BSGatlas

cotA

BSGatlas-gene-785

BSGatlas

Description	Information
Coordinates	683462..685003
Genomic Size	1542 bp
Name	cotA
Outside Links	SubtiWiki
	BsubCyc
Strand	-
Type	CDS

SubtiWiki

Description	Information
Alternative Name	cotA
	cotA
	pig
Category	SW 4 Lifestyles
	SW 4.2 Sporulation
	SW 4.2.1 Sporulation proteins
	SW 4.2.1.1 Spore coat proteins
	SW 4.2.1.1.5 Class V
	SW 6 Groups of genes
	SW 6.11 Efp-dependent proteins
Description	laccase, bilirubin oxidase, spore coat protein (outer)
Enzyme Classifications	EC 1.3.3.5: bilirubin oxidase
Function	resistance of the spore
Is essential?	no
Isoelectric point	5.89
Locus Tag	BSU_06300
Molecular weight	58.3312
Name	cotA
Product	laccase, bilirubin oxidase

RefSeq

Description	Information
Alternative Locus Tag	BSU06300
Description	Evidence 1a: Function from experimental evidencesin the studied strain; PubMedId: 11514528, 12637519,16391148, 17242517, 18031270, 19933362, 22112278,25259857, 27050268; Product type e: enzyme
Enzyme Classifications	EC 1.3.3.5: bilirubin oxidase
Functions	16.6: Maintain
	16.8: Protect
Locus Tag	BSU_06300
Name	cotA
Title	outer spore coat copper-dependent promiscuouslaccase
Type	CDS

BsubCyc

Description	Information
Alternative Name	pig
Citation	Bello M;Correa-Basurto J;Rudino-Pinera E Simulation of the cavity-binding site of three bacterial multicopper oxidases upon complex stabilization: interactional profile and electron transference pathways. J Biomol Struct Dyn 32(8);1303-17 (2014) PUBMED: 23859715
	Beneyton T;Wijaya IP;Salem CB;Griffiths AD;Taly V Membraneless glucose/O(2) microfluidic biofuel cells using covalently bound enzymes. Chem Commun (Camb) 49(11);1094-6 (2013) PUBMED: 23282987
	Bento I;Silva CS;Chen Z;Martins LO;Lindley PF;Soares CM Mechanisms underlying dioxygen reduction in laccases. Structural and modelling studies focusing on proton transfer. BMC Struct Biol 10(1);28 (2010) PUBMED: 20822511
	Cho EA;Seo J;Lee DW;Pan JG Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores. Enzyme Microb Technol 49(1);100-4 (2011) PUBMED: 22112278
	Esbelin J;Mallea S;Clair G;Carlin F Inactivation by Pulsed Light of Bacillus subtilis Spores with Impaired Protection Factors. Photochem Photobiol (2016) PUBMED: 26790838
	Fan L;Zhao M;Wang Y Expression of CotA laccase in Pichia pastoris and its electrocatalytic sensing application for hydrogen peroxide. Appl Microbiol Biotechnol 99(22);9483-93 (2015) PUBMED: 26062535
	Fernandes AT;Pereira MM;Silva CS;Lindley PF;Bento I;Melo EP;Martins LO The removal of a disulfide bridge in CotA-laccase changes the slower motion dynamics involved in copper binding but has no effect on the thermodynamic stability. J Biol Inorg Chem 16(4);641-51 (2011) PUBMED: 21369750
	Fowler ZL;Baron CM;Panepinto JC;Koffas MA Melanization of flavonoids by fungal and bacterial laccases. Yeast 28(3);181-8 (2011) PUBMED: 21360730
	Hong G;Ivnitski DM;Johnson GR;Atanassov P;Pachter R Design parameters for tuning the type 1 Cu multicopper oxidase redox potential: insight from a combination of first principles and empirical molecular dynamics simulations. J Am Chem Soc 133(13);4802-9 (2011) PUBMED: 21388209
	Ihssen J;Reiss R;Luchsinger R;Thony-Meyer L;Richter M Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli. Sci Rep 5;10465 (2015) PUBMED: 26068013
	Imamura D;Kuwana R;Takamatsu H;Watabe K Localization of proteins to different layers and regions of Bacillus subtilis spore coats. J Bacteriol 192(2);518-24 (2010) PUBMED: 19933362
	Kepp KP Halide binding and inhibition of laccase copper clusters: the role of reorganization energy. Inorg Chem 54(2);476-83 (2015) PUBMED: 25532722
	Liebeton K;Lengefeld J;Eck J The nucleotide composition of the spacer sequence influences the expression yield of heterologously expressed genes in Bacillus subtilis. J Biotechnol 191;214-20 (2014) PUBMED: 24997355
	Liu Z;Xie T;Zhong Q;Wang G Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site. Acta Crystallogr F Struct Biol Commun 72(Pt 4);328-35 (2016) PUBMED: 27050268
	Mendes S;Farinha A;Ramos CG;Leitao JH;Viegas CA;Martins LO Synergistic action of azoreductase and laccase leads to maximal decolourization and detoxification of model dye-containing wastewaters. Bioresour Technol 102(21);9852-9 (2011) PUBMED: 21890348
	Plomp M;Carroll AM;Setlow P;Malkin AJ Architecture and Assembly of the Bacillus subtilis Spore Coat. PLoS One 9(9);e108560 (2014) PUBMED: 25259857
	Sheng S;Jia H;Topiol S;Farinas ET Engineering CotA Laccase for Acidic pH Stability Using Bacillus subtilis Spore Display. J Microbiol Biotechnol (2016) PUBMED: 27780951
	Silva CS;Damas JM;Chen Z;Brissos V;Martins LO;Soares CM;Lindley PF;Bento I The role of Asp116 in the reductive cleavage of dioxygen to water in CotA laccase: assistance during the proton-transfer mechanism. Acta Crystallogr D Biol Crystallogr 68(Pt 2);186-93 (2012) PUBMED: 22281748
	Xie T;Liu Z;Liu Q;Wang G Structural insight into the oxidation of sinapic acid by CotA laccase. J Struct Biol 190(2);155-61 (2015) PUBMED: 25799944
	Zeng J;Zhu Q;Wu Y;Lin X Oxidation of polycyclic aromatic hydrocarbons using Bacillus subtilis CotA with high laccase activity and copper independence. Chemosphere 148;1-7 (2016) PUBMED: 26784443
Comment	16.6: Maintain 16.8: Protect
Description	spore copper-dependent laccase
Enzyme Classifications	EC 1.3.3.5: bilirubin oxidase
Gene Ontology	GO:0005507 copper ion binding
	GO:0016491 oxidoreductase activity
	GO:0030435 sporulation resulting in formation of a cellular spore
	GO:0052716 hydroquinone:oxygen oxidoreductase activity
	GO:0055114 oxidation-reduction process
Locus Tag	BSU06300
Molecular weight	58.499
Name	cotA

Nicolas et al. predictions

Description	Information
Expression neg. correlated with	BSU14270, BSU33210, BSU06330, BSU14280, new_3195466_3195557_c, BSU07370, new_1143506_1143576, BSU10640, BSU14290, BSU22360
Expression pos. correlated with	BSU10920, BSU10910, BSU30860, BSU30880, BSU30870, BSU19490, BSU10930, new_1171643_1171754_c, BSU10940, BSU10950
Highly expressed condition	(B36) 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].
	(B60) 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].
	(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].
	(M9stat) Cells were grown in M9 supplemented with glucose (0.3 %) at 37°C with vigorous shaking. The composition of the M9 minimal medium is (per liter): 8.5 g Na2HPO4.2H20, 3 g KH2PO4, 1 g NH4Cl and 0.5 g NaCl. The following solutions were individually sterilized and added (volumes per liter of medium): 1 ml 0.1 M CaCl2.2H2O, 1 ml 1 M MgSO4.7H2O, 1 ml 50 mM Fe-Citrate. Also added was 10 ml of a trace salts solution containing (per liter): 170 mg ZnCl2, 100 mg MnCl2.4H2O, 60 mg CoCl2.6H2O, 60 mg Na2MoO4.2H2O and 43 mg CuCl2.2H2O. Overnight cultures were diluted 2000-fold in pre-warmed M9 medium and samples were harvested during exponential growth [M9exp], at the transition phase [M9tran] and during stationary phase [M9stat].
	(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.
	(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	(dia0) 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].
	(dia5) 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].
	(Etha) Cells were grown in a synthetic medium (J. Stülke, R. Hanschke, M. Hecker, J Gen Microbiol 139, 2041, Sep, 1993) with 0.2 % glucose as carbon source (Belitsky Minimal Medium/BMM) at 37 °C with vigorous shaking. Stress was applied to exponentially growing cultures at OD500nm of 0.4. Samples were harvested before stress [BMM]; after a rapid temperature up-shift from 37 °C to 48 °C [Heat]; after a temperature down-shift from 37 °C to 18 °C [Cold]. Ethanol stress was imposed by adding ethanol to a final concentration of 4 % (v/v) and cells were harvested 10 minutes after ethanol addition [Etha].
	(Heat) Cells were grown in a synthetic medium (J. Stülke, R. Hanschke, M. Hecker, J Gen Microbiol 139, 2041, Sep, 1993) with 0.2 % glucose as carbon source (Belitsky Minimal Medium/BMM) at 37 °C with vigorous shaking. Stress was applied to exponentially growing cultures at OD500nm of 0.4. Samples were harvested before stress [BMM]; after a rapid temperature up-shift from 37 °C to 48 °C [Heat]; after a temperature down-shift from 37 °C to 18 °C [Cold]. Ethanol stress was imposed by adding ethanol to a final concentration of 4 % (v/v) and cells were harvested 10 minutes after ethanol addition [Etha].
	(LBexp) Cells were grown in Luria-Bertani medium (Sigma) [LB] at 37°C with vigorous shaking in flasks. Overnight cultures were diluted 2000-fold in fresh pre-warmed medium and samples were collected during the exponential [exp], transition [tran] and stationary [stat] phases of the growth cycle .
	(LBGexp) Cells were grown in Luria-Bertani medium (Sigma) supplemented with glucose 0.3 % [LBG] at 37°C with vigorous shaking in flasks. Overnight cultures were diluted 2000-fold in fresh pre-warmed medium and samples were collected during the exponential [exp], transition [tran] and stationary [stat] phases of the growth cycle .
	(LBGstat) Cells were grown in Luria-Bertani medium (Sigma) supplemented with glucose 0.3 % [LBG] at 37°C with vigorous shaking in flasks. Overnight cultures were diluted 2000-fold in fresh pre-warmed medium and samples were collected during the exponential [exp], transition [tran] and stationary [stat] phases of the growth cycle .
	(LBGtran) Cells were grown in Luria-Bertani medium (Sigma) supplemented with glucose 0.3 % [LBG] at 37°C with vigorous shaking in flasks. Overnight cultures were diluted 2000-fold in fresh pre-warmed medium and samples were collected during the exponential [exp], transition [tran] and stationary [stat] phases of the growth cycle .
	(M0t45) Cells were grown in LB medium at 37°C with vigorous shaking. An exponentially growing culture (O.D.600 approx. 0.25) was divided: one culture acted as the control [no mitomycin C , M0] while mitomycin was added to the second culture to a final concentration of 40 ng/ml [mitomycin, M40]. Samples were harvested at 0, 45 and 90 minutes after mitomycin addition [t0, t45 and t90].
	(Mt0) Cells were grown in LB medium at 37°C with vigorous shaking. An exponentially growing culture (O.D.600 approx. 0.25) was divided: one culture acted as the control [no mitomycin C , M0] while mitomycin was added to the second culture to a final concentration of 40 ng/ml [mitomycin, M40]. Samples were harvested at 0, 45 and 90 minutes after mitomycin addition [t0, t45 and t90].
Name	cotA

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