Advancing approaches to cultivate industrially and ecologically relevant microorganisms from termite guts.

Prompted by our limited understanding of the degradation of lignin and lignin-derived aromatic metabolites in termites, we studied the metabolism of monoaromatic model compounds by termites and their gut microflora. Feeding trials performed with [ring-U-(sup14)C]benzoic acid and [ring-U-(sup14)C]cinnamic acid revealed the general ability of termites of the major feeding guilds (wood and soil feeders and fungus cultivators) to mineralize the aromatic nucleus. Up to 70% of the radioactive label was released as (sup14)CO(inf2); the remainder was more or less equally distributed among termite bodies, gut contents, and feces. Gut homogenates of the wood-feeding termites Nasutitermes lujae (Wasmann) and Reticulitermes flavipes (Kollar) mineralized ring-labeled benzoic or cinnamic acid only if oxygen was present. In the absence of oxygen, benzoate was not attacked, and cinnamate was only reduced to phenylpropionate. Similar results were obtained with other, nonlabeled lignin-related phenylpropanoids (ferulic, 3,4-dihydroxycinnamic, and 4-hydroxycinnamic acids), whose ring moieties underwent degradation only if oxygen was present. Under anoxic conditions, the substrates were merely modified (by side chain reduction and demethylation), and this modification occurred at the same time as a net accumulation of phenylpropanoids formed endogenously in the gut homogenate, a phenomenon not observed under oxic conditions. Enumeration by the most-probable-number technique revealed that each N. lujae gut contained about 10(sup5) bacteria that were capable of completely mineralizing aromatic substrates in the presence of oxygen (about 10(sup8) bacteria per ml). In the absence of oxygen, small numbers of ring-modifying microorganisms were found (<50 bacteria per gut), but none of these microorganisms were capable of ring cleavage. Similar results were obtained with gut homogenates of R. flavipes, except that a larger number of anaerobic ring-modifying microorganisms was present (>5 x 10(sup3) bacteria per gut). Neither inclusion of potential cosubstrates (H(inf2), pyruvate, lactate) nor inclusion of hydrogenotrophic partner organisms resulted in anoxic ring cleavage in most-probable-number tubes prepared with gut homogenates of either termite. The oxygen dependence of aromatic ring cleavage by the termite gut microbiota is consistent with the presence, and uptake by microbes, of O(inf2) in the peripheral region of otherwise anoxic gut lumina (as reported in the accompanying paper [A. Brune, D. Emerson, and J. A. Breznak, Appl. Environ. Microbiol. 61:2681-2687, 1995]). Taken together, our results indicate that microbial degradation of plant aromatic compounds can occur in termite guts and may contribute to the carbon and energy requirement of the host.

Termites are global pests and can cause serious damage to buildings, crops, and plantation forests. The symbiotic intestinal flora plays an important role in the digestion of cellulose and nitrogen in the life of termites. Termites and their symbiotic microbes in the gut form a synergistic system. These organism work together to digest lignocellulose to make the termites grow on nitrogen deficient food. In this paper, the diversity of symbiotic microorganisms in the gut of termites, including protozoan, spirochetes, actinomycetes, fungus and bacteria, and their role in the digestion of lignocellulose and also the biotechnological applications of these symbiotic microorganisms are discussed. The high efficiency lignocellulose degradation systems of symbiotic microbes in termite gut not only provided a new way of biological energy development, but also has immense prospect in the application of cellulase enzymes. In addition, the study on the symbiotic microorganisms in the gut of termites will also provide a new method for the biological control of termites by the endophytic bacteria in the gut of termites.

The termite gut is an ideal ecosystem for studying hydrogen ecophysiology. Hydrogen is central to the obligate mutualism between termites and their gut microbes and is turned over at rates as high as 33 m3 H2 per m3 hindgut volume daily and maintained near saturation in some species. Acetogenic bacteria use hydrogen to produce up to 1/3 of the total flux of the termite’s primary carbon and energy source, acetate. We have taken a three-fold approach to investigate the hydrogen ecophysiology of the termite gut. In our first approach (Chapter 2) we completed a bioinformatic analysis of [FeFe] hydrogenase-like (H domain) proteins encoded in the genomes of three termite gut treponemes. Treponemes are among the most highly represented groups of gut bacteria. The remarkable diversity of H domain proteins encoded accentuates the importance of hydrogen to their physiology. Moreover, they encoded a poorly understood class hydrogen sensing H domain proteins and thereby present a unique opportunity for their further study. In our second approach (Chapters 3 and 4) we analyzed molecular inventories prepared from termite gut microbiomes of a class of [FeFe] hydrogenases found highly represented in a termite hindgut metagenome. The libraries of peptide sequences clustered with one another in a manner congruent with termite host phylogeny suggesting co-evolution. Interestingly, we observed that higher termite guts may harbor higher sequence diversity than lower termites. In our third approach (Chapter 5) we used microfluidic digital PCR to identify bacteria in the gut of Reticulitermes tibialis encoding [FeFe] hydrogenases. The majority of the 16S rRNA gene phylotypes observed to co-amplify with hydrogenase sequences were treponemal, and the only observed instances of the same 16S rRNA-hydrogenase gene pair co-amplifying in multiple microfluidic chambers corresponded to treponemal phylotypes. Therefore, treponemes may be an important or predominant bacterial group encoding an important family of [FeFe] hydrogenases in the termite gut. The above results provide support for an important role for treponemes in mediating hydrogen metabolism in the termite gut and accentuate the intimacy and stability of the association termites have maintained over the course of their evolution with their gut microbial communities.

In this study, screening and isolation of xylano-cellulolytic enzymes producing positive microbes from termitarium and termite gut microbiome were done using cost-effective agricultural wastes. The enrichment of xylano-cellulolytic microbes was done in three steps using wheat bran and waste paper. The qualitative screening of xylanase and cellulase producing micro-organisms was done on nutrient agar plates containing wheat bran and waste paper, respectively. Xylanase and cellulase positive colonies were analysed by observing the zone of substrate (wheat bran and waste paper) hydrolysis around the colonies. A total of 30 bacterial isolates were obtained from termite gut and termitarium, respectively. Xylan and cellulose degrading potential of the positive isolates was also quantitatively estimated using agro-wastes-based medium. All the bacterial isolates displayed cellulase and xylanase activities in the range of 0.45–6.80 and 51–380 IU/ml, respectively. This is the first report mentioning the isolation of xylano-cellulolytic microbes from termite gut and termitarium using very simple cost-effective methodology.

Genome-scale community modeling for deciphering the inter-microbial metabolic interactions in fungus-farming termite gut microbiome

The termite gut microbiome is a model system to investigate microbial interactions and their associations with host. For decades, extensive research with molecular tools and conventional cultivation method has been carried out to define the microbial diversity in termite gut. Yet, many bacterial groups of the termite gut microbiome have not been successfully cultivated in laboratory. In this study, we adapted the recently developed microfluidic streak plate (MSP) technique for cultivation of termite gut microbial communities at both aerobic and anaerobic conditions. We found that 99 operational taxonomic units (OTUs) were cultivable by MSP approach and 18 OTUs were documented first time for termite gut microbiota. Further analysis of the bacterial diversities derived by culture‐dependent MSP approach and culture‐independent 16S rRNA gene typing revealed that both methods have bias in recovery of gut microbiota. In total 396 strains were isolated with MSP technique, and potential new taxa at species and/or genus levels were obtained that were phylogenetically related to Burkholderia, Micrococcus, and Dysgonomonas. Results from this study indicate that MSP technique is applicable for cultivating previously unknown and new microbial groups of termite gut microbiota.

The recent application of culture-independent molecular approaches provides a new way to characterize the microbial populations in the symbiotic community in the termite gut. Now that the symbiotic community within the termite gut has been shown to be highly structured, many aspects of the interactions between the host and the symbionts and among the symbionts should be studied further. Beyond the mere description of phylogenetic diversity it is necessary to characterize the in situ localization of individual populations, and to directly link the identity of individual cells to their functions. New probes for functional marker genes and characterization of their expression in situ will lead to remarkable advances. In order to discuss evolution of symbiosis within the termite gut, the structure and functions of the gut community should be extensively characterized in some model termites. A more diverse range of termite species should be analyzed. Finally, it is noted that the symbiotic relationships between the gut protists and their endobionts or ectobionts are attractive research subjects in terms of the symbiosis-accelerated evolution of eukaryotic cells, since these protists represent early emerging groups of eukaryotes.

BackgroundThe microbial landscape within termite guts varies across termite families. The gut microbiota of lower termites (LT) is dominated by cellulolytic flagellates that sequester wood particles in their digestive vacuoles, whereas in the flagellate-free higher termites (HT), cellulolytic activity has been attributed to fiber-associated bacteria. However, little is known about the role of individual lineages in fiber digestion, particularly in LT.ResultsWe investigated the lignocellulolytic potential of 2223 metagenome-assembled genomes (MAGs) recovered from the gut metagenomes of 51 termite species. In the flagellate-dependent LT, cellulolytic enzymes are restricted to MAGs of Bacteroidota (Dysgonomonadaceae, Tannerellaceae, Bacteroidaceae, Azobacteroidaceae) and Spirochaetota (Breznakiellaceae) and reflect a specialization on cellodextrins, whereas their hemicellulolytic arsenal features activities on xylans and diverse heteropolymers. By contrast, the MAGs derived from flagellate-free HT possess a comprehensive arsenal of exo- and endoglucanases that resembles that of termite gut flagellates, underlining that Fibrobacterota and Spirochaetota occupy the cellulolytic niche that became vacant after the loss of the flagellates. Furthermore, we detected directly or indirectly oxygen-dependent enzymes that oxidize cellulose or modify lignin in MAGs of Pseudomonadota (Burkholderiales, Pseudomonadales) and Actinomycetota (Actinomycetales, Mycobacteriales), representing lineages located at the hindgut wall.ConclusionsThe results of this study refine our concept of symbiotic digestion of lignocellulose in termite guts, emphasizing the differential roles of specific bacterial lineages in both flagellate-dependent and flagellate-independent breakdown of cellulose and hemicelluloses, as well as a so far unappreciated role of oxygen in the depolymerization of plant fiber and lignin in the microoxic periphery during gut passage in HT.Ak1UNwnHwhcNN-Fj12kHobVideo

Bacterial species metabolic interaction network for deciphering the lignocellulolytic system in fungal cultivating termite gut microbiota

Nitrogen fixation by the microorganisms in the gut of termites is one of the crucial aspects of symbiosis, since termites usually thrive on a nitrogen-poor diet. The phylogenetic diversity of the nitrogen-fixing organisms within the symbiotic community in the guts of various termite species was investigated without culturing the resident microorganisms. A portion of the dinitrogenase reductase gene (nifH) was directly amplified from DNA extracted from the mixed population in the termite gut. Analysis of deduced amino acid sequences of the products of the clonally isolated nifH genes revealed the presence of diverse nifH sequences in most of the individual termite species, and their constituents were considerably different among termite species. A majority of the nifH sequences from six lower termites, which showed significant levels of nitrogen fixation activity, could be assigned to either the anaerobic nif group (consisting of clostridia and sulfur reducers) or the alternative nif methanogen group among the nifH phylogenetic groups. In the case of three higher termites, which showed only low levels of nitrogen fixation activity, a large number of the sequences were assigned to the most divergent nif group, probably functioning in some process other than nitrogen fixation and being derived from methanogenic archaea. The nifH groups detected were similar within each termite family but different among the termite families, suggesting an evolutionary trend reflecting the diazotrophic habitats in the symbiotic community. Within these phylogenetic groups, the sequences from the termites formed lineages distinct from those previously recognized in studies using classical microbiological techniques, and several sequence clusters unique to termites were found. The results indicate the presence of diverse potentially nitrogen-fixing microbial assemblages in the guts of termites, and the majority of them are as yet uncharacterized.

The objective of this review paper is to critically review the termite gut microbiome, which could offer a potential source of chemical frameworks and biosynthetic pathways for next-generation antimicrobials with potential applications in human health, agriculture, and beyond. The termite gut microbiome is a rich and largely untapped source of novel antibiotics driven by the diverse microbial communities that co-evolved with termites to facilitate complex digestion and host defense. These symbiotic gut bacteria, including prolific antibiotic producers such as Streptomyces spp., generate bioactive compounds that inhibit a range of pathogenic bacteria, making them promising candidates for antimicrobial drug discovery in the face of rising antibiotic resistance. Recent studies employing metagenomics, 16S rRNA metabarcoding, and transcriptomic analyses have revealed numerous antibiotic biosynthesis gene clusters within termite-associated microbes, indicating an active role in microbial competition and termite colony immunity. Moreover, functional investigations demonstrate that termite gut symbionts contribute to the host’s resilience against infections by fine-tuning gene expression related to immunity and metabolism. The ecological niche of the termite gut, characterized by intense microbial interactions and co-dependencies, drives the evolution of novel chemical scaffolds with potential applications in human health and agriculture. This review represents an exciting frontier for identifying new antibiotics and understanding symbiotic microbial ecology. Harnessing termite gut microbiota could provide innovative solutions against multidrug-resistant pathogens, promoting sustainable antimicrobial development.

Rampant Host Switching Shaped the Termite Gut Microbiome

The termite gut microbiota can include a variety of micro-organisms from the three domains: Bacteria, Archaea and Eucarya. The bacterial groups from the gut systems are mainly affiliated to the proteobacteria, the Gram-positive groups Bacterioiodes/Flavobacterium branch and the spirochetes, Firmicutes and Actinobacteria. However, culture independent molecular studies have revealed that the majority of these microbial gut symbionts have not yet been cultured, including actinobacterial clusters associated with termite guts. Accordingly, the aim of this study was to selectively isolate the actinofloral layers of gut associated microflora of the Coptotermes lacteus (Froggatt) species located at the Sunshine Coast Region of Queensland, Australia to increase our knowledge on the diversity of actinobacterial taxa present in the termite guts. Actinofloral layers associated with the guts of the wood-eating subterranean termite C. lacteus were investigated by exploiting the phage susceptibility of different gut associated bacteria which impede the growth of actinomycetes on isolation plates. These unwanted microbial taxa were removed by exposing the gut contents to polyvalent bacteriophages specifically targeting different background bacterial taxa and after their removal from the isolation plates previously undetected and novel actinomycetes were successfully cultured from the gut samples. Use of bacteriophages as a means of selective pressure successfully revealed the presence of novel actinomycete species within the guts of C. lacteus. Molecular ecology has undoubtedly revealed the fascinating diversity of micro-organisms, which cannot be cultured. However, these advances in the field still have not provided the ability to detect and isolate micro-organisms effectively from their ecological niches. Accordingly, studies like the one described here have importance in increasing the chances of uncultured taxa to be isolated to complement molecular microbial ecological efforts towards the establishment of an understanding on the diversity of termite gut microflora.

BackgroundTermites and their microbial gut symbionts are major recyclers of lignocellulosic biomass. This important symbiosis is obligate but relatively open and more complex in comparison to other well-known insect symbioses such as the strict vertical transmission of Buchnera in aphids. The relative roles of vertical inheritance and environmental factors such as diet in shaping the termite gut microbiome are not well understood.ResultsThe gut microbiomes of 66 specimens representing seven higher and nine lower termite genera collected in Australia and North America were profiled by small subunit (SSU) rRNA amplicon pyrosequencing. These represent the first reported culture-independent gut microbiome data for three higher termite genera: Tenuirostritermes, Drepanotermes, and Gnathamitermes; and two lower termite genera: Marginitermes and Porotermes. Consistent with previous studies, bacteria comprise the largest fraction of termite gut symbionts, of which 11 phylotypes (6 Treponema, 1 Desulfarculus-like, 1 Desulfovibrio, 1 Anaerovorax-like, 1 Sporobacter-like, and 1 Pirellula-like) were widespread occurring in ≥50% of collected specimens. Archaea are generally considered to comprise only a minority of the termite gut microbiota (<3%); however, archaeal relative abundance was substantially higher and variable in a number of specimens including Macrognathotermes, Coptotermes, Schedorhinotermes, Porotermes, and Mastotermes (representing up to 54% of amplicon reads). A ciliate related to Clevelandella was detected in low abundance in Gnathamitermes indicating that protists were either reacquired after protists loss in higher termites or persisted in low numbers across this transition. Phylogenetic analyses of the bacterial communities indicate that vertical inheritance is the primary force shaping termite gut microbiota. The effect of diet is secondary and appears to influence the relative abundance, but not membership, of the gut communities.ConclusionsVertical inheritance is the primary force shaping the termite gut microbiome indicating that species are successfully and faithfully passed from one generation to the next via trophallaxis or coprophagy. Changes in relative abundance can occur on shorter time scales and appear to be an adaptive mechanism for dietary fluctuations.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0067-8) contains supplementary material, which is available to authorized users.

Genomic insights into the metabolic potential of a novel lignin-degrading and polyhydroxyalkanoates producing bacterium Pseudomonas sp. Hu109A