- Email: [email protected]
Editorial overview: Cell regulation: When you think you know it all, there is another layer to be discovered
Available online at www.sciencedirect.com
ScienceDirect Editorial overview: Cell regulation: When you think you know it all, there is another layer to be discovered Carol A Gross and Angelika Gru¨ndling Current Opinion in Microbiology 2015, 24:v–vii For a complete overview see the Issue Available online 20th February 2015 http://dx.doi.org/10.1016/j.mib.2015.02.001 1369-5274/# 2015 Elsevier Ltd. All rights reserved.
Carol A Gross Department of Microbiology and Immunology, Department of Cell and Tissue Biology, California Insitute for Quantitative Biology, University of California at San Francisco Genentech Hall, Room S372E, San Francisco, CA 94158, USA e-mail: [email protected] Carol A Gross is a professor at the University of California at San Francisco, where she studies gene function and regulation. Historically, she addressed cellular integration of responses by studying the transcriptional apparatus and stress responses. At present she is combining functional genomics approaches with molecular dissection to dissect gene function and understand cellular connections between processes.
Angelika Gru¨ndling Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, Flowers Building Room 3.21, Armstrong Road, London SW7 2AZ, UK e-mail: [email protected] Angelika Gru¨ndling is a reader in molecular microbiology at Imperial College London. The research in her lab focuses on the investigation of fundamental processes that are essential for the growth of Gram-positive bacterial pathogens. She combines genetic, biochemical and structural approaches to provide mechanistic insight into cell wall synthesis and nucleotide signaling pathways in Staphylococcus aureus and Listeria monocytogenes.
www.sciencedirect.com
Welcome to the 2015 Cell regulation issue in Current Opinion in Microbiology. When we discussed what to include in the issue, we were struck by the palpable sense of excitement in the microbial community. Capturing the excitement of the moment was our primary motivation for putting together the contents of the issue. Four broad themes emerge in the work presented here. First, there are amazing new insights into important problems that have been studied since the beginning of modern microbiology. Second, and possibly even more surprising, even the best-studied processes are being found to have missing layers. Third, the importance of an evolutionary/crossorganism perspective is becoming increasingly evident. In this regard, we note that many of the articles in this issue deal with multiple types of bacteria and some even cross the prokaryotic–eukaryotic divide. We attribute some of this to modern technologies, which facilitate cross-species comparisons, with very important outcomes. Finally, only touched on in this issue, are the profound insights coming from studying interactions among bacteria, and between the microbiome and host organisms. The first six articles in this issue deal with features of bacterial growth that have been recognized for decades, but have recently reached new levels of understanding. We begin with the bacterial ‘growth law’ formulated over 50 years ago by microbiologists of the ‘Danish school’ [1]. This group observed that bacterial cell size and composition correlate with the growth rate of bacteria. Vadia and Levin now report on the molecular underpinnings of the growth law and raise new questions about the factors contributing to the law. In a closely related review, Tsang and Bernhardt tackle the cell division process mediated by the divisome. The definition of the divisome dates to the 1990s, when FtsZ was visualized at the midcell using immunogold labeling [2,3]. Unraveling the divisome mechanism of action is proving to be quite challenging, as the complex is dynamic and comprises of upwards of 35 proteins, some with semi-redundant functions, but as described here progress is being made. In addition to reporting on the molecular function of some of the divisome proteins, this review also considers the larger questions of how the divisome coordinates with replication and cell constriction. The idea that radiation-induced mutations in the genome are repaired differentially was first put forward by Evelyn Witkin in a seminal article almost 50 years ago [4]. Later, this phenomenon was traced to a specialized form of nucleotide excision repair (NER) called transcription coupled repair (TCR) and linked to the action of the protein Mfd [5]. Kamarthapu and Nudler revisit TCR and report compelling evidence that a major player in the TCR pathway in Escherichia coli is UvrD, a helicase component of the NER that binds RNA polymerase stoichiometrically — it’s a fascinating story! Transitioning to transcription, Current Opinion in Microbiology 2015, 24:v–vii
vi Cell regulation
Landick et al. take on the reality that prokaryotic transcription, like its eukaryotic counterpart, is carried out in vivo on DNA compacted by interacting with nucleoid associated proteins. First investigated in the 1980s [6], it was shown that the Histone-like nucleoid structuring protein H-NS sculpts DNA and usually negatively regulates gene expression. Using genomic approaches, Grainger, Landick and colleagues explore the often antagonistic relationship between H-NS and RNA polymerase. The discovery and characterization of polyphosphate was the last great scientific chapter in Arthur Kornberg’s life [7]. What was confusing was the multiplicity of phenotypes observed for cells that lacked the ability to make polyphosphate. Interestingly, that was the same early problem faced by pioneering investigators of protein chaperones (see for instance [8]). Gray and Jakob now lay out the evidence that polyphosphate is actually an ancient chaperone. Given its ancient lineage, polyphosphate chaperoning may have preceded the development of modern protein chaperones. Finally, Imlay discusses the mode of action of bactericidal antibiotics, a topic that has been investigated since the beginning of the antibiotic era. Recent evidence led James Collins to propose a unifying killing mechanism for antibiotics: release of reactive oxygen species. The data supporting this view have recently been reviewed [9]. However, the field has not yet converged on a unified view, and James Imlay cogently lays out the arguments for the opposing view here. Young students take note: many longstanding scientific issues are far from settled and worth pursuing. The next five articles report on new layers of long-known fundamental biological processes, by presenting either the evidence for these layers, or the methods that identified them. One thing is clear — lots of work remains to be done! The first two of these articles deal with transcription, and the fact that classic measurements assume that expression is evenly divided across cells. However, Martins and Locke now show how heterogeneous expression across single cells enables bet-hedging strategies and phenotypic diversity and how heterogeneity enhances functionality of the population as a whole. This is followed by an article by Castillo-Hair et al. discussing the tools used in synthetic biology that are enabling us to rigorously define circuits, including important population heterogeneity that facilitates survival. The final three articles in this section deal with RNA, in one guise or another. Herman and colleagues discuss a hitherto unacknowledged role of mRNAs: how errors in transcripts acquired as a consequence of the transcription process itself, or ‘epimutations’, can lead to phenotypic changes and sometimes even result in heritable changes in cellular behavior. Miyakoshi et al. tell us that to date, we have been examining only the role of a restricted fraction of the small non-coding RNAs (sRNAs) in bacteria — those that originate from intercistronic regions of the genome, as many sRNAs originate from the 30 ends of existing genes. Current Opinion in Microbiology 2015, 24:v–vii
Indeed, this location is attractive for evolution of new sRNAs because such transcripts will already have the intrinsic termination signal that is part of sRNAs. Finally, Li reviews results of genome-wide approaches to translation, which indicate that we currently are unable to predict translation rates. Synthetic gene libraries revealed that mRNA structure right around the start site influences translation. Ribosome profiling allows us to measure the rate of protein synthesis on a genome wide level, and has revealed that the number of protein molecules produced per mRNA, or translation efficiency, varies dramatically, even for two open reading frames (ORF) in the same mRNA. Although bacteria precisely specify the translation efficiency of each ORF, the two identified specifiers — strength of the Shino–Dalgarno sequence and the RNA structure around the start site account for only a small amount of the variability. Thus, at present, the rules governing translation are incompletely specified. Our third section points out the importance of an evolutionary perspective. Pisithkul et al. bring us up to date on metabolic control. Long a poster child for allosteric regulation, this review highlights that post-translational modifications common in eukaryotes are surprisingly often used to regulate the activity of metabolic enzymes in bacteria. Continuing on this theme, Dworkin tells us about the importance of eukaryotic-type serine threonine kinases (e-STKs), which are widely distributed in bacteria. These pathways differ from the well-studied 2-component systems (TCS), not only in the moiety phosphorylated, but also because they lack dedicated regulatory proteins. Instead, the e-STKs can phosphorylate TCS to achieve transcriptional effects, and can function in large networks. With the article by Salzar and Laub, we transition to a study on the evolution of TCS. Given that the cell contains multiple TCS, the question arises how a new TCS in a bacterial clade becomes insulated from the already existing TCS present in the cell. In addition, the dynamics of the response, which can sometimes result in population heterogeneity, are discussed in this article. Continuing in the transcription realm, Fimlaid and Shen compare sporulation in Clostridia to that in the better-studied Firmicute, Bacillus subtilis. Despite the fact that the sigma factors driving sporulation are conserved, their mechanism of action is surprisingly different, indicating that the rewiring of regulation demonstrated in eukaryotes is also true in prokaryotic species. Finally, Liu et al. compare the roles of ‘magic spot’ or (p)ppGpp in E. coli and B. subtilis, demonstrating the same theme — the molecule stays the same, but its mechanism of action may change. The final small section has two important articles on interactions above the single cell level. Lyons and Kolter probe the origins of multicellularity and the selective pressures that led to their evolution. Lastly, Yoon et al. look at the diversity of the microbiota, especially in www.sciencedirect.com
Editorial overview Gross and Gru¨ndling vii
simpler eukaryotic host organisms. The authors discussed how functional genomic and metagenomic approaches can reveal colonization strategies, nutritional processing and modulation of eukaryotic cell patterns of expression. We feel it is appropriate to end on this small window into the promise of very important investigations yet to come.
References 1.
Schaechter M, Maaloe O, Kjeldgaard NO: Dependency on medium and temperature of cell size and chemical composition during balanced grown of Salmonella typhimurium. J Gen Microbiol 1958, 19:592-606.
2.
Bi EF, Lutkenhaus J: FtsZ ring structure associated with division in Escherichia coli. Nature 1991, 354:161-164.
3.
Nanninga N: Cell division and peptidoglycan assembly in Escherichia coli. Mol Microbiol 1991, 5:791-795.
www.sciencedirect.com
4.
Witkin EM: Radiation-induced mutations and their repair. Science 1966, 152:1345-1352.
5.
Selby CP, Sancar A: Structure and function of transcriptionrepair coupling factor: I. Structural domains and binding properties. J Biol Chem 1995, 270:4882-4889.
6.
Spassky A, Rimsky S, Garreau H, Buc H: H1a, an E. coli DNAbinding protein which accumulates in stationary phase, strongly compacts DNA in vitro. Nucleic Acids Res 1984, 12:5321-5340.
7.
Ahn K, Kornberg A: Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate. J Biol Chem 1990, 265:11734-11739.
8.
Bardwell JC, Craig EA: Major heat shock gene of Drosophila and the Escherichia coli heat-induced danK gene are homologous. Proc Natl Acad Sci U S A 1984, 81:848-852.
9.
Dwyer DJ, Collins JJ, Walker GC: Unraveling the physiological complexities of antibiotic lethality. Annu Rev Pharmacol Toxicol 2015, 55:313-332.
Current Opinion in Microbiology 2015, 24:v–vii
ScienceDirect Editorial overview: Cell regulation: When you think you know it all, there is another layer to be discovered Carol A Gross and Angelika Gru¨ndling Current Opinion in Microbiology 2015, 24:v–vii For a complete overview see the Issue Available online 20th February 2015 http://dx.doi.org/10.1016/j.mib.2015.02.001 1369-5274/# 2015 Elsevier Ltd. All rights reserved.
Carol A Gross Department of Microbiology and Immunology, Department of Cell and Tissue Biology, California Insitute for Quantitative Biology, University of California at San Francisco Genentech Hall, Room S372E, San Francisco, CA 94158, USA e-mail: [email protected] Carol A Gross is a professor at the University of California at San Francisco, where she studies gene function and regulation. Historically, she addressed cellular integration of responses by studying the transcriptional apparatus and stress responses. At present she is combining functional genomics approaches with molecular dissection to dissect gene function and understand cellular connections between processes.
Angelika Gru¨ndling Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, Flowers Building Room 3.21, Armstrong Road, London SW7 2AZ, UK e-mail: [email protected] Angelika Gru¨ndling is a reader in molecular microbiology at Imperial College London. The research in her lab focuses on the investigation of fundamental processes that are essential for the growth of Gram-positive bacterial pathogens. She combines genetic, biochemical and structural approaches to provide mechanistic insight into cell wall synthesis and nucleotide signaling pathways in Staphylococcus aureus and Listeria monocytogenes.
www.sciencedirect.com
Welcome to the 2015 Cell regulation issue in Current Opinion in Microbiology. When we discussed what to include in the issue, we were struck by the palpable sense of excitement in the microbial community. Capturing the excitement of the moment was our primary motivation for putting together the contents of the issue. Four broad themes emerge in the work presented here. First, there are amazing new insights into important problems that have been studied since the beginning of modern microbiology. Second, and possibly even more surprising, even the best-studied processes are being found to have missing layers. Third, the importance of an evolutionary/crossorganism perspective is becoming increasingly evident. In this regard, we note that many of the articles in this issue deal with multiple types of bacteria and some even cross the prokaryotic–eukaryotic divide. We attribute some of this to modern technologies, which facilitate cross-species comparisons, with very important outcomes. Finally, only touched on in this issue, are the profound insights coming from studying interactions among bacteria, and between the microbiome and host organisms. The first six articles in this issue deal with features of bacterial growth that have been recognized for decades, but have recently reached new levels of understanding. We begin with the bacterial ‘growth law’ formulated over 50 years ago by microbiologists of the ‘Danish school’ [1]. This group observed that bacterial cell size and composition correlate with the growth rate of bacteria. Vadia and Levin now report on the molecular underpinnings of the growth law and raise new questions about the factors contributing to the law. In a closely related review, Tsang and Bernhardt tackle the cell division process mediated by the divisome. The definition of the divisome dates to the 1990s, when FtsZ was visualized at the midcell using immunogold labeling [2,3]. Unraveling the divisome mechanism of action is proving to be quite challenging, as the complex is dynamic and comprises of upwards of 35 proteins, some with semi-redundant functions, but as described here progress is being made. In addition to reporting on the molecular function of some of the divisome proteins, this review also considers the larger questions of how the divisome coordinates with replication and cell constriction. The idea that radiation-induced mutations in the genome are repaired differentially was first put forward by Evelyn Witkin in a seminal article almost 50 years ago [4]. Later, this phenomenon was traced to a specialized form of nucleotide excision repair (NER) called transcription coupled repair (TCR) and linked to the action of the protein Mfd [5]. Kamarthapu and Nudler revisit TCR and report compelling evidence that a major player in the TCR pathway in Escherichia coli is UvrD, a helicase component of the NER that binds RNA polymerase stoichiometrically — it’s a fascinating story! Transitioning to transcription, Current Opinion in Microbiology 2015, 24:v–vii
vi Cell regulation
Landick et al. take on the reality that prokaryotic transcription, like its eukaryotic counterpart, is carried out in vivo on DNA compacted by interacting with nucleoid associated proteins. First investigated in the 1980s [6], it was shown that the Histone-like nucleoid structuring protein H-NS sculpts DNA and usually negatively regulates gene expression. Using genomic approaches, Grainger, Landick and colleagues explore the often antagonistic relationship between H-NS and RNA polymerase. The discovery and characterization of polyphosphate was the last great scientific chapter in Arthur Kornberg’s life [7]. What was confusing was the multiplicity of phenotypes observed for cells that lacked the ability to make polyphosphate. Interestingly, that was the same early problem faced by pioneering investigators of protein chaperones (see for instance [8]). Gray and Jakob now lay out the evidence that polyphosphate is actually an ancient chaperone. Given its ancient lineage, polyphosphate chaperoning may have preceded the development of modern protein chaperones. Finally, Imlay discusses the mode of action of bactericidal antibiotics, a topic that has been investigated since the beginning of the antibiotic era. Recent evidence led James Collins to propose a unifying killing mechanism for antibiotics: release of reactive oxygen species. The data supporting this view have recently been reviewed [9]. However, the field has not yet converged on a unified view, and James Imlay cogently lays out the arguments for the opposing view here. Young students take note: many longstanding scientific issues are far from settled and worth pursuing. The next five articles report on new layers of long-known fundamental biological processes, by presenting either the evidence for these layers, or the methods that identified them. One thing is clear — lots of work remains to be done! The first two of these articles deal with transcription, and the fact that classic measurements assume that expression is evenly divided across cells. However, Martins and Locke now show how heterogeneous expression across single cells enables bet-hedging strategies and phenotypic diversity and how heterogeneity enhances functionality of the population as a whole. This is followed by an article by Castillo-Hair et al. discussing the tools used in synthetic biology that are enabling us to rigorously define circuits, including important population heterogeneity that facilitates survival. The final three articles in this section deal with RNA, in one guise or another. Herman and colleagues discuss a hitherto unacknowledged role of mRNAs: how errors in transcripts acquired as a consequence of the transcription process itself, or ‘epimutations’, can lead to phenotypic changes and sometimes even result in heritable changes in cellular behavior. Miyakoshi et al. tell us that to date, we have been examining only the role of a restricted fraction of the small non-coding RNAs (sRNAs) in bacteria — those that originate from intercistronic regions of the genome, as many sRNAs originate from the 30 ends of existing genes. Current Opinion in Microbiology 2015, 24:v–vii
Indeed, this location is attractive for evolution of new sRNAs because such transcripts will already have the intrinsic termination signal that is part of sRNAs. Finally, Li reviews results of genome-wide approaches to translation, which indicate that we currently are unable to predict translation rates. Synthetic gene libraries revealed that mRNA structure right around the start site influences translation. Ribosome profiling allows us to measure the rate of protein synthesis on a genome wide level, and has revealed that the number of protein molecules produced per mRNA, or translation efficiency, varies dramatically, even for two open reading frames (ORF) in the same mRNA. Although bacteria precisely specify the translation efficiency of each ORF, the two identified specifiers — strength of the Shino–Dalgarno sequence and the RNA structure around the start site account for only a small amount of the variability. Thus, at present, the rules governing translation are incompletely specified. Our third section points out the importance of an evolutionary perspective. Pisithkul et al. bring us up to date on metabolic control. Long a poster child for allosteric regulation, this review highlights that post-translational modifications common in eukaryotes are surprisingly often used to regulate the activity of metabolic enzymes in bacteria. Continuing on this theme, Dworkin tells us about the importance of eukaryotic-type serine threonine kinases (e-STKs), which are widely distributed in bacteria. These pathways differ from the well-studied 2-component systems (TCS), not only in the moiety phosphorylated, but also because they lack dedicated regulatory proteins. Instead, the e-STKs can phosphorylate TCS to achieve transcriptional effects, and can function in large networks. With the article by Salzar and Laub, we transition to a study on the evolution of TCS. Given that the cell contains multiple TCS, the question arises how a new TCS in a bacterial clade becomes insulated from the already existing TCS present in the cell. In addition, the dynamics of the response, which can sometimes result in population heterogeneity, are discussed in this article. Continuing in the transcription realm, Fimlaid and Shen compare sporulation in Clostridia to that in the better-studied Firmicute, Bacillus subtilis. Despite the fact that the sigma factors driving sporulation are conserved, their mechanism of action is surprisingly different, indicating that the rewiring of regulation demonstrated in eukaryotes is also true in prokaryotic species. Finally, Liu et al. compare the roles of ‘magic spot’ or (p)ppGpp in E. coli and B. subtilis, demonstrating the same theme — the molecule stays the same, but its mechanism of action may change. The final small section has two important articles on interactions above the single cell level. Lyons and Kolter probe the origins of multicellularity and the selective pressures that led to their evolution. Lastly, Yoon et al. look at the diversity of the microbiota, especially in www.sciencedirect.com
Editorial overview Gross and Gru¨ndling vii
simpler eukaryotic host organisms. The authors discussed how functional genomic and metagenomic approaches can reveal colonization strategies, nutritional processing and modulation of eukaryotic cell patterns of expression. We feel it is appropriate to end on this small window into the promise of very important investigations yet to come.
References 1.
Schaechter M, Maaloe O, Kjeldgaard NO: Dependency on medium and temperature of cell size and chemical composition during balanced grown of Salmonella typhimurium. J Gen Microbiol 1958, 19:592-606.
2.
Bi EF, Lutkenhaus J: FtsZ ring structure associated with division in Escherichia coli. Nature 1991, 354:161-164.
3.
Nanninga N: Cell division and peptidoglycan assembly in Escherichia coli. Mol Microbiol 1991, 5:791-795.
www.sciencedirect.com
4.
Witkin EM: Radiation-induced mutations and their repair. Science 1966, 152:1345-1352.
5.
Selby CP, Sancar A: Structure and function of transcriptionrepair coupling factor: I. Structural domains and binding properties. J Biol Chem 1995, 270:4882-4889.
6.
Spassky A, Rimsky S, Garreau H, Buc H: H1a, an E. coli DNAbinding protein which accumulates in stationary phase, strongly compacts DNA in vitro. Nucleic Acids Res 1984, 12:5321-5340.
7.
Ahn K, Kornberg A: Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate. J Biol Chem 1990, 265:11734-11739.
8.
Bardwell JC, Craig EA: Major heat shock gene of Drosophila and the Escherichia coli heat-induced danK gene are homologous. Proc Natl Acad Sci U S A 1984, 81:848-852.
9.
Dwyer DJ, Collins JJ, Walker GC: Unraveling the physiological complexities of antibiotic lethality. Annu Rev Pharmacol Toxicol 2015, 55:313-332.
Current Opinion in Microbiology 2015, 24:v–vii