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Three computer software packages were written in the C++ language for the analysis of numerical phenetic, 16S rRNA sequence and pyrolysis mass spectrometric data. The X program, which provides routines for editing binary data, for calculating test error, for estimating cluster overlap and for selecting diagnostic and selective tests, was evaluated using phenotypic data held on streptomycetes. The AL16S program has routines for editing 16S rRNA sequences, for determining secondary structure, for finding signature nucleotides and for comparative sequence analysis; it was used to analyse 16S rRNA sequences of mycolic acid-containing actinomycetes. The ANN program was used to generate backpropagation-artificial neural networks using pyrolysis mass spectra as input data. Almost complete 1 6S rDNA sequences of the type strains of all of the validly described species of the genera Nocardia and Tsukamurel!a were determined following isolation and cloning of the amplified genes. The resultant nucleotide sequences were aligned with those of representatives of the genera Corynebacterium, Gordona, Mycobacterium, Rhodococcus and Turicella and phylogenetic trees inferred by using the neighbor-joining, least squares, maximum likelihood and maximum parsimony methods. The mycolic acid-containing actinomycetes formed a monophyletic line within the evolutionary radiation encompassing actinomycetes. The "mycolic acid" lineage was divided into two clades which were equated with the families Coiynebacteriaceae and Mycobacteriaceae. The family Coiynebacteriaceae contained the genera Cotynebacterium, Dietzia and Turicella and the family Mycobacteriaceae the genera Gordona, Mycobacterium, Nocardia, Rhodococcus and Tsukamurella. It was clear from the 1 6S rDNA sequence data that Nocardia pinensis was misclassified in the genus Nocardia and that TsukamurelIa wratislaviensis belonged to the genus Rhodococcus. The genus Nocardia formed a distinct dade that was clearly associated with the genus Rhodococcus. Two sublines were recognised within the Nocardia dade; one consisted of Nocardia asteroides and related taxa and the other of Nocardia otitidiscaviarum and allied species. The two sublines are distinguished by nucleotide differences in helix 37-1. The type strains of all of the Nocardia species contained hexahydrogenated menaquinones with eight isoprene units in which the two end units were cyclised. Actinomycetes selectively isolated from an activated sludge plant showing extensive foaming were the subject of a polyphasic taxonomic study. The sludge isolates, which clearly belong to the genus Tsukamurella on the basis of 1 6S rRNA data, contained highly unsaturated long chain mycolic acids and unsaturated menaquinones with nine isoprene units, properties consistent with their classification in the genus Tsukamurella. Six representative isolates and marker strains of Tsukamurella paurometabola were the subject of a numerical phenetic taxonomic study. The test strains were assigned to four groups in the simple matching coefficient, unweighted pair group method with arithmetic averages analysis. The sludge isolates formed a homogeneous cluster with the three remaining clusters composed of Tsukamurella paurometabola strains. Excellent congruence was found between these numerical taxonomic data and results derived from corresponding studies based on Curie point pyrolysis mass spectrometric and whole-organism protein electrophoretic analyses. The combined data suggest that the sludge isolates form the nucleus of a new species of the genus Tsukamurella and that Tsukamure!!a paurometabola is a heterogeneous taxon. Representatives of three putatively novel streptomycete species isolated from soil were used to develop and evaluate an identification system based on Curie point pyrolysis mass spectromety and artificial neural network analysis. The test strains consisted of sixteen target Streptomyces strains and one hundred and thirty-eight actinomycetes belonging to the genera Actinomadura, Mycobacterium, Nocardia, Nocardiopsis, Saccharomonospora and Streptosporangium. It was clear from the identification results that artificial neural network analysis was superior to conventional procedure based on principal component and canonical variate statistics. The problem of misidentification of some of the non-target strains was solved by the development of a neural network topology which contained an output neuron designed to detect non-target pyrolysis mass spectrometric patterns. The pyrolysis mass spectrometry-artificial neural network system was evaluated using thirteen fresh streptomycete isolates and found to be capable of long-term identification of the target strains.

Results are reported from a series of experiments to determine the effects of certain environmental factors, namely, sunlight, temperature and , salinity on the survival of selected enteric bacteria which are of public health importance either as indicators of faecal pollution or The survival of the as pathogens. starved organisms in seawater and in freshwater microcosms exposed to artificial and natural sunlight, and in the dark, at a range of temperatures was investigated. An acridine orange direct viable count (AODVC) using an epifluorescent microscope was employed in addition to selective and non-selective cultural methods for estimation of decay rates of the test bacteria. The use of the AODVC allowed enumeration of those bacteria which respond to the stresses of the natural environment by entering into a viable but non-culturable form. These otherwise would not be detected, as they are, by definition, non-culturable using traditional enumeration techniques based upon the production of visible signs of growth. Two strategies were employed by the organisms in response to the unfavourable conditions. Prior growth of those members of the Enterobacteriaceae in a nutrient-poor medium before inoculation into the microcosms allowed adaptation of the cells to low nutrient concentrations, thus extending survival and resulting in higher resistance to other stresses such as visible light. This extension was, however, only temporary, the ultimate fate of the bacteria being death. Enterococci were particularly sensitive to low nutrient concentrations and died very rapidly in the light. All bacteria tested were able to adopt the viable but non-culturable strategy in the dark as a temporary measure, though it was only a matter of time before viability as well as culturability was also lost. This included enterococci, for which an AODVC using the antibiotic ciprofloxacin was developed by modifying the original nalidixic acid method. Responses of bacteria in the dark were influenced by temperature. Bright natural sunlight produced rapid death in bacteria exposed in seawater microcosms, a result of the synergistic interaction of salinity, UV light and possibly temperature too, whereas the presence of humic acids in freshwater afforded some protection to the cells by absorbing the damaging UV component of sunlight. Low intensities of UV and visible light typical of those found below the surface of water may induce bacteria to evolve towards a viable but non-culturable form.

Molecular systematic methods were applied in a series of studies designed to resolve the taxonomic relationships of thermophilic actinomycetes known to be difficult to classify using standard taxonomic procedures. The test strains included representatives of clusters defined in an extensiven umerical phenetic survey of thermophilic streptomycetesa nd twelve marker strains. The resultant genotypic data together with the results of corresponding phenotypic studies were used to highlight novel taxa and to improve the circumscription of validly described species. The most comprehensive study was undertaken to clarify relationships within and between representative alkalitolerant, thermophilic and neutrophilic, thermophilic streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA: DNA relatedness studies, supported the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and Streptomyces thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of the priority, Streptomyces thermonitrificans is a later subjective synonym of Streptomyces thermovulgaris. Similarly, eight out of eleven representative alkalitolerant, thermophilic isolates and three out of sixteen representative neutrophilic, thermophilic isolates had a combination of properties consistent with their classification as Streptomyces thermovulgaris. One of the remaining alkalitolerant, thermophilic isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic, thermophilic isolate, Streptomyces strain NAR85, was identified as Streptomyces thermodiastaticus. Four other neutrophilic thermophilic isolates assigned to a numerical phenetic cluster and a thermophilic isolates from poultry faeces were also considered to warrant species status; the names Streptomyces eurythermophilus sp. nov. and Streptomyces thermocoprophilus sp. nov. are proposed to accommodate these strains. It was also concluded that additional comparative taxonomic studies are required to clarify the relationships between additional thermophilic streptomycete strains included in the present investigation. A corresponding polyphasic approach was used to clarify the taxonomy of six thermophilic isolates provisionally assigned to either the genera Amycolatopsis or Excellospora. Two of the isolates, strain NT202 and NT303, had properties consistent with their classification in the genus Amycolatopsis. However, the genotypic and phenotypic data also showed that these strains formed a new centre of taxonomic variation for which the name Amycolatopsis eurythermus sp. nov. is proposed. Similarly, the four remaining strains formed two new centre of taxonomic variation within the genus Excellospora. It is proposed that isolates TA113 and TA114 be designated Excellospora alcalithermophilus sp. nov. Similarly, the name Excellospora thermoalcalitolerans sp. nov. is proposed for strains TA86 and TA111. An emended description is also given for the genus Excellospora.

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Bacterial contamination due to excessive levels of bacteria is a confounding problem and remediation of impaired watersheds relies on the detection of fecal indicator bacteria and then assessing the source of said bacteria. Bacterial source tracking (BST) is an approach for assessing potential sources of this contamination. The purpose of this study was to utilize both cultivation-independent and –dependent methods to improve the ability to track sources of fecal contamination. First, E. coli community composition was assessed across three standard water quality assessments including USEPA Methods 1603 and 1604, and Colilert®, to determine their impact on BST library-based performance. Results indicate that the three assessed methods of enumeration and isolation may select for different populations of E. coli and standardized methods may be warranted if library-dependent BST is part of a research plan. Next, BST techniques were used to enumerate and characterize E. coli communities across various dairy manure management techniques used in the Leon River watershed in central Texas to determine effectiveness of BST efforts in tracking contamination from dairy manure. Results of this study indicated that manure and effluent management strategies which employed means to remove solids from the manure tended to decrease the levels of E. coli in the effluent. Some E. coli genotypes were found across the managerial treatments even though there were no clear seasonal trends or site groupings among the dataset. The vast majority of the isolates classified using the Texas E. coli BST library were correctly classified back to their major source class, thus increasing confidence in the methods currently being utilized to track dairy fecal contributions in this Central Texas watershed. Finally, deer bacterial fecal communities from south and central Texas were analyzed using 454-pyrosequencing to assess the potential for the development of a deer-specific BST marker. Microbial communities did not cluster by site or year suggesting that deer fecal communities in these Texas regions are stable over time and could be amenable to marker development.

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No description available.