Bacillus: Cellular and Molecular Biology (Third edition)
"a one-stop shop for a huge range of Bacillus-focused molecular biology" (Microbiology Today)
Caister Academic Press
Peter L. Graumann
University of Freiburg, Germany
viii + 470
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The third edition of this authoritative reference work is indispensable to Bacillus scientists and invaluable for anyone involved in bacterial cell biology. Revised, updated and expanded with two new chapters on motility and nucleotide regulation, the new edition provides up-to-date reviews on current Bacillus research. Subjects covered include chromosome replication, DNA repair, chromosome segregation, cell division, transcription and translation, RNA-mediated regulation, general and regulatory proteolysis, the MreB cytoskeleton, membrane proteins, the cell wall, sporulation, biofilms, multicellularity and social behaviour, competence and transformation, motility and nucleotide regulation.
An essential book for anyone involved in Bacillus and an invaluable reference work for those working in fields as diverse as medicine, biotechnology, agriculture, food and industry. A recommended acquisition for all microbiology laboratories.
"logical and easy to follow ... a one-stop shop for a huge range of Bacillus-focused molecular biology ... good comprehensive coverage ... figures and tables are clear and concise ... a very detailed but useful overview book" from Microbiology Today
Table of contents
1. Replication of the Bacillus subtilis Chromosome
Heath Murray, Tomas T. Richardson, Marie-Françoise Noirot-Gros, Patrice Polard and Philippe Noirot
Eubacteria have evolved multicomponent protein machines, termed replisomes, which duplicate their chromosomes rapidly and accurately. Extensive studies in the model bacteria Escherichia coli and Bacillus subtilis have revealed that in addition to the core replication machinery, other proteins are necessary to form a functional replication fork. Specific subsets of proteins mediate: a) assembly of the replisome at the chromosomal origin of replication [initiation]; b) progression of the replication forks along the chromosome [elongation] and their maintenance by providing solutions for replication restart, which are adapted to overcome possible 'roadblocks' encountered on the DNA template; and c) physiological arrest of replication when chromosome duplication is completed [termination]. This review summarises recent knowledge about chromosomal replication in Bacillus subtilis and related Gram-positive bacteria. It is focused on the events governing the assembly and fate of the replication fork, describes protein networks connected with the replisome, and emphasises several novel aspects of DNA replication in this group of bacteria.
2. Dynamics of DNA Double-strand Break Repair in Bacillus subtilis
Begoña Carrasco, Paula P. Cárdenas, Ester Serrano, Rubén Torres, Elena M. Seco, Silvia Ayora and Juan C. Alonso
All organisms have developed a variety of DNA repair mechanisms to cope with DNA double strands breaks (DSBs). In replicating cells, homologous recombination (HR), which uses an intact homologous template to restore lost information at the break site, is the main pathway for error-free repair of one- or two-ended DSBs and for promoting the re-establishment of replication forks during vegetative growth. Genetic, cytological and biochemical approaches were used to analyse the requirements of exponentially growing Bacillus subtilis cells to survive broken ended and to visualize the choreography of DSB repair. The damage-induced multi-protein complex (recombinosome), organised into focal assemblies, has been confirmed by biochemical approaches. HR is coordinated with other essential processes, such as DNA replication, transcription and chromosomal segregation. When DSB recognition or end resection are severely impaired or when an intact homologous template is simply not available, the ends should be reconnected by template-independent mechanisms using minimal (single-strand annealing) or no-sequence-homology (non-homologous end joining). These effective repair mechanisms lead to loss of genetic information (if the DNA ends are altered before re-ligation), and to chromosomal rearrangements (if incorrect ends are rejoined).
3. Chromosome Arrangement and Segregation
Peter L. Graumann
After a bit more than a decade of the use of GFP - or immuno-fluorescence microscopy to study bacterial chromosome segregation, it has become clear that this process is highly organized, temporally as well as spatially, and that a machinery exists that mediates an overall gradual separation of sister chromosomes. Several key factors in this process have been identified, and at least a rough overall picture can be drawn on how chromosomes are separated so highly rapidly and efficiently. Bacillus subtilis has a circular chromosome. Replication initiates at the origin of replication that is defined as 0°, and two replication forks proceed bidirectionally to converge at the terminus region, which is defined as 180°. All other regions on the chromosome are defined as the corresponding site on a circle. DNA replication occurs in the cell centre, and duplicated regions are moved away from the cell centre towards opposite cell poles. How this process is energetically driven is still unknown, but entropic forces could play a major role. A dedicated protein complex called SMC forms two subcellular centres that organize newly duplicated chromosome regions within each cell half, setting up the spatial organization that characterizes bacterial chromosome segregation. Several proteins, including topoisomerases, DNA translocases and recombinases, ensure that entangled sister chromosomes or chromosome dimers can be completely separated into the future daughter cells shortly before cell division occurs at the middle of the cells.
4. Cell Division
Cell division in rod-shaped bacteria like Bacillus subtilis is carried out by a contractile protein ring, made up of about a dozen different polypeptides and known as the divisome. This sophisticated macromolecular machine, which is centered around the tubulin-like protein FtsZ, is capable of promoting the coordinated invagination of the cell membrane and cell wall to create the so-called division septum. The goal of this chapter is to provide an overview of the mechanism of septum formation in B. subtilis. Emphasis will be placed on describing the properties of the individual division proteins and how they assemble into the divisome complex, and on a discussion of the regulatory mechanisms that ensure that septum formation will happen with great spatial and temporal precision. In addition, the peculiar asymmetric division that happens during B. subtilis sporulation will be described.
5. The Organisation of Transcription and Translation
Peter Lewis and Xiao Yang
The traditional view of transcription and translation within the cell was that of a very closely coupled process where translating ribosomes assembled on the nascent transcript as it was produced by transcribing RNA polymerase. Whilst this close physical coupling is undoubtedly important, it seems clear now that a number of other events are significant with respect to the physical organization of these two processes within the cell. Transcription is crudely segregated into two regions within the nucleoid where either stable (r- and t-) RNA, or mRNA transcription predominate. Translation by polysomes is enriched at cell poles, whereas the assembly of initiation complexes, and some transcriptionally linked ribosomes may occur throughout the nucleoid.
6. RNA-mediated Regulation in Bacillus subtilis
Wade C. Winkler
Bacterial genetic regulation is generally assumed to occur at the level of transcription initiation through the use of transcription factors. Regulatory mechanisms that take place post-transcription initiation are sometimes treated as anomalies - as exceptions to the rule. However, the actual degree of usage for post-initiation regulatory strategies in bacteria still remains to be fully determined. As evidence to this fact, recent research has significantly expanded the general understanding of post-initiation regulation in Bacillus subtilis and other bacteria. Regulatory RNAs are now predicted to control expression of numerous fundamental biochemical pathways that together constitute greater than 4% of the B. subtilis genome. Therefore, post-initiation regulation is a vital layer of bacterial genetic circuitry that still remains to be fully revealed.
7. General and Regulatory Proteolysis in Bacillus subtilis
General and regulatory proteolysis is an important part of different cellular processes such as protein homeostasis and the specific control of regulatory networks for cellular development or stress response in B. subtilis. The intricate involvement of AAA+ proteins and their adaptor proteins and the role of regulated intra-membrane proteolysis in these processes are introduced and discussed.
8. The Actin-like MreB 'Cytoskeleton'
Prokaryotic cells possess filamentous proteins, analogous to eukaryotic cytoskeletal proteins, that play a key role in the spatial organization of essential cellular processes. The bacterial homologues of actin (MreB, ParM, MamK, AlfA and Alps proteins) are involved in cell shape determination, DNA segregation, cell polarity, cell motility and other functions that require the targeting and accurate positioning of proteins and molecular complexes in the cell. In Bacillus subtilis, MreB homologues (MreB, Mbl and MreBH) assemble into dynamic polymeric structures that move processively along peripheral tracks perpendicular to the cell axis together with other morphogenetic factors involved in growth of the cylindrical sidewall (elongation). The ultimate morphology of the cell is believed to depend on a dynamic interplay between the intracellular MreB proteins and the extracellular proteins that carry up cell wall biosynthesis, maturation and degradation, probably linked through MreCD and/or other membrane proteins such as RodZ. Peptidoglycan synthesis drives the circumferential movement of MreB filaments around the cell periphery, which in turn leads to spatial organization of the peptidoglycan elongation machinery. MreB isoforms of B. subtilis have also been implicated in the organization of the cell membrane and of viral DNA replication, in the inhibition of cell elongation during the escape from the competence state, and in chromosome segregation, although they do not seem to be essential for this process. The general properties of MreB proteins, relative to eukaryotic actin and to other prokaryotic homologues of actin, and the known functions of the MreB cytoskeleton in B. subtilis and other bacteria, will be discussed in this chapter.
9. Ins and Outs of the Bacillus subtilis Membrane Proteome
Jan Maarten van Dijl, Annette Dreisbach, Marcin J. Skwark, Mark J.J.B. Sibbald, Harold Tjalsma, Jessica C. Zweers and Girbe Buist
Bacterial homeostasis is largely determined by a phospholipid bilayer that encloses the cytoplasm. The proteins residing in this cytoplasmic membrane are responsible for communication between the cytoplasm and extracytoplasmic cell compartments or the extracellular milieu of the cell. This chapter deals with the cytoplasmic membrane proteome of Bacillus subtilis. Specifically, we address current views on the roles of membrane proteins in homeostasis, their membrane targeting and retention signals, machinery for membrane insertion, localization of membrane proteins, membrane protein degradation and, finally, the identified and predicted composition of the B. subtilis membrane proteome. Known mechanisms and knowledge gaps are discussed to give a comprehensive overview of the ins and outs of the B. subtilis membrane proteome.
Danae Morales Angeles and Dirk-Jan Scheffers
The cell wall of Bacillus subtilis
is a rigid structure on the outside of the cell that forms the first barrier between the bacterium and the environment, and at the same time maintains cell shape and withstands the pressure generated by the cell's turgor. In this chapter, the chemical composition of peptidoglycan, teichoic and teichuronic acids, the polymers that comprise the cell wall, and the biosynthetic pathways involved in their synthesis will be discussed, as well as the architecture of the cell wall. B. subtilis
has been the first bacterium for which the role of an actin-like cytoskeleton in cell shape determination and peptidoglycan synthesis was identified and for which the entire set of peptidoglycan synthesizing enzymes has been localised. The role of the cytoskeleton in shape generation and maintenance will be discussed and results from other model organisms will be compared to what is known for B. subtilis
. Finally, outstanding questions in the field of cell wall synthesis will be discussed.
11. Genomics and Cellular Biology of Endospore Formation
Many species of the classes Bacilli and Clostridia can be found in two distinct states. In the vegetative state, these bacteria are metabolically active and use available nutrients to grow and divide by binary fission, a process that generates two identical daughter cells. By contrast, when nutrients are scarce, a developmental program of endospore formation (sporulation) is initiated, resulting in the production of highly resistant spores. In the spore state, bacteria are metabolically dormant, and their genetic material, protected in the core of the spore, can endure a variety of challenges, including exposure to radiation, elevated temperatures and noxious chemicals. Sporulation is a complex process, which requires the generation of two distinct cell types: a forespore and a mother cell. The progression of the developmental program is controlled by two exquisitely regulated cell type-specific lines of gene expression that run in parallel and are connected through signaling systems. In the model organism Bacillus subtilis, various genetic screens and genome-wide transcriptional analyses have identified more than 600 genes that are expressed in the course of sporulation. The function of several of these genes has been characterized in detail and subcellular localization data are available for more than 100 sporulation proteins. Thus, sporulation constitutes one of the best-characterized developmental programs at the molecular and cellular levels.
12. Multicellularity and Social Behaviour in Bacillus subtilis
José Eduardo González-Pastor
Most of the knowledge about Bacillus subtilis derives from studies of laboratory strains growing as planktonic cultures, in which all the individual cells are considered identical. Recently, the study of a natural and undomesticated isolate has revealed that B. subtilis cells display multicellular and social features that were lost in the laboratory strains, which were selected over generations for easy manipulation. In undomesticated strains, certain environmental conditions trigger cells of this bacterium to form multicellular communities where sporulation takes place, and to exhibit some particular social traits, like swarming motility, the fratricide of sibling cells or cannibalism during sporulation, and the release of extracellular DNA. Interestingly, some of these behaviours are based in the heterogeneity of the B. subtilis populations, which has been determined using cell biological techniques like fluorescence and light microscopy. This chapter outlines the genetic pathways governing the transition from a unicellular to a multicellular stage, swarming motility and cannibalism. The biological relevance of these alternative lifestyles is discussed.
Transformation is the process of import and inheritable integration of DNA from the environment. As such, it is believed to be a major driving force for evolution. Competence for transformation is widespread among bacterial species. Recent findings draw a picture of a conserved molecular machine that binds DNA at the cell surface and subsequently transports it through the cell envelope. Within the cytoplasm the DNA is coated by proteins that mediate recombination or self-annealing. The regulatory mechanisms and environmental signals affecting competence are very diverse between different bacterial species. Competence in Bacillus subtilis
has become a paradigm for stochastic determination of cell-fate. Quantitative analysis at the single cell level in conjunction with mathematical modeling allowed understanding of induction and decline of competence at the systems level. Currently, the picture is emerging of stochastic differentiation as a fitness trade-off in fluctuating environments.
14. Swimming, Swarming and Sliding Motility in Bacillus subtilis
Anna C. Hughes and Daniel B. Kearns
Bacillus subtilis has the capacity for three different types of movement called swimming, swarming, and sliding motility. There has been renewed interest in bacterial motility due in large part to the complex relationship between motility, biofilm formation, and more generally, pathogenesis. Whereas flagellar-mediated motility like swimming and swarming appears to be oppositely regulated with biofilm formation, sliding motility and biofilms appear to be manifestations of the same phenomenon. Furthermore, the study of biofilm formation in ancestral strains of B. subtilis brought attention to the fact that motility of all kinds was severely impaired by domestication of commonly-used laboratory derivatives. In this chapter we will discuss the different motile behaviors found in B. subtilis and the genetic requirements for each. Biofilm formation will be introduced in relation to sliding motility and how regulators of biofilm inhibit the expression and function of flagella. To begin, we will briefly review flagellar structure, assembly, and function as flagella mediate swimming and swarming motility and are the target of motility regulation.
15. Nucleotide Second Messengers: (p)ppGpp and Cyclic Dinucleotides
Danny K. Fung, Brent W. Anderson, Jessica L. Tse and Jue D. Wang
Bacteria adapt to diverse environmental changes by intricate regulation of cellular processes, such as through regulation of gene expression by the alternative sigma factors and various two-component systems. In addition, bacteria produce various nucleotide derivatives that function as second messengers of environmental signals. These nucleotide signals, notably the 'alarmone' guanosine-5', 3'-tetraphosphate or guanosine-5', 3'-pentaphosphate ((p)ppGpp) and the cyclic nucleotides cyclic adenosine monophosphate (c-AMP), cyclic diadenylate monophosphate (c-di-AMP) and cyclic diguanylate monophosphate (c-di-GMP), regulate diverse cellular processes ranging from central dogma processes to cellular behavior such as motility and biofilm development. This chapter summarizes our current knowledge on the signaling principles, cellular targets, and physiological roles of these nucleotide second messengers, using Bacillus subtilis and other bacteria as examples.
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(EAN: 9781910190579 9781910190586 Subjects: [bacteriology] [microbiology] [molecular microbiology] )