The spatial evolution of the chemotaxis proteins of the Bacillus subtilis group
- Yssel, Anna Elizabeth Johanna
- Authors: Yssel, Anna Elizabeth Johanna
- Date: 2011
- Subjects: Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4027 , http://hdl.handle.net/10962/d1004087 , Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Description: The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit.
- Full Text:
- Date Issued: 2011
- Authors: Yssel, Anna Elizabeth Johanna
- Date: 2011
- Subjects: Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4027 , http://hdl.handle.net/10962/d1004087 , Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Description: The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit.
- Full Text:
- Date Issued: 2011
Isolation of xylanolytic multi-enzyme complexes from Bacillus subtilis SJ01
- Authors: Jones, Sarah Melissa Jane
- Date: 2010
- Subjects: Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3974 , http://hdl.handle.net/10962/d1004033 , Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Description: Cellulose and hemicellulose account for a large portion of the world‘s plant biomass. In nature, these polysaccharides are intertwined forming complex materials that require multiple enzymes to degrade them. Multi-enzyme complexes (MECs) consist of a number of enzymes working in close proximity and synergistically to degrade complex substrates with higher efficiency than individual enzymes. The cellulosome is a cellulolytic MEC produced by anaerobic bacteria that has been studied extensively since its discovery in 1983. The aim of this study was to purify a cellulolytic and/or hemicellulolytic MEC from an aerobic bacterium of the Bacillus genus. Several bacterial isolates were identified using morphological characteristics and 16S rDNA sequencing, and screened for their ability to degrade cellulose and xylan using a MEC. The isolate that produced a high molecular weight protein fraction with the greatest ability to degrade Avicel®, carboxymethyl cellulose (CMC) and birchwood xylan was identified as Bacillus subtilis SJ01. An optimised growth medium, consisting of vitamins, trace elements, birchwood xylan (as the carbon source), and yeast and ammonium sulphate (as the nitrogen sources), increased the production of CMCase and xylanase enzymes from this bacterium. The removal of a competing bacterial strain from the culture and the inhibition of proteases also increased enzyme activities. A growth curve of B. subtilis SJ01 indicated that xylanase production was highest in early stationary growth phase and thus 84 hours was chosen as the best cell harvesting time. To purify the MECs produced by B. subtilis SJ01 size-exclusion chromatography on a Sephacryl S-400 column was used. It was concluded that (for the purposes of this study) the best method of concentrating the culture supernatant prior to loading onto Sephacryl S-400 was the use of ultrafiltration with a 50 kDa cut-off membrane. Two MECs, named C1 and C2 of 371 and 267 kDa, respectively, were purified from the culture supernatant of B. subtilis SJ01. Electrophoretic analysis revealed that these MECs consisted of 16 and 18 subunits, respectively, 4 of which degraded birchwood xylan and 5 of which degraded oat spelt xylan. The MECs degraded xylan substrates (C1: 0.24 U/mg, C2: 0.14 U/mg birchwood xylan) with higher efficiency than cellulose substrates (C1: 0.002 U/mg, C2: 0.01 U/mg CMC), and could therefore be considered xylanosomes. Interestingly, the MECs did not bind to insoluble birchwood xylan or Avicel® and did not contain glycosylated proteins, which are common features of cellulosomes. This study is, therefore, important in revealing the presence of MECs that differ from the cellulosome and that may have particular application in industries requiring high xylanase activity, such as the paper and pulp industry. The abundant genetic information available on B. subtilis means that this organism could also be used for genetic engineering of cellulolytic/hemicellulolytic MECs.
- Full Text:
- Date Issued: 2010
- Authors: Jones, Sarah Melissa Jane
- Date: 2010
- Subjects: Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3974 , http://hdl.handle.net/10962/d1004033 , Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Description: Cellulose and hemicellulose account for a large portion of the world‘s plant biomass. In nature, these polysaccharides are intertwined forming complex materials that require multiple enzymes to degrade them. Multi-enzyme complexes (MECs) consist of a number of enzymes working in close proximity and synergistically to degrade complex substrates with higher efficiency than individual enzymes. The cellulosome is a cellulolytic MEC produced by anaerobic bacteria that has been studied extensively since its discovery in 1983. The aim of this study was to purify a cellulolytic and/or hemicellulolytic MEC from an aerobic bacterium of the Bacillus genus. Several bacterial isolates were identified using morphological characteristics and 16S rDNA sequencing, and screened for their ability to degrade cellulose and xylan using a MEC. The isolate that produced a high molecular weight protein fraction with the greatest ability to degrade Avicel®, carboxymethyl cellulose (CMC) and birchwood xylan was identified as Bacillus subtilis SJ01. An optimised growth medium, consisting of vitamins, trace elements, birchwood xylan (as the carbon source), and yeast and ammonium sulphate (as the nitrogen sources), increased the production of CMCase and xylanase enzymes from this bacterium. The removal of a competing bacterial strain from the culture and the inhibition of proteases also increased enzyme activities. A growth curve of B. subtilis SJ01 indicated that xylanase production was highest in early stationary growth phase and thus 84 hours was chosen as the best cell harvesting time. To purify the MECs produced by B. subtilis SJ01 size-exclusion chromatography on a Sephacryl S-400 column was used. It was concluded that (for the purposes of this study) the best method of concentrating the culture supernatant prior to loading onto Sephacryl S-400 was the use of ultrafiltration with a 50 kDa cut-off membrane. Two MECs, named C1 and C2 of 371 and 267 kDa, respectively, were purified from the culture supernatant of B. subtilis SJ01. Electrophoretic analysis revealed that these MECs consisted of 16 and 18 subunits, respectively, 4 of which degraded birchwood xylan and 5 of which degraded oat spelt xylan. The MECs degraded xylan substrates (C1: 0.24 U/mg, C2: 0.14 U/mg birchwood xylan) with higher efficiency than cellulose substrates (C1: 0.002 U/mg, C2: 0.01 U/mg CMC), and could therefore be considered xylanosomes. Interestingly, the MECs did not bind to insoluble birchwood xylan or Avicel® and did not contain glycosylated proteins, which are common features of cellulosomes. This study is, therefore, important in revealing the presence of MECs that differ from the cellulosome and that may have particular application in industries requiring high xylanase activity, such as the paper and pulp industry. The abundant genetic information available on B. subtilis means that this organism could also be used for genetic engineering of cellulolytic/hemicellulolytic MECs.
- Full Text:
- Date Issued: 2010
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