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Mechanisms of bacterial morphogenesis and their subversion by phages
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ScienceDirect Mechanisms of bacterial morphogenesis and their subversion by phages Editorial overview Thomas G Bernhardt and Waldemar Vollmer Current Opinion in Microbiology 2013, 16:728–730 For a complete overview see the Issue Available online 1st November 2013 1369-5274/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2013.10.004
Thomas G Bernhardt Department of Microbiology and Immunobiology, Harvard Medical School, HIM Building Room 1026, 4 Blackfan Circle, Boston, MA 02115, USA e-mail: [email protected] Thomas G Bernhardt is an Associate Professor in the Department of Microbiology and Immunobiology at Harvard Medical School. His interests are in understanding how bacterial cells assemble their cell wall and how the process can be disrupted for the development of novel antibacterial therapies. Research in the Bernhardt lab combines the use of molecular genetic screens and selections with physiology, biochemistry, and genomic approaches to uncover regulatory factors governing cellular morphogenic processes and determine their mechanism of action.
Waldemar Vollmer Institute for Cell and Molecular Biosciences, The Centre for Bacterial Cell Biology, Baddiley-Clark Building, Newcastle University, Richardson Road, Newcastle upon Tyne NE2 4AX, United Kingdom e-mail: [email protected] Waldemar Vollmer is Professor of Bacterial Biochemistry at the recently established Centre for Bacterial Cell Biology at Newcastle University, United Kingdom. His research aims to decipher the molecular mechanisms of peptidoglycan growth by studying the activities and interactions of peptidoglycan enzymes. He also investigates how bacterial cell shape is generated and maintained by the spatiotemporal regulation of peptidoglycan growth. His laboratory collaborates with other groups on various aspects of the bacterial cell wall including novel cell wall degrading enzymes and cell wall structure in Grampositive and Gram-negative bacteria.
Current Opinion in Microbiology 2013, 16:728–730
Cellular replication involves much more than the duplication and segregation of the genetic material. New cell surface must be built to house freshly minted chromosomes, and the expanded cellular compartment must be divided to generate daughter cells capable of initiating the cycle anew. These morphogenetic processes have fascinated scientists since cells were first visualized. Yet, even in relatively simple organisms like bacteria, the mechanisms underlying growth, cell division, and cell shape maintenance are only just beginning to be elucidated. Bacterial cell shape is maintained by the peptidoglycan (PG) sacculus, a netlike macromolecule that encases the cytoplasmic membrane to protect the cell from osmotic lysis. Next to the nucleoid, the PG sacculus is the only other cellular component that exists as a single molecule that must be faithfully duplicated in each cell cycle. Growth and division of the sacculus requires both synthetic and hydrolytic enzymes and is controlled from inside the cell by dynamic cytoskeletal structures. Although many of the players involved in these morphogenetic processes have been identified, we still know very little about how they all work together to build the cell wall and even less about how PG growth is coordinated with the replication of the nucleoid and the synthesis of the other cell envelope layers. The reviews in this issue highlight recent advances in our understanding of bacterial morphogenesis and how viruses co-opt or subvert the process during their replication cycle. Major progress has been stimulated by the study of nontraditional model organisms and the development and application of new methods and technologies. High-throughput, genome-wide screens for mutant phenotypes and genetic interactions have identified new factors involved in the cell growth and division machineries. Super-resolution and time-lapse fluorescence microscopy along with electron crytotomography have revealed the cellular localization and dynamics of cell surface assembly factors and cytoskeletal proteins at previously unthinkable resolution. Finally, the combination of biochemical and genetic studies with the steadily growing number of new protein structures is revealing how the cell is built in atomic detail. Some of the most exciting new tools for studying cell wall growth are based on old discoveries. For example, in the early 1990s Miguel de Pedro discovered that Escherichia coli incorporates certain D-amino acids like Dmethionine, D-leucine or D-tyrosine, into its PG sacculus. He subsequently developed the elegant technique of D-cysteine labeling of PG to follow the growth of the sacculus through the cell cycle. His work demonstrated www.sciencedirect.com
Editorial overview Bernhardt and Vollmer 729
that E. coli cells insert new PG material along their lateral wall in diffuse patches to elongate. This mode of elongation is followed by a brief phase of zonal elongation via the highly localized insertion of new material at midcell, which is best seen in cells with blocked septal PG synthesis, and finally the division phase in which the new daughter cell poles are assembled. The major drawback to the D-cysteine labeling method, however, is that it requires purification of sacculi prior to labeling. The VanNieuwenhze and Bertozzi labs have overcome this problem through the synthesis of a collection of ‘‘clickable’’ or fluorescently labeled D-amino acids capable of labeling sites of new cell wall growth in a wide variety of bacteria. The value of these novel labeling tools can hardly be underestimated. The review by Cava et al. highlights the different modes of cell wall growth in rod-shaped bacteria, expanding from the E. coli model via Caulobacter crescentus to other aproteobacteria. During the cell cycle, all of these species alternate between elongation and division modes of cell wall synthesis. However, the amount of the cell cycle dedicated to the different modes of elongation and even the mechanism of cell elongation itself varies from species to species. As discussed by Cava et al., both E. coli and C. crescentus insert new PG material along their cell length in a dispersed mode of growth directed by the actin-like cytoskeletal protein MreB. Where they differ is in the timing of the switch to the zonal mode of elongation from midcell under the direction of the tubulin-like cytoskeletal protein FtsZ. This zonal phase of growth accounts for a substantial fraction of total elongation in C. crescentus but hardly contributes to elongation in E. coli. Interestingly, other members of the a-proteobacteria like Agrobacterium tumefaciens elongate via an MreB-independent mechanism involving growth from the cell pole. Such distinct growth mechanisms can now be readily visualized using the fluorescently labeled D-amino acids described above. Current work is now focused on understanding how the various rod-shaped cells position and regulate their cell wall elongation machinery, and how they coordinate elongation with other major cell cycle events. Irrespective of their shape, most bacterial cells follow cell expansion with cell division. The review of den Blaauwen discusses the mechanisms by which the assembly of the division machinery, called the divisome, is regulated in different species. Although the core components of the divisome are highly conserved, the regulatory mechanisms governing Z-ring assembly and dynamics vary significantly between species, leading to differences in the timing of division and the placement of the division site. An extreme example that is discussed occurs in the rodshaped Laxus oneistus, a g-proteobacterium associated with the epidermis of a marine nematode that surprisingly divides along the long axis of the cell instead of the short www.sciencedirect.com
axis like E. coli and other commonly studied rod-shaped bacteria. Szwedziak and Lo¨we bring the discussion of cell elongation and cell division together through an exploration of the potential evolutionary relatedness of the underlying machineries driving the processes. The striking similarity between the two machines was recently revealed by structural and biochemical studies demonstrating that both MreB and the divisome factor FtsA are actin-like proteins forming polymers that associate with the membrane via amphipathic helical domains. Thus, as discussed in the review, these two proteins are likely playing equivalent roles within their respective complex, and the two machines may control PG biogenesis in a very similar way. The major difference apparently lies in the addition of FtsZ and several other factors to the equation in order for the divisome to promote a constrictive mode of growth as opposed to elongation. The cell division process generates a new cell pole. Many rod-shaped cells take advantage of the uniqueness of the cell pole relative to the lateral cell body to localize important cellular functions. Davis and Waldor review recent advances in our understanding of how cells establish polar identity. They discuss the discovery of new factors that govern the polar localization of chemotaxis and signaling proteins in Vibrio cholerae and C. crescentus, including the key role played by nucleotide-dependent switches. The interesting case of polarity switching in motile cells of Myxococcus xanthus is also covered. For many years, studies of cell wall assembly and cell shape have focused on the activity and regulation of PG synthases. However, in recent years there has been a growing appreciation for the critical roles played by enzymes that cleave bonds in the cell wall network in these processes. This shift in emphasis has been stimulated greatly by the discovery of essential sets of PG hydrolases required for cell growth in several organisms and the identification of related enzymes responsible for generating the helical shape of Helicobacter pylori and Campylobacter jejuni. Lee and Huang review the essential role played by PG hydrolases in cell wall growth and discuss how the relationship between cell wall synthesis and hydrolysis can be better understood through the combination of time-lapse imaging and computational modeling. Frirdich and Gaynor provide a complementary discussion of the role of PG hydrolases in cell shape determination and how the activities of these enzymes can impact immune signaling and disease outcome. In addition to cell wall, Gram-negative bacteria are surrounded by a second (outer) membrane. This membrane is unique in that it is asymmetric with the inner leaflet being composed of normal phospholipids and the outer leaflet of lipopolysaccharide (LPS). The review by Kahne Current Opinion in Microbiology 2013, 16:728–730
730 Growth and development: prokaryotes
and co-workers covers advances in our understanding of LPS transport from its site of synthesis in the inner membrane to its final destination in the outer membrane. Also discussed is the realization that, despite what was once commonly believed, LPS is not essential for the growth of all Gram-negative organisms. The authors provide possible explanations for this observation and its implications for the development of antimicrobial therapies based on the inhibition of LPS synthesis or transport. As in eukaryotic systems, the study of viruses that infect bacteria has provided many fundamental insights related to the growth and development of the host organism. Two reviews in this issue focus on the bacteriophage life cycle. Erb and Pogliano discuss how phages either co-opt host cytoskeletal proteins or use their own homologues to promote their efficient replication within bacterial cells,
Current Opinion in Microbiology 2013, 16:728–730
and Young updates us on how phage encoded proteins disrupt the cell surface to promote the release of progeny virions into the surrounding milieu to find fresh host cells to infect. Interestingly, in both instances, striking parallels to strategies used by eukaryotic viruses are revealed such as the formation of viral replication factories and the use of membrane fusion proteins. Based on the reviews presented in this issue, it is clear that our knowledge and understanding of bacterial morphogenesis is rapidly expanding, both from the perspective of the bacteria themselves and the viruses that infect them. With the advent of new tools and techniques as well as the continued expansion of the field into diverse organisms, we can certainly expect significant advances and exciting new discoveries in the years to come.
www.sciencedirect.com
ScienceDirect Mechanisms of bacterial morphogenesis and their subversion by phages Editorial overview Thomas G Bernhardt and Waldemar Vollmer Current Opinion in Microbiology 2013, 16:728–730 For a complete overview see the Issue Available online 1st November 2013 1369-5274/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2013.10.004
Thomas G Bernhardt Department of Microbiology and Immunobiology, Harvard Medical School, HIM Building Room 1026, 4 Blackfan Circle, Boston, MA 02115, USA e-mail: [email protected] Thomas G Bernhardt is an Associate Professor in the Department of Microbiology and Immunobiology at Harvard Medical School. His interests are in understanding how bacterial cells assemble their cell wall and how the process can be disrupted for the development of novel antibacterial therapies. Research in the Bernhardt lab combines the use of molecular genetic screens and selections with physiology, biochemistry, and genomic approaches to uncover regulatory factors governing cellular morphogenic processes and determine their mechanism of action.
Waldemar Vollmer Institute for Cell and Molecular Biosciences, The Centre for Bacterial Cell Biology, Baddiley-Clark Building, Newcastle University, Richardson Road, Newcastle upon Tyne NE2 4AX, United Kingdom e-mail: [email protected] Waldemar Vollmer is Professor of Bacterial Biochemistry at the recently established Centre for Bacterial Cell Biology at Newcastle University, United Kingdom. His research aims to decipher the molecular mechanisms of peptidoglycan growth by studying the activities and interactions of peptidoglycan enzymes. He also investigates how bacterial cell shape is generated and maintained by the spatiotemporal regulation of peptidoglycan growth. His laboratory collaborates with other groups on various aspects of the bacterial cell wall including novel cell wall degrading enzymes and cell wall structure in Grampositive and Gram-negative bacteria.
Current Opinion in Microbiology 2013, 16:728–730
Cellular replication involves much more than the duplication and segregation of the genetic material. New cell surface must be built to house freshly minted chromosomes, and the expanded cellular compartment must be divided to generate daughter cells capable of initiating the cycle anew. These morphogenetic processes have fascinated scientists since cells were first visualized. Yet, even in relatively simple organisms like bacteria, the mechanisms underlying growth, cell division, and cell shape maintenance are only just beginning to be elucidated. Bacterial cell shape is maintained by the peptidoglycan (PG) sacculus, a netlike macromolecule that encases the cytoplasmic membrane to protect the cell from osmotic lysis. Next to the nucleoid, the PG sacculus is the only other cellular component that exists as a single molecule that must be faithfully duplicated in each cell cycle. Growth and division of the sacculus requires both synthetic and hydrolytic enzymes and is controlled from inside the cell by dynamic cytoskeletal structures. Although many of the players involved in these morphogenetic processes have been identified, we still know very little about how they all work together to build the cell wall and even less about how PG growth is coordinated with the replication of the nucleoid and the synthesis of the other cell envelope layers. The reviews in this issue highlight recent advances in our understanding of bacterial morphogenesis and how viruses co-opt or subvert the process during their replication cycle. Major progress has been stimulated by the study of nontraditional model organisms and the development and application of new methods and technologies. High-throughput, genome-wide screens for mutant phenotypes and genetic interactions have identified new factors involved in the cell growth and division machineries. Super-resolution and time-lapse fluorescence microscopy along with electron crytotomography have revealed the cellular localization and dynamics of cell surface assembly factors and cytoskeletal proteins at previously unthinkable resolution. Finally, the combination of biochemical and genetic studies with the steadily growing number of new protein structures is revealing how the cell is built in atomic detail. Some of the most exciting new tools for studying cell wall growth are based on old discoveries. For example, in the early 1990s Miguel de Pedro discovered that Escherichia coli incorporates certain D-amino acids like Dmethionine, D-leucine or D-tyrosine, into its PG sacculus. He subsequently developed the elegant technique of D-cysteine labeling of PG to follow the growth of the sacculus through the cell cycle. His work demonstrated www.sciencedirect.com
Editorial overview Bernhardt and Vollmer 729
that E. coli cells insert new PG material along their lateral wall in diffuse patches to elongate. This mode of elongation is followed by a brief phase of zonal elongation via the highly localized insertion of new material at midcell, which is best seen in cells with blocked septal PG synthesis, and finally the division phase in which the new daughter cell poles are assembled. The major drawback to the D-cysteine labeling method, however, is that it requires purification of sacculi prior to labeling. The VanNieuwenhze and Bertozzi labs have overcome this problem through the synthesis of a collection of ‘‘clickable’’ or fluorescently labeled D-amino acids capable of labeling sites of new cell wall growth in a wide variety of bacteria. The value of these novel labeling tools can hardly be underestimated. The review by Cava et al. highlights the different modes of cell wall growth in rod-shaped bacteria, expanding from the E. coli model via Caulobacter crescentus to other aproteobacteria. During the cell cycle, all of these species alternate between elongation and division modes of cell wall synthesis. However, the amount of the cell cycle dedicated to the different modes of elongation and even the mechanism of cell elongation itself varies from species to species. As discussed by Cava et al., both E. coli and C. crescentus insert new PG material along their cell length in a dispersed mode of growth directed by the actin-like cytoskeletal protein MreB. Where they differ is in the timing of the switch to the zonal mode of elongation from midcell under the direction of the tubulin-like cytoskeletal protein FtsZ. This zonal phase of growth accounts for a substantial fraction of total elongation in C. crescentus but hardly contributes to elongation in E. coli. Interestingly, other members of the a-proteobacteria like Agrobacterium tumefaciens elongate via an MreB-independent mechanism involving growth from the cell pole. Such distinct growth mechanisms can now be readily visualized using the fluorescently labeled D-amino acids described above. Current work is now focused on understanding how the various rod-shaped cells position and regulate their cell wall elongation machinery, and how they coordinate elongation with other major cell cycle events. Irrespective of their shape, most bacterial cells follow cell expansion with cell division. The review of den Blaauwen discusses the mechanisms by which the assembly of the division machinery, called the divisome, is regulated in different species. Although the core components of the divisome are highly conserved, the regulatory mechanisms governing Z-ring assembly and dynamics vary significantly between species, leading to differences in the timing of division and the placement of the division site. An extreme example that is discussed occurs in the rodshaped Laxus oneistus, a g-proteobacterium associated with the epidermis of a marine nematode that surprisingly divides along the long axis of the cell instead of the short www.sciencedirect.com
axis like E. coli and other commonly studied rod-shaped bacteria. Szwedziak and Lo¨we bring the discussion of cell elongation and cell division together through an exploration of the potential evolutionary relatedness of the underlying machineries driving the processes. The striking similarity between the two machines was recently revealed by structural and biochemical studies demonstrating that both MreB and the divisome factor FtsA are actin-like proteins forming polymers that associate with the membrane via amphipathic helical domains. Thus, as discussed in the review, these two proteins are likely playing equivalent roles within their respective complex, and the two machines may control PG biogenesis in a very similar way. The major difference apparently lies in the addition of FtsZ and several other factors to the equation in order for the divisome to promote a constrictive mode of growth as opposed to elongation. The cell division process generates a new cell pole. Many rod-shaped cells take advantage of the uniqueness of the cell pole relative to the lateral cell body to localize important cellular functions. Davis and Waldor review recent advances in our understanding of how cells establish polar identity. They discuss the discovery of new factors that govern the polar localization of chemotaxis and signaling proteins in Vibrio cholerae and C. crescentus, including the key role played by nucleotide-dependent switches. The interesting case of polarity switching in motile cells of Myxococcus xanthus is also covered. For many years, studies of cell wall assembly and cell shape have focused on the activity and regulation of PG synthases. However, in recent years there has been a growing appreciation for the critical roles played by enzymes that cleave bonds in the cell wall network in these processes. This shift in emphasis has been stimulated greatly by the discovery of essential sets of PG hydrolases required for cell growth in several organisms and the identification of related enzymes responsible for generating the helical shape of Helicobacter pylori and Campylobacter jejuni. Lee and Huang review the essential role played by PG hydrolases in cell wall growth and discuss how the relationship between cell wall synthesis and hydrolysis can be better understood through the combination of time-lapse imaging and computational modeling. Frirdich and Gaynor provide a complementary discussion of the role of PG hydrolases in cell shape determination and how the activities of these enzymes can impact immune signaling and disease outcome. In addition to cell wall, Gram-negative bacteria are surrounded by a second (outer) membrane. This membrane is unique in that it is asymmetric with the inner leaflet being composed of normal phospholipids and the outer leaflet of lipopolysaccharide (LPS). The review by Kahne Current Opinion in Microbiology 2013, 16:728–730
730 Growth and development: prokaryotes
and co-workers covers advances in our understanding of LPS transport from its site of synthesis in the inner membrane to its final destination in the outer membrane. Also discussed is the realization that, despite what was once commonly believed, LPS is not essential for the growth of all Gram-negative organisms. The authors provide possible explanations for this observation and its implications for the development of antimicrobial therapies based on the inhibition of LPS synthesis or transport. As in eukaryotic systems, the study of viruses that infect bacteria has provided many fundamental insights related to the growth and development of the host organism. Two reviews in this issue focus on the bacteriophage life cycle. Erb and Pogliano discuss how phages either co-opt host cytoskeletal proteins or use their own homologues to promote their efficient replication within bacterial cells,
Current Opinion in Microbiology 2013, 16:728–730
and Young updates us on how phage encoded proteins disrupt the cell surface to promote the release of progeny virions into the surrounding milieu to find fresh host cells to infect. Interestingly, in both instances, striking parallels to strategies used by eukaryotic viruses are revealed such as the formation of viral replication factories and the use of membrane fusion proteins. Based on the reviews presented in this issue, it is clear that our knowledge and understanding of bacterial morphogenesis is rapidly expanding, both from the perspective of the bacteria themselves and the viruses that infect them. With the advent of new tools and techniques as well as the continued expansion of the field into diverse organisms, we can certainly expect significant advances and exciting new discoveries in the years to come.
www.sciencedirect.com