Browsing by Author "Makonde, Huxley M."
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Item 16S-rRNA-based analysis of bacterial diversity in the gut of fungus-cultivating termites (Microtermes and Odontotermes species)(Springer Netherlands, 2013-08-14) Makonde, Huxley M.; Boga, Hamadi I.; Mwirichia, Romano K.; Mackenzie, Lucy; Göker, Markus; Klenk, Hans-PeterThe interaction between termites and their gut symbionts has continued to attract the curiosity of researchers over time. The aim of this study was to characterize and compare the bacterial diversity and community structure in the guts of three termites (Odontotermes somaliensis, Odontotermes sp. and Microtermes sp.) using 16S rRNA gene sequencing of clone libraries. Clone libraries were screened by restriction fragment length polymorphism and representative clones from O. somaliensis (100 out of 330 clones), Odontotermes sp. (100 out of 359 clones) and Microtermes sp. (96 out 336 clones) were sequenced. Phylogenetic analysis indicated seven bacterial phyla were represented: Bacteroidetes, Spirochaetes, Firmicutes, Proteobacteria, Synergistetes, Planctomycetes and Actinobacteria. Sequences representing the phylum Bacteroidetes (>60 %) were the most abundant group in Odontotermes while those of Spirochaetes (29 %) and Firmicutes (23 %) were the abundant groups in Microtermes. The gut bacterial community structure within the two Odontotermes species investigated here was almost identical at the phylum level, but the Microtermes sp. had a unique bacterial community structure. Bacterial diversity was higher in Odontotermes than in Microtermes. The affiliation and clustering of the sequences, often with those from other termites’ guts, indicate a majority of the gut bacteria are autochthonous having mutualistic relationships with their hosts. The findings underscore the presence of termite-specific bacterial lineages, the majority of which are still uncultured.Item 454 Pyrosequencing‑based assessment of bacterial diversity and community structure in termite guts, mounds and surrounding soils(2015-09) Makonde, Huxley M.; Mwirichia, Romano; Osiemo, Zipporah; Boga, Hamadi I.; Klenk, Hans‑PeterTermites constitute part of diverse and economically important termite fauna in Africa, but information on gut microbiota and their associated soil microbiome is still inadequate. In this study, we assessed and compared the bacterial diversity and community structure between termites’ gut, their mounds and surrounding soil using the 454 pyrosequencing-based analysis of 16S rRNA gene sequences. A wood-feeder termite (Microcerotermes sp.), three fungus-cultivating termites (Macrotermes michaelseni, Odontotermes sp. and Microtermes sp.), their associated mounds and corresponding savannah soil samples were analyzed. The pH of the gut homogenates and soil physico-chemical properties were determined. The results indicated significant difference in bacterial community composition and structure between the gut and corresponding soil samples. Soil samples (Chao1 index ranged from 1359 to 2619) had higher species richness than gut samples (Chao1 index ranged from 461 to 1527). The bacterial composition and community structure in the gut of Macrotermes michaelseni and Odontotermes sp. were almost identical but different from that of Microtermes and Microcerotermes species, which had unique community structures. The most predominant bacterial phyla in the gut were Bacteroidetes (40–58 %), Spirochaetes (10–70 %), Firmicutes (17–27 %) and Fibrobacteres (13 %) while in the soil samples were Acidobacteria (28–45 %), Actinobacteria (20–40 %) and Proteobacteria (18–24 %). Some termite gut-specific bacterial lineages belonging to the genera Dysgonomonas, Parabacteroides, Paludibacter, Tannerella, Alistipes, BCf9-17 termite group and Termite Treponema cluster were observed. The results not only demonstrated a high level of bacterial diversity in the gut and surrounding soil environments, but also presence of distinct bacterial communities that are yet to be cultivated. Therefore, combined efforts using both culture and culture-independent methods are suggested to comprehensively characterize the bacterial species and their specific roles in these environments.Item Bacteria and Archaea diversity within the hot springs of Lake Magadi and Little Magadi in Kenya(BioMed Central, 2016-06) Mwirichia, Romano K.; Kasili, Remmy W.; Karanja, Edward N.; Makonde, Huxley M.; Boga, Hamadi I.; Kambura, Anne K.Background: Lake Magadi and little Magadi are hypersaline, alkaline lakes situated in the southern part of Kenyan Rift Valley. Solutes are supplied mainly by a series of alkaline hot springs with temperatures as high as 86 °C. Previous culture-dependent and culture-independent studies have revealed diverse groups of microorganisms thriving under these conditions. Previous culture independent studies were based on the analysis of 16S rDNA but were done on less saline lakes. For the first time, this study combined illumina sequencing and analysis of amplicons of both total community rDNA and 16S rRNA cDNA to determine the diversity and community structure of bacteria and archaea within 3 hot springs of L. Magadi and little Magadi. Methods: Water, wet sediments and microbial mats were collected from springs in the main lake at a temperature of 45.1 °C and from Little Magadi “Nasikie eng’ida” (temperature of 81 °C and 83.6 °C). Total community DNA and RNA were extracted from samples using phenol-chloroform and Trizol RNA extraction protocols respectively. The 16S rRNA gene variable region (V4 – V7) of the extracted DNA and RNA were amplified and library construction performed following Illumina sequencing protocol. Sequences were analyzed done using QIIME while calculation of Bray-Curtis dissimilarities between datasets, hierarchical clustering, Non Metric Dimensional Scaling (NMDS) redundancy analysis (RDA) and diversity indices were carried out using the R programming language and the Vegan package. Results: Three thousand four hundred twenty-six and one thousand nine hundred thirteen OTUs were recovered from 16S rDNA and 16S rRNA cDNA respectively. Uncultured diversity accounted for 89.35 % 16S rDNA and 87.61 % 16S rRNA cDNA reads. The most abundant phyla in both the 16S rDNA and 16S rRNA cDNA datasets included: Proteobacteria (8.33–50 %), Firmicutes 3.52–28.92 %, Bacteroidetes (3.45–26.44 %), Actinobacteria (0.98–28.57 %) and Euryarchaeota (3.55–34.48 %) in all samples. NMDS analyses of taxonomic composition clustered the taxa into three groups according to sample types (i.e. wet sediments, mats and water samples) with evident overlap of clusters between wet sediments and microbial mats from the three sample types in both DNA and cDNA datasets. The hot spring (45.1 °C) contained less diverse populations compared to those in Little Magadi (81–83 °C). Conclusion: There were significant differences in microbial community structure at 95 % level of confidence for both total diversity (P value, 0.009) based on 16S rDNA analysis and active microbial diversity (P value, 0.01) based on 16S rRNA cDNA analysis, within the three hot springs. Differences in microbial composition and structure were observed as a function of sample type and temperature, with wet sediments harboring the highest diversity.Item Diversity of fungi in sediments and water sampled from the hot springs of Lake Magadi and Little Magadi in Kenya(2016-02) Mwirichia, Romano K.; Kambura, Ann K.; Kasili, Remmy W.; Boga, Hamadi Iddi; Karanja, Edward N.; Makonde, Huxley M.Lake Magadi and Little Magadi are saline, alkaline lakes lying in the southern part of Kenyan Rift Valley. Their solutes are supplied by a series of alkaline hot springs with temperatures as high as 86°C. Previous culture-dependent and independent studies have revealed diverse prokaryotic groups adapted to these conditions. However, very few studies have examined the diversity of fungi in these soda lakes. In this study, amplicons of Internal Transcribed Spacer (ITS) region on Total Community DNA using Illumina sequencing were used to explore the fungal community composition within the hot springs. Operational taxonomic units (OTUs) were analyzed using QIIME 1.8.0, taxonomy assigned via BLASTn against SILVA 119 Database and hierarchical clustering was done using R programming software. A total of 334, 394 sequence reads were obtained from which, 151 OTUs were realized at 3% genetic distance. Taxonomic analysis revealed that 80.33% of the OTUs belonged to the Phylum Ascomycota, 11.48% Basidiomycota while the remaining consisted of Chytridiomycota, Glomeromycota and early diverging fungal lineages. The most abundant Ascomycota groups consisted of Aspergillus (18.75%), Stagonospora and Ramularia (6.25% each) in wet sediment at 83.6°C, while Penicillium and Trichocomaceae (14.29% each) were dominant in wet sediment at 45.1°C. The results revealed representatives of thermophilic and alkaliphilic fungi within the hot springs of Lake Magadi and Little Magadi. This suggests their ability to adapt to high alkalinity, temperature and salinity.Item Isolation and characterization of some gut microbial symbionts from fungus-cultivating termites (Macrotermes and Odontotermes spp.)(Academic Journals, 2016-07) Muwawa, Edith M.; Budambula, Nancy; Osiemo, Zipporah L.; Boga, Hamadi I.; Makonde, Huxley M.Microbiota of termites is crucial for nitrogen cycle activities and degradation of recalcitrant components of plant biomass that influence soil structure and carbon mineralization in tropical and subtropical regions. The aim of this study was to isolate and characterize gut bacteria that may be potentially associated with nitrogen metabolism from two fungus-feeding termites (Macrotermes and Odontotermes spp.). Twenty termites from the intact colony of each termite species were aseptically degutted. Gut homogenate was inoculated and cultured on selective media for the isolation of pure bacteria. Pure bacterial isolates were characterized using their morphological, biochemical and molecular characters. DNA was extracted from the isolates, polymerase chain reaction (PCR) amplified and sequenced. The 16S rDNA gene sequences were blastn analyzed against the Genbank database and phylogenetic analysis was performed using MEGA 5 software. All forty-six isolates reacted positive for the ammonifying, nitrate, catalase and nitrogenase tests. Phylogenetic analysis grouped the isolates into three phyla: Firmicutes, Proteobacteria and Actinobacteria. Isolates were affiliated with the genera Pseudomonas, Citrobacter, Enterobacter, Proteus, Klebssiella, Bacillus, Staphylococcus, Rhodococcus and Micrococcus. The results confirm that termites harbor diverse gut bacterial groups that have different physiological/enzymatic activities and might have functional implications in the termitemicrobe symbiotic association.Item Isolation and characterization of some gut microbial symbionts from fungus-cultivating termites (Macrotermes and Odontotermes spp.)(2016-07) Budambula, Nancy; Muwawa, Edith M.; Osiemo, Zipporah L.; Boga, Hamadi I.; Makonde, Huxley M.Microbiota of termites is crucial for nitrogen cycle activities and degradation of recalcitrant components of plant biomass that influence soil structure and carbon mineralization in tropical and subtropical regions. The aim of this study was to isolate and characterize gut bacteria that may be potentially associated with nitrogen metabolism from two fungus-feeding termites (Macrotermes and Odontotermes spp.). Twenty termites from the intact colony of each termite species were aseptically degutted. Gut homogenate was inoculated and cultured on selective media for the isolation of pure bacteria. Pure bacterial isolates were characterized using their morphological, biochemical and molecular characters. DNA was extracted from the isolates, polymerase chain reaction (PCR) amplified and sequenced. The 16S rDNA gene sequences were blastn analyzed against the Genbank database and phylogenetic analysis was performed using MEGA 5 software. All forty-six isolates reacted positive for the ammonifying, nitrate, catalase and nitrogenase tests. Phylogenetic analysis grouped the isolates into three phyla: Firmicutes, Proteobacteria and Actinobacteria. Isolates were affiliated with the genera Pseudomonas, Citrobacter, Enterobacter, Proteus, Klebssiella, Bacillus, Staphylococcus, Rhodococcus and Micrococcus. The results confirm that termites harbor diverse gut bacterial groups that have different physiological/enzymatic activities and might have functional implications in the termitemicrobe symbiotic association.