The adenylate energy charge values of twenty bacteria isolated from soil and cultured in a medium consisting of soil and distilled water were determined by the luciferin-luciferase bioluminescense method. The purpose of this study was to examine the growth and energy charge values of these organisms in soil extract medium, and to determine what effect the addition of glucose has on their energy charge values. Three of the organisms employed in this study showed energy charge values similar to those reported for bacteria grown in enriched media. The remainder of the isolates demonstrated low energy charge values, and scant growth in the soil medium.

A unique, previously undescribed Gram-negative bacterium was isolated from several soils in Texas and extensively characterized in this study. The cells measured 1-2 by 4-6 μm. The distinguishing characteristic of the bacterium is the extraordinary capsular material which surrounds the cells. The new isolates are aerobic, mesophilic, non motile and have the ability to utilize a variety of organic compounds as the sole source of carbon and energy. The organism grows optimally at 30° C and the optimal pH lies between 7.0-8.0. The isolates produce catalase but oxidase is not produced. They do not produce indole or hydrogen sulfide. The organism can hydrolyze gelatin and Tween 80 but not starch, esculin and casein. The major cellular fatty acid is anteiso 15:0. The guanine and cytosine content is 58-62 mole%. The organism's taxonomic position was further established by specific gene probes, 16S rRNA homology, DNA homology and "ribotyping." These data showed that it was most closely related to members of the genus Paenibacillus, although somewhat divergent from other species classified in this genus. After careful evaluation of the results obtained during this study, it is proposed that this unique bacterium be named Paenibacillus velasolus sp. nov.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / The soil environment is thought to contain a lot of the earth’s undiscovered biodiversity. The aim of this study was to understand the extent of microbial diversity in the unique ecosystem of the Western Cape’s fynbos biome. It is known that many processes give rise to this immense microbial diversity in soil. In addition the aim was to link microbial diversity with the soils physio-chemical properties as well as the plant community’s structure. Molecular methods especially automated ribosomal intergenic spacer analysis (ARISA) was used in the study. The most important property of environmental DNA intended for molecular ecology studies and other downstream applications is purity from humic acids and phenolic compounds. These compounds act as PCR inhibitors and need to be removed during the DNA extraction protocol. The fist goal in the study was to develop an effective DNA extraction protocol by using cationic locculation of humic acids. The combination of cationic flocculation with CuCl2 and the addition of PVPP and KCl resulted in a high yield of DNA, suitable for PCR amplification with bacterial and fungal specific primers. Determining the reproducibility and accuracy of ARISA and ARISA-PCR was important because these factors have an important influence on the results and effectiveness of these techniques. Primer sets for automated ribosomal intergenic spacer analysis, ITS4/ITS5, were assessed for the characterization of the fungal communities in the fynbos soil. The primer set delivered reproducible ARISA profiles for the fungal community composition with little variation observed between ARISAPCR’s. ARISA proved useful for the assessment and comparison of fungal diversity in ecological samples. The soil community composition of both fungal and bacterial groups in the Sand fynbos was characterized. Soil from 4 different Sand fynbos sites was compared to investigate diversity of eubacterial and fungal groups at the local as well as a the landscape scale. A molecular approach was used for the isolation of total soil genetic DNA. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified when performing bacterial ARISA from total soil community DNA (BARISA). Correspondingly, the internal transcribed spacers, ITS1, ITS2 and the 5.8S rRNA gene from the fungal rRNA operon were amplified when undertaking fungal ARISA (F-ARISA). The community structure from different samples and sites were statistically analysed. ARISA data was used to evaluate different species accumulation and estimation models for fungal and bacterial communities and to predict the total community richness. Diversity, evenness and dominance were the microbial communities were used to describe the extent of microbial iversity of the fynbos soils. The spatial ordination of the bacterial and fungal species richness and diversity was considered by determining the species area relationship and beta diversity of both communities. The correlation between the soil physio-chemical properties was determined. The plant community structure data was correlated with the fungal and the bacterial community structure. The results indicated that bacterial species numbers and diversity were continually higher at the local scale. Fungi however showed higher species turnover at the landscape scale. Bacterial community structure showed stronger links to the plant community structure whereas the fungi community structure conformed to spatial separation patterns. To further investigate the diversity of soil microbes the potential of genus specific primes was investigated. The genus Penicillium is widespread in the soil environment and the extent of its diversity and distribution is however not. For this reason Penicillium was chosen as a model organism. To expand the insight into the diversity of Penicillium species in the fynbos soil ecosystem, a rapid group specific molecular approach would be useful. Penicillium specific primers targeting the 18S rRNA ITS gene region were evaluated. Fungal specific primers ITS4 and ITS5, targeting the internal transcribed region (ITS) were used to target Penicillium specific in the soil sample. Nested PCR, using primer Pen-10 and ITS5, was then utilized to target Penicillium species specifically. The discrimination of Penicillium species was possible due to length heterogeneity of this gene region. Eight different peaks was detected in the soil sample with ARISA and eight different species could be isolated on growth media. The technique proved useful for the detection and quantification of Penicillium species in the soil.

Escherichia coli (E. coli) are common and typically innocuous copiotrophic bacteria found in the mammalian gut microbiome. However, over the past 30 years, pathogenic E. coli have been responsible for several outbreaks of foodborne illness linked to contaminated produce. The introduction of Escherichia coli to an agricultural soil, via contaminated water, compost, or raw manure, exposes the bacterium to a medley of ecological forces not found in a mammalian gut environment. This study assesses a variety of abiotic and biotic soil factors that influence the ability of an "invasive" copiotrophic coliform bacterium to survive in compost-amended agricultural soil. The study included both field and laboratory components. In the lab experiment, a cocktail of rifampicin-resistant generic E.coli strains was added to sterile and non-sterile extracts of eight different composts and one soil sample from the field sites. E. coli abundance was monitored over a one-week period and composts were analyzed for their nutrient profile. In the field experiment, the same E. coli cocktail was sprayed on plots with the following treatments: 1) dairy windrow compost, 2) dairy vermicompost, 3) poultry windrow compost, or 4) no compost. E. coli abundance, soil water potential, soil temperature, extracellular enzyme activity, microbial respiration, phospholipid fatty acid biomarker abundance, and genetic sequencing of the microbial community were measured over a six-month field season. The lab experiment showed that E. coli were able to grow well in sterile compost extracts, without microbial competition for nutrients. Conversely, E. coli populations were only able to survive in non-sterile soil extracts. These results suggest that copiotrophic organisms adapted for high-nutrient environments may depend on the extracellular enzyme activity of native oligotrophic organisms to acquire sufficient nutrients to survive in soils. Results of the field experiment showed clear and interdependent effects of soil moisture and nutrient availability on microbial community dynamics and E. coli survival. Data suggest that saturated soils cause a decrease in microbial extracellular enzyme activity, and drying-rewetting cycles can cause respiration bursts, nutrient mineralization, and shifts in community composition. The saturation of soils, which mobilizes nutrients and may result in a decrease in competition from aerobic organisms, correlated directly with increased survival of E. coli. Additionally, amendment with ammonium-rich poultry compost resulted in the maintenance of high levels of E. coli throughout the field season. Despite an increase in microbial biomass from dairy vermicompost amendment, poultry compost was the only compost that had a significant effect on E. coli survival. The results suggest that nitrogen availability and water potential are strong drivers of E. coli's survival in soils. Correlations among abiotic factors, community composition, and E. coli survival reveal insights into the complex relationships that occur in disturbed agricultural soil environments. Further research on E. coli's response to targeted organisms, abiotic soil properties, and nutrient inputs could have implications for agricultural considerations in food safety and microbial ecology.

<p>Soil from local beech-forest, spruce-forest and an industrial area was taken. Control- and test-microcosms containing 150 ml soil were spiked with cypermethrin 0,4 mg/ml soil. Cypermethrin residues were extracted on day seven and 14.</p><p>Cyclohexane and deionized water was utilized in multiple step extraction processes. Samples were analyzed in a Gas Chromatograph (GC) with electron capture detector (ECD). </p><p>Concentration values for the samples were highest for beech-forest soil and lower for the other two soil-types. Statistical differences in concentrations between control- and test-microcosms for each soil-type on day seven and day 14 were evaluated with Mann Whitney U tests. Significant result was only found in the industrial 14-day group. The small amounts of cypermethrin in the extracts could not only be ascribed to a bacterial degradation process. Used insecticide has a high bonding affinity for particles and is sequestered in soil.</p>

The aim of this research project was to test new collection and cultivation techniques that may increase the range of cultivable diversity of soil bacteria. Fortified BioSep beads were employed in situ to capture soil bacteria, and the success of the beads was analyzed using Phylochip microarray analysis. In the cultivation phase, three different media substrates and increased incubation period were evaluated for the ability to select novel or rare bacteria. Over 700 agricultural soil bacterial isolates were classified, including a rare Gemmatimonadetes sp., a rare Verrucomicrobia sp., several Acidobacteria sp., and many novel isolates. Land management, media, and incubation period each resulted in lineage specific preferences. The yeast fortified BioSep bead cultivation collection was significantly different from the bulk soil or acyl homoserine lactone (AHL) fortified bead cultivation collections, and there were lineage specific differences in all three collection types. Phylochip analysis showed a significant difference between bulk soil and all BioSep bead (water, yeast, or AHL fortified) communities based on microarray analysis of 16S rDNA. The yeast fortified BioSep bead community was richer in operational taxonomic units (OTU) than all others. The number of phyla determined by the Phylochip analysis was much higher than that seen in the overall cultivation collection. Prophage induction assays of 21 isolates were performed, using mitomycin C (mitC) and a mixture of six AHLs, to examine soil lysogenic phage-host interactions. The fraction induced by mitC was 29%, and 10% were induced by AHL. There was no correlation between induction and land management or host growth rate. This research showed that increases in cultivable diversity can be attained by the use of BioSep beads in the collection process, varying media substrates, and by extending incubation of inoculate cultures. Phylochip analysis, however, revealed that even with altered cultivation methods, there is still a wealth of soil bacterial diversity that remains to be cultivated from this site. We also found that AHLs impact the interactions between soil bacterial hosts and prophage.

Soil from local beech-forest, spruce-forest and an industrial area was taken. Control- and test-microcosms containing 150 ml soil were spiked with cypermethrin 0,4 mg/ml soil. Cypermethrin residues were extracted on day seven and 14. Cyclohexane and deionized water was utilized in multiple step extraction processes. Samples were analyzed in a Gas Chromatograph (GC) with electron capture detector (ECD). Concentration values for the samples were highest for beech-forest soil and lower for the other two soil-types. Statistical differences in concentrations between control- and test-microcosms for each soil-type on day seven and day 14 were evaluated with Mann Whitney U tests. Significant result was only found in the industrial 14-day group. The small amounts of cypermethrin in the extracts could not only be ascribed to a bacterial degradation process. Used insecticide has a high bonding affinity for particles and is sequestered in soil.

Abstract Based on maintaining healthy soil for sustainable agriculture and enhancing banana disease resistance, Taiwan Banana Research Institute began to conduct organic cultivation on a trial basis in 1998. It had been proved that the morbidity of banana Fusarial wilt disease at organic cultivation plots was significantly lower than that of conventional farming. In order to study the differences of soil microbiota between the organic cultivation plots and the conventional farming areas, physical and chemical properties of the rhizosphere and non- rhizosphere soil samples were assayed during the period of Aug. 2010 to May 2011. The bacterial diversity was analyzed by molecular biology methods, including PCR-DGGE to separate the 16S rDNA V6 ~ V8 region of various bacteria and the recombinant DNA technology by using pGEM-T Easy Vector System to separate and sequence the DNA fragments. The results showed that organic plots was loam soil, but the conventional farming soil was sandy loam with higher sand content. The soil pH in 13 years organic area was mildly alkaline, but in conventional farming area was mildly acidic to slightly acidic. The content of various nutrients in organic 13-year area soil was not necessarily higher than the conventional farming area soil. The available nutrient contents in organic areas trend to be more stable than that in the conventional areas. Fertilization may affect the content of available nutrients in the soil. No bacterial DNA could be extracted from the organic fertilizer. The bacterial microbiota in soil was very stable, and was not related to the sampling seasons. The Banana strains had little effect on soil bacterial microbiota. There was no difference on the bacterial microbiota between the rhizosphere and non-rhizosphere soil samples. It is not sure whether there were any differences on the bacterial microbiota between the nearby soil of banana Fusarial wilt plants and the nearby soil of the healthy plants. By analyzing the DNA fragment clone library, 43 strains correspond to known category, of which 28 belonged to the Proteobacteria, and 34 were uncultured strains. The role of these microbial strains might involve in various element cycles, such as N cycles, C cycles, and S cycles (including some photosynthetic bacteria). The systematic cladogram showed that organic 13-year areas, organic 3-year areas and conventional farming areas represented three major categaries. The organic 13-year area and conventional area possessed the highest difference on the microbiota composition.

The aim of this research project was to test new collection and cultivation techniques that may increase the range of cultivable diversity of soil bacteria. Fortified BioSep beads were employed in situ to capture soil bacteria, and the success of the beads was analyzed using Phylochip microarray analysis. In the cultivation phase, three different media substrates and increased incubation period were evaluated for the ability to select novel or rare bacteria. Over 700 agricultural soil bacterial isolates were classified, including a rare Gemmatimonadetes sp., a rare Verrucomicrobia sp., several Acidobacteria sp., and many novel isolates. Land management, media, and incubation period each resulted in lineage specific preferences. The yeast fortified BioSep bead cultivation collection was significantly different from the bulk soil or acyl homoserine lactone (AHL) fortified bead cultivation collections, and there were lineage specific differences in all three collection types. Phylochip analysis showed a significant difference between bulk soil and all BioSep bead (water, yeast, or AHL fortified) communities based on microarray analysis of 16S rDNA. The yeast fortified BioSep bead community was richer in operational taxonomic units (OTU) than all others. The number of phyla determined by the Phylochip analysis was much higher than that seen in the overall cultivation collection.Prophage induction assays of 21 isolates were performed, using mitomycin C (mitC) and a mixture of six AHLs, to examine soil lysogenic phage-host interactions. The fraction induced by mitC was 29%, and 10% were induced by AHL. There was no correlation between induction and land management or host growth rate. This research showed that increases in cultivable diversity can be attained by the use of BioSep beads in the collection process, varying media substrates, and by extending incubation of inoculate cultures. Phylochip analysis, however, revealed that even with altered cultivation methods, there is still a wealth of soil bacterial diversity that remains to be cultivated from this site. We also found that AHLs impact the interactions between soil bacterial hosts and prophage.

Pseudomonas fluorescens SBW25 is a gram-negative soil bacterium capable of growing on histidine as the sole source of carbon and nitrogen. Expression of histidine utilization (hut) genes is controlled by the HutC repressor with urocanate, the first intermediate of the histidine degradation pathway, as the direct inducer. Recent genome sequencing of P. fluorescens SBW25 revealed the presence of hutD in the hut locus, which encodes a highly conserved hypothetical protein. Previous genetic analysis showed that hutD is involved in hut regulation, in such a way that it prevents overproduction of the hut enzymes. Deletion of hutD resulted in a slow growth phenotype in minimal medium with histidine as the sole carbon and nitrogen source. While the genetic evidence supporting a role of hutD in hut regulation is strong, nothing is known of the mechanism of HutD action. Here I have cloned and expressed the P. fluorescens SBW25 hutD in E. coli. Purified HutD was subjected to chemical and structural analysis. Analytic size-exclusion chromatography indicated that HutD forms a dimer in the elution buffer. The crystal structure of HutD was solved at 1.80 Å (R = 19.3% and Rfree = 22.3%) by using molecular replacement based on HutD from P. aeruginosa PAO1. P. fluorescens SBW25 HutD has two molecules in an asymmetric unit and each monomer consists of one subdomain and two ß-barrel domains. Comparative structural analysis revealed a conserved binding pocket. The interaction of formate with a highly conserved residue Arg61 via salt-bridges in the pocket suggests HutD binds to small molecules with carboxylic group(s) such as histidine, urocanate or formyl-glutamate. The hypothesis that HutD functions via binding to urocanate, the hut inducer, was tested. Experiments using a thermal shift assay and isothermal titration calorimetry (ITC) analysis suggested that HutD binds to urocanate but not to histidine. However, the signal of HutD-urocanate binding was very weak and detected only at high urocanate concentration (53.23 mM), which is not physiologically relevant. The current data thus does not support the hypothesis of HutD-urocanate binding in vivo. Although the HutD-urocanate binding was not confirmed, this work has laid a solid foundation for further testing of the many alternative hypotheses regarding HutD function.