Savitr Trakulnaleamsai

Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host

ABSTRACT We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.

Intracolony variation of bacterial gut microbiota among castes and ages in the fungus‐growing termite Macrotermes gilvus

The fungus‐growing termites Macrotermes cultivate the obligate ectosymbiontic fungi, Termitomyces. While their relationship has been extesively studied, little is known about the gut bacterial symbionts, which also presumably play a crucial role for the nutrition of the termite host. In this study, we investigated the bacterial gut microbiota in two colonies of Macrotermes gilvus, and compared the diversity and community structure of bacteria among nine termite morphotypes, differing in caste and/or age, using terminal restriction fragment length polymorphism (T‐RFLP) and clonal analysis of 16S rRNA. The obtained molecular community profiles clustered by termite morphotype rather than by colony, and the clustering pattern was clearly more related to a difference in age than to caste. Thus, we suggest that the bacterial gut microbiota change in relation to the food of the termite, which comprises fallen leaves and the fungus nodules of Termitomyces in young workers, and leaves degraded by the fungi, in old workers. Despite these intracolony variations in bacterial gut microbiota, their T‐RFLP profiles formed a distinct cluster against those of the fungus garden, adjacent soil and guts of sympatric wood‐feeding termites, implying a consistency and uniqueness of gut microbiota in M. gilvus. Since many bacterial phylotypes from M. gilvus formed monophyletic clusters with those from distantly related termite species, we suggest that gut bacteria have co‐evolved with the termite host and form a microbiota specific to a termite taxonomic and/or feeding group, and furthermore, to caste and age within a termite species.

Comparison of bacterial communities in the alkaline gut segment among various species of higher termites

The first proctodeal (P1) segment in the hindgut of certain higher termites shows high alkalinity. We examined the bacterial diversity of the alkaline P1 gut segments of four species of higher termites by T-RFLP and phylogenetic analyses based on PCR-amplified 16S rRNA genes. The bacterial community of the P1 segment was apparently different from that of the whole gut in each termite. Sequence analysis revealed that Firmicutes (Clostridia and Bacilli) were dominant in the P1 segments of all four termites; however, the phylogenetic compositions varied among the termites. Although some of the P1 segment-derived sequences were related to the sequences previously reported from the alkaline digestive tracts of other insects, most of them formed phylogenetic clusters unique to termites. Such “termite P1 clusters” were distantly related to known bacterial species as well as to sequences reported from alkaline environments in nature. We successfully obtained enrichment cultures of Clostridia- and Bacilli-related bacteria, including putative novel species under anaerobic alkaline conditions from the termite guts. Our results suggest that the alkaline gut region of termites harbors unique bacterial lineages and are expected to be a rich reservoir of novel alkaliphiles yet to be cultivated.

Symbiotic Fungi Produce Laccases Potentially Involved in Phenol Degradation in Fungus Combs of Fungus-Growing Termites in Thailand

ABSTRACT Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.

Sphaerisporangium rufum sp. nov., an endophytic actinomycete from roots of Oryza sativa L.

An endophytic actinomycete, strain R10-82(T), isolated from surface-sterilized roots of rice (Oryza sativa L.) was studied using a polyphasic approach. Strain R10-82(T) produced branching substrate mycelia and developed spherical spore vesicles on aerial hyphae containing non-motile spores. The major cellular fatty acids were iso-C16 : 0, iso-C14 : 0 and 10-methyl C17 : 0. The predominant menaquinones were MK-9, MK-9(H2), MK-9(H4) and MK-9(H6). Rhamnose, ribose, madurose, mannose and glucose were detected in whole-cell hydrolysates. The diagnostic phospholipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol mannosides, hydroxylphosphatidylethanolamine and ninhydrin-positive phosphoglycolipids. These morphological and chemotaxonomic data were similar to those of the genus Sphaerisporangium. Analysis of the 16S rRNA gene sequence revealed that strain R10-82(T) was related most closely to Sphaerisporangium cinnabarinum JCM 3291(T) (98.3 % similarity). The DNA G+C content of strain R10-82(T) was 74 mol%. DNA-DNA relatedness data in combination with differences in the biochemical and physiological properties suggested that strain R10-82(T) should be classified as representing a novel species of the genus Sphaerisporangium, for which the name Sphaerisporangium rufum is proposed. The type strain is R10-82(T) ( = BCC 51287(T) = NBRC 109079(T)). An emended description of the genus Sphaerisporangium is also provided.

Streptomyces oryzae sp. nov., an endophytic actinomycete isolated from stems of rice plant

An actinomycete strain S16–07T, isolated from surface-sterilized stems of rice plant (Oryza sativa L.), was characterized using a polyphasic approach. Phylogenetic analysis of 16S rRNA gene sequences indicated affiliation of the strain belonged to the genus Streptomyces. The highest levels of sequence similarity were found with Streptomyces smyrnaeus SM3501T (97.7% similarity), S. abikoensis NBRC 13860T (97.6% similarity) and S. thermocarboxydovorans NBRC 16324T (97.5% similarity). The cell wall of strain S16–07T contained LL-diaminopimelic acid. The predominant menaquinones were MK-9(H6) and MK-9(H8). Phospholipids detected were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, hydroxy-phosphatidylethanolamine, hydroxy-phosphatidylmonomethylethanolamine and phosphatidylinositol mannosides. The major cellular fatty acids were ai-C15:0, i-C16:0 and ai-C17:0. The G+C content of strain S16–07T was 70.4 mol%. On the basis of the phylogeny of the isolate and its differences from the most closely related species, the isolate S16–07T represents a novel species for which the name S. oryzae sp. nov. is proposed. The type strain is S16–07T (=BCC 60400T=NBRC 109761T).