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Monitoring amino acid metabolism during fermentation has significant potential from the standpoint of strain selection, optimizing growth and production in host strains, and profiling microbial metabolism and growth state. A method has been developed based on rapid quantification of underivatized amino acids using liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) to monitor the metabolism of 20 amino acids during microbial fermentation. The use of a teicoplanin-based chiral stationary phase coupled with electrospray tandem mass spectrometry allows complete amino acid analyses in less than 4 min. Quantification is accomplished using five isotopically labeled amino acids as internal standards. Because comprehensive chromatographic separation and derivatization are not required, analysis time is significantly less than traditional reversed- or normal-phase LC-based amino acid assays. Intra-sample precisions for amino acid measurements in fermentation supernatants using this method average 4.9% (R.S.D.). Inter-day (inter-fermentation) precisions for individual amino acid measurements range from 4.2 to 129% (R.S.D.). Calibration curves are linear over the range 0-300 microg/ml, and detection limits are estimated at 50-450 ng/ml. Data visualization techniques for constructing semi-quantitative fermentation profiles of nitrogen source utilization have also been developed and implemented, and demonstrate that amino acid profiles generally correlate with observed growth profiles. Further, cellular growth events, such as lag-time and cell lysis can be detected using this methodology. Correlation coefficients for the time profiles of each amino acid measured illustrate that while several amino acids are differentially metabolized in similar fermentations, a select group of amino acids display strong correlations in these samples, indicating a sub-population of analytes that may be most useful for fermentation profiling.

A method has been developed for the direct determination of coenzyme A (CoA) and organic acid-CoA thioesters in mixtures using directly combined liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Mixtures of CoA and organic acid-CoA thioesters were analyzed by LC/ESI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of the CoA-thioesters were established based on LC retention times and simultaneously recorded mass spectra. Monitoring of the CoA specific fragment ion at m/z 428 throughout the chromatogram provides a unique fingerprint for CoA content in the samples that corroborates the identification of organic acid-CoA thioesters in the mixtures. Furthermore, fragment ions arising from the ester linkage portion of the molecule allow unambiguous identification of the CoA esters in the samples. A second LC elution system was developed that allows the simultaneous separation and identification of 2-hydroxypropionyl-CoA (lactyl-CoA) and 3-hydroxypropionyl CoA (3HP-CoA), which have the same mass and identical MS fragmentation behavior. The utility of LC/ESI-MS employing this elution system is demonstrated by the determination of 3HP-CoA and lactyl-CoA (converted to CoA-thioesters from their corresponding free acids using CoA-transferase) in fermentation broths from Escherichia coli strains engineered for the production of 3-hydroxypropionic acid (3HP). External calibration employing a purified 3HP-CoA standard allowed indirect quantification of 3HP content in the broth with a precision of 1% (RSD). The feasibility of extending the method described above to perform LC/selected reaction monitoring-tandem mass spectrometry for direct determination of organic acid-CoA thioesters in cells was also demonstrated.

Certain ancient Near Eastern texts develop over time towards a reasonably stable state of transmission. However, the development towards a single ‘stabilised’ transmitted form that marks the biblical manuscripts between the second century B.C.E. and second century C.E. is often considered to permit the Hebrew bible a unique position in the ancient Near Eastern textual corpus. The degree to which the wider body of ancient Near Eastern texts actually support or undermine this position is the topic of this dissertation. The study begins by formulating a methodology for comparing the accuracy with which ancient texts of varying genres and languages were transmitted. Exemplars from the first millennium B.C.E. cuneiform evidence are selected for analysis on the basis of genre. Texts that are preserved in more than one ancient copy are compared to determine how much variation occurs between manuscripts of the same text. The study begins with representative texts from the Neo-Assyrian, Neo-Babylonian and Late Babylonian periods that range in date from the late eighth century B.C.E. to the third century B.C.E. The study then turns to the Torah scrolls from the Dead Sea area that range in date from the third century B.C.E. to the second century C.E. The accuracy with which the cuneiform texts were transmitted is then compared with the biblical evidence. The study finds that the most stable texts surveyed are those containing ritual instructions. The mechanisms that may have led to the exact transmission of the Torah in the late Second Temple period are discussed in the conclusion.

Little is known about the survival mechanics of Mycobacterium tuberculosis within the human host. A number of genes are likely to be activated during infection, especially within macrophages, and these potential virulence determinants will be of great importance in the ultimate development of new vaccines and antimycobacterials. In an effort to identify some of these virulence determinants, a M.tuberculosis H37Rv gene promoter-probe library in a BCG host was constructed using an E.coli - mycobacterial shuttle vector with lacZ as the reporter gene. 4800 individual clones were arrayed in a 96 well microtitre(TM) format, enabling the testing of individual clones for promoter activity under a variety of environmental stressing conditions. Two separate in vitro screenings of the arrayed clones identified 53 clones that exhibited up-regulation of lacZ when stressed. 50 of these clones were bi-directionally sequenced to determine the cloned M.tuberculosis H37Rv DNA sequence. Identification of the location of the cloned DNA within the M.tuberculosis H37Rv genome identified 32 clones likely to containing potential promoter sequences. By measuring the levels of lacZ expression post-infection of macrophages, compared to in vitro expression, clones were identified which contained potential promoter sequences up-regulated within the macrophage. Quantitative Reverse Transcriptase-PCR was employed, using cDNA obtained from M.tuberculosis from infected and un-infected macrophages, to measure the levels of expression of 5 M.tuberculosis H37Rv genes identified as potentially up- regulated during infection. The results suggest that two of the five genes, genes Rv1265 and Rv2711, are indeed up-regulated during infection. The protein coded for by the gene Rv1265 has an unknown function whilst the gene Rv2711 encodes the protein IdeR, which is an iron-dependent repressor. Sequence and lacZ expression analysis identified four independently selected and screened clones which contained near identical sequences for the same promoter - that of the gene Rv0440 (groEL2). Gene Rv0440 codes for a 60KDa heat shock protein previously identified as up-regulated during infection. The identification of four independent clones containing the promoter for this gene validates this technology as a means of identifying M.tuberculosis H37Rv genes up-regulated during infection.