Isolation and characterisation of an extracellular ... · Highest activity of MvP1 was observed at...
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Conclusions Inhibition of caseinase activity Bacterial growth and caseinase production Thermostability of caseinase activity Materials and Methods The ECP of M. viscosa were produced by broth cultivation at 15°C and concentrated by precipitation. The MvP1 peptidase was isolated on ion exchange columns and gel columns, using fast protein liquid chromatography. Azocasein assay, zymograms and inhibitors were used to determine MvP1 activity. Partial sequencing of the MvP1 gene was performed, starting at the N-terminal end of the protein. Primers were based on the N-terminal amino acid sequence of MvP1 and on sequences found to be conserved within vibriolysins. The BLAST search program was used to compare sequences. Genetic relation of MvP1 to vibriolysins Sequence alignment Partial sequencing OPA; 1,10-phenanthroline, EDTA; ethylene glycol-bis(beta- aminoethyl ether)-N,N,N´,N´-tetraacetic acid, PMSF; phenylmethanesulfonyl fluoride. M. viscosa ECP were incubated with each inhibitor for 30 min. at 15°C before azocasein assay was performed. Protein mAU OD280 -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 min 25 20 15 Azocasein assay OD430 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Fractions C6+C7 Fractions B7+B6 on gel column. Caseinase activity of fractions is shown in red. Sequences with significant alignments identities / positives (%) metalloproteinase I Alteromonas sp. str. O-7 82 / 90 metalloproteinase Antarctic bacterium str. 643 81 / 89 metal protease Pseudoalteromonas sp. A28 79 / 87 M4:Peptidase Shewanella amazonensis 79 / 89 M4:Peptidase Shewanella baltica 74 / 87 Azocasein assay OD430 0.0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 Fractions B7+B6 V. anguillarum V. fluvialis A. punctata Antarctic bacterium S. baltica S. amazonensis V. vulnificus M. viscosa MvP1 Pseudoalteromonas sp. Alteromonas sp. Isolation and characterisation of an extracellular metallopeptidase from Moritella viscosa Bryndís Björnsdóttir 1 , Valgerður Andrésdóttir 1 , Guðmundur Ó. Hreggviðsson 2 and Bjarnheiður K. Guðmundsdóttir 1 1 Institute for Experimental Pathology, University of Iceland, Keldur, Reykjavík, Iceland, 2 Prokaria Ltd., Reykjavík, Iceland Moritella viscosa is the causative organism of winter ulcers in salmonids and cod (Gadus morhua). The mechanisms of infection are not well understood, but the extracellular products (ECP) of M. viscosa produce internal disease symptoms similar to those seen in fish challenged with live bacterium and the ECP are lethal to salmon (Salmo salar). Bacterial peptidases have been found to play a role in the virulence of several fish pathogenic bacteria. The aim of this study was to isolate and characterise an extracellular peptidase from M. viscosa. Introduction Casein zymogram of the purification steps, stained with Coomassie blue. 12% SDS-PAGE. A metallopeptidase, termed MvP1, with MW of ~39 kDa was isolated and partially characterised. MvP1 was produced during exponential growth and was stable in culture for four days. Highest activity of MvP1 was observed at 30°C and it was stable for up to 50°C. Partial sequencing of the Mvp1 gene suggested that MvP1 is a member of the thermolysin family, with highest homology with vibriolysins. Several thermolysins have been associated with bacterial virulence. MvP1 is the first peptidase from M. viscosa to be identified. Caseinase activity Superdex200 Fractions B7+B6 MonoQ min 25 20 15 10 5 0 30 40 50 60 70 Protein mAU OD280 0 10 20 M. viscosa ECP on ion exchange column. Caseinase activity of fractions is shown in red. min Buffer: 50mM phosphate buffer (pH 7.0) with 150 mM NaCl Buffer: 200mM Tris-HCl (pH 8.0) with 0-100% gradient of 1M NaCl. Precipitated M. viscosa ECP were fractionised on a MonoQ column and caseinase activity of fractions measured using azocasein assay. The major caseinase activity peak in fractions B7+B6 was then put on a Superdex200 column, where MvP1 was purificated to an apparent homogeneity. M. viscosa growth (blue) and caseinase production (red) in broth culture at 15°C. CFU; colony forming units. Translated partial sequence of Mvp1. The sequence obtained by N-terminal amino acid sequencing is shown in red letters. A HEXXH zinc-binding motif (red box) and a third zinc ligand-motif (yellow box) are shown. The MvP1 sequence is aligned with five vibriolysins. A part of the Mvp1 gene was sequenced and the translated sequence was used to perform a BLAST search. Antarctic bacterium str. 643 ADATGPGGNLKTGKYIYGTDFDSLNVSQTGSNCLMNTTNVKTINLNGGTSGSSAYSFTCP 60 Pseudoalteromonas sp. A28 ASATGPGGNLKTGKYIYGTDFDSLDVTQSGNTCTMNNANVRTINLNGSTSGSTAYSFTCP 60 Alteromonas sp. str. O-7 ANATGPGGNQKTGRYQYGTDYGHLDVAQSGNTCTMTNANVKTINLNHGSSGSTAHSFTCP 60 Moritella viscosa MvP1 ADATGFGGNEKTGKYHYGTDFGYLNVGQSGNNCIMNNTNVKTINLNHGTNGSSAFSFTCP 60 Shewanella amazonensis GTGTGPGGNAKTGQYEYGTDFGNLDVEVNGDTCTMNNANVKTVNLNHGTSGNTAFSYTCP 60 Shewanella baltica ASGTGPGGNIKTGQYEYGTDFSYLDVEVSGDTCTMNSPNVKTVNLNGATSGATAFSYTCP 60 . .** *** ***:* ****:. *:* .*..* *...**:*:*** .:.* :*.*:*** Antarctic bacterium str. 643 ENTFKEINGAYSPLNDAHYFGNVIFNMYNDWVGTPPLSFQLLMKVHYSSNYENAFWDGSA 120 Pseudoalteromonas sp. A28 ENTFKEINGAYSPLNDAHYFGNVIFNMYNDWVGTAPLTFQLKMRVHYGSNYENAFWDGSA 120 Alteromonas sp. str. O-7 ENTVKEINGAYSPLNDAHYFGNVVFNMYNDWLGTAPLSFQLKMRVHYSSNYENAFWDGSA 120 Moritella viscosa MvP1 ENTVKSINGAFSPLNDAHYFGGVVFDMYNDWINTAPLSFQLKMRVHYSKDYENAFWDGTA 120 Shewanella amazonensis RNTVKEINGAYSPLNDAHYFGGVVYDMYDQWYGTAPLSFQLTMRVHYSNNYENAFWDGSA 120 Shewanella baltica RNTVKEINGAYSPLNDAHYFGNVIYNMYSEWYNTAPLTFQLTMRVHYSSNYENAFWDGSA 120 .**.*.****:**********.*:::**.:* .*.**:*** *:***..:********:* Antarctic bacterium str. 643 MTFGDGQNTFYPLVSLDVSAHEVSHGFTEQNSGLVYSGKSGGLNEAFSDMAGE 173 Pseudoalteromonas sp. A28 MTFGDGQNTFYPLVSLDVSAHEVSHGFTEQNSGLIYSGKSGGLNEAFSDMAGE 173 Alteromonas sp. str. O-7 MTFGDGANTFYPLVSLDVSAHEVSHGFTEQNSGLVYRYKSGGLNEAFSDMAGE 173 Moritella viscosa MvP1 MTFGDGESYFYPLVSLDVSAHEVSHGFTEQNSGLVYEAKSGGLNEAFSDMAGE 173 Shewanella amazonensis MTFGDGQSYFYPLVSLDVSAHEVSHGFTEQNSGLVYANQSGGMNEAFSDMAGE 173 Shewanella baltica MTFGDGATTFYPLVSLDVSAHEVSHGFTEQNSGLIYDAQSGGMNEAFSDMAGE 173 ****** . *************************:* :***:********** A neighbour joining dendrogram based on the partial translated sequence of MvP1 (created by Jalview). All the peptidases are vibriolysins, except for the peptidase of Aeromonas punctata, which is used as an outgroup. ~39 kDa Protein staining Silver staining of the purification steps. 12% SDS-PAGE. Azocasein assay OD430 CFU/ml x 10 9 Caseinase activity Bacterial growth hours 92 ± 7.3 Inhibitor (mM conc.) Caseinase activity (%) None, ddH2O 100 ± 7.9 OPA (10) EDTA (10) 5 ± 0.5 53 ± 4.2 PMSF (5) The aligned sequences are vibriolysins, peptidases of the M4 thermolysin family (MEROPS database). Caseinase activity Azocasein assay OD430 temperature (°C) Caseinase activity of M. viscosa ECP at different temperatures during azocasein assay (red) and after 15 min. incubation at different temperatures prior to azocasein assay (blue).
Transcript of Isolation and characterisation of an extracellular ... · Highest activity of MvP1 was observed at...
Conclusions
Inhibition of caseinase activityBacterial growth and caseinase production Thermostability of caseinase activity
Materials and MethodsThe ECP of M. viscosa were producedby broth cultivation at 15°C andconcentrated by precipitation.The MvP1 peptidase was isolated onion exchange columns and gel columns,using fast protein liquid chromatography.Azocasein assay, zymograms andinhibitors were used to determine MvP1activity.Partial sequencing of the MvP1 genewas performed, starting at theN-terminal end of the protein. Primerswere based on the N-terminal aminoacid sequence of MvP1 and onsequences found to be conserved withinvibriolysins.The BLAST search program was usedto compare sequences.
Genetic relation of MvP1 to vibriolysinsSequence alignment Partial sequencing
OPA; 1,10-phenanthroline, EDTA; ethylene glycol-bis(beta-aminoethyl ether)-N,N,N´,N´-tetraacetic acid,PMSF; phenylmethanesulfonyl fluoride.
M. viscosa ECP were incubated with eachinhibitor for 30 min. at 15°C before azocaseinassay was performed.
ProteinmAUOD280
-1.0
0.0
1.0
2.0
3.0
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5.0
6.0
min252015
AzocaseinassayOD430
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
FractionsC6+C7
Fractions B7+B6 on gel column. Caseinaseactivity of fractions is shown in red.
Sequences with significant alignments identities / positives (%)
metalloproteinase I Alteromonas sp. str. O-7 82 / 90metalloproteinase Antarctic bacterium str. 643 81 / 89metal protease Pseudoalteromonas sp. A28 79 / 87M4:Peptidase Shewanella amazonensis 79 / 89M4:Peptidase Shewanella baltica 74 / 87
AzocaseinassayOD430
0.0
0.10
0.20
0.30
0.40
0.50
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0.70
FractionsB7+B6
V. anguillarum
V. fluvialis
A. punctata
Antarctic bacterium
S. baltica
S. amazonensis
V. vulnificus
M. viscosa MvP1
Pseudoalteromonas sp.
Alteromonas sp.
Isolation and characterisation of an extracellularmetallopeptidase from Moritella viscosa
Bryndís Björnsdóttir1, Valgerður Andrésdóttir1, Guðmundur Ó. Hreggviðsson2 and Bjarnheiður K. Guðmundsdóttir1
1Institute for Experimental Pathology, University of Iceland, Keldur, Reykjavík, Iceland, 2Prokaria Ltd., Reykjavík, Iceland
Moritella viscosa is the causative organism of winter ulcers insalmonids and cod (Gadus morhua). The mechanisms ofinfection are not well understood, but the extracellular products(ECP) of M. viscosa produce internal disease symptoms similarto those seen in fish challenged with live bacterium and theECP are lethal to salmon (Salmo salar).Bacterial peptidases have been found to play a role in thevirulence of several fish pathogenic bacteria.
The aim of this study was to isolate and characterisean extracellular peptidase from M. viscosa.
Introduction
Casein zymogram of the purification steps, stainedwith Coomassie blue. 12% SDS-PAGE.
A metallopeptidase, termed MvP1, with MW of ~39 kDa wasisolated and partially characterised.MvP1 was produced during exponential growth and wasstable in culture for four days.Highest activity of MvP1 was observed at 30°C and it wasstable for up to 50°C.Partial sequencing of the Mvp1 gene suggested that MvP1is a member of the thermolysin family, with highest homologywith vibriolysins.Several thermolysins have been associated with bacterialvirulence.MvP1 is the first peptidase from M. viscosa to be identified.
Caseinase activity
Superdex200
FractionsB7+B6
MonoQ
min2520151050
30
40
50
60
70
ProteinmAUOD280
0
10
20
M. viscosa ECP on ion exchange column. Caseinase activity of fractions is shown in red.min
Buffer:50mM phosphatebuffer (pH 7.0) with150 mM NaCl
Buffer:200mM Tris-HCl(pH 8.0) with 0-100%gradient of 1M NaCl.
Precipitated M. viscosa ECP were fractionised on a MonoQ column and caseinase activity of fractions measuredusing azocasein assay. The major caseinase activity peak in fractions B7+B6 was then put on a Superdex200column, where MvP1 was purificated to an apparent homogeneity.
M. viscosa growth (blue) and caseinase production (red) in brothculture at 15°C.CFU; colony forming units.
Translated partial sequence of Mvp1. The sequence obtained by N-terminal amino acid sequencing isshown in red letters. A HEXXH zinc-binding motif (red box) and a third zinc ligand-motif (yellow box) areshown. The MvP1 sequence is aligned with five vibriolysins.
A part of the Mvp1 gene was sequenced and the translatedsequence was used to perform a BLAST search.
Antarctic bacterium str. 643 ADATGPGGNLKTGKYIYGTDFDSLNVSQTGSNCLMNTTNVKTINLNGGTSGSSAYSFTCP 60Pseudoalteromonas sp. A28 ASATGPGGNLKTGKYIYGTDFDSLDVTQSGNTCTMNNANVRTINLNGSTSGSTAYSFTCP 60Alteromonas sp. str. O-7 ANATGPGGNQKTGRYQYGTDYGHLDVAQSGNTCTMTNANVKTINLNHGSSGSTAHSFTCP 60Moritella viscosa MvP1 ADATGFGGNEKTGKYHYGTDFGYLNVGQSGNNCIMNNTNVKTINLNHGTNGSSAFSFTCP 60Shewanella amazonensis GTGTGPGGNAKTGQYEYGTDFGNLDVEVNGDTCTMNNANVKTVNLNHGTSGNTAFSYTCP 60Shewanella baltica ASGTGPGGNIKTGQYEYGTDFSYLDVEVSGDTCTMNSPNVKTVNLNGATSGATAFSYTCP 60 . .** *** ***:* ****:. *:* .*..* *...**:*:*** .:.* :*.*:***
Antarctic bacterium str. 643 ENTFKEINGAYSPLNDAHYFGNVIFNMYNDWVGTPPLSFQLLMKVHYSSNYENAFWDGSA 120Pseudoalteromonas sp. A28 ENTFKEINGAYSPLNDAHYFGNVIFNMYNDWVGTAPLTFQLKMRVHYGSNYENAFWDGSA 120Alteromonas sp. str. O-7 ENTVKEINGAYSPLNDAHYFGNVVFNMYNDWLGTAPLSFQLKMRVHYSSNYENAFWDGSA 120Moritella viscosa MvP1 ENTVKSINGAFSPLNDAHYFGGVVFDMYNDWINTAPLSFQLKMRVHYSKDYENAFWDGTA 120Shewanella amazonensis RNTVKEINGAYSPLNDAHYFGGVVYDMYDQWYGTAPLSFQLTMRVHYSNNYENAFWDGSA 120Shewanella baltica RNTVKEINGAYSPLNDAHYFGNVIYNMYSEWYNTAPLTFQLTMRVHYSSNYENAFWDGSA 120 .**.*.****:**********.*:::**.:* .*.**:*** *:***..:********:*
Antarctic bacterium str. 643 MTFGDGQNTFYPLVSLDVSAHEVSHGFTEQNSGLVYSGKSGGLNEAFSDMAGE 173Pseudoalteromonas sp. A28 MTFGDGQNTFYPLVSLDVSAHEVSHGFTEQNSGLIYSGKSGGLNEAFSDMAGE 173Alteromonas sp. str. O-7 MTFGDGANTFYPLVSLDVSAHEVSHGFTEQNSGLVYRYKSGGLNEAFSDMAGE 173Moritella viscosa MvP1 MTFGDGESYFYPLVSLDVSAHEVSHGFTEQNSGLVYEAKSGGLNEAFSDMAGE 173Shewanella amazonensis MTFGDGQSYFYPLVSLDVSAHEVSHGFTEQNSGLVYANQSGGMNEAFSDMAGE 173Shewanella baltica MTFGDGATTFYPLVSLDVSAHEVSHGFTEQNSGLIYDAQSGGMNEAFSDMAGE 173 ****** . *************************:* :***:**********
A neighbour joining dendrogram based on the partial translated sequenceof MvP1 (created by Jalview). All the peptidases are vibriolysins, exceptfor the peptidase of Aeromonas punctata, which is used as an outgroup.
~39 kDa
Protein staining
Silver staining of the purification steps. 12% SDS-PAGE.
Azo
case
in a
ssa
y O
D4
30
CF
U/m
l x 1
09
Ca
sein
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act
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cte
ria
lg
row
th
hours92 ± 7.3
Inhibitor(mM conc.)
Caseinaseactivity (%)
None, ddH2O 100 ± 7.9OPA (10)EDTA (10)
5 ± 0.553 ± 4.2
PMSF (5)
The aligned sequences are vibriolysins, peptidases of the M4thermolysin family (MEROPS database).
Ca
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ase
act
ivity
Azo
case
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ssa
y O
D4
30
temperature (°C)Caseinase activity of M. viscosa ECP at different temperatures duringazocasein assay (red) and after 15 min. incubation at different temperaturesprior to azocasein assay (blue).
