Keywords

13.1 Introduction

Bacillus is a genus of Gram-positive , rod -shaped (bacillus) bacteria and a member of the phylum Firmicutes . Bacillus species can be obligate aerobes (oxygen reliant), or facultative anaerobes (having the ability to be aerobic or anaerobic ), and are ubiquitous in nature. They give positive test for the enzyme catalase when oxygen is used or present. Bacillus include both free-living (nonparasitic) and parasitic pathogenic species. Under stressful environmental conditions, bacteria can produce oval endospores that are not true spores but which the bacteria can reduce themselves and remain in a dormant state for very long periods. These characteristics originally defined the genus , but not all such species are closely related, and many have been moved to other genera of Firmicutes .

Many species of Bacillus can produce copious amounts of enzymes which are made use of in different industries. Some Bacillus species can form intracellular inclusions of polyhydroxyalkanoates under certain adverse environmental conditions, as in a lack of elements such as phosphorus , nitrogen or oxygen combined with an excessive supply of carbon sources.

B. subtilis has proved to be an invaluable model for research. Other species of Bacillus are important pathogens causing anthrax and food poisoning. Many Bacillus species are able to secrete large quantities of enzymes . B. amyloliquefaciens is the source of a natural antibiotic protein barnase (a ribonuclease), alpha amylase used in starch hydrolysis, the protease subtilisin used with detergents, and the Bam HI restriction enzyme used in DNA research. A portion of the Bacillus thuringiensis genome was incorporated into corn (brinjal and cotton ) crops . The resulting GMOs were then found to be resistant to some insect pests .

B. subtilis is one of the best understood prokaryotes , in terms of molecular biology and cell biology. Its superb genetic amenability and relatively large size have proved to be powerful tools required to investigate a bacterium from all possible aspects. Recent improvements in fluorescence microscopy techniques have provided novel and amazing insight into the dynamic structure of a single-cell organism . Research on B. subtilis has been at the forefront of bacterial molecular biology and cytology , and the organism is a model for differentiation, gene /protein regulation , and cell cycle events in bacteria .

Two Bacillus species are considered medically significant: B. anthracis , which causes anthrax and B. cereus , which causes food poisoning similar to that caused by Staphylococcus . A third species, B. thuringiensis , is an important insect pathogen and is sometimes used to control insect pests. The type species B. subtilis is an important model organism . It is also a notable food spoiler, causing ropiness in bread and related food. Some environmental and commercial strains such as B. coagulans may play a role in food spoilage of highly acidic tomato -based products.

An easy way to isolate Bacillus is by placing non-sterile soil in a test tube with water, then shaking it, ultimately plating it in melted mannitol salt agar and incubating at room temperature for at least a day. Colonies are usually large, spreading and irregularly shaped. Under the microscope, Bacillus cells appear as rods and a substantial portion usually contain an oval endospore at one end, making it bulge.

The cell wall of Bacillus is a structure on the outside of the cell that forms the second barrier between the bacterium and the environment and at the same time maintains the rod shape and withstands the pressure generated by the cell’s turgor. The cell wall is composed of teichoic and teichuronic acids. B. subtilis was the first bacterium for which the role of an actin-like cytoskeleton in cell shape determination and peptidoglycan synthesis was identified. It was also the first bacterium for which the entire set of peptidoglycan synthesizing enzymes was localized, thus, paving the way for understanding the role of cytoskeleton in shape generation and maintenance.

The genus Bacillus was named in 1835 by Christian Gottfried Ehrenberg to contain rod-shaped (bacillus) bacteria . He had 7 years earlier named the genus Bacterium. Bacillus was later amended by Ferdinand Cohn to further describe them as spore -forming, Gram-positive , aerobic or facultatively anaerobic bacteria (Xu and Côté 2003). Like other genera associated with the early history of microbiology, such as Pseudomonas and Vibrio , the 266 species of Bacillus are ubiquitous. The genus has very large ribosomal 16S diversity and is environmentally diverse.

One of the studies reconciles the exception that Lactobacillus plantarum does not entail iron . It might apparently manage to stimulate all its enzymatic functions with metals other than iron. After complexing iron, the ferric-siderophore complexes are taken up into the cell. The specific siderophore and its chirality are recognized by highly specific receptors in the outer membrane of bacteria . In an active and energy-dependent way, they transport the ferric complexes into the periplasm . Once the complexes are collected there, they are then handed over to the intracellular transport and storage components and finally integrated into proteins to accomplish their enzymatic functions (Archibald 1983).

Several studies have tried to reconstruct the phylogeny of the genus as mentioned in Fig. 13.1. The Bacillus-specific study with the most diversity covered is by Xu and Côté (2003) using 16S and the ITS region, where they divide the genus into ten groups, which includes the nested genera Paenibacillus , Brevibacillus , Geobacillus , Marinibacillus and Virgibacillus . However, the tree constructed by the living tree project, a collaboration between ARB-Silva and LPSN where a 16S (and 23S if available) tree of all validated species was constructed, the genus Bacillus contains a very large number of nested taxa and majorly in both 16S and 23S is paraphyletic to Lactobacillales ( Lactobacillus , Streptococcus , Staphylococcus , Listeria , etc.), due to Bacillus coahuilensis and others. A gene concatenation study found similar results to Xu and Côté (2003), but with a much more limited number of species in terms of groups, but used Listeria as an outgroup. So in light of the ARB tree, it may be “inside-out.”

Fig. 13.1
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Phylogenetic tree of genus Bacillus

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One clade , formed by B. anthracis , B. cereus , B. mycoides , B. pseudomycoides , B. thuringiensis and B. weihenstephanensis under current classification standards, should be a single species (within 97 % 16S identity), but due to medical reasons, they are considered separate species, an issue also present for four species of Shigella and Escherichia coli .

Much corroboration confirms that corynebactin was isolated from Gram-positive Corynebacterium glutamicum and B. subtilis incorporates a threonine trilactone and glycine spacers, which elongate the three chelating arms as compared with enterobactin . In B. subtilis (DNA with low G + C content), three Fur-like proteins have been characterized (Bsat et al. 1998). One, called Fur, regulates mainly iron uptake and siderophore biosynthesis. A second one, called PerR, regulates peroxide stress response genes and acts with manganese as corepressor . A third one, Zur, regulates genes for zinc uptake. The Zur protein found in E. coli shows only 25 % identity to the B. subtilis Zur, while the two Fur proteins have 32 % identical amino acids .

Apart from producing siderophores, some Bacillus spp. even help in the release of iron from siderophores by using ferri-reductase. Ferri-siderophore reductase of B. megaterium has been partially purified and it shows wide range of substrate specificity. The putative Mn oxidase CumA (Okazaki et al. 1997; Brouwers et al. 1999; Francis and Tebo 2001) of P. putida GB-1 and MnB1 is a multicopper-type oxidase enzyme that utilizes oxygen as an electron acceptor. Same is also true in other well-studied systems (Brouwers et al. 2000; Tebo et al. 2004) including Bacillus sp. spores (Van Waasbergen et al. 1996; Dick et al. 2006).

Webb et al. (2005) have elegantly demonstrated that the oxidation of Mn(II) to Mn(IV) by Bacillus spores is a two-step process involving a transient Mn(III) intermediate. Further, an enzymatically produced Mn(III) intermediate of Mn(II) oxidation by Bacillus sp. strain SG-1 spores has been trapped by pyrophosphate under conditions that minimized abiotic processes (Webb et al. 2005). The above observation suggests that ligands can trap Mn(III) when interacting with living systems. The differing origin of the Mn(III) in the Bacillus and Pseudomonas cases could involve the greater stability constant of PVD Mn(III) (Parker et al. 2004) than of pyrophosphate -Mn(III) (Webb et al. 2005). Beside chelation of ferric ions (iron chelator), degradation of textile dyes (Thakur et al. 2012; Joshi et al. 2013) and several other heavy metal ions such as nickel and chromium chelation have also been reported for B. megaterium in various studies.

Among pathogenic bacteria , characteristic features of the tubercle bacillus include its slow growth, dormancy , complex cell envelope, intracellular pathogenesis and genetic homogeneity . Generally bacteria are denoted as pathogenic because they have found out an easy way in animal system to survive where suitable temperature /environment (warmth) is always available for them. Plentiful of nutrition is also available for their survival and growth. Here the pathogenic organisms can acquire all the required minerals from the host body tissues apart from one, i.e., iron which is generally present in oxidized form Fe (III) at pH 7 and is difficult to utilize directly. The uptake mechanism of iron in Bacillus spp. is mentioned in Fig. 13.2.

Fig. 13.2
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Uptake mechanism of iron in Bacillus spp.

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13.2 Transport of Substrates into Bacillus spp.

The uptake of substrates into the best-studied organism, Bacillus subtilis , has been outlined in general terms. The uptake of substrates take place in three steps: (1) uptake across outer membrane, (2) transport across the cytoplasmic membrane and (3) periplasmic binding protein dependent transport.

13.3 Uptake of Ferri-siderophore Complex Across Outer Membrane

Gram-negative Bacillus sp. is surrounded by two membranes, the outer membrane and the cytoplasmic membrane (Braun and Hantke 1981; Braun et al. 1985; Lugtenberg and Van Alphen 1983). Hydrophilic substrate not larger than 600–700 Da diffuses through water-filled pores of the outer membrane found by the most abundant proteins in this membrane (Nikaido and Vaara 1987). However, for some substrates, stereochemical recognition takes place between the substrate and an outer membrane protein (Ferenci 1989). This has been demonstrated for maltodextrins , which are recognized by the LamB protein , and for nucleosides which interact with Tsx protein (Hantke 1976; Krieger-Brauer and Braun 1980; Maier et al. 1988; Benz et al. 1988). The PhoE protein forms more efficient pore than the porin (Lugtenberg and Van Alphen 1983) both for inorganic and organic phosphate but does not seem to specifically recognize phosphate, but rather it displays a broad specificity for anions (Benz and Bauer 1988). Before uptake studies had been performed, genetic evidence pointed to the role of these proteins in transport of certain substrates. Synthesis of these proteins was regulated at the transcriptional level by maltodextrins , nucleosides , and phosphates . Maltodextrins (the intracellular regulatory compound is maltotriose ) convert a protein (MalT) to an activator, nucleosides inactivates two repressor proteins (DeoR, CytR), and phosphate starvation induces a complex regulatory network resulting in PhoE synthesis. These proteins facilitate the diffusion of the substrate across the outer membrane. The smaller homologues of maltodextrins , phosphate, and nucleosides can also pass through the porins so that the specific porins are not absolutely required. They increase the rate of diffusion and are essential for the uptake of larger homologues across the outer membrane into the periplasmic space.

13.4 Transport Across the Cytoplasmic Membrane

Stereochemical recognition between the substrate and the transport proteins and actual transport against a concentration gradient occurs in the cytoplasmic membrane. Energy required for the active transport is provided in the form of an electrochemical potential across the cytoplasmic membrane by electron transport chain located therein or by the ATP hydrolysis through the membrane-bound ATPase . Certain substrates such as lactose are transported across the cytoplasmic membrane of B. subtilis by FhuBC protein (Ollinger et al. 2006), by a process driven by the electrochemical potential (Hengge and Boss 1983).

13.5 Periplasmic Binding Protein -Dependent Transport (PBT)

Transport of many amino acids , peptides , certain sugars and anions follows a so-called binding protein-dependent mechanism in which protein in the periplasmic space (located between the outer and the cytoplasmic membrane) is involved (Hengge and Boss 1983; Ames 1986). The binding proteins recognize the certain substrates and delivered them to the integral membrane proteins of the cytoplasmic membrane. They are essential constituents of the transport system. The periplasmic binding proteins can be released by an osmotic shock treatment involving plasmolysis of cells in 15 % sucrose (which counterbalances the internal osmotic pressure) in the presence of EDTA (releasing Mg(II) ions supposed to stabilize the outer membrane). Upon rapid dilution of the plasmolyzed cells in to a low-salt Tris/Mg(II) buffer, periplasmic proteins are released from the cells. Alternatively, cells are converted to spheroplasts by a similar procedure but with the inclusion of lysozyme to degrade the murein (peptidoglycan ) layer. Such treated cells show greatly reduced transport rates which can be restored by adding back the binding protein in the presence of Ca(II) (increases the permeability of the outer membrane) (Hengge and Boss 1983). Substrates bound to the binding protein rather than the free substrate are accepted by the cytoplasmic transmembrane protein . Usually the periplasmic proteins are synthesized in a large excess with respect to the membrane proteins and are the best characterized transport proteins. The structure of several such proteins (for arabinose , ribose , galactose , sulfate) has been resolved to the atomic scale by X-ray analysis. They exhibit similar conformations composed of two globular domains forming a cleft at the substrate binding sites, linked by a flexible hinge (Quiochio 1988). The very hydrophobic integral cytoplasmic membrane proteins accept substrates from the binding proteins. Usually two such proteins are found in a single system which exhibit the sequence similarity, for example, HisQ and HisM of histidine , MalF and MalG of maltose , PstA and PstC of phosphate , and OppB and OppC of the peptide transport system . But there are also exceptions to this rule. The high-affinity arabinose system contains only one hydrophobic protein of the usual size (34 kD) (Scripture et al. 1987), and variations also occur in the iron transport system.

Characteristic of the PBT system is the involvement of a polar but nevertheless membrane-bound protein which displays sequences also found in nucleotide-binding proteins. In fact, it has long been known that ATP either directly or indirectly serves as an energy source for PBT. ATP binding, but not ATP hydrolysis, has been demonstrated for the MalK , HisP, and OppD proteins (Higgins et al. 1988). There is evidence that these proteins are bound to the inside of the cytoplasmic membrane (Gallagher et al. 1989).

The PBT system is a high-affinity transport system (Km 1 μM and below) which concentrates substrates inside the cell against a very large gradient (in the order of 105).

13.6 Iron (III) Transport in B. subtilis

Iron in the ferric form in aerobic conditions and physiological pH (around 7.00) is extremely insoluble and only scarcely available for bacteria under all natural conditions. As a response to iron starvation , bacteria synthesize elaborate iron supply systems which are composed of low-molecular-weight ferric complexing compounds, termed siderophores and ferric-siderophore transport system. A number of iron(III) transport systems have been characterized in B. subtilis (Braun and Winkelmann 1987). Iron uptake in B. subtilis using various different siderophores such as bacillibactin , enantio-enterobactin , itoic acid , etc. has been reported by several researchers (May et al. 2001). Further this organism can recognize a variety of catecholate siderophores, but does so through the expression of several and sometimes overlapping membrane transport proteins .

Bacillus subtilis is prototypical for studying iron uptake in Gram-positive organisms. Recent studies of its iron metabolism have elucidated multiple aspects of siderophore synthesis, transport and regulation (Dertz et al. 2006; Miethke et al. 2006; Ollinger et al. 2006; Gaballa and Helmann 2007). Bacillus subtilis is a model system which produces a number of hydroxamate - and catecholate -type siderophores such as shizokinen , itoic acid, petrobactin , corynebactin , bacillibactin, etc. Corynebactin siderophore was produced by Corynebacterium glutamicum (Abergel et al. 2008), and later genomic data and biochemical analysis revealed that this organism is producing bacillibactin type of compound (Barbeau et al. 2002). Thus, the bacillibactin was the preferred siderophore name for the B. subtilis siderophore (Heinrichs et al. 2004; Koppisch et al. 2005).

Bacillibactin is a catecholate siderophore produced by many Bacilli (May et al. 2001). It is a cyclic trimeric ester made of three units of 2,3-dihydroxybenzoate-glycine -threonine (Fig. 13.3), joined by lactone linkages in a cyclic manner, similar to the enterobacterial siderophore enterobactin (May et al. 2001). The linear component 2,3-dihydroxybenzoylglycine , also known as itoic acid , also has iron-chelating capabilities (Ito 1993).

Fig. 13.3
figure 3

Chemical structure of bacillibactin

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13.7 Biosynthetic Pathway of Bacillibactin

Bacillibactin is synthesized by B. subtilis under iron -deficient conditions and secreted into the external environment where it binds with Fe(III) with high affinity and specificity. This Fe(III)-bound siderophore is called ferri-siderophore complex which is taken up into the cell by specific transport components.

Bacillibactin synthesis (Fig. 13.4) (http://www.metacyc.org/META/NEW-IMAGE?type=PATHWAY&object=PWY-5903&detail-level=3) can be divided into two parts:

Fig. 13.4
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Pathway for bacillibactin biosynthesis

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  1. 1.

    Conversion of chorismate to 2,3-dihydroxybenzoate: 2,3-dihydroxybenzoate is synthesized from chorismate through isochorismate and 2,3-dihydroxy-2,3-dihydrobenzoate . Chorismate plays a major role as a key intermediate and branch point in the biosynthesis of many aromatic compounds.

  2. 2.

    Synthesis of bacillibactin from 2,3-dihydroxybenzoate, glycine and L-threonine : Synthesis of bacillibactin is a complex process catalyzed by the bacillibactin synthetase multienzyme complex and is presented by a single pathway reaction. The synthesis starts with the activation of 2,3-dihydroxybenzoate, catalyzed by 2,3-dihydroxybenzoate-AMP ligase, encoded by dhbE (May et al. 2001) in the following reaction:

    $$ {\mathrm{2,3}\;\mathrm{dihydroxybenzoate} + \mathrm{ATP} + \mathrm{H}}^{+}\to\ \mathrm{2,3}\;\mathrm{dihydroxybenzoyladenylate} + \mathrm{PPi} $$

The product (2,3-dihydroxybenzoyl adenylate) is transferred onto the aryl carrier protein (ArCP) domain of a bifunctional protein dhbB, whose other function is isochorismatase (May et al. 2001), where it is attached to the free thiol group of its cofactor, 4′-phosphopantetheine . The 4′-phosphopantetheinyl transferase protein (sfp gene product) catalyzes the cofactor attachment (Grossman et al. 1993).

The glycine and L-threonine amino acids are initially bound to 4′-phosphopantetheine cofactors via thiol groups. These 4′-phosphopantetheine cofactors carrying glycine and L-threonine then bind to the seven-domain holo-DhbF protein , thereby activating the holo-DhbF protein. The 4′-phosphopantetheinyl transferase then catalyzes the transfer of glycine and L-threonine amino acids to two peptidyl-carrier-protein domains of the seven-domain holo-DhbF protein and subsequently to the activated 2,3-dihydroxybenzoate. The reaction for binding of glycine and L-threonine amino acids to the thiol groups of the 4′-phosphopantetheine cofactors are as follows:

$$ \mathrm{glycine} + \mathrm{ATP} = \mathrm{glycyl}-\mathrm{AMP} + \mathrm{PPi} $$
$$ \mathrm{L}-\mathrm{threonine} + \mathrm{ATP} = \mathrm{L}-\mathrm{threonyl}-\mathrm{AMP} + \mathrm{PPi} $$

Two additional domains of 4′-phosphopantetheinyl transferase enzyme catalyze the condensation of the activated amino acids to the activated 2,3-dihydroxybenzoate which ultimately form Dhb-glycine -threonine product (May et al. 2001). The product is transferred to the last domain of holo-DhbF protein, named as Te domain. This protein then trims off the three such moieties and releases the trilactone bacillibactin.

13.8 Mechanism of Ferri-bacillibactin Uptake

The bacillibactin synthesizing operon consists of five gene sets (dhbACEBF) whose function is given in Table 13.1 and Fig. 13.5.

Table 13.1 Functions of genes involved in biosynthesis of bacillibactin
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Fig. 13.5
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Operon for biosynthesis of bacillibactin

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The detailed molecular analysis of iron uptake pathways in B. subtilis was first described by Schneider and Hantke (1993). They suggested that ABC transporter subunits are needed for the uptake of iron siderophore compounds. This hypothesis was generated independently by an analysis of the protein similarities for all of the ABC transport systems (Fig. 13.6), and corresponding surface-binding proteins, in B. subtilis (Quentin et al. 1999). The uptake of iron(III) by B. subtilis using bacillibactin siderophore requires involvement of a number of membrane-bound proteins such as substrate-binding proteins, ATPase , permeases and transporters. FeuABC is one such membrane-bound bacillibactin transporter of B. subtilis. FepDG is another membrane-bound heterodimeric inner membrane permease involved in transport of bacillibactin. YusV acts as ATP-binding protein and provides energy for transport of ferri-bacillibactin. It is hypothesized that YuiI finally hydrolyzes bacillibactin and ferri-bacillibactin so that iron becomes available for cellular processes and bacillibactin may be recycled for further use. The whole process of iron uptake by bacillibactin in B. subtilis is regulated by a Fur homologue which binds directly to a Fur box. Another bacillibactin pathway regulator Mta has been recently discovered which is a MerR-type transcriptional regulator. It activates bacillibactin secretion (Miethke et al. 2008). The exact mechanism of Fur homologue and Mta binding with Fur box and metal ions is poorly understood. Hence, further studies are required in this direction to fully comprehend the regulation of metal uptake in B. subtilis or Gram-positive organisms in general.

Fig. 13.6
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Pathway for iron uptake in B. subtilis

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13.9 Role of Siderophore in Plant Growth Promotion

Siderophores are synthesized by microorganisms and released into the environment which then can bind to iron more specifically and lead the iron unavailable for other microorganisms in the vicinity and thereby limiting their growth. This approach may be used in the biological control of plant diseases (Raymond et al. 2003).

Microorganisms that prosper in the rhizosphere employ a number of different mechanisms to kill or evade pathogens . These microorganisms and their mechanisms can be used as first line of defense and hence become biocontrol agents for plants (Walsh et al. 1971). Plant roots normally exude a variety of different secondary metabolites which chemotactically attract bacteria and fungi toward itself. These bacteria and fungi produce secondary metabolites such as siderophores like bacillibactin which play an important role in competition between microorganisms in soil. These secondary metabolites may act as plant growth promoters (PGP) (Schneider and Hantke 1993). Fusarium wilt is widely reported to cause extensive damage to pepper crop . It was recently reported by Yu et al. (2011) that B. subtilis CAS15 significantly suppresses the spore germination of Fusarium wilt in pepper by 8–64 %. B. subtilis, B. amyloliquefaciens , B. cereus , and B. anthracis have been reported to produce siderophores such as bacillibactin , schizokinen , petrobactin , etc. and have also been used for biocontrol against soilborne pathogens , postharvest fungal pathogens, and even foliar pathogens (Ito and Neilands 1958; Xiong et al. 2000; Barbeau et al. 2002; Moore and Helmann 2005).