spoVA Operon Research Articles

The SpoVA membrane complex is required for dipicolinic acid import during sporulation and export during germination.

In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth in response to nutrients. The small molecule dipicolinic acid (DPA) plays a central role in both the stress resistance of the dormant spore and its exit from dormancy during germination. The spoVA locus is required for DPA import during sporulation and has been implicated in its export during germination, but the molecular bases are unclear. Here, we define the minimal set of proteins encoded in the Bacillus subtilis spoVA operon required for DPA import and demonstrate that these proteins form a membrane complex. Structural modeling of these components combined with mutagenesis and in vivo analysis reveal that the C and Eb subunits form a membrane channel, while the D subunit functions as a cytoplasmic plug. We show that point mutations that impair the interactions between D and the C-Eb membrane complex reduce the efficiency of DPA import during sporulation and reciprocally accelerate DPA release during germination. Our data support a model in which DPA transport into spores involves cycles of unplugging and then replugging the C-Eb membrane channel, while nutrient detection during germination triggers DPA release by unplugging it.

Clostridioides difficile SpoVAD and SpoVAE Interact and Are Required for Dipicolinic Acid Uptake into Spores.

Clostridioides difficile spores, like the spores from most endospore-forming organisms, are a metabolically dormant stage of development with a complex structure that conveys considerable resistance to environmental conditions, e.g., wet heat. This resistance is due to the large amount of dipicolinic acid (DPA) that is taken up by the spore core, preventing rotational motion of the core proteins. DPA is synthesized by the mother cell, and its packaging into the spore core is mediated by the products of the spoVA operon, which has a variable number of genes, depending on the organism. C. difficile encodes 3 spoVA orthologues, spoVAC, spoVAD, and spoVAE. Prior work has shown that C. difficile SpoVAC is a mechanosensing protein responsible for DPA release from the spore core upon the initiation of germination. However, the roles of SpoVAD and SpoVAE remain unclear in C. difficile. In this study, we analyzed the roles of SpoVAD and SpoVAE and found that they are essential for DPA uptake into the spore, similar to SpoVAC. Using split luciferase protein interaction assays, we found that these proteins interact, and we propose a model where SpoVAC/SpoVAD/SpoVAE proteins interact at or near the inner spore membrane, and each member of the complex is essential for DPA uptake into the spore core. IMPORTANCE C. difficile spore heat resistance provides an avenue for it to survive the disinfection protocols in hospital and community settings. The spore heat resistance is mainly the consequence of the high DPA content within the spore core. By elucidating the mechanism by which DPA is taken up by the spore core, this study may provide insight into how to disrupt the spore heat resistance with the aim of making the current disinfection protocols more efficient at preventing the spread of C. difficile in the environment.

Properties of spores of Bacillus subtilis with or without a transposon that decreases spore germination and increases spore wet heat resistance.

This work aimed to determine how genes on transposon Tn1546 slow Bacillus subtilis spore germination and increase wet heat resistance, and to clarify the transposon's 3 gene spoVA operon's role in spore properties, since the seven wild-type SpoVA proteins form a channel transporting Ca2+ -dipicolinic acid (DPA) in spore formation and germination. Deletion of the wild-type spoVA operon from a strain with Tn1546 gave spores with slightly reduced wet heat resistance but some large decreases in germination rate. Spore water content and CaDPA analyses found no significant differences in contents of either component in spores with different Tn1546 components or lacking the wild-type spoVA operon. This work indicates that the SpoVA channel encoded by Tn1546 functions like the wild-type SpoVA channel in CaDPA uptake into developing spores, but not as well in germination. The essentially identical CaDPA and water contents of spores with and without Tn1546 indicate that low core water content does not cause elevated wet heat resistance of spores with Tn1546. Since wet heat resistance of spores of Bacillus species poses problems in the food industry, understanding mechanisms of spores' wet heat resistance is of significant applied interest.

Genetic Characterization of the Exceptionally High Heat Resistance of the Non-toxic Surrogate Clostridium sporogenes PA 3679

Clostridium sporogenes PA 3679 is a non-toxic endospore former that is widely used as a surrogate for Clostridium botulinum by the food processing industry to validate thermal processing strategies. PA 3679 produces spores of exceptionally high heat resistance without botulinum neurotoxins, permitting the use of PA 3679 in inoculated pack studies while ensuring the safety of food processing facilities. To identify genes associated with this heat resistance, the genomes of C. sporogenes PA 3679 isolates were compared to several other C. sporogenes strains. The most significant difference was the acquisition of a second spoVA operon, spoVA2, which is responsible for transport of dipicolinic acid into the spore core during sporulation. Interestingly, spoVA2 was also found in some C. botulinum species which phylogenetically cluster with PA 3679. Most other C. sporogenes strains examined both lack the spoVA2 locus and are phylogenetically distant within the group I Clostridium, adding to the understanding that C. sporogenes are dispersed C. botulinum strains which lack toxin genes. C. sporogenes strains are thus a very eclectic group, and few strains possess the characteristic heat resistance of PA 3679.

High-Level Heat Resistance of Spores of Bacillus amyloliquefaciens and Bacillus licheniformis Results from the Presence of a spoVA Operon in a Tn1546 Transposon.

Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA2mob, present on a Tn1546 transposon in Bacillus subtilis, leads to profoundly increased wet heat resistance of B. subtilis spores. Such Tn1546 transposon elements including the spoVA2mob operon were also found in several strains of Bacillus amyloliquefaciens and Bacillus licheniformis, and these strains were shown to produce spores with significantly higher resistances to wet heat than their counterparts lacking this transposon. In this study, the locations and compositions of Tn1546 transposons encompassing the spoVA2mob operons in B. amyloliquefaciens and B. licheniformis were analyzed. Introduction of these spoVA2mob operons into B. subtilis 168 (producing spores that are not highly heat resistant) rendered mutant 168 strains that produced high-level heat resistant spores, demonstrating that these elements in B. amyloliquefaciens and B. licheniformis are responsible for high level heat resistance of spores. Assessment of growth of the nine strains of each species between 5.2°C and 57.7°C showed some differences between strains, especially at lower temperatures, but all strains were able to grow at 57.7°C. Strains of B. amyloliquefaciens and B. licheniformis that contain the Tn1546 elements (and produce high-level heat resistant spores) grew at temperatures similar to those of their Tn1546-negative counterparts that produce low-level heat resistant spores. The findings presented in this study allow for detection of B. amyloliquefaciens and B. licheniformis strains that produce highly heat resistant spores in the food chain.

Bacillus subtilis spore protein SpoVAC functions as a mechanosensitive channel

A critical event during spore germination is the release of Ca-DPA (calcium in complex with dipicolinic acid). The mechanism of release of Ca-DPA through the inner membrane of the spore is not clear, but proteins encoded by the Bacillus subtilis spoVA operon are involved in the process. We cloned and expressed the spoVAC gene in Escherichia coli and characterized the SpoVAC protein. We show that SpoVAC protects E. coli against osmotic downshift, suggesting that it might act as a mechanosensitive channel. Purified SpoVAC was reconstituted in unilamellar lipid vesicles to determine the gating mechanism and pore properties of the protein. By means of a fluorescence-dequenching assay, we show that SpoVAC is activated upon insertion into the membrane of the amphiphiles lysoPC and dodecylamine. Patch clamp experiments on E. coli giant spheroplast as well as giant unilamellar vesicles (GUVs) containing SpoVAC show that the protein forms transient pores with main conductance values of about 0.15 and 0.1 nS respectively. Overall, our data indicate that SpoVAC acts as a mechanosensitive channel and has properties that would allow the release of Ca-DPA and amino acids during germination of the spore.

Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developing spores as members of the spoVA operon, which encodes proteins essential for the uptake and release of dipicolinic acid (DPA) during spore formation and germination. SpoVAF is likely an integral inner spore membrane protein and exhibits sequence identity to A subunits of the spore's nutrient germinant receptors (GRs), while SpoVAEa is a soluble protein with no obvious signals to allow its passage across a membrane. However, like SpoVAD, SpoVAEa is present on the outer surface of the spore's inner membrane, as SpoVAEa was accessible to an external biotinylation agent in spores and SpoVAEa disappeared in parallel with SpoVAD during proteinase K treatment of germinated spores. SpoVAEa and SpoVAD were also distributed similarly in fractions of disrupted dormant spores. Unlike spoVAD, spoVAEa is absent from the genomes of some spore-forming members of the Bacillales and Clostridiales orders, although SpoVAEa's amino acid sequence is conserved in species containing spoVAEa. B. subtilis strains lacking SpoVAF or SpoVAEa and SpoVAF sporulated normally, and the spores had normal DPA levels. Spores lacking SpoVAF or SpoVAEa and SpoVAF also germinated normally with non-GR-dependent germinants but more slowly than wild-type spores with GR-dependent germinants, and this germination defect was complemented by ectopic expression of the missing proteins.

Germination of individual Bacillus subtilis spores with alterations in the GerD and SpoVA proteins, which are important in spore germination.

Release of Ca(2+) with dipicolinic acid (CaDPA) was monitored by Raman spectroscopy and differential interference contrast microscopy during germination of individual spores of Bacillus subtilis strains with alterations in GerD and SpoVA proteins. Notable conclusions about germination after the addition of nutrient were as follows. (i) Following L-alanine addition, wild-type and gerD spores and spores with elevated SpoVA protein levels (↑SpoVA spores) slowly released ∼10% of their CaDPA during a variable (6- to 55-min) period ending at T(lag), the time when faster CaDPA release began. (ii) T(lag) times were lower for ↑SpoVA spores than for wild-type spores and were higher for gerD spores. (iii) The long T(lag) times of gerD spores were partially due to slow commitment to germinate. (iv) The intervals between the commitment to germinate and CaDPA release were similar for wild-type and ↑SpoVA spores but longer for gerD spores. (v) The times for rapid CaDPA release, ΔT(release) = T(release) - T(lag) (with T(release) being the time at which CaDPA release was complete), were similar for wild-type, gerD, and ↑SpoVA spores. (vi) Spores with either one of two point mutations in the spoVA operon (spoVA(1) and spoVA(2) spores) exhibited a more rapid rate of CaDPA release beginning immediately after L-alanine addition leading to ∼65% CaDPA release prior to T(lag). (vii) T(lag) times for spoVA(1) and spoVA(2) spores were longer than for wild-type spores. (viii) The intervals between spoVA(1) and spoVA(2) spores' commitment and CaDPA release were similar to those for wild-type spores, but commitment occurred later. In contrast to germination after the addition of nutrient, T(lag) and ΔT(release) times were relatively similar during dodecylamine germination of spores of the five strains. These findings suggest the following. (i) GerD plays no role in CaDPA release during spore germination. (ii) SpoVA proteins are involved in CaDPA release during germination with nutrients, and probably with dodecylamine. (iii) Spores release significant CaDPA before commitment. (iv) CaDPA release during T(lag) and ΔT(release) may signal subsequent germination events.

Studies of the release of small molecules during pressure germination of spores of Bacillus subtilis

To measure rates of release of small molecules during pressure germination of Bacillus subtilis spores, and the role of SpoVA proteins in dipicolinic acid (DPA) release. Rates of DPA release during B. subtilis spore germination with pressures of 150 or 500 megaPascals were much higher in spores with elevated levels of SpoVA proteins, and spores with a temperature-sensitive mutation in the spoVA operon were temperature-sensitive in DPA release during pressure germination. Spores also released arginine and glutamic acid, but not AMP, during pressure germination. Pressure germination of B. subtilis spores causes release of many small molecules including DPA. SpoVA proteins are involved in the release of DPA, perhaps because SpoVA proteins are a component of a DPA channel in the spore's inner membrane. This work provides new insight into the mechanism of pressure germination of spores of Bacillus species, a process that has significant potential for usage in the food industry.

Analysis of interactions between nutrient germinant receptors and SpoVA proteins ofBacillus subtilisspores

Yeast two-hybrid and Far Western analyses were used to detect interactions between Bacillus subtilis spores' nutrient germinant receptor proteins and proteins encoded by the spoVA operon, all of which are involved in spore germination and located in the spores' inner membrane. These analyses indicated that two subunits of the GerA nutrient germinant receptor interact, consistent with previous genetic data, and that some GerA proteins interact with SpoVAD and some with SpoVAE. SpoVA proteins appear to be involved in the release of the spore's dipicolinic acid during spore germination, an event triggered by the binding of nutrient germinants to their receptors. Consequently, these new findings suggest that nutrient germinant receptors physically contact SpoVA proteins, and presumably this is a route for signal transduction during spore germination.

Analysis of the germination of spores of Bacillus subtilis with temperature sensitive spo mutations in the spoVA operon

A Bacillus subtilis strain with a base substitution in the ribosome-binding site of spoVAC was temperature sensitive (ts) in sporulation and spores prepared at the permissive temperature were ts in l-alanine-triggered germination, but not in germination with Ca 2+-dipicolinic acid (DPA) or dodecylamine. Spores of a ts spo mutant with a missense mutation in the spoVAC coding region were not ts for germination with l-alanine, dodecylamine or Ca 2+-DPA. These findings are discussed in light of the proposal that SpoVA proteins are involved not only in DPA uptake during sporulation, but also in DPA release during nutrient-mediated spore germination.

The products of the spoVA operon are involved in dipicolinic acid uptake into developing spores of Bacillus subtilis.

Bacillus subtilis cells with mutations in the spoVA operon do not complete sporulation. However, a spoVA strain with mutations that remove all three of the spore's functional nutrient germinant receptors (termed the ger3 mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation. ger3 spoVA and sleB spoVA spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or ger3 spores. These properties of ger3 spoVA and sleB spoVA spores are identical to those of ger3 spoVF and sleB spoVF spores that lack DPA due to deletion of the spoVF operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in ger3 spoVF spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into ger3 spoVA spores. These data indicate that one or more products of the spoVA operon are involved in DPA transport into the developing forespore during sporulation.

Cloning and sequencing of the Bacillus megaterium spoIIA operon

The spoIIA operon of Bacillus megaterium has been cloned and the nucleotide sequence determined. The spoIIA sequence contains three open reading frames coding for putative proteins of 116 aa, 147 aa, and 253 aa; the first and the third genes are preceded by a ribosomal binding site. The genes are in the same order as those of B subtilis and B licheniformis. The deduced amino acid sequences of these three open reading frames show 78–92% homology with SpoIIAA, SpoIIAB and SpoIIAC of B subtilis and B licheniformis. Northern hybridization revealed that B megaterium also has two spoIIA transcripts, 1.77 kb and 2.92 kb, attaining maximum expression at t 1 and t 3, respectively. In addition, homology to a possible penicillin binding protein gene upstream and the first part of a spoVA operon downstream has been identified on the 3.34-kb fragment. The spoIIA and the downstream spoVA promoter regions are highly conserved among these three species. Sequence analysis of the spoVA promoter revealed a region upstream to the −35 that is highly conserved across Bacillus species.

Identification of the promoter and the transcriptional start site of the spoVA operon of Bacillus subtilis and Bacillus licheniformis

The region upstream of the coding sequence of the spoVA operon was studied by several techniques to identify the promoter and to determine the start point for transcription of spoVA. The results of plasmid integration analysis in Bacillus subtilis showed that no more than 119 bp upstream of the coding sequence is needed for expression. A comparison of the sequence of this upstream region with the corresponding sequence from Bacillus licheniformis showed four stretches that were perfectly conserved, interspersed with poorly conserved stretches; the second and third of these conserved stretches appeared to represent the '-35' and '-10' regions of a promoter recognized by RNA polymerase containing sigma G. Primer extension analysis in B. subtilis revealed a spoVA transcript which had apparently initiated 6 bp downstream of the putative '-10' heptanucleotide CATACTA, that is, 27 bp upstream of the coding sequence. This transcript was observed 4 h and 5 h after the initiation of sporulation, but not at earlier times.

Use of a lacZ gene fusion to determine the dependence pattern and the spore compartment expression of sporulation operon spoVA in spo mutants of Bacillus subtilis.

A spoVAA::lacZ gene fusion has been used to study expression of the spoVA operon during sporulation in Bacillus subtilis. beta-Galactosidase activity, encoded by the fusion gene, begins to be produced about 2.5 h after the induction of sporulation, well before the phenotypic consequences of spoVA mutations are manifested. spoVA expression is dependent on all of the known spo0 and spoII loci and on some of the 'early' spoIII loci, but not on 'later' loci. Several lines of evidence suggest that spoVA expression occurs only in the spore compartment. The implications of this observation for models of the overall regulation of gene expression during sporulation are discussed.