Amino Acid Substitutions in Transmembrane Domains 9 and 10 of GerVB That Affect the Germination Properties of
            <i>Bacillus megaterium</i>
            Spores

Christie, Graham; Lowe, Christopher R.

doi:10.1128/jb.01073-08

Cited by 19 publications

(24 citation statements)

References 34 publications

(40 reference statements)

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“…We do not have a firm explanation for this result, although KBr germination of B. megaterium spores is mediated by one or more GRs, albeit perhaps fortuitously (6,7). However, given the chaotropic effects of KBr and the potential for nonspecific inhibition of germination at high KBr concentrations, perhaps very high KBr concentrations can both stimulate spore germination via GRs and nonspecifically inhibit germination, and the nonspecific effects decrease much more than the effects on GRs as KBr concentrations decrease.…”

Section: Discussionmentioning

confidence: 70%

Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

Zhang

Garner²,

Yi³

et al. 2010

J Bacteriol

105

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The simultaneous nutrient germination of hundreds of individual wild-type spores of three Bacillus species and a number of Bacillus subtilis strains has been measured by two new methods, and rates of release of the great majority of the large pool of dipicolinic acid (DPA) from individual spores of B. subtilis strains has been measured by Raman spectroscopy with laser tweezers. The results from these analyses and published data have allowed a number of significant conclusions about the germination of spores of Bacillus species as follows. (i) The time needed for release of the great majority of a Bacillus spore's DPA once rapid DPA release had begun (⌬T release ) during nutrient germination was independent of the concentration of nutrient germinant used, the level of the germinant receptors (GRs) that recognize nutrient germinants used and heat activation prior to germination. Values for ⌬T release were generally 0.5 to 3 min at 25 to 37°C for individual wild-type spores. (ii) Despite the conclusion above, germination of individual spores in populations was very heterogeneous, with some spores in wild-type populations completing germination >15-fold slower than others. (iii) The major factor in the heterogeneity in germination of individual spores in populations was the highly variable lag time, T lag , between mixing spores with nutrient germinants and the beginning of ⌬T release . (iv) A number of factors decrease spores' T lag values including heat activation, increased levels of GRs/spore, and higher levels of nutrient germinants. These latter factors appear to affect the level of activated GRs/spore during nutrient germination. (v) The conclusions above lead to the simple prediction that a major factor causing heterogeneity in Bacillus spore germination is the number of functional GRs in individual spores, a number that presumably varies significantly between spores in populations.Spores of various Bacillus species are metabolically dormant and can survive for years in this state (30). However, spores constantly sense their environment, and if appropriate small molecules termed germinants are present, spores can rapidly return to life in the process of germination followed by outgrowth (25,29,30). The germinants that most likely trigger spore germination in the environment are low-molecularweight nutrient molecules, the identities of which are strain and species specific, including amino acids, sugars, and purine nucleosides. Metabolism of these nutrient germinants is not needed for the triggering of spore germination. Rather, these germinants are recognized by germinant receptors (GRs) located in the spore's inner membrane that recognize their cognate germinants in a stereospecific manner (17,24,25,29). Spores have a number of such GRs, with three functional GRs in Bacillus subtilis spores and even more in Bacillus anthracis, Bacillus cereus, and Bacillus megaterium spores (6,29,30).Binding of nutrient germinants to some single GRs is sufficient to trigger spore germination, for example the triggering of B...

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Section: Discussionmentioning

confidence: 70%

Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

Zhang

Garner²,

Yi³

et al. 2010

J Bacteriol

105

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“…Plasmid pGEM⌬gerKA::Km was subsequently isolated from transformant E. coli and then used as a template to PCR amplify the ⌬gerKA::Km cassette, introducing MfeI sites at the 5= ends, before digestion and ligation with EcoRI-restricted pUCTV2 (21) to yield pUCTV-⌬gerKA::Km. This plasmid was introduced into B. megaterium PV361 via polyethylene glycol (PEG)-mediated protoplast transformation (19). A Tc s Km r transformant, which had undergone double homologous recombination to introduce an insertion and deletion at the gerKA locus, was isolated after incubation of several single-crossover colonies at 42°C in the absence of antibiotics.…”

Section: Construction Of B Megaterium Gr Mutant Strainsmentioning

confidence: 99%

“…In evolutionary terms, Bacillus megaterium is a key member of the Bacillus genus, forming a deep-rooted phylogenetic branch that is genetically and phenotypically distinct from the Bacillus subtilis and Bacillus cereus groups (11). Strains and species from all three groups have for several decades been employed in numerous studies on spore germination; however, whereas the different classes of GRs have been examined in vitro by mutational analyses in B. subtilis (2,12), B. cereus (13,14), and Bacillus anthracis (6,15), detailed analyses of B. megaterium's GRs, GerU apart (16)(17)(18)(19), have not been conducted. Hence, the objective of the present work was to characterize the expression, function, and germinant recognition profiles associated with novel B. megaterium spore germinant receptors.…”

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confidence: 99%

Investigating the Functional Hierarchy of Bacillus megaterium PV361 Spore Germinant Receptors

et al. 2013

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Spores of Bacillus megaterium QM B1551 germinate rapidly when exposed to a number of single-trigger germinant compounds, including glucose, proline, leucine, and certain inorganic salts. However, spores of strain PV361, a plasmidless QM B1551 derivative that lacks the GerU germinant receptor (GR) responsible for mediating germination in response to single-trigger compounds, can germinate efficiently when incubated in nutritionally rich media, presumably via activation of additional germinant receptors. In this work, we have identified five chromosomally encoded GRs and attempted to characterize, by mutational analysis, germinant recognition profiles associated with the respective receptors in strain PV361. Of strains engineered with single GR insertion-deletions, only GerK-null spores displayed significant defective germination phenotypes when incubated in 5% (wt/ vol) beef extract or plated on rich solid medium. Cumulative decreases in viability were observed in GerK-null spores that also lacked GerA or GerA 2 , indicating that these GRs, which exerted little effect on spore germination when disrupted individually, have a degree of functionality. Unexpectedly, an efficient germination response to combinations of germinants was restored in GerA ؉ spores, which lack all other functional GRs, providing evidence for negative cooperativity between some GRs within the spore. Tetrazolium-based germinative assays conducted with purified spores indicated that these newly characterized B. megaterium GRs are cognate for a wide and chemically diverse range of germinant molecules, but unlike GerU, can only be induced to trigger germination when stimulated by at least two different germinants.

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“…However, while a single residue in TM10 of GerVB (leucine 345) was shown to be crucial to the proline-mediated germinative response, amino acid substitution and kinetic analyses indicated that this position is not directly involved in binding to the germinant (5). Indeed, while a number of residues have been identified as being important to receptor function, particularly in response to proline-and leucine-stimulated germination, the residues involved directly in binding to germinants-and, in particular, those involved in binding to glucose-have yet to be identified.…”

mentioning

confidence: 99%

“…In particular, experiments conducted in our own laboratory using cross-homologue chimeric constructs and site-directed mutagenesis (SDM) experiments with two closely related receptor B proteins have begun to yield insights into the structure-function relationships in the Bacillus megaterium GerU receptor (4,5). GerU structural genes, which are plasmid-borne on the wild-type QM B1551 strain, are required to initiate the B. megaterium QM B1551 germinative response to glucose, proline, leucine, and inorganic salts (6,36).…”

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confidence: 99%

Identification of a Receptor Subunit and Putative Ligand-Binding Residues Involved in the Bacillus megaterium QM B1551 Spore Germination Response to Glucose

2010

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The molecular basis for the recognition of glucose as a germinant molecule by spores of Bacillus megaterium QM B1551 has been examined. A chromosome-located locus (BMQ_1820, renamed gerWB) is shown to encode a receptor B-protein subunit that interacts with the GerUA and GerUC proteins to form a receptor that is cognate for both glucose and leucine. GerWB represents the third receptor B protein that binds to glucose in this strain. Site-directed mutagenesis (SDM) experiments conducted on charged proline and aromatic residues predicted to reside in the transmembrane domains of a previously characterized receptor B protein, GerVB, reveal the importance to receptor function of a cluster of residues predicted to reside in the middle of the transmembrane 6 (TM6) domain. Reductions in the region of 70-to 165-fold in the apparent affinity of receptors for glucose in which Glu196, Tyr191, and Phe192 are individually replaced by SDM indicate that some or all of these residues may be directly involved in the binding of glucose and perhaps other germinants to the germinant receptor.Spores of Bacillus species are formed via the process of sporulation in response to nutrient starvation and are characterized by metabolic dormancy and extreme properties of resistance to a range of stress factors. Reinitiation of metabolism and the generation of a new vegetative cell necessitate the dormant spore to undergo the process of germination, which can be stimulated by a number of biochemical and/or physical means (19,33).Two pathways to germination that are probably the most relevant under physiological conditions have been identified.

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Amino Acid Substitutions in Transmembrane Domains 9 and 10 of GerVB That Affect the Germination Properties of Bacillus megaterium Spores

Cited by 19 publications

References 34 publications

Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

Factors Affecting Variability in Time between Addition of Nutrient Germinants and Rapid Dipicolinic Acid Release during Germination of Spores of Bacillus Species

Investigating the Functional Hierarchy of Bacillus megaterium PV361 Spore Germinant Receptors

Identification of a Receptor Subunit and Putative Ligand-Binding Residues Involved in the Bacillus megaterium QM B1551 Spore Germination Response to Glucose

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