Romano Mwirichia

Isolation and characterisation of bacteria from the haloalkaline Lake Elmenteita, Kenya

Culture-independent studies show that soda lake environments harbour diverse groups of bacteria and archaea. In this study different enrichment and isolation media were used in an attempt to isolate novel groups of bacteria from Lake Elmenteita. Different media were prepared using filter-sterilised water from the lake. The isolates recovered were purified on tryptic soy agar supplemented with 1% sodium carbonate and 4% sodium chloride. Phylogenetic analysis of 181 partial 16S rRNA gene sequences with excellent quality showed that the majority of the isolates were affiliated to the class Gammaproteobacteria and to the genus Bacillus. Isolates from the genus Halomonas and Bacillus constituted 37 and 31% of the total sequenced isolates, respectively. Other groups recovered were related to Marinospirillum, Idiomarina, Vibrio, Enterococcus, Alkalimonas, Alkalibacterium, Amphibacillus, Marinilactibacillus and the actinobacteria Nocardiopsis and Streptomyces. Fifty-one different genera were represented with 31 and 15 cultures scoring with their nearest neighbour similarities below 98 and 97%, respectively. Some novel taxa were identified which had not been isolated previously from the soda environment. The results show that the use of different media with varying compositions can help retrieve novel bacterial diversity from the soda lake environment.

Metabolic traits of an uncultured archaeal lineage -MSBL1- from brine pools of the Red Sea

The candidate Division MSBL1 (Mediterranean Sea Brine Lakes 1) comprises a monophyletic group of uncultured archaea found in different hypersaline environments. Previous studies propose methanogenesis as the main metabolism. Here, we describe a metabolic reconstruction of MSBL1 based on 32 single-cell amplified genomes from Brine Pools of the Red Sea (Atlantis II, Discovery, Nereus, Erba and Kebrit). Phylogeny based on rRNA genes as well as conserved single copy genes delineates the group as a putative novel lineage of archaea. Our analysis shows that MSBL1 may ferment glucose via the Embden–Meyerhof–Parnas pathway. However, in the absence of organic carbon, carbon dioxide may be fixed via the ribulose bisphosphate carboxylase, Wood-Ljungdahl pathway or reductive TCA cycle. Therefore, based on the occurrence of genes for glycolysis, absence of the core genes found in genomes of all sequenced methanogens and the phylogenetic position, we hypothesize that the MSBL1 are not methanogens, but probably sugar-fermenting organisms capable of autotrophic growth. Such a mixotrophic lifestyle would confer survival advantage (or possibly provide a unique narrow niche) when glucose and other fermentable sugars are not available.

Archaeal Diversity in the Haloalkaline Lake Elmenteita in Kenya

A non-culture approach was used to study the archaeal diversity in Lake Elmenteita, Kenya. Five different sampling points were selected randomly within the lake. Wet sediments and water samples were collected from each sampling point. In addition, dry mud cake was collected from three points where the lake had dried. DNA was extracted from these samples and the 16S rRNA genes were amplified using primers described to be Domain-specific for Archaea. Eleven clone libraries were constructed using PCR-amplified 16S rRNA genes. A total of 1,399 clones were picked and analysed via ARDRA. 170 ARDRA patterns were unique and the respective clones were selected for sequencing. 149 clones gave analysable sequences. BLAST analysis showed that 49 belong to the Domain Archaea while the others were either chimera or affiliated to eukaryotic taxa. Comparative sequence analysis of archaeal clones affiliated them to a wide range of genera. The order Halobacteriales was represented by members of the genera Natronococcus, Halovivax, Halobiforma,Halorubrum, and Halalkalicoccus. The highest percentage (46%) of the clones, however, belonged to uncultured members of the Domain Archaea in the order Halobacteriales. The results show that the archaeal diversity in the lake could be higher than previously reported.

Potential of indigenous bradyrhizobia versus commercial inoculants to improve cowpea (Vigna unguiculata L. walp.) and green gram (Vigna radiata L. wilczek.) yields in Kenya

Limited information is available on reduced cowpea (Vigna unguiculata L. Walp.) and green gram (Vigna radiata L.Wilczek.) yields in Kenya. Declining soil fertility and absence or presence of ineffective indigenous rhizobia in soils are assumptions that have been formulated but still require to be demonstrated. In this study, soils were collected from legume growing areas of Western (Bungoma), Nyanza (Bondo), Eastern (Isiolo), Central (Meru) and Coast (Kilifi) provinces in Kenya to assess indigenous rhizobia in soils nodulating cowpea and green gram under greenhouse conditions. Our results showed that highest nodule fresh weights of 4.63 and 3.32 g plant−1 for cowpea and green gram were observed in one soil from Isiolo and another from Kilifi, respectively, suggesting the presence of significant infective indigenous strains in both soils. On the other hand, the lowest nodule fresh weights of 2.17 and 0.72 g plant−1 were observed in one soil from Bungoma for cowpea and green gram, respectively. Symbiotic nitrogen (N) fixation by cowpea and green gram was highest in Kilifi soil with values of 98% and 97%, respectively. A second greenhouse experiment was undertaken to evaluate the performance of commercial rhizobial inoculants with both legumes in Chonyi soil (also from Coast province) containing significant indigenous rhizobia [>13.5 × 103 Colony Forming Units (CFU) g−1]. Rhizobial inoculation did not significantly (P < 0.05) affect nodulation, biomass yield and shoot N content in cowpea and green gram compared with controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 16S-23S rDNA intergenic spacer (IGS) region analysis of nodules revealed six groups of which only IGS Group IV corresponded with those from commercial inoculants applied, indicating a lower competitiveness of inoculated strains. In cowpea, IGS III was dominant in nodules of plants inoculated with Biofix and Rizoliq commercial inoculants, and the uninoculated control treatment (63.2, 60 and 52.9%, respectively). Similarly, in green gram, IGS Group III was dominant in nodules of plants inoculated with Biofix 704 and Rizoliq commercial inoculants, and the uninoculated control treatment (75, 73.7 and 61.1%, respectively). Our results suggest that the systematic inoculation of both legumes with current available commercial inoculants to improve biomass yields is not necessary in these regions of Kenya. Also, according to our study, it would make sense to promote the utilization of indigenous strains performing well with both legumes.

Complete genome sequence of the gliding freshwater bacterium Fluviicola taffensis type strain (RW262T)

Fluviicola taffensis O’Sullivan et al. 2005 belongs to the monotypic genus Fluviicola within the family Cryomorphaceae. The species is of interest because of its isolated phylogenetic location in the genome-sequenced fraction of the tree of life. Strain RW262T forms a monophyletic lineage with uncultivated bacteria represented in freshwater 16S rRNA gene libraries. A similar phylogenetic differentiation occurs between freshwater and marine bacteria in the family Flavobacteriaceae, a sister family to Cryomorphaceae. Most remarkable is the inability of this freshwater bacterium to grow in the presence of Na+ ions. All other genera in the family Cryomorphaceae are from marine habitats and have an absolute requirement for Na+ ions or natural sea water. F. taffensis is the first member of the family Cryomorphaceae with a completely sequenced and publicly available genome. The 4,633,577 bp long genome with its 4,082 protein-coding and 49 RNA genes is a part of the GenomicEncyclopedia ofBacteria andArchaea project.

Diversity of Termitomyces associated with fungus-farming termites assessed by cultural and culture-independent methods

Background Fungus-cultivating termites make use of an obligate mutualism with fungi from the genus Termitomyces, which are acquired through either vertical transmission via reproductive alates or horizontally transmitted during the formation of new mounds. Termitomyces taxonomy, and thus estimating diversity and host specificity of these fungi, is challenging because fruiting bodies are rarely found. Molecular techniques can be applied but need not necessarily yield the same outcome than morphological identification. Methodology Culture-dependent and culture-independent methods were used to comprehensively assess host specificity and gut fungal diversity. Termites were identified using mitochondrial cytochrome oxidase II (COII) genes. Twenty-three Termitomyces cultures were isolated from fungal combs. Internal transcribed spacer (ITS) clone libraries were constructed from termite guts. Presence of Termitomyces was confirmed using specific and universal primers. Termitomyces species boundaries were estimated by cross-comparison of macromorphological and sequence features, and ITS clustering parameters accordingly optimized. The overall trends in coverage of Termitomyces diversity and host associations were estimated using Genbank data. Results and Conclusion Results indicate a monoculture of Termitomyces in the guts as well as the isolation sources (fungal combs). However, cases of more than one Termitomyces strains per mound were observed since mounds can contain different termite colonies. The newly found cultures, as well as the clustering analysis of GenBank data indicate that there are on average between one and two host genera per Termitomyces species. Saturation does not appear to have been reached, neither for the total number of known Termitomyces species nor for the number of Termitomyces species per host taxon, nor for the number of known hosts per Termitomyces species. Considering the rarity of Termitomyces fruiting bodies, it is suggested to base the future taxonomy of the group mainly on well-characterized and publicly accessible cultures.

Complete genome sequence of the thermophilic sulfur-reducer Desulfurobacterium thermolithotrophum type strain (BSA T ) from a deep-sea hydrothermal vent

Desulfurobacterium thermolithotrophum L’Haridon et al. 1998 is the type species of the genus Desulfurobacterium which belongs to the family Desulfurobacteriaceae. The species is of interest because it represents the first thermophilic bacterium that can act as a primary producer in the temperature range of 45–75 °C (optimum 70°C) and is incapable of growing under microaerophilic conditions. Strain BSAT preferentially synthesizes high-melting-point fatty acids (C18 and C20) which is hypothesized to be a strategy to ensure the functionality of the membrane at high growth temperatures. This is the second completed genome sequence of a member of the family Desulfurobacteriaceae and the first sequence from the genus Desulfurobacterium. The 1,541,968 bp long genome harbors 1,543 protein-coding and 51 RNA genes and is a part of the GenomicEncyclopedia ofBacteria andArchaea project.

Diversity of fungi in sediments and water sampled from the hot springs of Lake Magadi and Little Magadi in Kenya

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. Key words: Fungi, hot springs, temperature, DNA, diversity.

Complete genome sequence of Oceanithermus profundus type strain (506T)

Oceanithermus profundus Miroshnichenko et al. 2003 is the type species of the genus Oceanithermus, which belongs to the family Thermaceae. The genus currently comprises two species whose members are thermophilic and are able to reduce sulfur compounds and nitrite. The organism is adapted to the salinity of sea water, is able to utilize a broad range of carbohydrates, some proteinaceous substrates, organic acids and alcohols. This is the first completed genome sequence of a member of the genus Oceanithermus and the fourth sequence from the family Thermaceae. The 2,439,291 bp long genome with its 2,391 protein-coding and 54 RNA genes consists of one chromosome and a 135,351 bp long plasmid, and is a part of the GenomicEncyclopedia ofBacteria andArchaea project.

Isolation, characterization and identification of Enterobacteriaceae from well water in Juja town in Kenya.

Water for docmestic use is increasingly becoming a scarce resource forcing communities to resort to underground water sources. However, increasing wells and boreholes are facing threats of pullution from sewage. Water samples were collected from 12 different wells in Juja town, Kenya using pre-sterilized 100 ml plastic bottles. Sampling was done in May 2004 during the long rains and in September 2004 during the dry season. All the water samples had total coliform counts >1100 per 100 ml of water. The Enterobacteriaceae isolated from the wells were from the following genera: Escherichia, Enterobacter, Citrobacter, Proteus, Edwardsiella, Erwinia, Kluyvera, Klebsiella, Salmonella, Shigella, Serratia, Rahnella, Cedecea, Morganella, and Yersinia . Bacteria detected but not belonging to Enterobacteriaceae included the genera Vibrio, Acinetobacter, and Chromobacterium. Indiscriminate refuse disposals as well as the location of septic tanks, soak away pits and pit latrines in proximity to the wells could be the most probable cause of the overall high number of coliforms in the wells. Water treatment through chlorination or boiling was shown to reduce the microbial load to zero. Therefore it is recommended that water from the shallow wells be treated through chlorination or boiling before it can be used for domestic purposes. Keywords: shallow wells, coliforms, fecal pollution, water treatment