BSGatlas

spoVAC

BSGatlas-gene-2754

BSGatlas

Description	Information
Coordinates	2441804..2442256
Genomic Size	453 bp
Name	spoVAC
Outside Links	SubtiWiki
	BsubCyc
Strand	-
Type	CDS

SubtiWiki

Description	Information
Alternative Name	spoVAC
Category	SW 4 Lifestyles
	SW 4.2 Sporulation
	SW 4.2.1 Sporulation proteins
	SW 4.2.1.4 Sporulation proteins/ other
	SW 4.3 Coping with stress
	SW 4.3.7 Coping with hypo-osmotic stress
	SW 6 Groups of genes
	SW 6.2 Membrane proteins
Description	mechanosensitive channel, essential for the uptake of the 1:1 chelate of pyridine-2,6-dicarboxylic acid (DPA(2,6)) and Ca(2 ) into developing spores, required for spore maturation
Function	spore maturation
Is essential?	no
Isoelectric point	9.46
Locus Tag	BSU_23420
Molecular weight	15.9668
Name	spoVAC
Product	mechanosensitive channel

RefSeq

Description	Information
Alternative Locus Tag	BSU23420
Description	Evidence 1a: Function from experimental evidencesin the studied strain; PubMedId: 15849754, 16077113,16850406, 24666282, 26604257, 27105070; Product type cp :cell process
Functions	16.5: Explore
Locus Tag	BSU_23420
Name	spoVAC
Title	mechanosensitive channel; stage V sporulationprotein AC
Type	CDS

BsubCyc

Description	Information
Citation	Moir A;Cooper G Spore Germination. Microbiol Spectr 3(6) (2015) PUBMED: 27337279
	Wang G;Yi X;Li YQ;Setlow P Germination of individual Bacillus subtilis spores with alterations in the GerD and SpoVA proteins, which are important in spore germination. J Bacteriol 193(9);2301-11 (2011) PUBMED: 21398556
Comment	SpoVAC appears to act as a mechanosensitive membrane channel and has properties that would allow the release of calcium-dipicolinic acid (Ca-DPA) and amino acids during germination of the spore \|CITS: [24666282]\|. From GenBank annotation: Evidence 1a: Function experimentally demonstrated in the studied strain; PubMedId: 15849754, 16077113, 16850406; Product type cp: cell process
Description	stage V sporulation protein AC, mechanosensitive channel protein
Gene Ontology	GO:0005886 plasma membrane
	GO:0016020 membrane
	GO:0016021 integral component of membrane
	GO:0030435 sporulation resulting in formation of a cellular spore
Locus Tag	BSU23420
Molecular weight	16.109
Name	spoVAC

Nicolas et al. predictions

Description	Information
Expression neg. correlated with	BSU06330, BSU35480, BSU06780, BSU10630, BSU37600, BSU10640, BSU10620, BSU03569, BSU03570, BSU40400, new_1143506_1143576
Expression pos. correlated with	BSU23440, BSU23430, BSU23410, BSU03920, BSU22930, BSU05530, BSU23402, BSU03930, BSU18720, BSU34470
Highly expressed condition	(BT) A fresh colony grown on an LB plate was used to inoculate 10 ml of LB and grown for 10 hoursat 30°C. This culture wasused to inoculate 10 ml of MSgg medium (S.S. Branda et al., J Bacteriol 186, 3970, Jun, 2004) and incubated with vigorous shaking. The cultures in MSgg were diluted to the same extent in 96 wells microtiterplates (5 μl for 1.5 ml of medium) and incubated without shaking at 30°C. Cells from the control cultures were harvested after 24 hours of incubation [BT]. Biofilms were harvested from 96 well plates after incubation for 36 hours [B36] and 60 hours [B60].
	(ferm) Cells were grown in a synthetic medium (E. Härtig, A. Hartmann, M. Schätzle, A. M. Albertini, D. Jahn, Appl Environ Microbiol 72, 5260, 2006) at 37 °C. For aerobic growth, an overnight culture was used to inoculate 100 ml of the synthetic medium to a starting OD578 of 0.05. The culture was then incubated in a 500 ml baffled flask with shaking at 250 rpm [aero]. Anaerobic growth was carried out (i) in the presence of 10 mM potassium nitrate (nitrate respiration) [nit]; or (ii) in the absence of 10 mM postassium nitrate (fermentative growth) [ferm]. The procedure for anaerobic growth was: medium was inoculated to an OD578 nm of 0.1 in flasks completely filled with medium and sealed with rubber stoppers. They were shaken at 100 rpm to minimize cell aggregation. These cultures were inoculated aerobically with an aerobically grown overnight culture. Anaerobic conditions were achieved in the stoppered flasks after a short time through the consumption of residual oxygen. Cells were harvested during the exponential growth phase.
	(nit) Cells were grown in a synthetic medium (E. Härtig, A. Hartmann, M. Schätzle, A. M. Albertini, D. Jahn, Appl Environ Microbiol 72, 5260, 2006) at 37 °C. For aerobic growth, an overnight culture was used to inoculate 100 ml of the synthetic medium to a starting OD578 of 0.05. The culture was then incubated in a 500 ml baffled flask with shaking at 250 rpm [aero]. Anaerobic growth was carried out (i) in the presence of 10 mM potassium nitrate (nitrate respiration) [nit]; or (ii) in the absence of 10 mM postassium nitrate (fermentative growth) [ferm]. The procedure for anaerobic growth was: medium was inoculated to an OD578 nm of 0.1 in flasks completely filled with medium and sealed with rubber stoppers. They were shaken at 100 rpm to minimize cell aggregation. These cultures were inoculated aerobically with an aerobically grown overnight culture. Anaerobic conditions were achieved in the stoppered flasks after a short time through the consumption of residual oxygen. Cells were harvested during the exponential growth phase.
	(Pyr) A 5 ml aliquot of LB medium was inoculated using frozen culture stocks. After a few hours growth at 37°C, precultures were prepared by inoculating 5 ml of M9 with this LB culture at several different dilutions usually ranging from 500- to 2000-fold. The dilution range was chosen so that one of these precultures had grown to and OD600 of 0.5 - 1.0 after overnight inculation. The chosen M9 medium precultures [at OD600 of 0.5 - 1.0] were used to inoculate 100 mL of M9 medium in 500 mL non-baffled shake flasks to an OD600 of 0.02. Filter-sterilized carbon sources were added separately to the medium M9 at following concentration: D-Glucose 3g/L[Glu], L-Malic acid 4.5g/L[Mal], L-Malic acid + D-Glucose 3 and 2g/L[M+G], D-Fructose 3g/L[Fru], D-Gluconate 4g/L[Glucon], Pyruvate 6g/L[Pyr], Glycerol 6g/L[Gly], Glutamic acid + Succinic acid 2 and 2g/L[G+S]. Where necessary, carbon source solutions were pH neutralized with 4 M NaOH prior to addition to the medium. Cells were harvested during the exponential growth phase.
	(S4) Cells were grown in CH medium at 37°C and sporulation was induced by resuspension in warm sporulation medium as described by Sterlini and Mandelstam (J. M. Sterlini, J. Mandelstam, Biochem J 113, 29, Jun, 1969). The initiation of sporulation was designated T0, the time of resuspension. Samples were harvested at hourly intervals for 6 hours [S0 to S6] for the first set of experiments and for 8 hours [S0 to S8] for a second set of experiments.
	(S5) Cells were grown in CH medium at 37°C and sporulation was induced by resuspension in warm sporulation medium as described by Sterlini and Mandelstam (J. M. Sterlini, J. Mandelstam, Biochem J 113, 29, Jun, 1969). The initiation of sporulation was designated T0, the time of resuspension. Samples were harvested at hourly intervals for 6 hours [S0 to S6] for the first set of experiments and for 8 hours [S0 to S8] for a second set of experiments.
	(S6) Cells were grown in CH medium at 37°C and sporulation was induced by resuspension in warm sporulation medium as described by Sterlini and Mandelstam (J. M. Sterlini, J. Mandelstam, Biochem J 113, 29, Jun, 1969). The initiation of sporulation was designated T0, the time of resuspension. Samples were harvested at hourly intervals for 6 hours [S0 to S6] for the first set of experiments and for 8 hours [S0 to S8] for a second set of experiments.
	(S7) Cells were grown in CH medium at 37°C and sporulation was induced by resuspension in warm sporulation medium as described by Sterlini and Mandelstam (J. M. Sterlini, J. Mandelstam, Biochem J 113, 29, Jun, 1969). The initiation of sporulation was designated T0, the time of resuspension. Samples were harvested at hourly intervals for 6 hours [S0 to S6] for the first set of experiments and for 8 hours [S0 to S8] for a second set of experiments.
	(S8) Cells were grown in CH medium at 37°C and sporulation was induced by resuspension in warm sporulation medium as described by Sterlini and Mandelstam (J. M. Sterlini, J. Mandelstam, Biochem J 113, 29, Jun, 1969). The initiation of sporulation was designated T0, the time of resuspension. Samples were harvested at hourly intervals for 6 hours [S0 to S6] for the first set of experiments and for 8 hours [S0 to S8] for a second set of experiments.
Lowely expressed condition	(C90) Cellsgrown overnight on LB agar plates at 30°Cwere harvested and used to inoculate pre-warmed minimal medium at OD600 of 0.5 (D. Dubnau, R. Davidoff-Abelson, J Mol Biol 56, 209, Mar 14, 1971). After growth at 37°C with vigorous shaking, cells were diluted ten times in fresh pre-warmed minimal medium and samples were harvested after a period of 30 minutes [C30] , i.e. before maximal induction of competence, and after a period of 90 minutes [C90], i.e. when competence induction was maximal.
	(dia15) Diamide was added to an exponentially growing culture (OD600 approx. 0.6) at a sub-lethal concentration(0.5 mM) and growth continued at 37°C with vigorous shaking. Samples were collected 0, 5 and 15 minutes after diamide addition [dia0, dia5 and dia15].
	(G150) Purified spores were obtained by growing cells in DSM medium (P. Schaeffer, J. Millet, J. P. Aubert, Proc Natl Acad Sci U S A 54, 704, Sep, 1965) at 37°C for 48 hours after which they were washed ten times in ice cold distilled waterover a period of 5 days. Purified spores were heat activated at 70°C in Tris 10 mM pH8.4 and germination was initiated by the addition of L-alanine 10 mM (A. Moir, J Bacteriol 146, 1106, Jun, 1981). After incubation for one hour at 37°C, the culture was diluted with an equal volume of 2X LBmedium and germinating cells were harvested at 135, 150 or 180 minutes after addition of L-alanine [G135, G150 and G180].
	(H2O2) Cells were grown in LB medium at 37°C. At OD540 of 0.3, the culture were divided into four subcultures and diamide 0.6 mM [Diami], paraquat 0.4 mM [Paraq], H2O2 0.1mM [H2O2] or no oxidative drug [Oxctl] were added to the medium. Samples were taken 10 minutes after addition
	(Mal) A 5 ml aliquot of LB medium was inoculated using frozen culture stocks. After a few hours growth at 37°C, precultures were prepared by inoculating 5 ml of M9 with this LB culture at several different dilutions usually ranging from 500- to 2000-fold. The dilution range was chosen so that one of these precultures had grown to and OD600 of 0.5 - 1.0 after overnight inculation. The chosen M9 medium precultures [at OD600 of 0.5 - 1.0] were used to inoculate 100 mL of M9 medium in 500 mL non-baffled shake flasks to an OD600 of 0.02. Filter-sterilized carbon sources were added separately to the medium M9 at following concentration: D-Glucose 3g/L[Glu], L-Malic acid 4.5g/L[Mal], L-Malic acid + D-Glucose 3 and 2g/L[M+G], D-Fructose 3g/L[Fru], D-Gluconate 4g/L[Glucon], Pyruvate 6g/L[Pyr], Glycerol 6g/L[Gly], Glutamic acid + Succinic acid 2 and 2g/L[G+S]. Where necessary, carbon source solutions were pH neutralized with 4 M NaOH prior to addition to the medium. Cells were harvested during the exponential growth phase.
	(MG+25) A culture of LB medium was inocualted from a frozen glycerol stock of B. subtilis. After few hours at 37oC when the culture was growing exponentially, this culture was used to inoculate M9 minimal medium at several different dilutions usually in the range of 500- to 2000-fold. The dilution range was chosen to ensure that at least one of these M9 precultures had reached an OD600 between 0.5 - 1.0 after overnight incubation. These precultures were then used to inoculate 2.5 L of M9 medium in a 3.1 L KLF bioreactor (Bioengineering AG, Wald, Switzerland) to a starting OD600 of 0.03 – 0.05. Condiions in the bioreactor were rigorously controlled as follows: temperature was controlled at 37 °C; the pH was maintained at exactly 7.2 by automatic titration with 2.0 M KOH and 2.0 M H2SO4, and the dissolved oxygen tension was maintained above 50%. In each nutritional shift experiment cells were grown on the single substrate until the OD600 reached 0.50, at which point the second substrate was added instantaneously (4 g/L L-malate or 3 g/L glucose). The nutrient shifts performed were from glucose to glucose+malate [GM] and from malate to malate+glucose [MG] (Buescher et al., accompanying paper). Cell growth during the course was monitored throughout the experiment by measuring OD600.
	(MG+t5) A culture of LB medium was inocualted from a frozen glycerol stock of B. subtilis. After few hours at 37oC when the culture was growing exponentially, this culture was used to inoculate M9 minimal medium at several different dilutions usually in the range of 500- to 2000-fold. The dilution range was chosen to ensure that at least one of these M9 precultures had reached an OD600 between 0.5 - 1.0 after overnight incubation. These precultures were then used to inoculate 2.5 L of M9 medium in a 3.1 L KLF bioreactor (Bioengineering AG, Wald, Switzerland) to a starting OD600 of 0.03 – 0.05. Condiions in the bioreactor were rigorously controlled as follows: temperature was controlled at 37 °C; the pH was maintained at exactly 7.2 by automatic titration with 2.0 M KOH and 2.0 M H2SO4, and the dissolved oxygen tension was maintained above 50%. In each nutritional shift experiment cells were grown on the single substrate until the OD600 reached 0.50, at which point the second substrate was added instantaneously (4 g/L L-malate or 3 g/L glucose). The nutrient shifts performed were from glucose to glucose+malate [GM] and from malate to malate+glucose [MG] (Buescher et al., accompanying paper). Cell growth during the course was monitored throughout the experiment by measuring OD600.
	(Oxctl) Cells were grown in LB medium at 37°C. At OD540 of 0.3, the culture were divided into four subcultures and diamide 0.6 mM [Diami], paraquat 0.4 mM [Paraq], H2O2 0.1mM [H2O2] or no oxidative drug [Oxctl] were added to the medium. Samples were taken 10 minutes after addition
	(Paraq) Cells were grown in LB medium at 37°C. At OD540 of 0.3, the culture were divided into four subcultures and diamide 0.6 mM [Diami], paraquat 0.4 mM [Paraq], H2O2 0.1mM [H2O2] or no oxidative drug [Oxctl] were added to the medium. Samples were taken 10 minutes after addition
Name	spoVAC

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