Bacillus Subtilis

Defining Statement

Bacillus subtilis is a Gram-positive, rod-shaped bacterium that forms heat-resistant, dormant spores. It is not pathogenic. It produces important commercial products. The sequenced genome contains 4 214 630 base pairs. Its genome is easily manipulated genetically. It serves as a model organism for studies of sporulation and of the behavior of low GC Gram-positive bacteria.

Bacillus subtilis is a Gram-positive, rod-shaped bacterium that forms heat-resistant spores. It is commonly found in the soil. It is nonpathogenic. It received its name in 1872 from Ferdinand Cohn, who also demonstrated its ability to form spores that were heat-resistant. It produces several commercially important products, most notably proteases and amylases. In part because of its commercial importance, and more because of the ease of its genetic manipulation, B. subtilis has been intensively studied. It has a single circular genome (chromosome). The sequenced genome contains 4 214 630 base pairs (bp) with a 43.5% GC content; it encodes about 4100 proteins. B. subtilis is the best characterized of the low GC Gram-positive bacterial species. As is typical of Gram-positive species, it has a cytoplasmic membrane and a thick cell wall, but no outer membrane. This structure contrasts with Gram-negative species, which have a cytoplasmic membrane, a thin cell wall, and an outer membrane.

Phylogeny

Bacillus subtilis is a typical gram-positive eubacterium. As such it is significantly more similar to Archaea than is E. coli. Many metabolic genes have a distinct archaeal flavour, in particular genes involved in the synthesis of polyamines, but it is rare to find genes in B. subtilis that are similar to eukaryotic genes. This led Gupta to propose that ancestral bacteria comprised a monoderm organism that diverged into gram-positive bacteria and Archaea, and that gram-positive bacteria further led to gram-negative bacteria with their typical double membrane (diderms). This hypothesis stirred a very heated, but interesting, debate about the origin of the first cell(s). As such, bacilli form a heterogenous family of bacteria that can be split into at least five distinct groups. Bacillus subtilis is part of group 1 and is strongly linked to B. licheniformis (which is often found on the cuticle of insects), and to the group of animal pathogens formed by B. thuringiensis, B. cereus, and B. anthracis. In this classification B. sphaericus is typical of group 2, B. polymyxa of group 3, and B. stearothermophilus of group 5. The pathogen Listeria monocytogenes (in between groups 2 and 5) is related to B. subtilis, and, indeed, its genome has many features in common with that of the genome of B. subtilis. Accordingly, B. subtilis is an excellent model for these groups of bacteria.

Ecology and Significance

Bacillus subtilis is a ubiquitous organism. In the laboratory, B. subtilis is easy to grow and manipulate. In nature, B. subtilis inhabits the soil, roots of plants, and aquatic environments. Although B. subtilis can grow in the gastrointestinal (GI) tract of animals, it is not considered a human pathogen. In fact, B. subtilis along with other species of Bacillus is considered a GRAS (Generally Regarded As Safe) organism by the Food and Drug Administration (FDA). Moreover, B. subtilis has been widely used in biotechnology. Recently, improved expression and secretion of proteins by B. subtilis has become an efficient tool for enzyme production. It is estimated that Bacillus species, including B. subtilis, produce 60% of commercially available enzymes. Additionally, B. subtilis plays a major role in the production of food (fermented products, flavor enhancers, sweeteners, and animal feed additive), household detergents, antibiotics, and vitamins and in the development of vaccines; it serves as a model organism for the development of sporicides, chemical agents that kill spores (see section ‘Sporulation’). The biotechnology industry has helped drive the research of molecular genetics and cell biology forward, using B. subtilis as one of its greatest workhorses.

II. Plasmid Transformation

The transformation of competent cells (which can be maintained frozen for several years) by plasmid DNA occurs at high frequency (~10⁶ transformants/μgm DNA) but low efficiency. About 10³–10⁴ plasmid molecules are taken up per transformant (Contente and Dubnau, 1979a). Linear and open circular plasmid molecules do not transform (Contente and Dubnau, 1979a). Remarkably, the transforming activity of native plasmid preparations is entirely owing to the presence of oligormeric forms (Canosi et al., 1978). In fact, covalently closed circular (CCC) plasmid monomer is completely inactive in transformation, even at very high input concentrations (R. Villafane and D. Dubnau, unpublished). Various models have been presented to explain these and other properties of plasmid transformation (Dubnau et al., 1980a; Canosi et al., 1981; Haykinson et al., 1982). These models are beyond the scope of this chapter. However, the properties of plasmid transformation just discussed place a severe limitation on the efficiency of shotgun-cloning in B. subtilis. The requirement for CCC DNA prevents use of techniques that leave gaps or nicks in ligated recombinant DNA (e.g., homopolymer tailing or use of alkaline phosphatase to enrich for recombinant molecules). This is not a severe limitation, because a new vector permits direct selection for recombinant molecules (see Section III,D). A more serious limitation in practice is imposed by the requirement for plasmid oligomers. In a complex ligation mixture, the probability of forming a head-to-tail vector oligomer carrying a desired “foreign” fragment is low. Although shotgun cloning of fragments from simple mixtures (e.g., restriction digests or plasmid or bacteriophage DNA) is readily accomplished, the straightforward cloning of chromosomal fragments has proven very difficult. This problem has been surmounted using stratagems based on certain further properties of the plasmid transformation system. If the competent culture carries a plasmid that is homologous to the vector, then plasmid monomer can transform. The properties of this “helper” transformation system have been explored in some detail (Contente and Dubnau, 1979b), and they form the basis of a successful cloning stratagem (Gryczan et al., 1980a). Another form of homology-facilitated transformation also permits the use of plasmid monomers and is potentially useful for cloning (Bensi et al., 1981; Lopez et al., 1982). Michel et al. (1981) and Haykinson et al. (1982) have reported that plasmid monomers carrying short direct or indirect repeats can transform with high efficiency. The latter authors have adapted this observation to the construction of several potentially useful plasmid vectors. We discuss these various cloning stratagems in more detail in Section IV.

I Introduction

Bacillus subtilis is a gram-positive, spore-forming soil bacterial species that has several features useful for the study of chromosomal replication: (1) the ability to grow in a minimal medium, (2) highly efficient DNA-mediated transformation, (3) a completely replicated chromosome with all loci occurring at equal frequency in the spore and in the stationary-phase cells of some strains (e.g., W23), and (4) a single lipid bilayer membrane. This review will cover mainly the association of the chromosome with the membrane and the role of the cell membrane proteins and their genes in the regulation of initiation and termination of chromosome replication. Details of earlier work on this subject have been reviewed by Winston and Sueoka (1), and studies on in vitro DNA replication using membrane fractions have been reviewed by Firshein (2). More general reviews have been made by Yoshikawa and Wake (3) on the various aspects of B. subtilis chromosome replication, and by Wake (4) on chromosomal partition and cell division. For other aspects of bacterial chromosome replication, the readers are referred to reviewed on Escherichia coli (5) and B. subtilis (6). Here the emphasis is on the points that this author has thought about over the years based on the observations generated mainly in his laboratory studies on B. subtilis, and the points that have not been treated explicitly by the previous reviewers. This reviewer also includes his own views of the subjects, some of which may not agree with the previous reviewers.

1 Introduction

Bacillus subtilis is one of the best characterized bacteria and is used as a model organism for Gram-positive bacteria. B. subtilis is a rod-shaped bacterium, which produces endospores that allow the survival of extreme environmental conditions including heat and desiccation. In the soil, the natural environment of B. subtilis, the bacterium continuously encounters various changing environmental conditions including drastic differences in oxygen tension. For example, a rain shower reduces the accessibility of oxygen, as the diffusion rate of oxygen in water is approximately 10,000 times lower than in the gaseous phase. As oxygen is the essential electron acceptor during aerobic respiration, B. subtilis has adopted various alternative strategies for anaerobic growth.

Historically, B. subtilis was classified as strict aerobic organism. A first indication for the utilization of nitrate as an alternative electron acceptor under microaerophilic growth conditions was obtained 40 years ago (Michel, Piechaud, & Schaeffer, 1970). However, it took another 25 years before anaerobic nitrate respiration was demonstrated for B. subtilis, and the corresponding nitrate reductase genes were cloned (Glaser, Danchin, Kunst, Zuber, & Nakano, 1995; Hoffmann, Troup, Szabo, Hungerer, & Jahn, 1995). The elucidation of various fermentation processes sustaining anaerobic growth followed (Cruz-Ramos et al., 2000; Nakano, Dailly, Zuber, & Clark, 1997). Nowadays, anaerobic growth and regulation of anaerobically induced genes are understood in great detail (Nakano & Hulett, 1997; Nakano & Zuber, 1998, 2001). Here, we intent to draw a state of the art picture of the anaerobic metabolism of B. subtilis and its regulation.

1 Introduction

Bacillus subtilis, a low %G+C, Gram-positive, endospore-forming member of the bacterial phylum Firmicutes, is found predominately in the soil and in association with plants. B. subtilis is the type species for the genus Bacillus, and, following the discovery (Spizizen, 1958) that strain 168 exhibited natural genetic competence, this bacterium has been developed as a high tractable model for Gram-positive bacteria and for the study of basic metabolic and cellular differentiation processes such as sporulation, genetic competence and biofilm formation. The accumulation, over more than half a century, of knowledge of the biochemistry, genetics and physiology of B. subtilis has been enhanced in recent years by a number of systematic ‘omics’ analyses. As a result, B. subtilis is one of the most intensively studied and genetically amenable microorganisms and a suitable chassis for a wide range of synthetic biology applications.

B. subtilis is able to grow both in nutrient media and in chemically defined salt media in which glucose, malate and other simple sugars provide sources of carbon and ammonium salts or certain amino acids as sources of nitrogen (Harwood & Archibald, 1990). B. subtilis strain 168, on which most studies are performed, is a tryptophan auxotroph (trpC2) and therefore requires the addition of tryptophan to the growth media, even those containing acid-hydrolysed proteins such as casein. An analysis of the origins of B. subtilis 168 indicates that it was a derived from B. subtilis Marburg (ATCC 6051^T), the type strain of both B. subtilis and B. subtilis subsp. subtilis (Zeigler et al., 2008). More recently, a prototrophic variant of strain 168, called BSB1, has been isolated by transformation with DNA from strain W23 (Nicolas et al., 2012). BSB1 is increasingly replacing strain 168 in more systematic research programmes.

Strains of B. subtilis are available from a variety of international culture collections including the Bacillus Genetic Stock Center (http://www.bgsc.org), which has an extensive collection of wild-type and mutant B. subtilis strains, bacteriophages and cloning vectors and the Japanese National BioResource Project (www.shigen.nig.ac.jp/bsub).

When subject to nutrient or physical stress, B. subtilis initiates a series morphological and physiological responses in what has been described as a bet-hedging strategy (Veening et al., 2008). These include the induction of macromolecular hydrolases (e.g. proteases and carbohydrases), chemotaxis and motility and competence. If these responses fail to reestablish growth, sporulation is induced in portion (typically ≤ 10%) of the population, while another part is condemned to cell lysis (cannibalism) to provide the nutrients required to sustain sporulation (Gonzalez-Pastor, Hobbs, & Losick, 2007). Approximately 5% of the B. subtilis genome is devoted to the processes of sporulation and germination and its ability to differentiate into highly resistant endospores provides an enormous competitive advantage in environments such as the soil, where long periods of drought and nutrient deprivation are common.

Abstract

Successful respiration in Bacillus subtilis using oxygen or nitrate as the terminal electron acceptor requires the ResD–ResE signal transduction system. Although transcription of ResDE‐controlled genes is induced at the stationary phase of aerobic growth, it is induced to a higher extent upon oxygen limitation. Furthermore, maximal transcriptional activation requires not only oxygen limitation, but also nitric oxide (NO). Oxygen limitation likely results in conversion of the ResE sensor kinase activity from a phosphatase‐dominant to a kinase‐dominant mode. In addition, low oxygen levels promote the production and maintenance of NO during nitrate respiration, which leads to elimination of the repression exerted by the NO‐sensitive transcriptional regulator NsrR. ResD, after undergoing ResE‐mediated phosphorylation, interacts with the C‐terminal domain of the α subunit of RNA polymerase to activate transcription initiation at ResDE‐controlled promoters.

Abstract

The soil-dwelling organism Bacillus subtilis is able to form multicellular aggregates known as biofilms. It was recently reported that the process of biofilm formation is activated in response to the presence of various, structurally diverse small-molecule natural products. All of these small-molecule natural products made pores in the membrane of the bacterium, causing the leakage of potassium cations from the cytoplasm of the cell. The potassium cation leakage was sensed by the membrane histidine kinase KinC, triggering the genetic pathway to the production of the extracellular matrix that holds cells within the biofilm. This chapter presents the methodology used to characterize the leakage of cytoplasmic potassium as the signal that induces biofilm formation in B. subtilis via activation of KinC. Development of novel techniques to monitor activation of gene expression in microbial populations led us to discover the differentiation of a subpopulation of cells specialized to produce the matrix that holds all cells together within the biofilm. This phenomenon of cell differentiation was previously missed by conventional techniques used to monitor transcriptional gene expression.

Introduction

The YycFG two‐component system is arguably the most intriguing signal transduction system in Bacillus subtilis and other gram‐positive bacteria due to its essential role for cell viability and its high amino acid conservation (Fabret and Hoch, 1998; Martin et al., 1999). Since our discovery of its essential nature, this system has been studied in some detail in various organisms, and several laboratories have contributed nicely, clarifying the important role this system plays (Fukuchi et al., 2000; Howell et al., 2006; Ng et al., 2003). The regulon controlled by this two‐component system among different organisms is diverse, but a common theme is the control of genes for cell wall metabolic processes, cell membrane composition, and cell division (Dubrac and Msadek, 2004; Fukuchi et al., 2000; Howell et al., 2003; Mohedano et al., 2005).

Two important questions to answer when studying a novel two‐component system are what are the input signals feeding into the system and what is the output regulated by this system? The input is defined as the signals sensed by the histidine kinase, which can be as diverse as a nutrient, pH, temperature, or interaction with other proteins (Kaspar and Bott, 2002; Mansilla et al., 2005; Neiditch et al., 2006; Tiwari et al., 1996). The output is defined as the genes controlled by this system (the regulon) for the standard DNA‐binding response regulator. In well‐studied organisms the regulon is identified by microarray analysis of a deletion strain in comparison to the wild‐type strain. The procedure is more complicated when the two‐component system is essential. Because the system cannot be inactivated, a technique has to be designed allowing activity control of the two‐component system to change expression levels of the regulon. The most straightforward approach is either overexpression of the two‐component system of interest or construction of a strain depleted for the sensor kinase or response regulator (Mohedano et al., 2005). Particularly, the overexpression of the signaling proteins can lead to secondary effects and complicate the analysis.

Howell and colleagues (2003) designed an interesting approach that led to the identification of the consensus DNA‐binding sequence for B. subtilis YycF. This approach utilized the fact that B. subtilis expresses a phylogenetically closely related two‐component system to YycFG, the PhoPR system. This system has been well studied by Hulett and colleagues (1996). Activity of the kinase PhoR is induced under phosphate limitation conditions (Hulett et al., 1994). Construction of a hybrid response regulator consisting of the PhoP response regulator domain and the YycF DNA‐binding domain allowed for the phosphate‐dependent regulation of YycF‐dependent gene expression.

Had a signal been known for the YycFG system, identification of the regulon would have been simpler. Unfortunately, the identification of input signals for two‐component systems has been difficult and is not as straightforward as identification of the regulon. Indeed, signals controlling histidine kinase activity remain unknown for most two‐component systems currently under investigation. With some exceptions, well‐defined signals are available only for systems responsible for utilization of nutrient sources.

Our studies, described here, were designed to help the identification of signals feeding into the YycFG two‐component system. Some of these methods are necessary because of the essentiality of the YycFG system, whereas others are widely applicable to the study of many two‐component systems. Certainly, these studies are complementary to those identifying the output of a two‐component system and present an alternative approach when first studying a novel two‐component system.