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Diversity and function of bacteria related to the newly isolated
organism JT5, a possible lignocellulose-degrading species from the
gut of an evolutionarily higher termite
Brandi Deptula
University of Wisconsin Oshkosh
Dr. Eric Matson, Mentor
Department of Biology/Microbiology
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Abstract
Termites are one of the most successful groups of insect on the planet and are one of only two
insects known capable of subsisting solely on lignocellulosic plant biomass. The remarkable
digestive capability of termites is attributed to the highly complex microbial communities
inhabiting their guts, and yet the vast majority of these microbes are not well understood due to
an inability to cultivate them in the laboratory. Recently, a novel bacterium (strain JT5) related to
the genus Dysgonomonas was isolated from an evolutionarily-higher termite, Gnathamitermes
perplexus. This bacterium was determined to be an abundant member of the gut community and
to possess genes for enzymes involved in lignocellulose degradation. We hypothesized that
numerous bacteria related to JT5 would reside in G. perplexus and in a variety of other termite
species. To test the hypothesis, we created inventories of SSU rRNA genes (a marker gene used
for bacterial species identification) from G. perplexus gut community DNA. We targeted subsets
of SSU rRNA genes in the gut bacterial population that are closely related to JT5 as well as
subsets that are more broadly associated with the Dysgonomonas genus. We used similar
methods to determine whether or not these SSU rRNA genes were present in gut community
DNA samples from other termite species. Our investigations revealed a lower than expected
diversity of bacteria predicted to share species-level relationships with JT5 in G. perplexus but a
higher diversity at the genus level. We also found that many of the termite samples we analyzed
contained bacteria predicted to be related to JT5. These results indicate that JT5 represents one
of potentially several bacterial species within the genus Dysgonomonas that inhabit the guts of
termite species. Thus, research on JT5 may broadly provide information on the role of
bacterially-mediated lignocellulose degradation occurring in termites.
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Introduction
Investigating natural systems that break down plant biomass can provide us with new
biotechnological applications (Okhuma, 2003). Due to the availability of large quantities of
cellulosic plant materials, it is an attractive substrate for production of industrially important
products. Most plant biomass is currently disposed of by burning (Gupta et al., 2011). The
discovery of new approaches and systems for lignocellulose degradation would result in a
cheaper and more favorable processing of plant materials into useful products. Producing
biofuels out of woody crops, agricultural waste, and grasses poses the challenge of finding ways
to breakdown the lignocellulose/xylan material. Hydrolytic enzymes are already being applied in
industries to aid with agricultural and plant waste, and in chiral separation and ligand binding
studies (Gupta et al., 2011). Recombinant organisms containing cellulose degrading and ethanol
producing genes have been created, but as of yet they do not produce enough ethanol for
commercial scale production (Vasan et al., 2010). New discoveries in this area would lead the
way to more efficient renewable energies, and may also aid in better processing of paper
materials in mills. When searching for ways to breakdown and extract plant polysaccharides, it is
reasonable to look at the microbial systems found in the guts of organisms such as termites that
flourish on a diet of lignocelluloses plant biomass (Gupta et al., 2011).
Termites are subdivided into seven families. Six of these families are called lower
termites and the seventh family, called higher termites, is made up of about 85% of the known
species of termites (Matsui et al., 2009). Lower termites feed mainly on wood, utilizing the
enzymes they make themselves, as well as those from bacteria, archaea, and protists in their guts
to digest the wood (Matsui et al., 2009). Higher termites show a more diverse diet, including
soil, grass, and self-cultivated fungi. Those subsisting on wood alone, are the minority of this
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family. For this reason, higher termites are a good place to search for previously undiscovered
gene activity. It is especially desirable to examine the worker class of termites, as they play the
major role of foraging and breakdown of plant material (Matsui et al., 2009).
The diversity of food sources utilized by higher termites is assumed to be related to the
diversity of the prokaryotes they harbor in their gut. The variety of geographic habitats in which
higher termites live was not found to change bacterial diversity among members of the same
termite species (Husseneder et al., 2010). This suggests that termites need to retain a symbiotic
gut community composed of a similar number and composition of species to fulfill their
nutritional requirements. These species need not be identical in each termite; closely related
microbial species or microbes with overlapping functions suffice. However, in laboratory termite
colonies, a rapid shift in community structure was seen when varying the food source between
high and low molecular weight carbon sources. In one such study, it was found that the gut
population of a wood-feeding higher termite was dominated by spirochetes when fed wood or
wood powder, Bacteroidetes when fed xylan, cellobiose, or glucose, and Firmicutes when fed
xylose (Husseneder et al., 2010).
While the activity of microbial species inhabiting the termite gut is largely responsible
for the degradation of lignocellulose, and crucial to the ability to derive carbon and energy from
plant biomass, the termite gut microbial community is responsible for many other important
functions. For example, the gut microbiome is responsible for the production of acetate (the
principal source of nutrition taken up by the termite) from hydrogen and carbon dioxide, as well
as nitrogen fixation and recycling of uric acid (Ohkuma & Kudo, 1996). Wood is a poor source
of nitrogen, thus, most termite species that live solely on woody material derive their nitrogen
from nitrogen-fixing bacteria in their hindgut. Nitrogen fixing bacterial species have been
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isolated from both higher and lower termites. The waste product, uric acid, is converted by
bacteria into ammonia in order to recycle nitrogen containing compounds (Matsui et al., 2009).
The termite gut is generally composed of three major compartments: the foregut, the
midgut, and the hindgut or lumen. The hindgut is the largest section, comprising a large amount
of the termite's weight, and contains the most dense and diverse quantity of symbionts (Matsui
et al., 2009). Additional gut compartments vary based on species of termite. One example is that
the digestive tracts of termites that belong to the genus Cubitermes, members of the higher
termite family. In these species, the gut is further separated into distinct hindgut compartments
containing variations in microbial community makeup (Schmitt-Wagner et al., 2003). The large
number of potential niches allows for the possibility of species overlap between the sections.
However, solid evidence on the role of possible functional redundancy of microbial communities
in the termite gut is as of yet lacking (Yang et al., 2005).
The hindgut paunch is an example of one gut compartment displaying varied
microenvironments. Due to oxygen consumption by different prokaryotes within, there is a
decrease in oxygen concentration inward from the gut wall towards a completely anoxic center
(Brune et al., 1995). Hydrogen concentrations, consumption, and production are similarly
stratified throughout the termite gut. The presence of oxygen impacts the hydrogen
concentrations, which is explained by the presence of different species existing in different areas
of the gut. Some species present are oxidizing hydrogen, while others are producing hydrogen as
a byproduct of fermentative metabolism (Ebert & Brune, 1997). Studies using microsensors and
radiotracers show that the gut compartments are separate functional zones for microbial activity.
Such processes include hydrogen production, methanogenesis, and acetogenesis. It was noted
that hydrogen accumulates in the anterior hindgut of all Cubitermes species investigated. The
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population of bacteria related to clostridia, which are known fermentative bacteria, may explain
the source of increased hydrogen (Schmitt-Wagner et al., 2003). The special organization of
different bacteria in the termite guts have also been shown to control intestinal carbon flux (Yang
et al., 2005). Such features of the gut underscore the variety of roles microbial species perform in
the termite gut and their interdependence on each other (Ebert & Brune, 1997).
The termite gut epithelium is the most probable location for oxygen diffusion into the
gut. Oxygen concentration drops steadily on approaching the center of the gut which is anoxic.
In between the wall and center is a microoxic environment of decreasing oxygen concentration.
The anaerobic central portion allows for the presence of strict anaerobes to inhabit this niche.
Many bacterial inhabitants of the oxygenic gut wall are aerobes or facultative anaerobes,
however, members of Bacteroidetes and Clostridiales, which are often strict anaerobes, are
found to be abundant along the gut wall. This suggests they may be at least partially tolerant to
microoxic conditions (Nakajima et al, 2005). These findings highlight the importance of the
challenging task of investigating the locations of different bacterial species in situ, in a way that
doesn't disturb the internal structure of the gut community.
Studies have been conducted in an attempt to categorize the microbial diversity from a
variety of different termites. Very few numerically significant bacteria from higher termites have
ever been grown in pure culture (Okhuma, 2003). Most of the isolated organisms belong to one
of four phylogenetic divisions within the domain Bacteria. These included Proteobacteria,
Spirochetes, Bacteriodetes, and low G-C content gram positive bacteria. More current studies
state that the prokaryote inhabitants of termite guts are represented by organisms belonging to
the phyla Actinobacteria, Firmicutes, Bacteroides, Proteobacteria, and Spirochetes, as well as
various Archea (Matteotti et al., 2012; Yang et al., 2005; Schmitt-Wagner et al. ,2003). One
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study identified species that couldn't be assigned to any known major groups of bacteria. This
study also found that two thirds of the distinct species identified had less than 90% sequence
identity to any known 16s rDNA sequence data of cultured organisms while another 10 species
had no close similarity to any recognized bacterial phylum in rRNA databases (Ohkuma &
Kudo, 1996). Additionally, analysis of bacterial clone lineages showed they were most closely
related to bacteria isolated from closely related termites, suggesting co-evolution between
termites and their gut microbes (Hongoh, 2010; Schmitt-Wagner et al., 2003; Yang et al., 2005).
A study of the phylogeny and termite gut niche location in Reticulitermes santonensis
analyzed fractions of gut sections to compare the diversity and community structure of each
section. More than 200 different restriction patterns were found by analyzing the gut of this
wood-feeding termite using restriction fragment length polymorphism analysis. Species richness
of hindgut fluid was highest, while in contrast, it was lowest in the midgut. Of all the sections
sampled, it was implied that only in the hindgut would further sampling increase the number of
new species identified (Yang et al., 2005). Regarding phylogenic location studies, it has been
seen that the steep increase in gut pH in the midgut to hindgut transition correlates with a steep
drop in microbial density. This evidence of changing physiochemical conditions between the
mid and hindgut represents a probable barrier for microbes, preventing passage between gut
compartments (Schmitt-Wagner et al., 2003). Figure 1 shows the phylogenic placement of
isolate JT5.
Of all species studies, Reticulitermes speratus, a lower termite, has been the most studied
in regards to bacterial diversity in the gut. More than 300 bacterial phylotypes have been
reported, and has an estimated species per termite as high as up to 700 different species per
individual termite. This study especially focused on the microbial community niche of the gut
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epithelium. In this species, methanogenic Archaea exclusively colonized the gut wall, whereas,
in other species they are found within the cells of gut protists. Due to the oxygen and hydrogen
gradients originating at the gut epithelium, bacteria living in this niche are considered unlike
ones that are able to freely move throughout the gut. Findings such as these also show proof of
the formation of distinct niches within the termite guts (Nakajima et al., 2005). Abundant groups
found along the gut epithelium, including Actinobacteria, Bacteroidetes, Clostridiales, and
Lactococcus, are non-motile species. However, Bacteroidetes were found in significant numbers
in all gut segments analyzed, except for the protozoan fraction (Nakajima et al., 2005; Yang et
al., 2005). Groups found in the gut lumen, such as Spirochaetes and Desulfovibrio, have a high
level of motility. This may be necessary to counteract the fluid dynamics of the gut and stay in
the niche they're adapted to (Nakajima et al., 2005).
Comparisons of two soil-feeding higher termites of the genus Cubitermes were
undertaken to compare the abundance and distribution of bacterial species in their gut tract with
the intent to discover changes in microbiota along the passage through the gut and to compare
the different bacterial species in the homologous gut compartments of the two species (SchmittWagner et al., 2003). Cocci and short rods were found to be dominant in all gut sections.
Spirochetes were also found in significant number in the P 1 , P3, and P4 hindgut compartments.
The first section of the hindgut, P 1, contained mainly low G+C content gram positive bacteria.
The amount of this type of bacteria decreased progressively through the posterior hindgut
sections, while more diversity of species in the Proteobacteria, Cytophaga-FlexibacterBacteriodetes, and spirochetes was noted. Highest species diversity was observed in the P4
hindgut compartment. Despite the decrease in low G+C content gram positive bacteria upon
moving toward the posterior gut, one half of the clones in the P5 section were found to be in this
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category. One third of the total from the P5 section were associated with Proteobacteria,
Cytophaga-Flexibacter, and Bacteriodetes.
The increase in these phyla along the hindgut
correlates with the increase of acetate, lactate, and succinate in the P3 segment of the Cubitermes
gut. Finally, it was indicated that Actinomycetes did not play a major role in the guts of these
termites, as no clones of this type were found in this study. This is curious because
Actinomycetes showing cellulolytic and lignin-solubilizing activity, have been isolated in the
past from soil feeding termites (Schmitt-Wagner et al. 2003).
While it has been proven that termites produce their own celluloltytic enzymes in their
salivary glands or midgut epithelium to initiate the digestive process, microbial flora in the
digestive tract is necessary for termite survival (Matsui et al., 2009; Scharf et al., 2010; Yang et
al., 2005). It is well known that phylogenetically lower termites contain symbiotic protozoan
flagelletes that are contributing a significant amount of cellulases, as several cellulase genes have
been found in termite gut protozoa (Matsui et al., 2009; Wenzel et al., 2002). Termites that
cultivate fungus are known to consume fungal cellulases to aid in the breakdown of
lignocellulose. It is presumed that in higher termites, which lack flagellate protozoa, the
contribution of lignocelluloses-degrading enzymes from bacteria must be higher (Matsui et al.,
2009; Warneke et al., 2009).
Cellulose is broken down by a series of reactions involving three separate cellulases:
endo-beta-1,4-glucanses (EG), cellobiohydrolases (CBH), and beta-glucosidases (BGL). Hemicellulose is broken down by hemicellulases: endo-beta-1-4-xylanase, beta-xylosidase, and alphaglucuronidase (Matteotti et al., 2010). Endo-beta-1,4-glucanase is an essential enzyme in the
breakdown of cellulose because it begins the breakdown of cellulose into smaller cellulose
molecules (Cho et al., 2010). This is the enzyme at the beginning of the chain of cellulose
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breakdown, and is the enzyme most often found produced by the termite itself. Endo-beta-1-4glucanase is found in highest concentrations in the salivary glands of lower termites, and in the
mid-gut of higher termites (Matsui et al., 2009). This provides reaction sites for CBH to break
cellulose down further into oligosaccharides or cellobiose. Finally, the products of the CBH
reaction are hydrolyzed by BGL to yield glucose (Cho et al., 2010).
Investigations on termite gut communities have identified over 700 genes predicted to
encode enzymes likely contributing to the breakdown of lignocellulose, spanning at least 45
different carbohydrate active enzyme families (Warnecke et al., 2007). For example, two genera
of lower termites have yielded diverse species of Gram-positive and Gram-negative bacteria that
were able to be cultivated, and possessed beta-glucosidase, cellobiohydrolase, beta-xylosidase,
and Carboxymethylcellulase enzyme activity (Matteotti et al., 2012). Cellulose-degrading
bacteria from the gut of Zootemopsis angusticollis have been identified and quantified. Serial
dilutions were preformed from the gut contents of termites that were fed either wood or fiber
paper. After enrichment on carboxylmethylcellulose or filter paper, 119 cellulolytic bacterial
strains belonging to 23 different groups were isolated. Many of the isolates hydrolyzed both
carboxylmethylcellulose and the filter paper (Wenzel et al., 2002). Many other species are
unable to be cultivated as of yet, and while attempts to sequence the entire genome of
uncultivable bacteria from termite guts have been successful, enzyme activity is often presumed
by genomic analysis (Hongoh, 2010).
The guts of termites contain a wealth of highly specialized co-evolved symbionts.
Further research on culturing these species and analyzing their functional roles in the termite gut
will likely divulge a tremendous amount of information on how these gut communities function.
Combined with cultivation-independent approaches, this research will lead to a better
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understanding of termite ecology. Such pursuits advance our understanding of complex
symbiosis found in the guts of various termite species and may ultimately lead to new design
approaches to biomaterial processing inspired by termite gut systems. The termite gut systems
may even provide us with superior organisms and enzymes for biofuels production, nitrogen
processing, and methane generation.
Methods
Bacterial Cultivation
To examine the growth rate of isolate JT5 on four different carbon sources, anaerobic
tubes were amended with 50 ul of vitamin/cofactor mix (Leadbetter et al., 1999). Two tubes
were amended with 400 ul of 250 mM Cellobiose, two tubes with 200 ul of 0.5 M Glucose, two
tubes with 200 ul of 0.5M Xylose, and two tubes with 400u1 Carboxymethylcellulose(CMC) to
equal 36mg CMC per tube, providing a carbon source equivalent to the other tubes . Absorption
readings were taken using a Spectronic 21D (Milton Roy) Spectrophotometer. 200 ul of a log
phase culture of JT5 was added to each tube and another absorption reading was taken. Tubes
were incubated at 27°C. Daily readings were taken for 14 days. Logarithmic Growth curves
were plotted and generation time during log-phase growth was calculated using Microsoft excel.
PCR Amplification
Whole gut community DNA samples were diluted to equal 1Ong/u1 concentrations. Each
sample was combined with 10 ul fail-safe Buffer D (Epicentre, Madison,WI), 0.25 ul Taq
polymerase (New England Biolabs, Ipswich, MA), 7.25u1 molecular grade water, and 1 ul each
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forward and reverse primer, into a 20 ul reaction volume. The samples were amplified by
Polymerase Chain Reaction (PCR) using 27F/1492R universal bacterial primers with the ability
to amplify all bacterial DNA present (Lane et al., 1991). A second PCR reaction was prepared
using 591F/1392R specific dysgonomonas primers to amplify only DNA similar to isolate JT5
(5'-ACTGCGGGTCTTGAGTATGG-3' and 5'-GCGGTTACGCACTTCAGATA-3',
respectively). A third PCR reaction was prepared using 627F/1492R to amplify DNA
representative of the genus Dsygonomonas, which includes isolate JT5. A control tube of PCR
water was created for the universal primer set as a negative control.
PCR was performed using a T100 Thermocycler (Bio-rad, Hercules, CA). PCR
conditions were 95°C denaturation, 58°C annealing, and 72°C elongation temperatures for 30
cycles. PCR products were visualized using gel electrophoresis through 0.7% agarose gels. Gel
was post stained for visualization with 0.15mg/m1 Ethidium Bromide for 20 minutes. The image
was viewed using Biorad Gel Doc set to UV filter.
Cellular transformation & Clone library amplification
Topo TA Cloning Kits were used to clone PCR products from 591F/1392R and
627F/1492R. Cells were plated on LB agar containing 10Oug/m1 Ampicillian too select for
transformants. After growth at 37°C for 24 hours, 96 transformant clones were selected. PCR
was performed on each colony that resulted in successful transformation, using T7 and T3
primers designed to target Topo vector. A denaturation temperature of 95°C, annealing
temperature of 55°C, and elongation temperature of 72°C were used for 30 cycles.
Restriction Digest
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For each set of PCR products, a restriction digest was performed using 1.6u1 molecular grade
water, 0.2 ul MSPI enzyme (New England Biolabs, Ipswich, MA), Buffer 4 (New England
Biolabs, Ipswich, MA), and BSA (New England Biolabs, Ipswich, MA) to equal a 2 ul reaction
mixture. To the mixture, 5 ul of each PCR product was added, and the reaction proceeded for 3
hours at 37°C. Restriction digest products were visualized using gel electrophoresis through
2.5% agarose gels. Gels were post stained for visualization with Ethidium Bromide for 30
minutes. The image was viewed using Biorad molecular imaging FX scanner to visualize
restriction fragment length polymorphism (RFLP) patterns
Vector extraction
Unique RFLP types were cultured from the stored libraries in LB broth containing
10Oug/m1 Ampicillian and shaken at 190 rpm for 17 hours. Each culture was divided into sterile
ependorf microfuge tubes and centrifuged for 3 minutes at 8,000 x g. Supernatent was removed
and the remaining pellet was processed using QIA prep Spin Miniprep Kit (QIAGEN). Final
extracted vector samples were sent for genome sequencing by The University of Wisconsin
Biotechnology Center, Madison, WI.
Termite gut comparisons
A whole gut sample °Ong/up from 8 termite species had DNA amplified using PCR
using JT5 DNA Ong/0 as a positive control and water as a negative control. Each sample was
prepared in triplicate using 27F/1492R, 627F/1492R, and 591F/1392R primers. Temperatures
were 95°C denaturation, 58°C annealing, and 68°C elongation for 28 cycles. Products were
visualized using gel electrophoresis through 1% agarose gel. Gel was post stained for
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visualization with Ethidium Bromide for 20 minutes. The image was viewed using Biorad
molecular imaging FX scanner and Biorad GelDoc.
Results and Discussion
Analysis of generation times was performed by measuring optical density in three
separate experiments for 13 days, 10 days, and 12 days respectively. For the first experiment,
cellobiose readings were recorded for 13 days, whereas the glucose samples showed growth up
to 21 days. Generation time was calculated by plugging the formula (LOGI° (reading 2) —
LOGio( reading 1))/(0.301 * (time 2-time 1)) into Microsoft Excell. For each substrate, 4
separate generation times were calculated, and averaged together. Generation times were as
follows: Glucose 28.56 hours, Cellobiose 25.78 hours, Xylose 32.2 hours, and CMC 30.72 hours.
JT5 is capable of growth on all carbon sources we provided. Readings for CMC did not appear
to follow the standard bacterial growth curve as did the others. It appeared to have a short rapid
log phase, followed by a steady decline in optical density. It is unsure if this is due to bacterial
cell death or decrease in substrate due to bacterial consumption. As CMC is used in experiments
to induce expression of cellulolytic enzymes, possible upregulation of these enzymes could be
increasing consumption of the carbon source used. After an initial log phase growth,
demonstrated by increased optical density readings, the readings showed more fluctuation up and
down before decreasing steadily. The CMC media appeared to lose viscosity over the course of
bacterial growth. Regardless, we have shown that JT5 can be cultured on various carbon
sources, which suggests a variety of enzymes required for the breakdown of plant-based sugars.
PCR amplification of a whole gut sample was run against 27F/1492R, 591F/1392R, and
627F/1492R. Product from each reaction was cloned into E. coli and plated out on LB agar
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containing 10Oug/m1 Ampicillian, followed by the selection of 96 individual colonies.
Restriction digest was performed, resulting in an 84% yield for the universal bacteria library,
89% yield for the Dysgonomonas library, and 66% yield for the JT5-specific library. (Table 1)
The universal bacterial library was made up of 70 unique clones. The Dysgonomonas library
contained 17 unique clones. The JT5-specific library showed only 2 unique types of clones. A
universal bacterial library of 70 different clones, reinforces our expectations that the termite gut
habitat is comprised of a wide variety of bacteria. Finding 17 unique clones in the
Dysgonomonas library suggests that members of the genus Dysgonomonas are abundant and
diverse members of G. perplexus' gut community. (Table 1) JT5-like species were found to be
present in only 2 unique clones, dismissing our hypothesis that they are diverse in termite guts.
However, it still appears that they are abundant in the gut of G. perplexus.
PCR amplification of a whole gut sample from 8 different termites was run against
27F/1492R, 591F/1392R, and 627F/1492R. Higher termites sampled were: Gnathamitermes
perplexus, Nasutitermes sp., Rhynchotermes sp., Microcerotermes sp., and Amitermes sp.
Lower
termites sampled were: Insisitermes minor, Reticulitermes hesperus, & Zootermopsis nevadensis.
Positive results were seen in all specimens for the universal 27F/1492R bacterial primer set as
expected. Positive results for Dysgonomonas specific primers (627F/1492R) were present in all 5
higher termites, and the lower termite Reticulitermes hesperus, as well as a faintly positive result
in the lower termite Insisitermes minor. Dysgonomonas presence in higher termites matches with
our expectation that they are abundant and play a role in lignocellulose breakdown. A low
positive signal in the two lower termites shows that this genus is not only contained in members
of higher termites, but is also present to a lesser degree in some lower termites. A positive result
for presence of JT5 was found only in the higher termite Gnathamitermes perplexus. According
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to these results, Dsygonomonas appears to be widespread in the guts of numerous termites, to a
higher degree in higher termites, and a lower degree in lower termites. In contrast, JT5 was only
identified in G. perplexus gut habitat, suggesting that it is likely unique to this termite. (Figure 3)
PCR amplification of a sample from the proctedal segment 2A (P2A), diverticulum (DV),
and proctedal segment3 (P3P) sections of G. perplexus gut was run against 27F/1492R,
591F/1392R, and 627F/1492R. All 3 sections showed positive results for the universal bacterial
primer set as expected and for Dysgonomonas specific primers. Dysgonomonas signal was
weakest in the P2A segment and strongest in the DV segment. Indication of Dysgonomonas in
all three tested gut sections supports our idea that the genus is widespread and abundant. A
positive result for JT5 specific primer was seen only in the P3P segment (Figure 3) indicating
that the P3P section is this bacterium's specialized niche. As expected, universal bacterial
presence was seen in all three gut segments. Dysgonomonas was also present in all three
sections, suggesting the genus is an important player in the digestion of lignocellulose by
termites. Likely it plays more of a role in the DV segment, as the signal was strongest there, and
a lesser role in the P2A segment, where the signal was weakest. JT5 presence was only identified
in the P3P segment, suggesting its role in lignocellulose breakdown occurs after other processing
has occurred in the fore- and mid-gut.
Conclusions
We have observed that representatives of the genus Dysgonomonas are abundant, diverse,
and widespread in the gut tracts of termites. This is based on diversity analysis of the G.
perplexus whole gut, and comparison testing of various termite guts for the presence of
Dysgonomonas and JT5. We also found, contrary to our belief, that JT5-like species are not
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diverse, but it is likely they are abundant based on our studies. It has also possible that JT5 may
be unique to the termite G. perplexus since we only obtained a positive result for JT5-like
species in this termite. We identified that JT5-like species are specifically located in the hindgut
(posterior) portion of the G. perplexus gut. JT5 is could play a significant role in lignocellulose
degradation and warrants additional research into its metabolic capabilities. We are interested in
further studies of glycohydrolyase gene expression. We would like to understand if the genes are
being expressed in the termite, and under what conditions they are being expressed. There is a
lot of potential for future studies in cloning these genes to develop better processing of plant
materials in the creation of biofuels.
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21
Figure 1. Isolate JT5 is a member of phylum Bacteroidetes and groups with genus
Dysgonomonas within the family Porphyromonadacaea. The DNA distance tree was
constructed in ARB using the Kimura algorithm and was based on bacterial SSU rRNA
sequences withl, 204 aligned nucleotides for each sequence. Bootstrap values (based on 100
replicates) are given when >75%. Scale bar represents 10% difference in nucleotide sequences.
Bacteria from the phyla Proteobacteria and Spirochaetes are used as outgroups.
22
Figure 2. RFLP analysis of Dysgonomonas library.
PCR products and restriction digested PCR products are shown in the upper panel. The lower
panel shows the analysis of the library, indicating 17 different RFLP types. The most abundant
of which (DI) comprised over 25% of the inventory.
23
Figure 3. Isolate JT5 inhabits the anterior portion of the G. perplexus gut.
PCR amplifications of different gut sections show a JT5 signal only in the posterior section using
JT5-specific primers (J) while a strong signal was observed for the posterior section, the P2
section and, to a lesser extent, the anterior gut section using Dysgonomonas-specific primers. As
expected, all sections produced a strong signal when universal bacterial primers (U) were used.
24
Figure 1.
Dysgonomonas gadei
uncultured Bacteroidetes bacterium
Dysgonomonas hoistadii
Dysgonomonas mossii
Dysgonomonas wimpennyt
Isolate JT5
Dysgonomonas capnocytophagoides
uncultured Bacteroidetes bacterium
uncultured bacterium
1.
r
Bacteroides cellulosilyticus
Parabacteroides goldsteinii
Famil y:Porphyromonadaceae
uncultured Bacteroidetes bacterium
Porphyromonas gingival's
Cytophaga xylanolytica
Marinifilum fragile
Rikenella microfusus
Sphingobacterium multivorum
Jo"
IOU
Cellulophaga lytica
Flavobacteriumjohnsoniae
Flexibacter polymorphus
Kiebsiella pneumoniae subsp. pneumoniae
Pseudomonas fluorescens
Ralstonsa pickethi
Variovorax paradoxus
Pelagibactenum halotolerans
Rhodopseudomonas rhenobacensrs
Desulfobulbus propionicus
Campylobactercoh
Helicobacter pylon
1 00
Borreha burgdorien
Spirochaeta aura nha subsp aurantia
Treponema azotonutricium
Treponema succinifaciens
Leptospira interrogans serovar Icterohaemorrhagiae
0.1
25
Figure 2.
****** -0** * * * **.*************.* *****
**0
-***
1***** -** 4***** * w 01* ..
YIP
. W. MWM M.
Qu a ntity of e ac h
1 %..4 140
w
.
•
M
11:koniftpos.,I,
: 8 11401 411 X-
RR
25
20
a) 15
a
10
5
0
1
I
2
V=41
I r
3
4
5
6
7
8
as
4■■■4
1 41.4
4iwp-
9 10 11 12 13 14 15 16 17
Number of unique clones
26
Figure 3.
P2 Sample
Posterior Sample
Anterior Sample
1 mm
Anterior
Sample
P2
Sample
Posterior
Sample
27
Table 1.
Table 1
Termite Species
Ecosystem
Food
Warm temperate desert
Dry grass, soil
Soil Contact
Universal Bacterial
Dysgonomonas
High
+++
+++
+++
+++
+++
++
++
Higher Termites
Gnathamitermes perplexus
Rhynchotermes sp. Cost004
Wet rainforest transition Leaf litter
Wet rainforest transition Roots, Soil
Amitermes sp. Cost010
Nasutitermes sp. Cost003
Wet rainforest transition Wood
Microcerotermes sp. Cost008 Lowland moist forest
Palm
High
High
Low
Low
Lower Termites
lnsisitermes minor
Low-altitude residential
Wood
High
Reticulitermes hesperus
Zoo termopsis nevadensis
High-altitude pine forrest Wood
High
High-altitude pine forrest Wood
Low
+++
+++
+++
Relative signal intensity given by ( + ) symbols. Where the signal is indicated as ( - ), no PCR amplification was observed.
JTS-Specific