Orthophosphate turnover in East African lakes

Summary Turnover rates of ³²P–PO₄ and concentrations of orthophosphate as soluble reactive phosphorus (SRP) were measured in five East African waters. Rapid incorporation of ³²P–PO₄ by the seston and orthophosphate concentrations below the limit of detectibility were found in Lakes Elmenteita, Naivasha, and Naivasha Crater Lake. Turnover was slow and orthophosphate concentration high in both Lake Nakuru and the Crescent Island Crater basin of Lake Naivasha. Further experiments in Lake Nakuru indicated that colloidal binding of orthophosphate was limited and that particles retained by an 8.0 filter incorporated 66% as much tracer as particles retained by a 0.1 filter. These experiments strengthen our conclusion that a large quantity of orthophosphate is available for algal use in Lake Nakuru.     

Bacterial Diversity in the Haloalkaline Lake Elmenteita, Kenya

Lake Elmenteita is one of the alkaline saline lakes within the Kenyan Rift valley. The lake is situated on the floor of the Kenyan Rift Valley at 1,776?m above sea level and has no direct outlet. The microbial diversity of the lake was investigated using a culture-independent approach. 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 the samples and the 16S rRNA genes amplified using universal primers for Bacteria. Thirteen clone libraries were constructed using the PCR amplified 16S rRNA genes. A total of 1,663 clones were picked. Representative clones were selected using ARDRA technique for sequencing. 655 partial and non-chimeric clone sequences indicated the presence of 37 orders in the Domain Bacteria. Cyanobacteria were the most abundant clones in terms of numbers whereas members of the phylum Firmicutes group were the second in terms of numbers but the most diverse in terms of genera represented. All clones affiliated to the class Betaproteobacteria originated from DNA obtained from the water samples. Analysis using BLAST showed that 93.1% of the sequenced clones had similarity values below 98% to both cultured and as yet uncultured bacteria, resulting in 596 phylotypes. Therefore, it can be concluded that Lake Elmenteita harbours phylogenetically diverse groups of bacteria involved in complex metabolic interactions within the Lake??s ecosystem.     

Molecular Cloning and Expression of Cellulase Genes from Ruminococcus albus 8 in Escherichia coli Bacteriophage lambda

A genomic library of Ruminococcus albus 8 DNA was constructed by using the Escherichia coli bacteriophage lambdaDASH. Recombinants were screened for cellulolytic activity by plating in soft agar (0.7%) overlays containing either 1% (wt/vol) carboxymethyl cellulose (CMC), 4-methylumbelliferyl-beta-d-cellobioside (MUC, 1 mg/ml), or 1% (wt/vol) Ostazin brilliant red-hydroxyethyl cellulose (OBR-HEC). One hundred and three recombinant phage exhibiting activity against OBR-HEC were found, and these fell into different classes based on the size of the zone of hydrolysis. Twenty-one recombinant phage exhibiting activity against CMC and 19 recombinant phage exhibiting activity against MUC were isolated. Four OBR-HEC, five CMC, and seven MUC clones were further analyzed by restriction endonuclease mapping and cellulase substrate specificity to identify unique clones and to determine their cellulase type. Three different clone types representing endoglucanase activity were identified. Three clones that appeared to encode exoglucanase type activity and four clones that had a mixed specificity, including beta-glucosidase activity, were also identified.     

Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment

A novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria. Prokaryotic DNA was extracted and cloned into a copy control fosmid vector (pCC1FOS) generating a library of >7000 clones with inserts of 24-39 kb. Screening for clones expressing lipolytic activity based on the hydrolysis of tributyrin and p-nitrophenyl esters, identified 1% of the fosmids as positive. An insert of 29 kb was fragmented and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4-kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomonas putida esterase (BAD07370) was identified. Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was over expressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C14. Hydrolysis of the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) confirmed that h1Lip1 was a lipase. The apparent optimal temperature for h1Lip1, by hydrolysis of p-nitrophenyl butyrate, was 35 degrees C. Thermal stability analysis showed that h1Lip1 was unstable at 25 degrees C and inactivated at 40 degrees C with t1/2 <5 min.     

A phylogenetic analysis of Wadi el Natrun soda lake cellulase enrichment cultures and identification of cellulase genes from these cultures

Samples of sediments and surrounding soda soils (SS) from the extremely saline and alkaline lakes of the Wadi el Natrun in the Libyan Desert, Egypt, were obtained in October 2000. Anaerobic enrichment cultures were grown from these samples, DNA isolated, and the bacterial diversity assessed by 16S rRNA gene clone analysis. Clones derived from lake sediments (LS) most closely matched Clostridium spp., Natronoincola histidinovorans, Halocella cellulolytica, Bacillus spp., and the Cytophaga–Flexibacter–Bacteroides group. Similar clones were identified in the SS, but Bacillus spp. predominated. Many of the clones were most closely related to organisms already identified in alkaline or saline environments. Two genomic DNA libraries were made from the pooled LS enrichments and a single SS-enrichment sample. A novel cellulase activity was identified and characterized in each. The lake cellulase ORF encoded a protein of 1,118 amino acids; BLASTP analysis showed it was most closely related to an endoglucanase from Xanthomonas campestris. The soil-cellulase ORF encoded a protein of 634 amino acids that was most closely related to an endoglucanase from Fibrobacter succinogenes.     

Trends in waterbird numbers in the southern Rift Valley of Kenya

Bennun, L. & Nasirwa, O. 2000. Trends in waterbird numbers in the southern Rift Valley of Kenya. Ostrich 71 (1 & 2): 220–226.

Each January since 1991, volunteer teams have counted waterbirds at major wetlands in the southern Kenyan Rift Valley. There has been consistent coverage at Lakes Naivasha, Elmenteita, Nakuru and (since 1992) Bogoria. These lakes are shallow and, except for Bogoria, fluctuate greatly in extent; all but Naivasha are saline. Lake levels were moderately high in 1991–1993 but have been generally low since. Flamingo totals for the three saline lakes combined were more than one million from 1992–1994, but roughly halved each year since then. Greater Flamingos Phoenicopterus ruber made up between 0.7 and 4.1% of total flamingo numbers; other waterbirds made up between 2.7 and 10.2% of the overall total. Lakes Naivasha, Elmenteita and Nakuru together hold most of the non-flamingo waterbirds in the southern Rift; to compare trends for other species, we pooled totals for these sites. Significant, or near-significant, declines were evident for grebes, pelicans, cormorants, storks, gulls, rallids, kingfishers, terns and raptors. No group showed an overall increasing trend. At Dandora, a smaller site with stable water levels, these groups showed large annual fluctuations but no obvious declines. In most waterbird groups where numbers decreased, the probable cause was sustained low lake levels at Lake Nakuru; numbers at Lake Naivasha remained stable. Exceptions were rallids, kingfishers and raptors, where numbers steadily declined at Naivasha. There is a need to investigate local environmental causes of these changes for kingfishers and raptors, and to assess possible loss of breeding sites for rallids.     

Cloning of three endoglucanase genes from Thermomonospora curvata into Escherichia coli.

A BamHI genomic library from Thermomonospora curvata was constructed in E. coli using cosmid vector pHC79. Four clones able to hydrolyze CMC were isolated. Restriction digests and Southern gel analysis revealed the presence of three different endoglucanase genes. DNA fragments contained in all of the endoglucanase cosmids hybridized to T. curvata chromosomal DNA. The cellulase genes were expressed in E. coli, but at rather low levels.     

Representative freshwater bacterioplankton isolated from Crater Lake, Oregon

High-throughput culturing (HTC) methods that rely on dilution to extinction in very-low-nutrient media were used to obtain bacterial isolates from Crater Lake, Oregon. 16S rRNA sequence determination and phylogenetic reconstruction were used to determine the potential ecological significance of isolated bacteria, both in Crater Lake and globally. Fifty-five Crater Lake isolates yielded 16 different 16S rRNA gene sequences. Thirty of 55 (55%) Crater Lake isolates had 16S rRNA gene sequences with 97% or greater similarity to sequences recovered previously from Crater Lake 16S rRNA gene clone libraries. Furthermore, 36 of 55 (65%) Crater Lake isolates were found to be members of widely distributed freshwater groups. These results confirm that HTC is a significant improvement over traditional isolation techniques that tend to enrich for microorganisms that do not predominate in their environment and rarely correlate with 16S rRNA gene clone library sequences. Although all isolates were obtained under dark, heterotrophic growth conditions, 2 of the 16 different groups showed evidence of photosynthetic capability as assessed by the presence of puf operon sequences, suggesting that photoheterotrophy may be a significant process in this oligotrophic, freshwater habitat.     

Cyanobacteria and cyanobacterial toxins in three alkaline Rift Valley lakes of Kenya--Lakes Bogoria, Nakuru and Elmenteita

For decades frequent mass mortalities of Lesser Flamingos (Phoeniconaiasminor Geoffroy) have been observed at alkaline-saline KenyanRift Valley lakes. To estimate the potential influence of toxiccyanobacteria on these mass deaths, the phytoplankton communitieswere investigated in Lakes Bogoria, Nakuru and Elmenteita. Cyanobacterialtoxins were analyzed both in the phytoplankton from the threelakes and in isolated monocyanobacterial strains of Arthrospirafusiformis, Anabaenopsis abijatae, Spirulina subsalsa and Phormidiumterebriformis. Lake Bogoria was dominated by the cyanobacteriumA. fusiformis. In L. Nakuru and L. Elmenteita the phytoplanktonmainly consisted of A. fusiformis, A. abijatae and Anabaenopsisarnoldii, and in L. Nakuru an unknown Anabaena sp. was alsofound. Furthermore, this is the first time A. abijatae and theunknown Anabaena sp. have been found in Kenyan lakes. Phytoplanktonwet weight biomass was found to be high, reaching 777 mg L^–1in L. Bogoria, 104 mg L^–1 in L. Nakuru and 202 mg L^–1in L. Elmenteita. Using HPLC, the cyanobacterial hepatotoxinsmicrocystin-LR, -RR -YR, -LF and -LA and the neurotoxin anatoxin-awere detected in phytoplankton samples from L. Bogoria and L.Nakuru. Total microcystin concentrations amounted to 155 µgmicrocystin-LR equivalents g^–1 DW in L. Bogoria, and 4593µg microcystin-LR equivalents g^–1 DW in L. Nakuru,with anatoxin-a concentrations at 9 µg g^–1 DW inL. Bogoria and 223 µg g^–1 DW in L. Nakuru. In L.Elmenteita phytoplankton, no cyanobacterial toxins were found.A. fusiformis was identified as one source of the toxins. Theisolated strain of A. fusiformis from L. Bogoria was found toproduce both microcystin-YR (15.0 µg g^–1 DW) andanatoxin-a (10.4 µg g^–1 DW), whilst the A. fusiformisstrain from L. Nakuru was found to produce anatoxin-a (0.14µg g^–1 DW). Since A. fusiformis mass developmentsare characteristic of alkaline-saline lakes, health risks towildlife, especially the Arthrospira-consuming Lesser Flamingo,may be expected.     

Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library

A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.     

Representative Freshwater Bacterioplankton Isolated from Crater Lake, Oregon

High-throughput culturing (HTC) methods that rely on dilution to extinction in very-low-nutrient media were used to obtain bacterial isolates from Crater Lake, Oregon. 16S rRNA sequence determination and phylogenetic reconstruction were used to determine the potential ecological significance of isolated bacteria, both in Crater Lake and globally. Fifty-five Crater Lake isolates yielded 16 different 16S rRNA gene sequences. Thirty of 55 (55%) Crater Lake isolates had 16S rRNA gene sequences with 97% or greater similarity to sequences recovered previously from Crater Lake 16S rRNA gene clone libraries. Furthermore, 36 of 55 (65%) Crater Lake isolates were found to be members of widely distributed freshwater groups. These results confirm that HTC is a significant improvement over traditional isolation techniques that tend to enrich for microorganisms that do not predominate in their environment and rarely correlate with 16S rRNA gene clone library sequences. Although all isolates were obtained under dark, heterotrophic growth conditions, 2 of the 16 different groups showed evidence of photosynthetic capability as assessed by the presence of puf operon sequences, suggesting that photoheterotrophy may be a significant process in this oligotrophic, freshwater habitat.     

Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).     

Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector

Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.     

Isolation of novel lipolytic genes from uncultured bacteria of pond water

Metagenomic libraries give access to gene pool of bacteria present in environmental samples avoiding the culture bias. A metagenomic library of pond water microbial assemblage in plasmid vector containing about 532 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 11 unique lipolytic clones with an ability to hydrolyze tributyrin. DNA sequence of the lipolytic genes varied in G+C composition from 57% to 75%. Twelve lipolytic genes encoding proteins with 25-70% amino acid identity with proteins in the databases were identified. Ten of the encoded proteins belonged to seven known lipolytic protein families. One of the proteins was similar to recently identified esterase BioH. A lipolytic protein with high similarity to yet uncharacterized alpha/beta hydrolase protein family abh_upf0017 was identified from one of the clones. Conserved motif for lipolytic enzymes GXSXG, conserved aspartic and histidine residues were identified in this encoded protein.     

Construction of tomato genomic DNA libraries in a binary-BAC (BIBAC) vector

This is the first report of large insert genomic DNA libraries constructed in a binary-BAC (BIBAC) vector. Genomic DNA libraries containing approximately 4.6 haploid nuclear genomic equivalents were constructed for Lycopersicon esculentum (cv. Mogeor) and Lycopersicon pennellii (LA716) in the BIBAC2 vector. The L. esculentum library has an average insert size of 125 kb and is comprised of 42 272 individual colonies stored as frozen cultures in a 384-well format (108 plates). The L. pennellii library has an average insert size of 90 kb and is comprised of 53 760 individual clones (140 384-well plates). In each of the libraries, it is estimated that 90% of the colonies contain genomic DNA inserts. The composition of the L. esculentum and L. pennellii libraries was determined by analyzing a series of randomly selected clones. The L. esculentum library was surveyed for clones containing chloroplast DNA (1.4%), mitochondrial DNA (0.012%) and repetitive DNA motifs. BIBAC clones that may contain a gene of interest can be identified from these libraries by colony hybridization with homologous or heterologous probes or by PCR pooling techniques. Once identified, BIBAC genomic DNA library clones are immediately suitable for Agrobacterium tumefaciens-mediated plant transformation.     

Cloning and identification of cellulase genes from uncultured microorganisms in pulp sediments from paper mill effluent]

The metagenomic DNA of pulp sediments from paper mill effluent was extracted and purified. The 16S rDNA was amplified using the purified metagenomic DNA as template and a 16S rDNA library was prepared. Sequence analysis of 16S rDNA clones showed that diverse of uncultured bacteria inhabit in this environment, which can be classified into 4 clusters as Spirochaetes, Proteobacteria, Bacteroidetes and Firmicutes. A metagenomic library containing 10000 clones was constructed into cosmid vector, and the capacity of inserted DNA of which was 3.53 x 10(8) bp. Functional screening of the library resulted in isolation of two independent clones expressing endoglucanase activity, three independent clones expressing exoglucanase activity and two independent clones expressing beta-glucosidase activity. One clone expressing strongest enzyme activity from each activity category was chosen to be further analyzed. Three novel cellulase genes designated as umcel5L, umcel5M and umbgl3D were identified by subcloning, sequencing and expression. The umcel5L encodes an endoglucanase belonging to glycosyl hydrolase family 5, which is most related to an endoglucanase from Bradyrhizobium japonicum at 43% identity and 59% similarity. The umcel5M encodes a cellodextrinase belonging to glycosyl hydrolase family 5, which is most similar to a cellodextrinase from Fibrobacter succinogenes at 48% identity and 69% similarity. The umbgl3D encodes a putative beta-glucosidase belonging to glycosyl hydrolase family 3, which shares highest homology with a beta-glucosidase from Thermotoga maritima at 46% identity and 61% similarity. It is the first time to reveal the bacterial diversity of pulp sediments from paper mill effluent and clone novel cellulase genes from the bacteria by culture-independent method.     

Isolation and characterization of 70 novel microsatellite markers from ostrich (Struthio camelus) genome.

Microsatellite markers are widely used in linkage mapping, parentage testing, population genetic studies, and molecular evolution studies in many agricultural species, while only a limited number of ostrich (Struthio camelus) microsatellites have been isolated. Thus, we constructed a random small-insert genomic library and a microsatellite-enriched library containing CA repeats. Fourteen clones containing CA repeats were isolated from 3462 clones in the non-enriched library by radioactive screening and 248 positive clones were isolated from 300 sequenced clones from the enriched library by PCR screening. After the enrichment procedures, the proportion of clones containing CA repeats was raised to 78.8%, compared with 0.4% in the non-enriched libraries, indicating that the enrichment value approaches 200 fold, which decreased the time and cost of cloning. The number of complete simple CA repeats in these positive clones ranged from 5 to 29. The primers for 94 of these microsatellites were developed and used to detect polymorphisms, of which 61 loci exhibited length polymorphisms in 17 unrelated ostrich individuals. The new polymorphic microsatellite markers we have identified and characterized will contribute to the ostrich genetic map, parentage testing, and comparative genomics between avian species.     

An untapped bacterial cellulolytic community enriched from coastal marine sediment under anaerobic and thermophilic conditions

A bacterial community with strong cellulose [filter paper (FP) and microcrystalline cellulose] degradation ability was isolated from the coastal marine environment. They were isolated under thermophilic (60?°C) and anaerobic cultivation conditions. The library of 16S rRNA gene clones revealed a total of 16 operational taxonomic units after 50 clones were surveyed. Sixty percent of the clones were most related to the type strain of Clostridium thermocellum with 16S rRNA gene identity around 87-89%. All of them showed extremely low sequence similarities and were novel at least in species level. The gene clone libraries of glycosyl hydrolase family 48 showed low gene and amino acid sequence similarities around 70-72%. The results indicated that the cellulose degradation systems in the specific environment have not been well studied. The enrichment could disrupt FP within 3?days in a basal medium. The cellulase activity of the community was comparable to that of C.?thermocellum LQR1. The main fermentation products were ethanol, acetic acid and butyric acid. This work identified a novel microbial resource with a potential in lignocellulose conversion and biofuel production.     

Metagenomic profiling: microarray analysis of an environmental genomic library

Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing approximately 1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.