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The Influence of Wall Teichoic Acids on Cell Viability and Morphology in Bacillus Subtilis

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The Influence of Wall Teichoic Acids on Cell Viability and Morphology in Bacillus Subtilis The influence of Wall Teichoic Acids on cell viability and morphology in Bacillus subtilis by Jan Wintgens (S2504618) Master research Project I November 2014 Supervision: Marielle van den Esker Prof. Dr. O.P Kuipers 1 2 Abstract Wall teichoic acids (WTA’s) are negatively charged polymers that play an important role in the cell envelope. Among other functions they are influencing cell morphology and autolysin activity. The mechanism of the latter is only shown in Staphylococcus aureus and is poorly understood. We here show the impact on morphology by WTA’s in detail and started investigating the role of WTA’s in lysis in Bacillus subtilis. Lacking the first step in WTA biosynthesis the ΔtagO conditional knockout mutant was used to characterise growth under different wall teichoic acid concentrations. We found that growth is generally impaired and initially cannot be complemented to WT level. The mutant however, surprisingly regrows after a lysis phase of diverging length, a phenomenon that requires further investigation. Measuring the morphology of the mutant, we were able to show in a detailed length to width ratio analysis that WTA are important to maintain the rod shape of the bacteria. We also detected an increase of volume by up to 330% caused by the lack of WTA’s. Both results show that wall teichoic acids are a structure that is partially responsible for cell shape maintenance in gram positive bacteria. A difference in charge or size between WTA in the WT and in the mutant is apparent in our results. It gives insight in the reaction of the cell to a lack of WTA’s and the regulation of their biosynthesis. It also has an impact on all other results we obtain with this mutant. Finally we were able to show that WTA’s are responsible for up to 23% of the cation binding capacity in the cellular envelope of Bacillus subtilis. This result is similar to the ones achieved using Staphylococcus aureus, in which it has been shown that this influences autolysin activity. 3 Table of Contents Abstract ................................................................................................................................................... 3 Introduction ............................................................................................................................................. 6 The Cell Envelope ................................................................................................................................ 6 Wall Teichoic Acid Structure ............................................................................................................... 8 Wall Teichoic Acids Biosynthesis ......................................................................................................... 9 Intracellular assembly ..................................................................................................................... 9 Tailoring modifications of WTA’s .................................................................................................. 10 Polymer export .............................................................................................................................. 10 Necessity and conditional necessity of tag genes ......................................................................... 11 Roles of Wall Teichoic Acids .............................................................................................................. 11 Regulation of Ion Homeostasis ...................................................................................................... 11 Regulation of cell morphology and division .................................................................................. 12 Purpose and experimental setup ...................................................................................................... 12 Material and Methods ........................................................................................................................... 14 Strain description and growth ........................................................................................................... 14 Growth curves ................................................................................................................................... 14 Sequencing ........................................................................................................................................ 15 Cytochrome C test ............................................................................................................................. 16 Microscopy and ImageJ ..................................................................................................................... 16 Polyacrylamide gel of Wall teichoic acids ......................................................................................... 17 WTA extraction .............................................................................................................................. 17 polyacrylamide gel electrophoresis and staining .......................................................................... 18 Gram staining .................................................................................................................................... 18 Results ................................................................................................................................................... 20 Deletion strains growth characterisation ......................................................................................... 20 Sequencing ........................................................................................................................................ 25 Gram staining .................................................................................................................................... 25 Measurement of cell size .................................................................................................................. 25 WTA assay ......................................................................................................................................... 28 Cytochrome C test ............................................................................................................................. 29 Discussion .............................................................................................................................................. 30 Deletion strains growth characterisation .......................................................................................... 30 Cell wall integrity ............................................................................................................................... 31 4 Wall teichoic acids in ΔtagO .............................................................................................................. 33 Influence on cation binding ............................................................................................................... 34 Outlook .............................................................................................................................................. 35 References ............................................................................................................................................. 36 Acknowledgments ................................................................................................................................. 42 Appendix ................................................................................................................................................ 43 5 Introduction The Cell Envelope The cell envelope of bacteria is a complex structure that shows a great variety in composition and the functions it fulfils, like maintaining cell shape and interaction with the exocytoplasm[1]. The absence of an outer membrane in gram-positive bacteria, like the here studied Bacillus subtilis 168 is one of the main differences compared to gram-negative ones, in which two membranes create the periplasm[2]. This compartment filters toxic molecules and serves as an anchoring scaffold for proteins involved in respond systems to the environment. Functions fulfilled by this outer membrane have to be replaced by structures present in and bound to the peptidoglycan (PG) matrix, the dominant component of gram-positive cell envelopes(Figure 1)[3]. Figure 1: Schematic overview of gram positive and gram negative cell envelopes. In contrast to gram-negative organisms, gram-positive organisms do not show a distinct periplasm . Proteins are not shown. LTA: lipoteichoic acid; LPS: lipopolysaccharide; WTA: wall teichoic acid [2]. Peptidoglycan is a cross-linked matrix of glycan chains. This carbohydrate network is linked to one another via covalently bound peptide side chains[4]. In gram-positive bacteria the layer is especially thick and its organization serves several purposes, such as binding proteins as well as other components and withstanding high internal osmotic pressure. The cell wall of Bacillus subtilis for example defies a pressure of approximately 2.43 bar compared to 0.5 of the one of E.coli (gram- negative)[5]. 6 The architecture of PG is differing between bacterial strains[5]. In general, PG consists of strands made from two alternating amino sugars, namely N-acetylglucosamine (GlcNAc or NAG) and N- acetylmuramic acid (MurNAc or NAM). The strands are linked by a 4-5 residue amino acid unit between the chains. Different polymers are linked to the peptidoglycan.
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