Molecular Microbiology (1992) 6(2), 179-187
Cloning and characterization of a phospholipase gene from Erwinia chiysanthemi EC'\6 N. T. Keen,* D. Ridgway and C. Boyd Department of Plant Pathology and Genetics Graduate Group. University of California, Riverside, California 92521. USA. Summary A single gene ipIcA) was cloned from a cosmid library of Erwinia chrysanthemi EC16 DNA that encoded an extracellular phospholipase. The gene was subcloned and DNA sequence data showed the presence of a single open reading frame encoding a protein with a predicted size of 39 kDa. The coding region was G+C-rich and the protein had a predicted basic isoelectric point. The protein showed no significant homology with others in the PIR library, including other phospholipases. When overexpressed in Escherichia coli cells, the pIcA gene directed production of a c. 39kDa protein that was largely localized in the periplasm, but its N-terminal amino acid sequence was that of the native protein predicted from DNA sequence data. Unlike the wild-type bacterium, an E. chrysanthemi EC16 marker exchange mutant of the pIcA gene did not secrete extracellular phospholipase activity into the medium. However, no detectable change was observed in terms of the virulence of the mutant strain on potato tubers or chrysanthemum stems. Introduction Phospholipases have recently attracted considerable attention in eukaryotes because of their role as modulators of signal transduction (Burgess etai, 1990; Einspahr and Thompson. 1990; Rhee et ai, 1989). Several microorganisms also produce phospholipases (see. e.g., Homma et ai, 1984; Johansen et ai, 1988; Prichard and Vasil, 1986) and some of them have the ability to lyse mammalian erythrocytes (Ostroff et ai. 1990; Yamada et ai, 1988) or otherwise contribute to virulence. However, relatively few bacterial genes encoding phospholipases have been cloned and characterized.
Received 12 June. 1991; revised 7 October, 1991. 'For correspondence. Tel, (714) 787 4134; Fax (714) 787 4294; BItnet PIPath^fUCRVMS.
Erwinia spp. are soft-rotting pathogens of plants and produce a battery of extracellular enzymes including several pectate lyases. proteases and cellulases (Collmer and Keen. 1986; Kotoujansky, 1987). The genes encoding certain of these enzymes have been cloned and their roles in bacterial virulence tested by mutation experiments (see, e.g., Ried and Collmer, 1988; Boccara and Chatain, 1989). Erwinia spp. also produce extracellular phospholipase C enzymes (Tseng and Bateman, 1968; Tseng and Mount, 1974) but the genes encoding them have not been cloned and their role in bacterial virulence has not been critically evaluated. We therefore cloned and characterized a gene from Erwinia chrysanthemi EC 16 which encodes an extracellularly secreted phospholipase and tested its role in bacterial virulence in plants.
Results E. chrysanthemi EC16 colonies did not produce detectable haloes on YC tributyrin plates, suggesting that the bacteria did not produce an extracellular lipase. However, pronounced opaque haloes surrounded bacterial colonies growing on YC egg-yolk agar plates, indicating the liberation of an extracellular phospholipase activity (Fig. 1, top row). A cosmid library of E. chrysanthemi EC16 DNA was screened in order to select ciones containing the gene(s) encoding the extracellular phospholipase activity. Of approximately 1500 pLAFR3 library clones screened by plating Escherichia coli DH5a transfectants on YC eggyolk plates, 10 yielded cloudy, opaque haloes around the colonies. Digestion of these cosmids with Psfl revealed that all of them contained fragments of c. 6.8 and 0.9 kb, This suggested that the clones all contained a putative phospholipase gene(s) present on the 6.8 kb Psfl fragment. Subcloning of various regions of one cosmid clone (13-7C. subsequently called pNTKOI. Table 1) showed that phospholipase aotivity was only detected from E. coli cells carrying the far-left portion of the cosmid insert (Fig. 2). Similarly, shotgun' subcloning of M/ul fragments and Psfl fragments from the original cosmid clone showed that all phospholipase-positive subclones originated from the left-most portion of the insert of pNTKOI (Fig. 2). All active Psfl subclones carried c, 6.8 kb DNA inserts, while allactive Mtu\ subclones carried c. 3.8 kb inserts. Further characterization of this region from pNTKOI by restriction
180
N. T. Keen, D. Ridgway and C. Boyd
m FJg. 1, YC egg-yolK plate assay for phospholipase production. Colonies were tooth-picked onto the plates and grown for 24 h al 37"C before being photographed. Top row. E. c/i/>san//iemiEC16 wild-type; second row. EC 16 mutant strain P-1; third row. E. coli DH5ii expressing pNTK35 without promoter inversion; fourth row. B. coli DH5a cells containing pNHISa (vector controi); fifth row, E. coti DH5a cells carrying pNTK33,
mapping and subcloning delimited the putative phospholipase gene to a c. 1.3 kb Psf l-C/a! fragment {Fig. 2). Much larger haloes were observed when £ co//cells were grown on YC egg-yolk plates if the fragment was oloned in pUC129 instead of pUC128, suggesting that the putative phospholipase gene was oriented from right to left. Subclones involving the Sacll site (Fig. 2) were devoid of phosphotipase activity, indicating that this site occurred within the putative structural gene. Several subclones prepared in pUC or pMTL plasmids were somewhat unstable following successive transfers in E. coti DH5a cells, such that the bacteria grew slowly and mutant colonies subsequently appeared which grew faster but had either reduced or no phospholipase activity. This was true of pNTK12 and pNTK23. but pNTK25 was relatively stable in £ coti DH5« cells. Some of the unstable clones did not involve the 1.3kb Pst\-Cta\ region encoding phospholipase (Fig. 2). For instance. pNTK09. cloned in pMTL20p, reduced the growth of £ coli cells and led to altered piasmid variants, despite the fact that it mapped some distance from the putative phospholipase gene in pNTK24 and pNTK25. However, satisfactory quantities of CsCI-purified piasmid DNA of this and all other constructs were obtained by growing DH5a cells soon after the constructs were made. DNA sequence of the pIcA gene Sequencing was complicated by the occurrence of several G+C-rich regions in the c. 1.3kb fragment of pNTK24/25 which caused compressions on at least one
strand. These necessitated the subcloning of selected fragments for resequencing and the construction of oligonucteotide primers to obtain satisfactory data for both strands. The Cla\ terminus of the DNA insert of pNTK21 was also sequenced. The results disclosed the presence, in the sequenced region, of only a single long open reading frame on either strand that initiated at base 130 and terminated at base 1204 (Fig. 3). The orientation of this reading frame from the C/al end towards the Pst\ end o( pNTK25 was consistent with the vector promoter observations discussed above. The 5' end of the pIcA gene was also confirmed by the Seal deletion clone. pNTK32. constructed by recloning a c. 1.2kb Sca\/Pst\ fragment (Fig. 3) into the Smal and Pst\ sites of pMTL20p (Table 1). The creation of an out-of-frame fusion with the tac alpha fragment was confirmed by DNA sequencing. As predicted, the phospholipase activity of £ coli transformants carrying pNTK32 was greatly reduced relative to the analogous construct. pNTK25. However, very low-level activity was observed on YC egg-yolk plates from cells carrying pNTK32. presumably resulting from low-level upstream translational initiation. The extreme reduction in phospholipase activity directed by this piasmid. however,
tfTK 01
I ir if
r I f
f
If
I
—'
1 —I
NTK03
+
NTK 04
•*-
NTK 0 5 / 0 6
-
NTK 0 7 / 0 8
-
NTK 09/10
-
NTK It NTK 12
+
MTK 13/14 •+ STK 21
-
NTK 22 NTK 23
+
NTK 24/25
*
NTK 26/27
-
NTK 28/29 NTK 3 0 / 3 i
-
Fig. 2. Restriction map of the insert DNA ol pNTKOI (cosmid cone 13-7C) with distances shown as kb (top); an expanded restriction map ot the left-mosi portion is shown below (pNTK03). The bars below denote vanous additional subclones. described in Table 1. and their phospholipase (Pt_ase) activity in E, coll cells plated on YC egg-yolk plates. The H/ndlll and Psf! sites shown at the left border of pNTKOl are from tiie polyfinker of the cloning vector, pLAFRS, The H/ndlll site is unique but additional Psfl s i t ^ occur m the insert ONA of pNTKOI.
Phospholipase gene from Erwinia chrysanthemi EC16 181 Table 1, Bacterial strains and plasmids employed. Strain/ Piasmid
pNTK13/pNTK14 Reference/
Characteristics
Source pNTK21
strain
E.coll
pNTK22
DH5n
E, chrysanthemi EC16
P-1
BRL Hasan and Szybalski (1987)
WHd-type strain Marker exchange mutant deficient \t\plcA
A. Chatterjee This study
pUC128/pUC129 pRK415 PDSK509 pMTL20p/ pMTL21p pINKI
pNH18a
PCPP2006
pNTKOI
PNTK03
PNTK04
pNTK05/pNTK06
pNTK07/pNTK08
pNTK09/pNTK10
pNTKll
pNTK12
pNTK26/pNTK27
pNTK28/pNTK29
Piasmid
pGSD6
pNTK24/pNTK25
Clone expressing genes involved in the extracellular secretion of proteases from E. chrysanthemi Clone containing the out genes required for extracellular secretion of E. chrysanthemi pectate lyases =cosmid clone 13-7c from the E. chrysanthemi library expressing PLase activity in £. coli c. 9.0kb Hind\\\-BamHl fragment from cosmid clone NTKOI cloned into the same sites of pUC129; PLase-positive 4.6kb H/ndlll-eg/ll fragment from pNTK03 cioned in the same sites of pMTI_21 p; PLasepositive 2.5 kb Nsi\ fragment from pNTK03. cioned in botti orientations in the same site of pUC129; PLase-negative 3.9kb Bgt\\-Sac\ fragment cloned into the same sites of pMTL20p and pMTL21p. respectively; PLase-negative 2,9kb Sac\~Bgl\\ fragment from pNTKOI. cloned into the same sites of pMTL20p and pMTL21p. respectively; PLasenegative 3,8kb /Vsfl-BamHI fragment cloned into the same sites of pUC129; PLase-negative 3.2kb Psfl-8g/il fragment cloned into the same sttes of pMTL21 p; PLase-positive
Keen el al. (1988) Keen et ai. (1988) Keen ef al. (1988) Chambers ef al. (1988) Keen and Tamaki (1986) Hasan and Szybalski (1987) Dahler ef al. (1990)
pNTK30/pNTK31
pNTK32
PNTK33
He efa/. (1991) PNTK35 This work
This work
pNTK37 This work
This work
pNTK38 This work
This work pNTK39
This work
This work
pNTK40
1.7kb Pst\-Nsi\ fragment from pNTK04 cloned in both orientations into the Psf i site of pUC129; PLase-positive 2,0kb Clal-BglW fragment from pNTKH cioned into pUC128: PLase-negative 1.7kb Sacli-EcoRV fragment cioned into the same sites of pUC129; PLase-negatJve 1.4 kb Psti-Cla\ fragment from pNTK04 cloned into the same sites ot pUC128 or pUC129, respectively; both PLasepositive 2.2kb H/ndiii-SacIl fragments from pNTK04, cioned into the same sites of pUC128/pUC129; both PLase-negative 0.8kb Psfl-Sacii fragment from pNTK24 cioned into the same sitesof pUC128 and pUC129. respectiveiy; PLase-negative 0,3kb SacII-C/ai fragment from pNTK24 cioned into the same sites of PUC128 or pUC129. respectively; PLase-negative c. 1,4kb Scat-Pst\ fragment from pNTK25 cioned into the SmalPSfi sites of pMTL20p; weakiy Pi-as8-positive 1.4 kb C/a!-Psfl fragment from pNTK25 was end-filied with Klenow fragment and the resulting DNA ligated into the Smal site of piNKI such that the PLase coding region was oriented downstream from the vector promoters; strongiy PLase-positive 1,4kb Xt)al--Sacl fragment from pNTK33 cioned into the same sites of pNH18a. such that the PLase coding region was oriented downstream from the vector promoters foiiowmg promoter inversion; strongiy PLase-positive pNTK04 was restricted with Sacll, and foilowing Kienow endfilling. the blunt-end DNA fragment was iigated to a 1.6 kb Pvull fragment from pDSK509 carrying the npti kanamycinresistance gene; PLasenegative The 6.4 kb insert from pNTK37 was removed with EcoRi and partial H/ndlll digestion and the fragment was recloned into the same sites in pRK416; PLasenegative 1.1 kb Hincm\-Mlu\ fragment from pNTK25 cioned into the same sitesof pMTL21p; phosphoiipase-positive c. 1.0kb Clai-Sal\ fragment from pNTK25 cioned into the same sitesof pUC128; phosphoiipase-negative
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
W. T. Keen, D. Ridgway and C. Boyd w « u 10
o
Cloning and characterization of a phospholipase gene from Erwinia chiysanthemi EC'\6 N. T. Keen,* D. Ridgway and C. Boyd Department of Plant Pathology and Genetics Graduate Group. University of California, Riverside, California 92521. USA. Summary A single gene ipIcA) was cloned from a cosmid library of Erwinia chrysanthemi EC16 DNA that encoded an extracellular phospholipase. The gene was subcloned and DNA sequence data showed the presence of a single open reading frame encoding a protein with a predicted size of 39 kDa. The coding region was G+C-rich and the protein had a predicted basic isoelectric point. The protein showed no significant homology with others in the PIR library, including other phospholipases. When overexpressed in Escherichia coli cells, the pIcA gene directed production of a c. 39kDa protein that was largely localized in the periplasm, but its N-terminal amino acid sequence was that of the native protein predicted from DNA sequence data. Unlike the wild-type bacterium, an E. chrysanthemi EC16 marker exchange mutant of the pIcA gene did not secrete extracellular phospholipase activity into the medium. However, no detectable change was observed in terms of the virulence of the mutant strain on potato tubers or chrysanthemum stems. Introduction Phospholipases have recently attracted considerable attention in eukaryotes because of their role as modulators of signal transduction (Burgess etai, 1990; Einspahr and Thompson. 1990; Rhee et ai, 1989). Several microorganisms also produce phospholipases (see. e.g., Homma et ai, 1984; Johansen et ai, 1988; Prichard and Vasil, 1986) and some of them have the ability to lyse mammalian erythrocytes (Ostroff et ai. 1990; Yamada et ai, 1988) or otherwise contribute to virulence. However, relatively few bacterial genes encoding phospholipases have been cloned and characterized.
Received 12 June. 1991; revised 7 October, 1991. 'For correspondence. Tel, (714) 787 4134; Fax (714) 787 4294; BItnet PIPath^fUCRVMS.
Erwinia spp. are soft-rotting pathogens of plants and produce a battery of extracellular enzymes including several pectate lyases. proteases and cellulases (Collmer and Keen. 1986; Kotoujansky, 1987). The genes encoding certain of these enzymes have been cloned and their roles in bacterial virulence tested by mutation experiments (see, e.g., Ried and Collmer, 1988; Boccara and Chatain, 1989). Erwinia spp. also produce extracellular phospholipase C enzymes (Tseng and Bateman, 1968; Tseng and Mount, 1974) but the genes encoding them have not been cloned and their role in bacterial virulence has not been critically evaluated. We therefore cloned and characterized a gene from Erwinia chrysanthemi EC 16 which encodes an extracellularly secreted phospholipase and tested its role in bacterial virulence in plants.
Results E. chrysanthemi EC16 colonies did not produce detectable haloes on YC tributyrin plates, suggesting that the bacteria did not produce an extracellular lipase. However, pronounced opaque haloes surrounded bacterial colonies growing on YC egg-yolk agar plates, indicating the liberation of an extracellular phospholipase activity (Fig. 1, top row). A cosmid library of E. chrysanthemi EC16 DNA was screened in order to select ciones containing the gene(s) encoding the extracellular phospholipase activity. Of approximately 1500 pLAFR3 library clones screened by plating Escherichia coli DH5a transfectants on YC eggyolk plates, 10 yielded cloudy, opaque haloes around the colonies. Digestion of these cosmids with Psfl revealed that all of them contained fragments of c. 6.8 and 0.9 kb, This suggested that the clones all contained a putative phospholipase gene(s) present on the 6.8 kb Psfl fragment. Subcloning of various regions of one cosmid clone (13-7C. subsequently called pNTKOI. Table 1) showed that phospholipase aotivity was only detected from E. coli cells carrying the far-left portion of the cosmid insert (Fig. 2). Similarly, shotgun' subcloning of M/ul fragments and Psfl fragments from the original cosmid clone showed that all phospholipase-positive subclones originated from the left-most portion of the insert of pNTKOI (Fig. 2). All active Psfl subclones carried c, 6.8 kb DNA inserts, while allactive Mtu\ subclones carried c. 3.8 kb inserts. Further characterization of this region from pNTKOI by restriction
180
N. T. Keen, D. Ridgway and C. Boyd
m FJg. 1, YC egg-yolK plate assay for phospholipase production. Colonies were tooth-picked onto the plates and grown for 24 h al 37"C before being photographed. Top row. E. c/i/>san//iemiEC16 wild-type; second row. EC 16 mutant strain P-1; third row. E. coli DH5ii expressing pNTK35 without promoter inversion; fourth row. B. coli DH5a cells containing pNHISa (vector controi); fifth row, E. coti DH5a cells carrying pNTK33,
mapping and subcloning delimited the putative phospholipase gene to a c. 1.3 kb Psf l-C/a! fragment {Fig. 2). Much larger haloes were observed when £ co//cells were grown on YC egg-yolk plates if the fragment was oloned in pUC129 instead of pUC128, suggesting that the putative phospholipase gene was oriented from right to left. Subclones involving the Sacll site (Fig. 2) were devoid of phosphotipase activity, indicating that this site occurred within the putative structural gene. Several subclones prepared in pUC or pMTL plasmids were somewhat unstable following successive transfers in E. coti DH5a cells, such that the bacteria grew slowly and mutant colonies subsequently appeared which grew faster but had either reduced or no phospholipase activity. This was true of pNTK12 and pNTK23. but pNTK25 was relatively stable in £ coti DH5« cells. Some of the unstable clones did not involve the 1.3kb Pst\-Cta\ region encoding phospholipase (Fig. 2). For instance. pNTK09. cloned in pMTL20p, reduced the growth of £ coli cells and led to altered piasmid variants, despite the fact that it mapped some distance from the putative phospholipase gene in pNTK24 and pNTK25. However, satisfactory quantities of CsCI-purified piasmid DNA of this and all other constructs were obtained by growing DH5a cells soon after the constructs were made. DNA sequence of the pIcA gene Sequencing was complicated by the occurrence of several G+C-rich regions in the c. 1.3kb fragment of pNTK24/25 which caused compressions on at least one
strand. These necessitated the subcloning of selected fragments for resequencing and the construction of oligonucteotide primers to obtain satisfactory data for both strands. The Cla\ terminus of the DNA insert of pNTK21 was also sequenced. The results disclosed the presence, in the sequenced region, of only a single long open reading frame on either strand that initiated at base 130 and terminated at base 1204 (Fig. 3). The orientation of this reading frame from the C/al end towards the Pst\ end o( pNTK25 was consistent with the vector promoter observations discussed above. The 5' end of the pIcA gene was also confirmed by the Seal deletion clone. pNTK32. constructed by recloning a c. 1.2kb Sca\/Pst\ fragment (Fig. 3) into the Smal and Pst\ sites of pMTL20p (Table 1). The creation of an out-of-frame fusion with the tac alpha fragment was confirmed by DNA sequencing. As predicted, the phospholipase activity of £ coli transformants carrying pNTK32 was greatly reduced relative to the analogous construct. pNTK25. However, very low-level activity was observed on YC egg-yolk plates from cells carrying pNTK32. presumably resulting from low-level upstream translational initiation. The extreme reduction in phospholipase activity directed by this piasmid. however,
tfTK 01
I ir if
r I f
f
If
I
—'
1 —I
NTK03
+
NTK 04
•*-
NTK 0 5 / 0 6
-
NTK 0 7 / 0 8
-
NTK 09/10
-
NTK It NTK 12
+
MTK 13/14 •+ STK 21
-
NTK 22 NTK 23
+
NTK 24/25
*
NTK 26/27
-
NTK 28/29 NTK 3 0 / 3 i
-
Fig. 2. Restriction map of the insert DNA ol pNTKOI (cosmid cone 13-7C) with distances shown as kb (top); an expanded restriction map ot the left-mosi portion is shown below (pNTK03). The bars below denote vanous additional subclones. described in Table 1. and their phospholipase (Pt_ase) activity in E, coll cells plated on YC egg-yolk plates. The H/ndlll and Psf! sites shown at the left border of pNTKOl are from tiie polyfinker of the cloning vector, pLAFRS, The H/ndlll site is unique but additional Psfl s i t ^ occur m the insert ONA of pNTKOI.
Phospholipase gene from Erwinia chrysanthemi EC16 181 Table 1, Bacterial strains and plasmids employed. Strain/ Piasmid
pNTK13/pNTK14 Reference/
Characteristics
Source pNTK21
strain
E.coll
pNTK22
DH5n
E, chrysanthemi EC16
P-1
BRL Hasan and Szybalski (1987)
WHd-type strain Marker exchange mutant deficient \t\plcA
A. Chatterjee This study
pUC128/pUC129 pRK415 PDSK509 pMTL20p/ pMTL21p pINKI
pNH18a
PCPP2006
pNTKOI
PNTK03
PNTK04
pNTK05/pNTK06
pNTK07/pNTK08
pNTK09/pNTK10
pNTKll
pNTK12
pNTK26/pNTK27
pNTK28/pNTK29
Piasmid
pGSD6
pNTK24/pNTK25
Clone expressing genes involved in the extracellular secretion of proteases from E. chrysanthemi Clone containing the out genes required for extracellular secretion of E. chrysanthemi pectate lyases =cosmid clone 13-7c from the E. chrysanthemi library expressing PLase activity in £. coli c. 9.0kb Hind\\\-BamHl fragment from cosmid clone NTKOI cloned into the same sites of pUC129; PLase-positive 4.6kb H/ndlll-eg/ll fragment from pNTK03 cioned in the same sites of pMTI_21 p; PLasepositive 2.5 kb Nsi\ fragment from pNTK03. cioned in botti orientations in the same site of pUC129; PLase-negative 3.9kb Bgt\\-Sac\ fragment cloned into the same sites of pMTL20p and pMTL21p. respectively; PLase-negative 2,9kb Sac\~Bgl\\ fragment from pNTKOI. cloned into the same sites of pMTL20p and pMTL21p. respectively; PLasenegative 3,8kb /Vsfl-BamHI fragment cloned into the same sites of pUC129; PLase-negative 3.2kb Psfl-8g/il fragment cloned into the same sttes of pMTL21 p; PLase-positive
Keen el al. (1988) Keen et ai. (1988) Keen ef al. (1988) Chambers ef al. (1988) Keen and Tamaki (1986) Hasan and Szybalski (1987) Dahler ef al. (1990)
pNTK30/pNTK31
pNTK32
PNTK33
He efa/. (1991) PNTK35 This work
This work
pNTK37 This work
This work
pNTK38 This work
This work pNTK39
This work
This work
pNTK40
1.7kb Pst\-Nsi\ fragment from pNTK04 cloned in both orientations into the Psf i site of pUC129; PLase-positive 2,0kb Clal-BglW fragment from pNTKH cioned into pUC128: PLase-negative 1.7kb Sacli-EcoRV fragment cioned into the same sites of pUC129; PLase-negatJve 1.4 kb Psti-Cla\ fragment from pNTK04 cloned into the same sites ot pUC128 or pUC129, respectively; both PLasepositive 2.2kb H/ndiii-SacIl fragments from pNTK04, cioned into the same sites of pUC128/pUC129; both PLase-negative 0.8kb Psfl-Sacii fragment from pNTK24 cioned into the same sitesof pUC128 and pUC129. respectiveiy; PLase-negative 0,3kb SacII-C/ai fragment from pNTK24 cioned into the same sites of PUC128 or pUC129. respectively; PLase-negative c. 1,4kb Scat-Pst\ fragment from pNTK25 cioned into the SmalPSfi sites of pMTL20p; weakiy Pi-as8-positive 1.4 kb C/a!-Psfl fragment from pNTK25 was end-filied with Klenow fragment and the resulting DNA ligated into the Smal site of piNKI such that the PLase coding region was oriented downstream from the vector promoters; strongiy PLase-positive 1,4kb Xt)al--Sacl fragment from pNTK33 cioned into the same sites of pNH18a. such that the PLase coding region was oriented downstream from the vector promoters foiiowmg promoter inversion; strongiy PLase-positive pNTK04 was restricted with Sacll, and foilowing Kienow endfilling. the blunt-end DNA fragment was iigated to a 1.6 kb Pvull fragment from pDSK509 carrying the npti kanamycinresistance gene; PLasenegative The 6.4 kb insert from pNTK37 was removed with EcoRi and partial H/ndlll digestion and the fragment was recloned into the same sites in pRK416; PLasenegative 1.1 kb Hincm\-Mlu\ fragment from pNTK25 cioned into the same sitesof pMTL21p; phosphoiipase-positive c. 1.0kb Clai-Sal\ fragment from pNTK25 cioned into the same sitesof pUC128; phosphoiipase-negative
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
W. T. Keen, D. Ridgway and C. Boyd w « u 10
o
