Studies on glutamine synthetase and glutamate synthase during growth and sporulation in Bacillus subtilis

Pan, Fui-lin (1978) Studies on glutamine synthetase and glutamate synthase during growth and sporulation in Bacillus subtilis. MSc(R) thesis, University of Glasgow.

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The purpose of this vork was to investigate the activity of two enzymes, glutamine synthetase (GNS) and glutamate synthase (GTS) during growth and sporulation in Bacillus subtilis 168. The coupled activity of these two enzymes is known to be one route by which ammonia is assimilated in Bacillus spp. Ammonia is known to be a repressor of sporulation and the overall aim of the work was to assess the possibility that one or both of these enzymes may play a role in the repression of sporulation. The investigation adopted two broad approaches. 1. Determination of the specific activities of the enzymes; firstly, during growth of the organism in minimal medium with various nitrogen sources and secondly, during sporulation induced by either carbon or nitrogen limitation. 2. Characterisation of a GTS-defective mutant and observations on its ability to sporulate under conditions of carbon and nitrogen deprivation. Assay procedures for GNS and GTS were developed based on previously published methods. Both enzymes were found almost entirely in the soluble fraction of broken cell suspensions. GTS activity was found to be slightly unstable on storage and for this reason all enzyme assays were performed on freshly prepared cell-free extracts. B. subtilis 168 grew well in a minimal medium with glucose as carbon source and either ammonia, L-arginine or L-glutamine as nitrogen source. It grew more slowly with L-glutamate or nitrate and only very slowly with L-histidine as source of nitrogen. The specific activity of either GNS or GTS could be altered by varying the source of nitrogen supplied for growth. GNS was repressed when either ammonia, L-glutamine or L-arginine were used as nitrogen source, but was derepressed when L-glutamate or nitrate were used. GTS was repressed by L-glutamate or L-arginine and to a lesser extent by L-glutamine, but was relatively derepressed in the presence of ammonia or nitrate. These observations supported previous suggestions that GNS was repressed by L-glutamine and GTS repressed by L-glutamate in Bacillus spp. Sporulation was obtained using the replacement technique whereby exponentially growing cells in a rich medium were resuspended in a poor medium to induce sporulation. This procedure gives a clearly defined point of initiation to the process and allows good synchrony throughout the cell population during sporulation. The composition of the resuspension medium was adjusted so that sporulation could be induced either by starvation for an adequate carbon source or by starvation for a source of nitrogen. No significant alterations in GNS or GTS specific activities were detected at 20 min or 40 min after initiation to sporulation by either carbon or nitrogen starvation. This result indicated that repression or derepression of GNS or GTS played no role in the onset of sporulation, although transient changes in enzyme specific activity could not be excluded by these experiments. A mutant strain (glt-100), isolated originally as an auxotroph requiring L-glutamate for growth, was more fully characterised and shown to lack GTS activity. This strain was unable to grow in minimal medium with ammonia as source of nitrogen. This fact, coupled with a failure to detect glutamate dehydrogenase activity in cell-free extracts, indicated that B. subtilis l68 lacks glutamate dehydrogenase capable of assimilating ammonia. Ammonia assimilation was assumed to occur via the coupled reactions catalysed by GNS and GTS as strain git-100, which lacked a functional GTS, was unable to grow with ammonia. Strain glt-100 was unable to sporulate under conditions where the process was assumed to be initiated by starvation for an adequate nitrogen supply, although it sporulated normally under conditions of carbon deprivation. The deficiency in GTS activity in strain glt-100, which prevented successful sporulation under conditions of nitrogen deprivation, was active during the initial 2.5 hours of sporulation. L-glutamine has previously been implicated-as a repressor of sporulation, but the activities of GNS found during the onset of sporulation in the wild-type and strain glt-100 and the sporulation behaviour of the mutant strain in various resuspension media tended to conflict with this hypothesis.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Additional Information: Adviser: J G Coote
Keywords: Microbiology
Date of Award: 1978
Depositing User: Enlighten Team
Unique ID: glathesis:1978-72086
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:11
Last Modified: 24 May 2019 15:11

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