Molecular Analysis of the Chlamydial AnomalyRole of the cytoskeleton protein MreB, the serine hydroxymethyl transferase GlyA and the penicillin binding proteins from Chlamydophila pneumoniae
Molecular Analysis of the Chlamydial Anomaly
Role of the cytoskeleton protein MreB, the serine hydroxymethyl transferase GlyA and the penicillin binding proteins from Chlamydophila pneumoniae
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DissertationDate of Exam
21.09.2012Date of Publication
27.09.2012Advisor
Sahl, Hans-GeorgCo-Referee
König, Gabriele M.Metadata
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Abstract
Chlamydiae are Gram-negative, obligate intracellular pathogens. Because of the isotonic intracellular niche of the host, a functional cell wall is dispensable in these endobacteria. Surprisingly, a nearly complete pathway for the biosynthesis of cell wall precursor lipid II is conserved in chlamydiae. Moreover, the organism is susceptible to penicillin and other antibiotics that inhibit cell wall biosynthesis. This paradox, which is known as chlamydial anomaly, raises the question about the essential role of cell wall building block lipid II in maintaining bacterial growth and replication.
Cell wall biosynthesis and cell division in prokaryotes are driven by tightly coordinated, partially overlapped machineries. Therefore, it was hypothesized that lipid II, which is an essential part of both processes, is required to maintain a functional divisome in chlamydiae.
The bacterial actin homolog MreB, which is found exclusively in non-spherically shaped bacteria, is considered to organize the incorporation of cell wall precursors into the side-wall, whereas the tubulin homolog FtsZ, which is ubiquitously distributed in almost all bacteria, is known to tether incorporation of cell wall building blocks at the developing septum. Unexpectedly, chlamydial genomes lack the essential central organizer of cell division ftsZ, but harbor despite of its spherical shape the rod-shape determining mreB.
In this project, it was investigated how chlamydiae can divide in absence of FtsZ. It was demonstrated that cytoskeletal protein MreB might compensate for FtsZ for maintaining a functional divisome machinery in chlamydiae. It was shown that chlamydial MreB polymerizes in vitro and that polymerization is not inhibited by the blocking agent A22. As observed for MreB from B. subtilis, chlamydial MreB does not require ATP for polymerization but is capable of ATP hydrolysis in phosphate release assays. Co-pelleting and bacterial two-hybrid experiments indicate that MreB from C. pneumoniae interacts with MurF, MraY and MurG, three key components in lipid II biosynthesis. In addition, MreB polymerization is improved in the presence of MurF. These findings suggest that MreB is involved in tethering biosynthesis of lipid II and as such may be necessary for maintaining functional divisome machinery in Chlamydiaceae.
While chlamydial genomes lack the alr gene encoding for alanine racemase that is responsible for D-alanine synthesis in other bacteria, several findings suggest that D-alanine should be part of chlamydial lipid II. Preliminary results on the racemization activity of the chlamydial GlyA show that the protein may provide D-alanine in chlamydiae.
The sensitivity of chlamydiae to penicillin and other β-lactam antibiotics is attributed to the presence of three PBPs encoded in the chlamydial genome. Chlamydiae have three PBPs homologous to PBP2, 3 and 6a from E. coli. PBP2 and PBP3 are essential mono-functional transpeptidases in E. coli. On the other hand, the E. coli PBP6a shows a weak in vitro carboxypeptidase activity. To get insights into the functionality of the chlamydial PBPs, the three proteins were cloned, over-produced and purified successfully. Further optimizations for the established assays are still required to get clear insights into the functionality of these proteins in Chlamydiaceae.
Cell wall biosynthesis and cell division in prokaryotes are driven by tightly coordinated, partially overlapped machineries. Therefore, it was hypothesized that lipid II, which is an essential part of both processes, is required to maintain a functional divisome in chlamydiae.
The bacterial actin homolog MreB, which is found exclusively in non-spherically shaped bacteria, is considered to organize the incorporation of cell wall precursors into the side-wall, whereas the tubulin homolog FtsZ, which is ubiquitously distributed in almost all bacteria, is known to tether incorporation of cell wall building blocks at the developing septum. Unexpectedly, chlamydial genomes lack the essential central organizer of cell division ftsZ, but harbor despite of its spherical shape the rod-shape determining mreB.
In this project, it was investigated how chlamydiae can divide in absence of FtsZ. It was demonstrated that cytoskeletal protein MreB might compensate for FtsZ for maintaining a functional divisome machinery in chlamydiae. It was shown that chlamydial MreB polymerizes in vitro and that polymerization is not inhibited by the blocking agent A22. As observed for MreB from B. subtilis, chlamydial MreB does not require ATP for polymerization but is capable of ATP hydrolysis in phosphate release assays. Co-pelleting and bacterial two-hybrid experiments indicate that MreB from C. pneumoniae interacts with MurF, MraY and MurG, three key components in lipid II biosynthesis. In addition, MreB polymerization is improved in the presence of MurF. These findings suggest that MreB is involved in tethering biosynthesis of lipid II and as such may be necessary for maintaining functional divisome machinery in Chlamydiaceae.
While chlamydial genomes lack the alr gene encoding for alanine racemase that is responsible for D-alanine synthesis in other bacteria, several findings suggest that D-alanine should be part of chlamydial lipid II. Preliminary results on the racemization activity of the chlamydial GlyA show that the protein may provide D-alanine in chlamydiae.
The sensitivity of chlamydiae to penicillin and other β-lactam antibiotics is attributed to the presence of three PBPs encoded in the chlamydial genome. Chlamydiae have three PBPs homologous to PBP2, 3 and 6a from E. coli. PBP2 and PBP3 are essential mono-functional transpeptidases in E. coli. On the other hand, the E. coli PBP6a shows a weak in vitro carboxypeptidase activity. To get insights into the functionality of the chlamydial PBPs, the three proteins were cloned, over-produced and purified successfully. Further optimizations for the established assays are still required to get clear insights into the functionality of these proteins in Chlamydiaceae.
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500 NaturwissenschaftenGaballah, Ahmed Hassan Ahmed: Molecular Analysis of the Chlamydial Anomaly : Role of the cytoskeleton protein MreB, the serine hydroxymethyl transferase GlyA and the penicillin binding proteins from Chlamydophila pneumoniae. - Bonn, 2012. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-29973
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-29973
@phdthesis{handle:20.500.11811/5391,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-29973,
author = {{Ahmed Hassan Ahmed Gaballah}},
title = {Molecular Analysis of the Chlamydial Anomaly : Role of the cytoskeleton protein MreB, the serine hydroxymethyl transferase GlyA and the penicillin binding proteins from Chlamydophila pneumoniae},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2012,
month = sep,
note = {Chlamydiae are Gram-negative, obligate intracellular pathogens. Because of the isotonic intracellular niche of the host, a functional cell wall is dispensable in these endobacteria. Surprisingly, a nearly complete pathway for the biosynthesis of cell wall precursor lipid II is conserved in chlamydiae. Moreover, the organism is susceptible to penicillin and other antibiotics that inhibit cell wall biosynthesis. This paradox, which is known as chlamydial anomaly, raises the question about the essential role of cell wall building block lipid II in maintaining bacterial growth and replication.
Cell wall biosynthesis and cell division in prokaryotes are driven by tightly coordinated, partially overlapped machineries. Therefore, it was hypothesized that lipid II, which is an essential part of both processes, is required to maintain a functional divisome in chlamydiae.
The bacterial actin homolog MreB, which is found exclusively in non-spherically shaped bacteria, is considered to organize the incorporation of cell wall precursors into the side-wall, whereas the tubulin homolog FtsZ, which is ubiquitously distributed in almost all bacteria, is known to tether incorporation of cell wall building blocks at the developing septum. Unexpectedly, chlamydial genomes lack the essential central organizer of cell division ftsZ, but harbor despite of its spherical shape the rod-shape determining mreB.
In this project, it was investigated how chlamydiae can divide in absence of FtsZ. It was demonstrated that cytoskeletal protein MreB might compensate for FtsZ for maintaining a functional divisome machinery in chlamydiae. It was shown that chlamydial MreB polymerizes in vitro and that polymerization is not inhibited by the blocking agent A22. As observed for MreB from B. subtilis, chlamydial MreB does not require ATP for polymerization but is capable of ATP hydrolysis in phosphate release assays. Co-pelleting and bacterial two-hybrid experiments indicate that MreB from C. pneumoniae interacts with MurF, MraY and MurG, three key components in lipid II biosynthesis. In addition, MreB polymerization is improved in the presence of MurF. These findings suggest that MreB is involved in tethering biosynthesis of lipid II and as such may be necessary for maintaining functional divisome machinery in Chlamydiaceae.
While chlamydial genomes lack the alr gene encoding for alanine racemase that is responsible for D-alanine synthesis in other bacteria, several findings suggest that D-alanine should be part of chlamydial lipid II. Preliminary results on the racemization activity of the chlamydial GlyA show that the protein may provide D-alanine in chlamydiae.
The sensitivity of chlamydiae to penicillin and other β-lactam antibiotics is attributed to the presence of three PBPs encoded in the chlamydial genome. Chlamydiae have three PBPs homologous to PBP2, 3 and 6a from E. coli. PBP2 and PBP3 are essential mono-functional transpeptidases in E. coli. On the other hand, the E. coli PBP6a shows a weak in vitro carboxypeptidase activity. To get insights into the functionality of the chlamydial PBPs, the three proteins were cloned, over-produced and purified successfully. Further optimizations for the established assays are still required to get clear insights into the functionality of these proteins in Chlamydiaceae.},
url = {https://hdl.handle.net/20.500.11811/5391}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-29973,
author = {{Ahmed Hassan Ahmed Gaballah}},
title = {Molecular Analysis of the Chlamydial Anomaly : Role of the cytoskeleton protein MreB, the serine hydroxymethyl transferase GlyA and the penicillin binding proteins from Chlamydophila pneumoniae},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2012,
month = sep,
note = {Chlamydiae are Gram-negative, obligate intracellular pathogens. Because of the isotonic intracellular niche of the host, a functional cell wall is dispensable in these endobacteria. Surprisingly, a nearly complete pathway for the biosynthesis of cell wall precursor lipid II is conserved in chlamydiae. Moreover, the organism is susceptible to penicillin and other antibiotics that inhibit cell wall biosynthesis. This paradox, which is known as chlamydial anomaly, raises the question about the essential role of cell wall building block lipid II in maintaining bacterial growth and replication.
Cell wall biosynthesis and cell division in prokaryotes are driven by tightly coordinated, partially overlapped machineries. Therefore, it was hypothesized that lipid II, which is an essential part of both processes, is required to maintain a functional divisome in chlamydiae.
The bacterial actin homolog MreB, which is found exclusively in non-spherically shaped bacteria, is considered to organize the incorporation of cell wall precursors into the side-wall, whereas the tubulin homolog FtsZ, which is ubiquitously distributed in almost all bacteria, is known to tether incorporation of cell wall building blocks at the developing septum. Unexpectedly, chlamydial genomes lack the essential central organizer of cell division ftsZ, but harbor despite of its spherical shape the rod-shape determining mreB.
In this project, it was investigated how chlamydiae can divide in absence of FtsZ. It was demonstrated that cytoskeletal protein MreB might compensate for FtsZ for maintaining a functional divisome machinery in chlamydiae. It was shown that chlamydial MreB polymerizes in vitro and that polymerization is not inhibited by the blocking agent A22. As observed for MreB from B. subtilis, chlamydial MreB does not require ATP for polymerization but is capable of ATP hydrolysis in phosphate release assays. Co-pelleting and bacterial two-hybrid experiments indicate that MreB from C. pneumoniae interacts with MurF, MraY and MurG, three key components in lipid II biosynthesis. In addition, MreB polymerization is improved in the presence of MurF. These findings suggest that MreB is involved in tethering biosynthesis of lipid II and as such may be necessary for maintaining functional divisome machinery in Chlamydiaceae.
While chlamydial genomes lack the alr gene encoding for alanine racemase that is responsible for D-alanine synthesis in other bacteria, several findings suggest that D-alanine should be part of chlamydial lipid II. Preliminary results on the racemization activity of the chlamydial GlyA show that the protein may provide D-alanine in chlamydiae.
The sensitivity of chlamydiae to penicillin and other β-lactam antibiotics is attributed to the presence of three PBPs encoded in the chlamydial genome. Chlamydiae have three PBPs homologous to PBP2, 3 and 6a from E. coli. PBP2 and PBP3 are essential mono-functional transpeptidases in E. coli. On the other hand, the E. coli PBP6a shows a weak in vitro carboxypeptidase activity. To get insights into the functionality of the chlamydial PBPs, the three proteins were cloned, over-produced and purified successfully. Further optimizations for the established assays are still required to get clear insights into the functionality of these proteins in Chlamydiaceae.},
url = {https://hdl.handle.net/20.500.11811/5391}
}
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