The EMBO Journal Vol.2 No. 10 pp. 1831 - 1838, 1983 Construction of a correlated physical and genetic map of the Klebsiella pneumoniae hisDGO region using transposon Tn5 mutagenesis F.J. de Bruijnl*, I.L. Stroke2, D.J. Marvel1 and F.M. Ausubel1 Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA 02138, USA Communicated by J. Schell Received on 11 July 1983 Multicopy plasmids containing the hisDG region of Klebsiella pneumoniae were mutagenized with transposon Tn5. The resulting plasmids were examined for their ability to comple- ment hisD and hisG mutations in Escherichia coli. The physical location of Tn5 on each of the hisD::Tn5 and hisG::Tn5 plasmids was determined by restriction endo- nuclease analysis. By combining the two types of data, a precise correlated physical and genetic map of the K. pneu- moniae hisDG region was constructed. Based on this analysis, the minimum sizes of the hisD and the hisG genes were calculated to be 1100 bp and 900 bp, respectively. The hisO(P) region was also identified. The insertional specificity of transposon Tn5 was shown to be very low. One unantici- pated result was obtained: Tn5 insertions in the plasmid- borne hisG gene were not polar on hisD. Key words: Klebsiella pneumoniae/hisDGO/Tn5/polarity/ insertional specificity Introduction The biosynthesis of histidine in Salmonella typhimurium and Escherichia coli is catalysed by nine enzymes, coded for by a cluster of nine genes arranged in an operon (Hartman et al., 1971; Blasi and Bruni, 1981). The order of the nine his genes is the same in S. typhimurium and in E. coli. A com- parison of the nucleotide sequences of the his control regions of S. typhimurium and E. coli (Barnes, 1978; Dinocera et al., 1978; Verde et al., 1981) led to the proposal of a translation- dependent attenuation mechanism, originating at the his pro- moter and regulating transcription of the his operon in both species (for a review see Kolter and Yanofsky, 1982). In contrast to the his operons of S. typhimurium and E. coli, the his region of Klebsiella pneumoniae has not been extensively characterized, although it is likely that it will con- tain a his operon structurally and functionally similar to the analogous S. typhimurium and E. coli operons. The K. pneu- moniae hisD and hisG genes, coding for enzymes involved in the last and first steps of the histidine biosynthetic pathway (Hartman et al. 1971), as well as the his operon regulatory region, are located immediately adjacent to the nif (nitrogen fixation) gene cluster (Streicher et al., 1972; Dixon and Post- gate, 1972; see Figure 1). K. pneumoniae DNA fragments carrying the hisD and hisG structural genes, the hisO(P) 'Present address: Department of Molecular Biology, Massachusetts General Hospital, Jackson Research Building, Boston, MA 02114. 2Present address: Department of Biology, Yale University, New Haven, CT 06520. *To whom reprint requests should be sent at present address: Max Planck Institut, Abt. Schell 5000 Koln 30, FRG. regulatory region, and in some cases nif genes have been clon- ed into muticopy plasmids (Cannon et al., 1977, 1979; Riedel, 1980; Rodriguez et al., 1982). The cloned his and nif genes have been used to determine that the direction of transcrip- tion of K. pneumoniae his operon and at least the majority of genes in the nif gene cluster is the same (Janssen et al., 1980). This maps the location of the hisO(P) regulatory region bet- ween hisG and nifQ (see Figure 1). In order to be able to compare the structure and regulation of the his operon of K. pneumoniae with those of S. typhi- murium and E. coli, and to extend the physical and genetic analysis of the K. pneumoniae his-nif region (Riedel et al., 1979; Merrick et al., 1980), we have constructed a correlated physical and genetic map of the K. pneumoniae hisDG and hisO(P) region using transposon Tn5 mutagenesis. Trans- poson Tn5 confers resistance to certain aminoglycoside anti- biotics such as kanamycin (Km) and neomycin (Neo) (Berg et al., 1978). Tn5 has been shown to transpose at a frequency of - 5 x 105 (Kmr colonies/p.f.u.) of the X::Tn5 phage (Johnson et al., 1982) and Tn5 has been shown to display a rather low level of insertional specificity (Shaw and Berg, 1979; Berg et al., 1980; Miller et al., 1980). When Tn5 transposes into DNA sequences comprising a structural gene (or its regulatory region) it abolishes gene function through insertional inac- tivation and can exert a polar effect on genes within an operon located distally to its insertion point (Berg, 1977; Berg et al., 1980). These properties of Tn5 induced mutations, combined with their low reversion frequency ( < 10-6, Berg, 1977) have made Tn5 a useful tool for genetic and physical mapping of chromosomal genes and operons (see Kleckner et al., 1977; Riedel et al., 1979; Merrick et al., 1980). More recently, Tn5 mutagenesis has been adapted for use in the rapid generation of correlated physical and genetic maps of DNA segments which have been cloned into multi- copy plasmid vectors (Laird and Young, 1980; Pannekoek et al., 1980; de Bruijn and Ausubel, 1981; Lupski et al., 1982; de Bruijn, 1983). Here we present a physical and genetic analysis of the hisDGO(P) region of K. pneumoniae using transposon Tn5 mutagenesis of multicopy plasmids carrying cloned hisDGO(P) DNA sequences. A total of 170 Tn5 insertions in hisD, hisG and hisO(P) were isolated and characterized. Ap- proximately 100 of the insertions were physically mapped with the use of restriction endonucleases resulting in the con- struction of a correlated physical and genetic map of the hisDGO(P) region. Transposon Tn5 insertions into the plasmid-borne hisG gene did not exert a polar effect on the hisD gene carried on the same plasmid. The distribution of 1500 transposon Tn5 insertions into one of the plasmids car- rying the cloned hisDGO(P) region was shown to be essential- ly random. The results of the physical mapping of 100 hisDGO(P)::Tn5 insertions and the distribution study suggest that the insertional specificity of transposon Tn5 is extremely low. This supports our previous observations regarding the usefulness of Tn5 mutagenesis for the rapid construction of correlated physical and genetic nmaps of DNA segments clon- ed into multicopy plasmids. (c) IRL Press Limited, Oxford, England. 1831
Transcript
The EMBO Journal Vol.2 No. 10 pp. 1831 - 1838, 1983
Construction of a correlated physical and genetic map of theKlebsiella pneumoniae hisDGO region using transposon Tn5mutagenesis
F.J. de Bruijnl*, I.L. Stroke2, D.J. Marvel1 andF.M. Ausubel1Department of Cellular and Developmental Biology, Harvard University,Cambridge, MA 02138, USA
Communicated by J. SchellReceived on 11 July 1983
Multicopy plasmids containing the hisDG region of Klebsiellapneumoniae were mutagenized with transposon Tn5. Theresulting plasmids were examined for their ability to comple-ment hisD and hisG mutations in Escherichia coli. Thephysical location of Tn5 on each of the hisD::Tn5 andhisG::Tn5 plasmids was determined by restriction endo-nuclease analysis. By combining the two types of data, aprecise correlated physical and genetic map of the K. pneu-moniae hisDG region was constructed. Based on this analysis,the minimum sizes of the hisD and the hisG genes werecalculated to be 1100 bp and 900 bp, respectively. ThehisO(P) region was also identified. The insertional specificityof transposon Tn5 was shown to be very low. One unantici-pated result was obtained: Tn5 insertions in the plasmid-borne hisG gene were not polar on hisD.Key words: Klebsiella pneumoniae/hisDGO/Tn5/polarity/insertional specificity
IntroductionThe biosynthesis of histidine in Salmonella typhimurium
and Escherichia coli is catalysed by nine enzymes, coded forby a cluster of nine genes arranged in an operon (Hartman etal., 1971; Blasi and Bruni, 1981). The order of the nine hisgenes is the same in S. typhimurium and in E. coli. A com-parison of the nucleotide sequences of the his control regionsof S. typhimurium and E. coli (Barnes, 1978; Dinocera et al.,1978; Verde et al., 1981) led to the proposal of a translation-dependent attenuation mechanism, originating at the his pro-moter and regulating transcription of the his operon in bothspecies (for a review see Kolter and Yanofsky, 1982).
In contrast to the his operons of S. typhimurium andE. coli, the his region of Klebsiella pneumoniae has not beenextensively characterized, although it is likely that it will con-tain a his operon structurally and functionally similar to theanalogous S. typhimurium and E. coli operons. The K. pneu-moniae hisD and hisG genes, coding for enzymes involved inthe last and first steps of the histidine biosynthetic pathway(Hartman et al. 1971), as well as the his operon regulatoryregion, are located immediately adjacent to the nif (nitrogenfixation) gene cluster (Streicher et al., 1972; Dixon and Post-gate, 1972; see Figure 1). K. pneumoniae DNA fragmentscarrying the hisD and hisG structural genes, the hisO(P)
'Present address: Department of Molecular Biology, Massachusetts GeneralHospital, Jackson Research Building, Boston, MA 02114.
2Present address: Department of Biology, Yale University, New Haven, CT06520.*To whom reprint requests should be sent at present address: Max PlanckInstitut, Abt. Schell 5000 Koln 30, FRG.
regulatory region, and in some cases nifgenes have been clon-ed into muticopy plasmids (Cannon et al., 1977, 1979; Riedel,1980; Rodriguez et al., 1982). The cloned his and nif geneshave been used to determine that the direction of transcrip-tion of K. pneumoniae his operon and at least the majority ofgenes in the nifgene cluster is the same (Janssen et al., 1980).This maps the location of the hisO(P) regulatory region bet-ween hisG and nifQ (see Figure 1).
In order to be able to compare the structure and regulationof the his operon of K. pneumoniae with those of S. typhi-murium and E. coli, and to extend the physical and geneticanalysis of the K. pneumoniae his-nif region (Riedel et al.,1979; Merrick et al., 1980), we have constructed a correlatedphysical and genetic map of the K. pneumoniae hisDG andhisO(P) region using transposon Tn5 mutagenesis. Trans-poson Tn5 confers resistance to certain aminoglycoside anti-biotics such as kanamycin (Km) and neomycin (Neo) (Berg etal., 1978). Tn5 has been shown to transpose at a frequency of- 5 x 105 (Kmr colonies/p.f.u.) of the X::Tn5 phage (Johnsonet al., 1982) and Tn5 has been shown to display a rather lowlevel of insertional specificity (Shaw and Berg, 1979; Berg etal., 1980; Miller et al., 1980). When Tn5 transposes into DNAsequences comprising a structural gene (or its regulatoryregion) it abolishes gene function through insertional inac-tivation and can exert a polar effect on genes within anoperon located distally to its insertion point (Berg, 1977; Berget al., 1980). These properties of Tn5 induced mutations,combined with their low reversion frequency ( < 10-6, Berg,1977) have made Tn5 a useful tool for genetic and physicalmapping of chromosomal genes and operons (see Kleckner etal., 1977; Riedel et al., 1979; Merrick et al., 1980).More recently, Tn5 mutagenesis has been adapted for use
in the rapid generation of correlated physical and geneticmaps of DNA segments which have been cloned into multi-copy plasmid vectors (Laird and Young, 1980; Pannekoek etal., 1980; de Bruijn and Ausubel, 1981; Lupski et al., 1982;de Bruijn, 1983).
Here we present a physical and genetic analysis of thehisDGO(P) region of K. pneumoniae using transposon Tn5mutagenesis of multicopy plasmids carrying clonedhisDGO(P) DNA sequences. A total of 170 Tn5 insertions inhisD, hisG and hisO(P) were isolated and characterized. Ap-proximately 100 of the insertions were physically mappedwith the use of restriction endonucleases resulting in the con-struction of a correlated physical and genetic map of thehisDGO(P) region. Transposon Tn5 insertions into theplasmid-borne hisG gene did not exert a polar effect on thehisD gene carried on the same plasmid. The distribution of1500 transposon Tn5 insertions into one of the plasmids car-rying the cloned hisDGO(P) region was shown to be essential-ly random. The results of the physical mapping of 100hisDGO(P)::Tn5 insertions and the distribution study suggestthat the insertional specificity of transposon Tn5 is extremelylow. This supports our previous observations regarding theusefulness of Tn5 mutagenesis for the rapid construction ofcorrelated physical and genetic nmaps of DNA segments clon-ed into multicopy plasmids.
(c) IRL Press Limited, Oxford, England. 1831
F.J. de Bruijn et al.
his*-_____
n/f4 4- 44- - -4 4 - -- Approximate
(E I FAHBC)DGO QB A L FMVSUXN E Y K DH J gene locations
Fig. 1. Physical and genetic map of the K. pneumoniae his-nif region. The order, location, approximate boundaries (vertical broken lines), and organizationinto transcription units (horizontal arrows) of the nif genes and the EcoRI (R) sites are derived from Riedel et al. (1983). The direction of transcription of thehis operon was determined by Janssen et al. (1980). The order of the hisC-E genes (between brackets) are based on analogy with the gene order in S. typhi-murium and E. coli (Brenner and Ames, 1971); the order and location of the hisDGO region was determined in this work.
ResultsTransposon Tn5 mutagenesis ofplasmids carrying the clonedK. pneumoniae hisDG region: identification of HisD-pGRIOO::Tn5 and pCRAIO::Tn5 plasmidsWe used two plasmids, pGR100 and pCRA10 (Riedel,
1980; Cannon et al., 1977) for our initial transposon Tn5mutagenesis experiments in order to generate two indepen-dent collections of Tn5 insertions and to circumvent thepossibility of insertional 'hot-spots' for Tn5 within plasmidvector sequences (for bacterial strains, phages and plasmidsused, see Table I). Plasmid pGR100 contains a 2.3-kb EcoRIfragment of K. pneumoniae DNA cloned into the multicopyplasmid vector pBR322 (Bolivar et al., 1977). PlasmidpCRA1O contains the same fragment cloned into the multi-copy plasmid vector pMB9 (Bolivar et al., 1977). The struc-tures of pGR100 and pCRA1O are shown in Figure 2. Thefollowing observations indicate that plasmids pGR100 andpCRA10 carry the hisD and hisG structural genes as well asthe hisO(P) regulatory region.
Firstly, plasmids pGR100 and pCRA10 as well as severalderivative plasmids with the 2.3-kb EcoRI fragment cloned ineither orientation in vectors pBR322, pACYC184 (Chang andCohen, 1978) and pPV33 (West and Rodriguez, 1982) com-plement hisD - and hisG - mutant strains of E. coli andK. pneumoniae (Cannon et al., 1977; Riedel, 1980;Rodriguez et al., 1982).
Secondly, recombinant bacteriophage P4 derivatives(P4H5 and P4H1O) containing the hisDG 2.3-kb EcoRI frag-ment cloned in both orientations transduce hisD - and hisG -strains of E. coli and K. pneumoniae to His+ at high fre-quencies (Ow and Ausubel, 1980).
Thirdly, the synthesis of the hisG gene product by theP4H5 and P4H10 recombinant phages increases 2.1-fold inthe absence of exogenous amino acids (Ow and Ausubel,1980), a phenomenon analogous to hisG derepressionreported by Stephens et al. (1975). The latter observation sug-gests that the complete his regulatory region, hisO(P), is pre-sent on the 2.3-kb EcoRI fragment.
Transposon Tn5 mutagenesis of plasmids pGR100 andpCRA1O was carried out as described by de Bruijn andAusubel (1981; see Figure 3 and Materials and methods). ThehisD- strain D18FG was used to screen for pGRIOO::Tn5and pCRA1O::Tn5 plasmids unable to complement theHisD- phenotype. Among 500 pGRIOO::Tn5 plasmids andamong 500 pCRAIO::Tn5 plasmids analyzed, 50 and 11 werefound to be HisD - respectively. These 61 plasmids were used
Table I. Bacterial strains, phages and plasmids
Genotype Relevant pheno- Source/type reference
E. coli strainsD18FG hisD pro thr leu recA HisD- H. Greer
hspR
NK5875 F' 141/ /euB6 hisGI HisG- N. KlecknerargG6 metBI lacYl ga/6xy/7 mt/2 ma/AI rpsL 104tonA2 tsxl supE44recA I Xr X -
HB1I1 hsdS20 recA 13 aral4 For X::Tn5 H. BoyerproA2 lacYl galK2 rpsL20 infectionsxy/5 mtll X -
Ql C600: thrl /eu6 lacYl Preparation E. Signerthil supE44 tonA21 T5r of X::Tn5080r
PhagesX::Tn5 X467: Xb22I rex::Tn5 For Tn5 N. Kleckner
cI857 Oam29 Pam8O mutagenesis
PlasmidspGRIOO Tcr Apr hisDG' pBR322 with the Riedel (1980)
2.3-kb HisD+G+EcoRI fragment
pCRA1O Tcr hisDG + pMB9 with the Cannon et al.2.3-kb HisD +G + (1977)EcoRI fragment
pCRA13 Tcr hisDGO+ pMB9 with the Cannon et al.2.3-kb HisD+G+ (1977)and the adjacent1.4-kb EcoRIfragments
pBR322 Tcr Apr Plasmid vector Bolivar et al.of pGRIOO (1977)
pMB9 Tcr Plasmid vector Bolivar et al.of pCRA1O and (1977)pCRA13
for further analysis.Physical mapping of thepCRAIO::Tn5 plasmids
HisD - pGR10:: TnS and
To determine the physical location of each Tn5 insertion in
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Analysis of the K. pneumoniae hisDGO (P) region using Tn5 mutagenesis
isolate plasmid ____scrape cells IR Risolate ofosmid, off plat s3-5000 Tc Km /plate
transformDN offGhiD plteosfr NK 8 5hs )
a
dqri
t,
t,
I
f
2f
r
S
f
S
Inr
f
S
t
f
ti
I
II
transformn D18FG (hisDV transform NK5875 ( hisG-)H S H
Tn5 select Km select Km
Kmr * I
screen for HisD- phenotype screen for HisG- phenotype
Scale ' ' ' ' ' ' I I012 3 4 5 6 7 8 9 I 0Kb isolate plasmid DNA from isolate plasmid DNA from
HisD- clones HisG- clones
Fig. 2, Physical maps of plasmids pGR100, pCRA1O,pCRA13 and I Iransposon Tn5. Restriction endonuclease cleavage sites for EcoRI,S transform NK5875(fhsG) cleave withEcRI transform DI8FG(hisD)md HindlII are indicated by R, S and H, respectively. The sites in Tn5 are l lelerived from Jorgensonet al. (1979). The open box represents pBR322se- select Km cleave with HindselectKmRluences, the hatched boxes pMB9 sequences, and the arrows underline 4 I I Iepresent the inverted repeats (IRs) of Tn5 (Jorgenson et at., 1979). screen for His G phenotype measure junction screen for His D phenotype
fragments (polarity test)
:heHisD-pGRIOO::Tn5 and pCRAlO::Tn5 plasmids, each determine His D, His G determine physical determine HisD,HisG)lasmid DNA was isolated using a rapid, small-scale prepara- phenotypes location phenotypes.ion method (outlined in Materials and methods) and theninalyzed with restriction endonucleases EcoRI and HindIII. correlated physical and genetic map
3ecause transposon Tn5 does not contain a recognition siteFor EcoRI (Jorgenson et al., 1979), a Tn5 insertion in the Fig. 3. Flow chart of transposon TnS mutagenesis protocol.2.3-kb EcoRI fragment will result in the disappearance of thisFragment coupled with the appearance of a new EcoRI frag- fall into two classes: HisD -G + Tn5 insertions andnent 2.3 + 5.7 kb in length (Jorgenson et al., 1979). Fifty- HisD -G - insertions. To determine the HisG phenotype of a
,ix of the 61 HisD - pGRlOO::Tn5 and pCRAlO::Tn5 representative group of our HisD - pGRIOO::Tn5 insertions,,lasmids contained a Tn5-size insertion in the 2.3-kb EcoRI we transformed the hisG - strain NK5875 with 15 HisD-Fragment (data not shown). The remaining five plasmids pGRIOO::Tn5 plasmids containing Tn5 insertions which,ither displayed an anomalous restriction pattern, or were spanned the 1.08-kb mutagenized region. All 15 HisD-,hown to be HisD + upon retesting (data not shown). pGRIOO::Tn5 plasmids tested, as well as the parental pGR100
The 56 plasmids containing a Tn5 insertion in the 2.3-kb plasmid, restored the HisG + phenotype to strain NK5875
EcoRI fragment were further analyzed with HindIII to deter- (see Materials and methods section and Figure 3). Because nonine the precise location of the Tn5 insertion. HindlII cleaves HisD -G - plasmids were found, we concluded that the
)lasmids pGR100 and pCRAlO once within the vector DNA HisD - phenotype of the 56 pGRIOO::Tn5 and pCRAIO::Tn5,equences (see Figure 2) and twice within Tn5, -1150 bp plasmids analyzed was due to the insertion of Tn5 into the
From each end in the inverted repeats (Jorgenson et al., 1979; plasmid borne hisD gene and that the 1.08-kb segment of;ee Figure 2). Thus, cleavage of the pGRIOO::Tn5 and DNA, in which all 56 Tn5 insertions map, comprises the hisD
)CRA1O::Tn5 plasmids with HindIII resulted in the genera- structural gene (see Figure 5).ion of three restriction fragnents, the (constant) internal Identification and physical mapping ofHisG - pGRJIXOi::Tn51500-bp HindIII fragment of Tn5, and two 'junction' plasmidsFragments, consisting of 1150 bp of Tn5 sequences attached Since no HisG -(D ) Tn5 insertion mutations were foundo varying lengths of pGRIOO and pCRA1O DNA sequences. in the initial screen for HisD- pGRIOO::Tn5 andFrom the size of the junction fragments, the location of a Tn5 pCRAIOn5tlasiscr afor eprmnp was andnsetio wi hi th pl s m d ca be ca c l t d wi h a . c pCRA 1O::Tn5 plasmiLds, a second experiment was carriLed out
uracyio fw h 1 0 bp. asu h cana re veal edu that , ith ian th for HisG - Tn5 insertion utations directly.
luracy of 100 bp.Suh analysis reveale that, withinthe pGRIOO::Tn5 plasmid DNA was used to transform the
iccuracy limits of the experiment, Tn5 had inserted into at hisG - strain NK5875 and 200 Kmr transformants wereeast 18 independent sites in pGRIOO and seven independent screened for their HisG phenotype (see Figure 3; Materials;ites in pCRAIO. and methods). Eight HisG - pGRIOO::Tn5 plasmids wereWhen the data on the pGRIOO:Tn5 and pCRAIO::Tn5 identified and the physical location of the Tn5 insertion was
plasmids were combined, it was found that all of the HisD - determined as described above (data not shown; see Figure 5,Fn5 insertions mapped within a 1.08-kb segment of the filled in triangles on line 1). All eight Tn5 insertions were2.3-kb EcoRI fragment (see Figure 5, open triangles of lines 1 shown to map within a 500-bp segment of the 2.3-kb EcoRInd 2). fragment, on the nifproximal side of the previously identified
HIisG complementation tests with the HisD - pGR1OO:: Tn5 hisD gene. We concluded that the HisG - phenotype of thesePlasmids pGRIOO::Tn5 plasmids was due to the insertion of Tn5 intoBecause chromosomal transposon TnlO and Tn5 induced the plasmid borne hisG gene or into the hisO(P) regulatory
insertion mutations in the hisG gene of S. typhimurium exert region.apolar effect on the expression of hisD leading to HisD -G - To determine the HisD phenotype of the eight HisG-phenotype (Kleckner et al., 1975, 1977, 1979), we expected pGR100::Tn5 plasmids, each HisG - plasmid was transform-Lhat our collection of HisD - Tn5 insertion mutations would ed into the hisD- strain D18FG and transformants were
1833
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pCRAI
RH ? RI/
F.J. de Bruijn et al.
screened for their HisD phenotype (see Figure 3; Materialsand methods). All eight HisG - pGRI00::Tn5 plasmids com-plemented the HisD- phenotype of strain D18FG. Thus itappears that the HisG- pGRIOO::Tn5 (hisG::Tn5 andhisO(P)::Tn5) insertion mutations isolated in this experimentmust exert a minor, if any, polar effect on the hisD genelocated on the same plasmid. This result contrasts with thechromosomal hisG::TnlO(Tn5) and hisO::TnlO(Tn5) inser-tion mutations isolated by Kleckner et al. (1975, 1977, 1979),all of which were polar on hisD.Is the observed non-polar phenotype ofHisG - pGRIOO:Tn5plasmids correlated wih the orientation of TnS?
Transposon Tn5 insertion mutations, like most transposoninduced insertion mutations, are reported to exert a strong,polar effect on genes located distally to their insertion siteswithin an operon due to transcription termination sites car-ried by the transposon (Berg et al., 1980; Kleckner, 1981).However, incomplete polarity has been observed with someTnlO insertion mutations (Morgan, 1980; Kleckner, 1981)and Tn5 insertion mutations in the lac operon (Berg etal., 1980). Berg et al. (1980) observed that approximately onethird of a collection of Tn5 insertions in the lacZ gene resultedin low level, constitutive expression of the lacY gene, whilethe remaining two thirds of the lacZ::Tn5 insertions abolishedlacY expression. It was postulated by these authors that, inthe case of the non-polar lacZ::Tn5 insertions, the expressionof the lacY gene was due to promoter activity originatingfrom sequences within the ends of the Tn5 transposon andthat this effect was independent of the relative orientation ofthe Tn5. This contrasts with the orientation dependent polari-ty effects observed with insertion mutations caused by inser-tion sequence IS2 (Saedler et al., 1974; Sommer et al., 1979).
In order to investigate whether the lack of polarity observ-ed with the HisG- pGR1OO::Tn5 insertion mutations wascorrelated with a particular orientation of the inserted trans-poson, we determined the orientation of Tn5 in two sets ofthree hisG::Tn5 insertion mutations of pGR100 by cleavingthe plasmids with both EcoRI and Sall. Both orientationswere represented by three insertions (data not shown) andthus no correlation between a given orientation of Tn5 in theHisG - pGRIOO::Tn5 plasmids analyzed and the lack ofpolarity on hisD was observed. It is possible that the expres-sion of the hisD gene in HisG - pGRIOO::Tn5 plasmids iscaused by a promoter introduced when the Tn5 inserts intothe hisG gene, analogous to the Tn5 mediated effect seen byBerg et al. (1980).
Analysis of the hisO(P) region: identification of HisG -hisO(P)::Tn5 pCRA13::TnS plasmids
In S. typhimurium and E. coli the mol. wt. of the hisDgene product has been reported to be between 47 000 and55 000 daltons (Bitar et al., 1977; Bruni et al., 1980). InS. typhimurium the mol. wt. of the hisG gene product hasbeen determined to be 33 216 daltons (Piszkiewicz et al.,1979). Assuming that the mol. wts. of the hisD and hisG geneproducts of K. pneumoniae are approximately the same asthose of S. typhimurium and E. coli, the approximateminimum sizes of the K. pneumoniae hisD and hisG genesare 1.3 kb and 0.9 kb, respectively. The 1.3-kb estimate cor-responds well to the 1.08 4 0.1 kb determined to be theminimum size of the hisD gene by the Tn5 mutagenesis ex-periments described above (see Figure 5). With a minimum of
0.9 kb assigned to the hisG gene, only 100-200 bp remainwithin the 2.3-kb EcoRI hisDG fragment to code for thehisO(P) regulatory region. In S. typhimurium and E. coli thecorresponding his regulatory region, consisting of the his pro-moter and attenuator sequences, comprises 100 bp for thepromoter region plus 150 bp from the transcription initiationsite to the 3' end of the attenuator stem structure (Verde etal., 1981; Barnes, 1978; Kolter and Yanofsky, 1982). Thus,the minimum length of DNA sequences required to code forthe hisD and hisG genes plus the his regulatory (promoter-attenuator) region appears to exceed the coding capacity ofthe 2.3-kb EcoRI hisDG fragment of K. pneumoniae.
Similar observations were made independently by Rod-riguez et al. (1982) who determined the DNA sequence of the2.3-kb EcoRI hisDG fragment starting at the hisG proximalEcoRI site. They found that only 41 bp were present betweenthe EcoRI site and the presumptive translational start of thehisG gene (Rodriguez et al., 1982; West and Rodriguez, inpreparation).The above findings led us to re-evaluate the conclusion that
the hisO(P) region, and therefore the proper his regulatory se-quences and signals, were located on the 2.3-kb EcoRI hisDGfragment.To determine whether hisO(P) regulatory sequences could
be located outside the 2.3-kb EcoRI hisDG fragment on anadjacent K. pneumoniae DNA fragment, we made use ofplasmid pCRA13 (Cannon et al., 1977) which contains, in ad-dition to the 2.3-kb EcoRI fragment, the adjacent 1.4-kbEcoRI fragment containing the intergenic DNA sequencesbetween hisG and nifQ (see Figure 1).
Plasmid pCRA13 was mutagenized with Tn5 as describedabove (see also Figure 3 and Materials and methods) andpCRA13::Tn5 DNA was used to transform hisD- or hisG-strains D18FG or NK5875. A total of 21 HisD- and 10HisG- pCRA13::Tn5 plasmids were identified and thephysical location of the Tn5 insertions on these plasmids wasdetermined as described above. The results of the analysiswith HindIII of some of the pCRA13::Tn5 plasmids areshown in Figure 4 and the mapping results are summarized inFigure 5, line 3.
All 21 HisD- pCRA13::Tn5 plasmids were shown to con-tain Tn5 insertions within the 1.08-kb region previously iden-tified as the hisD structural gene (see Figure 5). Among the 10HisG - pCRA13::Tn5 plasmids, seven were shown to containTn5 insertions in the same region previously defined by theHisG- pGRIOO::Tn5 insertions. However, the remainingthree plasmids were shown to carry Tn5 insertions in the adja-cent 1.4-kb EcoRI fragment, within 100 bp of the EcoRI sitewhich constitutes the junction of the 2.3-kb and the 1.4-kbEcoRI fragments cloned in pCRA13 (see Figure 5). Theseresults confirm the hypothesis that the his regulatory region,hisO(P), including the his promoter and attenuator se-quences, is not located in its entirety on the 2.3-kb EcoRIhisDG fragment cloned in pGR100 and pCRA1O, but strad-dles the EcoRI site at the junction of the 2.3-kb and adjacent1.4-kb fragments. This result extends the observations madeby Rodriguez et al. (1982) and raises some interesting ques-tions about previous findings regarding the ability ofplasmids containing only the 2.3-kb EcoRI fragment to com-plement hisD- and hisG - strains of E. coli and K. pneumo-niae (Cannon et al., 1977; Riedel, 1980; Rodriguez et al.,1982) and of P4 recombinant phages containing only the2.3-kb EcoRI fragment to direct the elevated synthesis of
1834
Analysis of the K. pneumoniae hisDGO (P) region using Tn5 mutagenesis
1 2 3 4 5 6 7 8 9 10 11I) pGRIOO::Tn5
6.2 2) pCRAIO::Tn5
vv v
v v vvv9 v vwv vv vvv99 v v9vv V I
j vv vgm v v VWg vvvwgm if TVv
R R0 2.3
V VV V9gVV
R0
vvv v
R2.3
H H R- I
R HHt-.
-2.72.0
-1 .5-1.15
R
L___-~JL~ iL____ ____A C B A
Fig. 4. Physical mapping of Tn5 insertions into plasmid pCRA13. Lanes1-10 show the HindIII restriction endonuclease cleavage patterns of 10 in-dependent pCRA13::TnS plasmids. Lane 11 shows the migration pattern ofthe mol. wt. size markers (indicated in kb). Fragment C represents the in-ternal HindIII fragment of Tn5; fragments A and B represent the Tn5-pCRA13 junction fragments, as diagrammed. By determining the mol. wts.of junction fragments B, and subtracting 1150 bp, the physical location ofeach Tn5 transposon, relative to the HindlII site within the vector portionof pCRA13 (pMB9; cross-hatched), was determined (see text). Restrictionendonuclease cleavage sites for EcoRI and HindlII are indicated by R andH respectively.
hisG polypeptides in response to amino acid starvation (Owand Ausubel, 1980).The 10 HisG- pCRA13::Tn5 plasmids isolated were also
tested for their HisD phenotype as described above. All 10plasmids were shown to be able to complement the HisD-phenotype of E. coli strain D18FG. Thus, as observed withthe HisG- pGRlOO::Tn5 plasmids, none of the plasmidborne Tn5 insertions in hisG or hisO(P) had a polar effect onthe hisD gene located on the same plasmid.Construction of a correlated physical and genetic map of thehisDGO(P) region of K. pneumoniaeThe physical and genetic mapping data obtained from
transposon Tn5 mutagenesis of plasmids carrying the clonedK. pneumoniae hisDGO(P) region, as outlined in Figure 3,were combined to construct a correlated physical and geneticmap of this region, as shown in Figure 5, line 4. All of thehisD::Tn5 insertions are indicated by open triangles, thehisG::Tn5 and hisO(P)::Tn5 insertions by closed triangles.The larger number of hisD::Tn5 versus hisG::Tn5 insertionsreflects the much smaller numbers of Kmr transformantsscreened for HisG phenotype and does not necessarily in-dicate an insertional preference of Tn5 in the hisD gene. Thearrows underneath line 4 in Figure 5 indicate the proposedminimal sizes and locations of the hisD and hisG structuralgenes as well as the hisO(P) regulatory region.Insertional specificity of transposon TnS
Different transposable elements, such as Tn3, Tn5, Tn9,TnlO and bacteriophage Mu, have been examined for theirinsertional specificity or insertion site preference (for areview, see Kleckner, 1981). Transposon Tn5 has been ranked
3) pCRA13::Tn5
4) Correlatedphysical +
genetic map
.*
vv v I
9 999 99 9 1 IV IVvvvv vv yy v v v v ..TV.
R R R0 23 37
99 9 999 999V V
99 99 VV99V
99
V ~VIVVVVV9 VW VV9V999w IV IV,
. .
WVWIV VV VVWVVVVWVWV Ws tsw . .wiR0
R23
hls D hisG 0
R3.7
Fig. 5. Physical location of the transposon Tn5 insertions in pGRIO0,pCRA10 and pCRA13 and correlated physical and genetic map of theK. pneumoniae hisDGO region. The open triangles on lines 1-3 representthe position of HisD- and the closed triangles HisG- Tn5 insertions inplasmids pGRIOO, pCRAI0 and pCRA13 mapped with an accuracy of
100 bp. Line 4 represents a correlated physical and genetic map of theK. pneumoniae hisDGO(P) region, based on a composite of the datashown in lines 1-3. The minimum length and physical location of thehisD and the hisG genes, as well as their direction of transcription, are in-dicated by the arrows; the location of the hisO(P) region is indicated by the0. Restriction endonuclease cleavage sites for EcoRI are indicated by Rand their relative map positions are indicated below in kb.
amongst the transposons with the lowest insertional specifici-ty. Shaw and Berg (1979) determined the insertional specifici-ty of Tn5 on a 'macro-level' by examining the percentages ofauxotrophic mutations caused by transposition of Tn5 intothe E. coli chromosome. These authors concluded that,although a large variety of auxotrophic mutations caused byTn5 insertional inactivation could be readily identified, never-theless some preference for insertion into certain genes couldbe observed, suggesting low level insertional specificity ofTn5.
Berg et al. (1980) extended the analysis on a 'micro-level'by examining the distribution of Tn5 insertions into theE. coli lac operon and concluded that < 5% of the Tn5 inser-tions isolated were separable by genetic recombination, sug-gesting a low level of insertional specificity as well. The resultsfrom the experiments by Berg et al. (1980) were confirmed byMiller et al. (1980).We attempted to analyze the question of insertional
specificity of Tn5 by evaluating the results of three indepen-dent Tn5 mutagenesis experiments using pGR100 as targetplasmid. Plasmid pGR100 carries, in addition to the clonedK. pneumoniae hisD and hisG genes, vector-borne genescoding for tetracycline resistance (Tcr) and ampicillinresistance (Apr). In three independent experiments HB101cells harboring plasmid pGR100 were infected with X::Tn5phage and the plasmid DNA preparations from the resultingKmr 'infected' cells were used to transform hisD strainD18FG (as described above; see Materials and methods). Atotal of 500 Kmr transformants of D18FG from each experi-ment were screened for their HisD, Tcr and Apr phenotypes.This enabled us to evaluate the relative frequency with whichTn5 transposes into each of the three genes. The results ofthis experiment are summarized in Table II.The Tcr and Apr genes located on plasmid pGR100 derive
from the plasmid vector pBR322 (Bolivar et al., 1977). The
1835
A
B
C-1
F.J. de Bruijn et al.
Table H. Insertional specificity of transposon Tn5: distribution of Tn5 inser-tions in plasmid pGRIOO
Wo Tptb % Tn5 insertionsaExp. I Exp. II Exp. III Average
a500 Kmr colonies were screened in each experiment.b%Tpt = 7o total plasmid target DNA sequences (see text).CAssuming hisD= 1.1 kb (this work).dAssuming Tcr gene = 1.2 kb (Sutcliffe, 1979).eAssuniing Apr gene = 680 bp (Sutcliffe, 1979).
approximate size of the pBR322 Apr gene has been determin-ed to be 680 bp and the size of the pBR322 Tcr gene isestimated to be 1.21 kb (Sutcliffe, 1979). In addition, it canbe estimated that the region of pBR322 essential for replica-tion and plasmid maintenance is at least 400 bp (Sutcliffe,1979). The approximate size of the K. pneumoniae hisD genehas been determined to be 1.1 kb (this work). From thesedata we calculated the percentage of 'total plasmid target(tpt)' that the Tcr, Apr and hisD genes of pGR100 occupy.Since Tn5 insertions into the origin of replication (or,) of thepBR322 vector segment would be lost from the population ofpGRIOO:Tn5 plasmids, 400 bp is subtracted from the totalsize of pGR100 (4.35-kb pBR322 + 2.3-kb hisDG sequences)in order to determine the 'tpt' of this plasmid to be 6.25 kb.The percentage 'tpt' values calculated for the Tcr, Apr andhisD genes of pGR100 are listed in column 1 of Table II.The values for the percentage of Tn5 insertions/500 Kmr
transformants in the three genes in question are given foreach of the three independent experiments and average valuesare listed in column 5 of Table II. The percentage of Tn5 in-sertions into the Tcr gene appears to be quite close to thevalue expected from calculated percentage 'tpt'. Tn5 inser-tions into the cloned hisD appear to be under-represented aswell as Tn5 insertions into the Apr gene. However, clearlythere appear not to be any obvious 'hot spots' for Tn5 inser-tion in pGR100, considering that the genes examined com-prise almost half of the total DNA sequences of pGR100. Thecombination of these data with the physical map of allhisD::Tn5 insertions as shown in Figure 5 strongly supportsour previous finding that transposon Tn5 has a very low in-sertional specificity and therefore is particularly useful for thegeneration of random collections of insertion mutations incloned DNA fragments (de Bruijn and Ausubel, 1981; deBruijn, 1983).
DiscussionThe combined physical and genetic analysis of - 100 Tn5
insertions in the hisDGO(P) region of K. pneumoniae hasenabled us to construct a correlated physical and genetic mapof this segment of the his operon, extending our analysis ofthe his-nif region.The overall structure of the K. pneumoniae hisDGO(P)
region appears to be essentially the same as that of theanalogous regions from S. typhimurium and E. coli. TheK. pneumoniae hisD and hisG structural genes are located inthe expected order on a 2.3-kb EcoRI fragment which, when
cloned in either orientation in a variety of cloning vectorssuch as pBR322, pMB9, pACYC184, pPV33 and phage P4,complements both hisD and hisG strains of E. coli andK. pneumoniae (Cannon et al., 1977; Riedel, 1980;Rodriguez et al., 1982; Ow and Ausubel, 1980). In addition,Ow and Ausubel (1980) have shown that the synthesis of thehisG gene product, directed by recombinant P4 phages con-taining only the 2.3-kb hisDG EcoRI fragment, is subject toinduction by amino acid starvation conditions, characteristicof his [hisO(P)] specific regulation. In the light of these find-ings, one paradoxical result was obtained: the his regulatoryregion hisO(P) is located at least in part on an adjacent 1.4-kbEcoRI fragment. These results suggest the followingpossibilities. (i) A (his-specific) minor promoter is present onthe 2.3-kb EcoRI hisDG fragment, located between the startof the hisG gene and the EcoRI site. (ii) Expression of thehisD and hisG genes, located on the 2.3-kb EcoRI fragmentand cloned in a variety of plasmid and phage vectors is due toread-through transcription originating from vector-bornepromoters and the observed induction of the hisG productsynthesis is due to some form of post-transcriptional controlof K. pneumoniae his genes.The DNA sequence of the region between the (putative)
translational start site of the hisG gene and the EcoRI site wasdetermined by Rodriguez et al. (1982). No typical prokaryoticpromoter-like sequences are present in the 41-bp stretch ofDNA between the ATG start codon of hisG and the EcoRIsite. Nevertheless, experiments analyzing the 2.3-kb EcoRIhisDG fragment using the promoter-probe plasmid pPV33(West and Rodriguez, 1982) did reveal the presence of lowlevel promoter activity on this fragment (West andRodriguez, in preparation). Thus, although the existence of aminor promoter between the start of the hisG gene and theEcoRI site seems unlikely, it can not be rigorously excluded atthis time.The possibility of readthrough from vector promoters
seems unlikely because of the large number of differentplasmid and phage vectors in which the cloned 2.3-kb EcoRIhisDG fragment is expressed. However, in both pBR322 andpACYC184, promoters have been identified flanking theEcoRI site and reading into the direction of the EcoRI sitefrom opposite directions (Stuber and Bujard, 1981). In thecase of the P4 phage cloning vector, the presence of vectorpromoters in the regions surrounding the EcoRI cloning sitehas not been investigated (D. Ow, personal communication).
It should be noted that very little transcription of the hisoperon ( <10% of the fully repressed level) is needed to resultin a His+ phenotype (Johnston et al., 1980). Therefore,especially in the case of multicopy plasmids carrying thehisDG region, a very weak promoter or even random plasmidtranscription initiation events may result in sufficient expres-sion of the hisG and hisD genes to give rise to a HisD +G +phenotype. The observations described above do suggest,however, that caution should be exercised when analyzingmulticopy plasmids carrying cloned genes. The assumptionthat a cloned gene of interest is expressed from its own pro-moter, based on the observation that gene expression is in-dependent of the orientation of the cloned fragment relativeto the vector, may not always be correct.The following observations can be made regarding the
failure of Tn5 insertions in the cloned hisG gene and thehisO(P) region to exert a (noticeable) polar effect on the hisDgene, located immediately downstream on the same plasmid.
1836
Analysis of the K. pneumoniae hisDGO (P) region using Tn5 mutagenesis
(i) In general, insertion mutations caused by transposons suchas Tn3, Tn5, TnlO and bacteriophage Mu are strongly polaron genes located downstream within the same operon (seeKleckner, 1981). In the case of transposon Tn5, strong polareffects induced by Tn5 insertion mutations have been observ-ed in the chromosomal argCBH, ilvGEDA and his operonsof E. coli (Shaw and Berg, 1979; Kleckner et al., 1977), in thechromosomal lac operon of E. coli (Berg et al., 1980), and inoperons cloned into multicopy plasmid vectors such as the entgenes of E. coli (Laird and Young, 1980), the uvrB locus ofE. coli (Pannekoek et al., 1980), and the gInALG operon ofK. pneumoniae (de Bruijn and Ausubel, 1981). On the otherhand, non-polar Tn5 insertion mutations in the lac operonwere also described by Berg et al. (1980). Approximately onethird of the Tn5 insertions in the lacZ gene were shown bythese authors to induce low level, constitutive expression ofthe lacY gene, presumably due to promoter activity introduc-ed by the Tn5. This effect was shown to be independent of theorientation of Tn5 (Berg et al., 1980). Since very littletranscription of the his operon is needed to give rise to a His +phenotype (Johnston et al., 1980), orientation-independent,Tn5-mediated expression of the plasmid-bome hisD genecould be responsible for the HisD + phenotype of the HisG -pGR100::Tn5 and pCRA13::Tn5 plasmids. It remainscurious, however, that all hisG::Tn5 and hisO(P)::Tn5 inser-tions examined reveal the non-polar phenotype. (ii) The lackof Tn5 induced polarity could also be due to a paucity oftranscription termination factors necessary for Tn-mediatedpolarity (see Kleckner, 1981) in cells harboring multicopyplasmids carrying the Tn5 mutated genes. (iii) Finally, thelack of polarity could be due to the presence of a previouslyunidentified minor promoter located in the inter-cistronicregion between the hisD and hisG genes, the effect of whichcan only be observed with multicopy plasmids carrying thehisDG region.To analyze further the potential 'multicopy effects', EcoRI
restriction fragments carrying the hisDG region of twoHisG-D+ pGR100::Tn5 plasmids, with Tn5 insertions inhisG, were recloned into the low copy number plasmid vectorpRK290 (Ditta et al., 1980), and the resulting plasmids weretested for their HisD phenotype (de Bruijn, unpublishedresults). In spite of the fact that pRK290 derivatives only existin 5-10 copies per cell (Ditta et al., 1980), as opposed to the40-60 copies per cell observed for pBR322 derivatives(Bolivar et al., 1977), no difference in polarity was observed;hisG::Tn5 insertions remained non-polar on hisD. Ex-periments to reduce the copy number of some of thehisG::Tn5 hisD fragments to one, by integration into thechromosome, are in progress.When considering the usefulness of a given transposon for
the generation of large numbers of randomly distributed in-sertion mutations, two major criteria should be examined:transposition frequency and insertional specificity. In thiswork we generated - 3400 Tn5 insertions in multicopyplasmids carrying the K. pneumoniae hisDGO(P) region.This number of Tn5 insertions could be readily generated dueto the high frequency of transposition of Tn5 from its condi-tionally defective X phage vector (X::Tn5; Berg, 1977). Weroutinely infected 1 x 109 cells with 1- 10 x 109 p.f.u. ofX::Tn5 and obtained 1 -2 x 104 Kmr colonies, representingan approximate transposition frequency of2 x 10-5 - 1 X 10-6 Kmr colonies/p.f.u. of X::Tn5 and cor-responding well to values published by Johnson et al. (1982).
The insertional specificity of Tn5 constitutes the secondmajor criterion. Both the genetic analysis of 1500 Tn5 inser-tions in plasmid pGR100 (Table II) and the physical analysisof 70 Tn5 insertions in the hisD structural gene (Figure 5),strongly support the notion that the insertional specificity ofTn5 is extremely low. Our experiments confirm and extendobservations made in a number of laboratories regarding thelack of insertional specificity when TnS transposes intochromosomal genes (Berg, 1977; Shaw and Berg, 1979; Berget al., 1980; Miller et al., 1980) or into genes carried bymulticopy plasmids (Laird and Young, 1980; Pannekoek etat., 1980; Shanabruch and Walker, 1980; de Bruijn andAusubel, 1981; Hakkaart et al., 1981; Lupski et al., 1982; deBruijn, 1983).
Materials and methodsBacterial strains, phages and plasmids
The bacterial strains, phages and plasmids used in this work are listed inTable I.Media and chemicalsThe rich (LB) and minimal (BS or Min) media and plates used have been
described by Cannon et al. (1979). Amino acids were added to a final concen-tration of 20 1tg/ml. Antibiotics were added to the following final concentra-tions: tetracycline (Tc) 10 ug/ml; ampicillin (Ap) 40 ;g/ml; kanamycin20 Ag/ml. The media and plates used for growing bacteriophage X have beendescribed by Miller (1972).Transformations
Transformations were carried out using the procedure described by Cohenet al. (1972). For routine transformations, competent cells were prepared andcryogenically preserved as described by Morrison (1979).Preparation of kX:Tn5
Plate stocks of X::Tn5 (Xb22lrex::Tn5c18570arn29Pam80) were preparedby infection of the E. cofi supE44 strain Ql as described by Miller (1972).Transposon Tn5 mutagenesis protocol
Transposon Tn5 mutagenesis of plasmids pGRIOO, pCRAIO and pCRA13was carried out essentially as described by de Bruijn and Ausubel (1981).Cultures of E. coli strain HBIOI harboring plasmids pGRIOO, pCRAIO orpCRA13 were grown in LB broth, supplemented with 0.2% maltose and Tc,to a density of 5 x 108 cells/ml and infected with X::Tn5 phage at a multiplici-ty of infection (m.o.i.) of 0.1-1 phage particle/cell. After 1-2 h of phageadsorption at 32°C, the infected cells were plated out on LB Km20 plates andincubated at 32°C for 48 h. Since the bacteriophage X DNA is unable toreplicate or integrate after injection into a host cell, the Km' colonies resultingfrom the infection experiment represent Tn5 transpositions from the defectiveX::Tn5 vector into the chromosome of the host cell or into a resident plasmid.To select plasmids containing a copy of Tn5, the Km' cells (3-5000/plate)were scraped off 5-10 plates, and plasmid DNA was prepared from thesecells using the method of Klein et al. (1980). In some cases, an aliquot of theKmr cells was first grown to saturation in 75 mi LB Km medium beforeplasmid DNA isolation. The plasmid DNA thus isolated was used totransform E. cofi strains D18FG (hisD-) or NK5875 (hisG-); Kmr transfor-mants we.e selected on LB Km plates and examined for their HisD, HisG(and Apr or TcT) phenotypes.
Plasmid DNA was prepared from the transformants of interest, using oneof the small-scale, plasmid DNA preparation protocols listed below, and thelocation of the TnO on the plasmid DNA was determined by the use of restric-tion endonuclease analysis with EcoRI and HindlII (see text). A flow-diagramof the TnO mutagenesis protocol is shown in Figure 3.Complementation testsTo screen for the HisD phenotype, D18FG (hisD-) cells harboring
pGRlOO::Tn5, pCRAlO::Tn5, pCRA13::Tn5 and the parental plasmids werestreaked out on Min plates, supplemented with proline, leucine, arginine andthreonine (Min Pro Leu Arg Thr) and growth was scored after 2-3 days ofincubation at 37°C. The hisD- mutation of strain D18FG was checked by ex-amination of growth on Min Pro Leu Arg Thr plates supplemented with200 kg/ml of histidinol.To screen for the HisG phenotype, NK5875 (hisG-) cells harboring the
pGRlOO::Tn5, pCRAlO::Tn5, pCRA13::Tn5 and the parental plasmids were
streaked on Min plates supplemented with leucine and methionine (Min LeuMet) and growth was scored after 3-4 days of incubation at 37°C.
1837
F.J. de Bruijn et al.
Plasmid DNA isolationSupercoiled plasmid DNA was prepared using the cleared lysate, cesium
chloride-ethidium bromide gradient protocol as described by Clewell andHelinski (1969). Small scale preparations of plasmid DNA were carried out asdescribed by Klein et al. (1980) or by Holmes and Quigley (1981). The DNAobtained by these procedures was routinely desalted before restriction endo-nuclease analysis or transformation by passage through a 0.4 ml SephadexG-50 (medium) column equilibrated with sterile H20.Restriction endonuclease analysis
Restriction endonucleases HindIII and Sall were purchased from BethesdaResearch Laboratories (Rockville, MD) and used as specified by the manufac-turer. Restriction endonuclease EcoRI was prepared by G. Riedel, using asimplified purification procedure developed by Phyllis Myers (Cold SpringHarbor Laboratories, Cold Spring Harbor, NY). Gel electrophoresis was car-ried out in horizontal 0.7-1.4% agarose gels in SEB buffer (Hayward andSmith, 1972) as described by Riedel et al. (1979). The gels were stained in SEBbuffer containing 1 ytg/ml of ethidium bromide and photographed undershort wave length u.v. light with a Polaroid camera.
AcknowledgementsWe thank Bob West and Rod Riedel for their critical reading of themanuscript and sharing unpublished results, and Rachel Hyde for help withthe preparation of this manuscript. This work was supported by NationalScience Foundation grant PCM81-04193, awarded to F.M.A. and an NIHtraining grant awarded to D.J.M.
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