Year: 2016
Type: dissertation
Abstract: Enterococcus faecalis is a Gram-positive commensal bacterium forming diplococci/short chains that can also be an opportunistic pathogen.Some infections caused by E. faecalis such as endocarditis and bacteraemia can be life-threatening and difficult to treat due the resistance of this organism to several classes of antibiotics.Recent data revealed that the morphology of E. faecalis is an important factor for pathogenesis.Whilst strains forming diplococci or short chains can evade phagocytes and cause lethality in the zebrafish model of infection, isogenic derivatives forming long cell chains are more susceptible to phagocyte uptake and become avirulent.The enzyme dedicated to septum cleavage in E. faecalis, N-acetylglucosaminidase AtlA therefore has a key role during pathogenesis.The aim of this project was to identify genes involved in the control of AtlA activity during cell division.We used two distinct phenotypic screens to isolate spontaneous and transposon mutants impaired in septum cleavage.Our results indicate that several mechanisms ensure spatial and temporal control of AtlA enzymatic activity during growth.i 1.3.2.3.Peptidases 1.3.3.The physiological role of the PGHs 1.3.3.1.Contribution to peptidoglycan biosynthesis 1.3.3.2.Role during sporulation and germination 1.3.3.3.Septum cleavage during cell division 1.3.3.4.Resuscitation of dormant cells 1.3.3.5.Contribution to natural competence 1.3.3.6.PGHs as toxins to lyse competitors 1.3.4.E. faecalis PGHs 1.3.4.1.AtlA 1.3.4.2.AtlB 1.3.4.3.AtlC 1.3.4.4.EnpA 1.4.Aims and objectives Chapter 2 Material and Methods 2.1.Chemicals and enzymes 2.2.Antibiotics 2.3.Media 2.3.1.Brain heart infusion (BHI) 2.3.2.Super optimal broth with catabolite repression (SOC) 2.3.3.Semi-solid medium for the isolation of spontaneous mutants 2.3.4.Agar plates to detect AtlA activity 2.3.5.M17-Glu 2.3.6.SGM17 2.4.Bacterial strains, plasmids and oligonucleotides 2.5.Growth conditions 2.5.1.E. faecalis 2.5.1.1.Routine cultures of E. faecalis 2.5.1.2.Selection of spontaneous mutants in semi-solid medium 2.5.1.3.Analysis of sedimentation profiles in standing cultures 2.5.2.Micrococcus luteus 2.5.3.Escherichia coli 2.6.Buffers and stock solutions 2.6.1.10× DNA loading buffer 2.6.2.Phosphate buffered saline (PBS) 2.6.3.TAE (50x) 2.6.4.SDS-PAGE solutions 2.6.4.1.4× SDS-PAGE loading buffer 2.6.4.2.SDS-PAGE 2.6.4.3.10× SDS-PAGE electrophoresis buffer 2.6.4.4.Coomassie Blue staining solution 2.6.4.5.Destain solution 2.6.4.6.Zymogram solutions renaturing solution 2.6.5.TE 2.6.6.Solutions to prepare Escherichia coli competent cells 2.6.7.Suc-Gly 2.6.8.SM17MC viii 2.7.Molecular biology techniques 2.7.1.Polymerase chain reaction (PCR) 2.7.1.1.High-fidelity PCR using Phusion DNA polymerase (Thermofisher Scientific) 2.7.1.2.Routine PCR Taq DNA polymerase 2.7.2. PCR purification 2.7.3.Gel extraction 2.7.4.Plasmid purification 2.7.5.Genomic DNA extraction 2.7.6.Enzymatic digestion of DNA 2.7.7.DNA ligation 2.7.8.Gibson assembly 2.7.9.Agarose gel electrophoresis 2.7.10.Agarose gel photography 2.8.Spectrophotometric measurement (OD 600 ) 2.9.Protein expression and purification 2.9.1. Expression of recombinant proteins inEscherichia coli BL21(DE3) 2.9.2.Preparation of protein extracts 2.9.3.Protein purification using immobilized metal affinity chromatography (IMAC) 2.9.4.Protein purification using size exclusion chromatography 2.10.Preparation of cell walls as a substrate in agar plates 2.10.1.M. luteus cells 2.10.2.E. faecalis cells 2.10.3.Unbroken cell walls 2.10.4.E. faecalis broken cells 2.10.5.Preparation of broken cell walls 2.11.Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) 2.11.1.SDS-PAGE 2.11.2.Coomassie blue staining 2.11.3.Renaturing SDS-PAGE
Author: Fathe Elsarmane
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2577185677