Anaerobic carbon monoxide dehydrogenase (CODH) is a key enzyme in the Wood-Ljungdahl (acetyl-CoA) pathway for acetogenesis performed by homoacetogenic bacteria. Acetate generated by gut bacteria via the acetyl-CoA pathway provides considerable nutrition to wood-feeding dictyopteran insects making CODH important to the obligate mutualism occurring between termites and their hindgut microbiota. To investigate CODH diversity in insect gut communities, we developed the first degenerate primers designed to amplify cooS genes, which encode the catalytic (β) subunit of anaerobic CODH enzyme complexes. These primers target over 68 million combinations of potential forward and reverse cooS primer-binding sequences. We used the primers to identify cooS genes in bacterial isolates from the hindgut of a phylogenetically lower termite and to sample cooS diversity present in a variety of insect hindgut microbial communities including those of three phylogenetically-lower termites, Zootermopsis nevadensis, Reticulitermes hesperus, and Incisitermes minor, a wood-feeding cockroach, Cryptocercus punctulatus, and an omnivorous cockroach, Periplaneta americana. In total, we sequenced and analyzed 151 different cooS genes. These genes encode proteins that group within one of three highly divergent CODH phylogenetic clades. Each insect gut community contained CODH variants from all three of these clades. The patterns of CODH diversity in these communities likely reflect differences in enzyme or physiological function, and suggest that a diversity of microbial species participate in homoacetogenesis in these communities.
Peptidoglycan Remodeling and Conversion of an Inner Membrane into an Outer Membrane During Sporulation
Two hallmarks of the Firmicute phylum, which includes the Bacilli and Clostridia classes, are their ability to form endospores and their "Gram-positive" single-membraned, thick-cell-wall envelope structure. Acetonema longum is part of a lesser-known family (the Veillonellaceae) of Clostridia that form endospores but that are surprisingly "Gram negative," possessing both an inner and outer membrane and a thin cell wall. Here, we present macromolecular resolution, 3D electron cryotomographic images of vegetative, sporulating, and germinating A. longum cells showing that during the sporulation process, the inner membrane of the mother cell is inverted and transformed to become the outer membrane of the germinating cell. Peptidoglycan persists throughout, leading to a revised, "continuous" model of its role in the process. Coupled with genomic analyses, these results point to sporulation as a mechanism by which the bacterial outer membrane may have arisen and A. longum as a potential "missing link" between single- and double-membraned bacteria.
Genes for Selenium Dependent and Independent Formate Dehydrogenase in the Gut Microbial Communities of Three Lower, Wood-feeding Termites and a Wood-feeding Roach
The bacterial Wood-Ljungdahl pathway for CO(2)-reductive acetogenesis is important for the nutritional mutualism occurring between wood-feeding insects and their hindgut microbiota. A key step in this pathway is the reduction of CO(2) to formate, catalysed by the enzyme formate dehydrogenase (FDH). Putative selenocysteine- (Sec) and cysteine- (Cys) containing paralogues of hydrogenase-linked FDH (FDH(H)) have been identified in the termite gut acetogenic spirochete, Treponema primitia, but knowledge of their relevance in the termite gut environment remains limited. In this study, we designed degenerate PCR primers for FDH(H) genes (fdhF) and assessed fdhF diversity in insect gut bacterial isolates and the gut microbial communities of termites and cockroaches. The insects examined herein represent three wood-feeding termite families, Termopsidae, Kalotermitidae and Rhinotermitidae (phylogenetically 'lower' termite taxa); the wood-feeding roach family Cryptocercidae (the sister taxon to termites); and the omnivorous roach family Blattidae. Sec and Cys FDH(H) variants were identified in every wood-feeding insect but not the omnivorous roach. Of 68 novel alleles obtained from inventories, 66 affiliated phylogenetically with enzymes from T. primitia. These formed two subclades (37 and 29 phylotypes) almost completely comprised of Sec-containing and Cys-containing enzymes respectively. A gut cDNA inventory showed transcription of both variants in the termite Zootermopsis nevadensis (family Termopsidae). The gene patterns suggest that FDH(H) enzymes are important for the CO(2)-reductive metabolism of uncultured acetogenic treponemes and imply that the availability of selenium, a trace element, shaped microbial gene content in the last common ancestor of dictyopteran, wood-feeding insects, and continues to shape it to this day.
