当前位置：文档库 › Binding and Spreading of ParB on DNA Determine Its Biological

Binding and Spreading of ParB on DNA Determine Its Biological

J OURNAL OF B ACTERIOLOGY,July2011,p.3342–3355Vol.193,No.13 0021-9193/11/$12.00doi:10.1128/JB.00328-11

Function in Pseudomonas aeruginosa??

Magdalena Kusiak,1Anna Gapczyn′ska,1Danuta P?ochocka,1

Christopher M.Thomas,2and Graz˙yna Jagura-Burdzy1*

The Institute of Biochemistry and Biophysics,PAS,02-106Warsaw,Pawin′skiego5A,Poland,1and School of Biosciences, The University of Birmingham,Edgbaston,Birmingham B152TT,United Kingdom2

Received8March2011/Accepted15April2011

ParB protein of Pseudomonas aeruginosa belongs to a widely represented ParB family of chromosomally and

plasmid-encoded partitioning type IA proteins.Ten putative parS sites are dispersed in the P.aeruginosa

chromosome,with eight of them localizing in the oriC domain.After binding to parS,ParB spreads on the DNA,

causing transcriptional silencing of nearby genes(A.A.Bartosik et al.,J.Bacteriol.186:6983–6998,2004).We

have studied ParB derivatives impaired in spreading either due to loss of DNA-binding ability or oligomer-

ization.We de?ned speci?c determinants outside of the helix-turn-helix motif responsible for DNA binding.

Analysis con?rmed the localization of the main dimerization domain in the C terminus of ParB but also

mapped another self-interactive domain in the N-terminal domain.Reverse genetics were used to introduce?ve

parB alleles impaired in spreading into the P.aeruginosa chromosome.The single amino acid substitutions in

ParB causing a defect in oligomerization but not in DNA binding caused a chromosome segregation defect,

slowed the growth rate,and impaired motilities,similarly to the pleiotropic phenotype of parB-null mutants,

indicating that the ability to spread is vital for ParB function in the cell.The toxicity of ParB overproduction

in Pseudomonas spp.is not due to the spreading since several ParB derivatives defective in oligomerization were

still toxic for P.aeruginosa when provided in excess.

Accurate segregation of genomes is important for genetic stability.The prokaryotic DNA partitioning process is best understood for low-copy-number plasmids.It relies on two partitioning proteins,A and B,and a cis-acting centromere-like sequence(parS),speci?cally recognized by the B compo-nent.Protein A with a weak NTPase activity forms dynamic spatial structures,and the interactions between protein A and protein B bound to DNA facilitate the separation and the movement of plasmid molecules to opposite poles before cell division(13).Plasmid partitioning operons are classi?ed into four different groups(I to IV)based on the type of NTPase (Walker-type ATPase,actin-like ATPase,and tubulin-like GTPase),DNA-binding protein structure,and localization of parS(12,13,17,29,51).The majority of bacterial chromosomes encode ParA-ParB systems classi?ed as IA on the basis of pos-session of ParA Walker-type ATPase and ParB,a large DNA-binding protein with an helix-turn-helix(H-T-H)motif.The mul-tiple copies of the highly conserved parS sequence are dispersed throughout the chromosomes(34,55).Despite the conservation in the binding sites and overall homology of chromosomal Par proteins,the effects of par de?ciencies are genus speci?c.The effects range from lethality in Caulobacter crescentus(34,39),to impairments in cell cycle regulation,chromosome segregation, and sporulation processes in Streptomyces coelicolor and Bacillus subtilis(8,19,23,24,31,36),to mild defects in chromosome segregation at a speci?c growth phase in Pseudomonas putida(14,33).

We are interested in the chromosome partitioning process in the opportunistic pathogen Pseudomonas aeruginosa.It en-codes the parA-parB operon in the proximity of oriC.Ten putative parS sequences are dispersed in the P.aeruginosa chromosome,with eight of them localizing in the oriC domain (20%of the nucleoid around oriC).The parA-or parB-null mutants exhibit pleiotropic phenotypes:slower growth rate, increase in cell size,frequent production of anucleate cells independently of the growth phase,and defects in swarming and swimming motilities(4,30).The lack of one partner in-creases the susceptibility of another for proteolytic degrada-tion,making it dif?cult to separate the roles of individual proteins.However,some parB mutants with short deletions (e.g.,H-T-H motif)produce normal levels of ParA and still demonstrate similar pleiotropic phenotypes,which strongly suggests that inactive ParB is the primary cause of the observed defects(4).The wide spectrum of parB de?ciency effects sug-gests that ParB may play the regulatory role in the expression of different operons either through interaction with DNA or with other proteins,including ParA.

Chromosomally encoded ParBs belong to the highly homol-ogous ParB family.ParBs of subtype IA(12)are relatively large proteins(300to400amino acids)with a number of well-conserved motifs(5,54).The H-T-H motif present in the middle of the proteins is involved in interactions with DNA(3, 5).The C-terminal dimerization domain with conserved region 4(5)is the main dimerization domain(5,10,21,52).The functions of the two motifs ParB box I and ParB box II(54),as well as those of three other regions(1to3),remain unspeci?ed (5).The best-studied members of ParB family are the plasmid-

*Corresponding author.Mailing address:Institute of Biochemistry and Biophysics,PAS,02-106Warsaw,Pawin′skiego5A,Poland.Phone: 48228237192.Fax:48226584636.E-mail:gjburdzy@ibb.waw.pl.

?Supplemental material for this article may be found at http://jb

https://www.wendangku.net/doc/2a9783145.html,/.

?Published ahead of print on29April2011.

3342

encoded ParB of P1prophage(35,53),KorB of RK2/RP4

plasmid(6,21,36),and SopB of F plasmid(1,16,37).The crystallographic studies have shown that ParB,KorB,and SopB interact with the speci?c DNA sequences parS,O

,and sopC,respectively,in different ways.ParB of P1recognizes a complex combination of heptameric and hexameric motifs in parS(A and B sequences)through two distinct DNA-binding domains:an H-T-H structure(A-box)and a new type of DNA-binding domain formed by dimerized C termini on DNA(B-box)(47).KorB of the RK2/RP4plasmid(partition protein acting as the global transcriptional regulator)recognizes a sim-ple palindromic motif of13bp,12copies of which are scattered through the plasmid genome.It has been concluded that al-though the H-T-H motif is the important determinant of op-

erator O

recognition,other residues downstream of H-T-H are also involved in speci?c DNA interactions(26).SopB of F plasmid,on the other hand,recognizes an18-bp core of the 43-bp sequence directly repeated12times in the sopC region (40).SopB wrapped around DNA forms an extended partition complex with sopC in which the SopA interacting domains are aligned on one face of the complex(48).Although the primary dimerization domain of SopB was mapped to the C-terminal 48residues(275to323),the crystal structure revealed the secondary dimerization domain to be located between residues 245and272.This secondary dimerization domain is supposed to be responsible for plasmid pairing and in trans spreading on the bridged DNA molecules(48).Superimposition of crystal-lographic data on SopB,ParB,and KorB in this region ex-cluded the formation of such secondary dimerization domains in ParB of the P1plasmid and KorB of the RK2/RP4plasmid (48).

The unique feature of ParBs of type IA is the ability to spread on DNA starting from the speci?cally recognized se-quence of parS,which may result in the transcriptional silenc-ing of the nearby promoters(5,7,24,35,37,44).The over-

production of ParB

Pa.

in P.aeruginosa and P.putida but not in Escherichia coli(one of the exceptions among bacteria since it does not possess a par system)is“toxic”for the cells since their division time becomes longer(5).One hypothesis was that toxicity might be the direct result of ParB spreading on DNA around multiple ParB binding sites(parS s)and silencing of important genes.

We describe here an analysis of P.aeruginosa parB mutants unable to spread and transcriptionally silence a promoter lo-cated close to parS.In vitro analysis of puri?ed products of mutated alleles correlated the spreading de?ciency either with impairment in DNA binding or with the ability to form higher-order structures.It allowed us to de?ne the DNA-binding determinants outside of the H-T-H motif and demonstrate the role of N-terminal residues in the oligomerization process. Several ParB spreading-de?cient derivatives are still toxic for P.aeruginosa if overproduced,indicating that DNA binding and spreading on DNA is not the main cause of toxicity.The introduction of analyzed mutant parB alleles with single nucle-otide substitutions into the P.aeruginosa chromosome leads to phenotypes observed for a parB-null mutant,strongly suggest-ing that spreading on DNA starting from the parS sequences is required for the biological function of ParB in P.aeruginosa.

MATERIALS AND METHODS

Bacterial strains and growth conditions.Strains used in the present study are listed in Table1.Bacteria were grown in L broth(25)at37or30°C or on L agar (L broth with1.5%[wt/vol]agar).The antibiotics used were as follows:ben-zylpenicillin(150?g/ml in liquid medium and300?g/ml on agar plates),strep-tomycin(30?g/ml),kanamycin(50?g/ml),chloramphenicol(10?g/ml in E.coli and100?g/ml in P.aeruginosa),carbenicillin(300?g/ml),and rifampin(300?g/ml).The L agar used for blue-white screening contained0.1mM IPTG (isopropyl-?-D-thiogalactopyranoside)and X-Gal(5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside)at40?g ml?1.MacConkey agar base supplemented with 2%galactose was used for screening of the Gal?phenotype.Growth was mon-itored by measurement of the optical density at600nm(OD600).

Motility assays.For motility assays,cells of P.aeruginosa PAO1161strains were taken from a deep-frozen stock spread on L-agar plates and grown over-night at37°C.Bacteria from single colonies were then used to inoculate test plates(43).Plates were incubated for24h or48h either at27or37°C.All sets of plates were standardized by using the same volume of medium.

Plasmids and DNA manipulations.The plasmids used in the present study are listed in Tables2and3.Plasmid DNA isolation and genetic manipulations were based on standard techniques(45).Standard PCRs were performed on plasmid DNA templates with the mutated parB alleles using2.5pmol of the primers parB1and parB2(see Table S1in the supplemental material).DNA sequencing was performed by the internal sequencing facility(Institute of Biochemistry and Biophysics,Warsaw,Poland)using the dye terminator method in conjunction with an ABI373automated DNA sequencer.

TABLE1.Bacterial strains used in this study

Strain Genotype a Source or reference Escherichia coli

BL21(DE3)F?ompT hsdS B(r B?m B?)gal dcm(?DE3)Novagen

DH5?recA1endA1gyrA96thi-1hsdR17(r K?m K?)supE44

relA1deoR?(lacZYA-argF)U169(?80d lacZ?M15)

NR9786?(pro-lac)thi galK2R.Schaaper

S17-1pro?hsdR hsdM?recA;Tp r Sm r?RP4-Tc::Mu-Kn::Tn750

Pseudomonas aeruginosa

PAO1161leu r? B.M.Holloway PAO1161Rif leu r?Rif r4

PAO1161parB null leu r?Rif r parB1-18::TcR4

PAO1161parB3leu r?Rif r parB G2893A(ParB A97T)*This study

PAO1161parB7leu r?Rif r parB C4733T(ParB A158V)*This study

PAO1161parB9leu r?Rif r parB C2803T(ParB R94C)*This study

PAO1161parB55leu r?Rif r parB G2113A(ParB G71S)*This study

PAO1161parB62leu r?Rif r parB C4943T(ParB T165I)*This study

a*,Amino acid substitutions in ParB protein derivatives are indicated in parentheses.Tp r,trimethoprim resistance;Sm r,streptomycin resistance,Rif r,rifampin resistance;r?,restriction-negative strain.

V OL.193,2011parB SPREADING MUTANTS OF PSEUDOMONAS AERUGINOSA3343

PCR site-directed mutagenesis was used to obtain P.aeruginosa parB (parB Pa )alleles with single amino acid substitutions in pMKB4,a pAKE600derivative (11)(a modi?cation of Stratagene QuikChange site-directed mutagenesis).Pairs of primers were designed to introduce the desired mutation and remove or introduce restriction site (without changing amino acid sequence)in close prox-imity to mutagenized sequence (see Table S1in the supplemental material).The PCR ampli?cation was performed according to the following thermocycler pro-gram:30s of initial denaturation at 95°C,followed by 16cycles of denaturation at 95°C for 30s,annealing at 55°C for 1min,and elongation at 68°C for 16min,with a ?nal elongation step at 68°C for 30min.After the temperature cycling,the product was treated with 10U of DpnI endonuclease in order to digest the parental DNA template and used for transformation.The plasmid DNA was sequenced to verify the presence of the mutation.

E.coli and P.aeruginosa https://www.wendangku.net/doc/2a9783145.html,petent cells of E.coli were prepared by the standard CaCl 2method (45).Competent P.aeruginosa cells were prepared as described previously (18).

Isolation of ParB mutants impaired in spreading.Plasmid DNA of pKLB2tacp -parB (20?g)was treated for 20h with hydroxylamine solution (580?l of 0.1M sodium phosphate [pH 6.0],2mM EDTA,and 400?l of hydroxylamine)at 37°C and then dialyzed overnight against TEN buffer (10mM Tris-HCl [pH 8.0],1mM EDTA [pH 8.0],100mM NaCl).Mutated DNA was used to transform E.coli NR9786(pABB811).The transformation mixture was plated on selective MacConkey medium with galactose containing streptomycin (selection for resi-dent plasmid),penicillin (selection for incoming plasmid),and 0.5mM IPTG (ParB overproduction).Transformants forming red colonies were analyzed as carrying pKLB2with parB mutant alleles incapable of transcriptional silencing.Transformants were tested for plasmid integrity and overproduction of ParB forms by Western blotting.

Determination of plasmid compatibility (silencing test).The recipient strain DH5?(pABB811)was transformed with pGBT30expression vector,pKLB2,and its mutated derivatives.Next,100?l of undiluted and serially diluted transfor-mation mixtures was plated in repetition on three different selection plates.The selection was either for incoming plasmid only (L agar with penicillin)for both resident and incoming plasmids (L agar with penicillin and streptomycin),and the double selection plates were supplemented with 0.5mM IPTG.

Puri?cation of His 6-tailed polypeptides.Exponentially growing BL21(DE3)strains with pET28mod derivatives (36)were induced with 0.5mM IPTG at a cell density of ?108cells ml ?1and grown for an additional 2h with shaking at 37°C.The cells were harvested by centrifugation and sonicated.Overproduced His-tagged proteins were puri?ed on Ni-agarose columns with an imidazole gradient in phosphate buffer (pH 8.0)as recommended by the manufacturer (Qiagen)for soluble native proteins.The puri?cation procedure was monitored by SDS-PAGE using a Pharmacia PHAST gel system.

Circular dichroism spectra.Puri?ed His 6-ParB and its derivatives in 100mM NaF were analyzed at wavelengths between 190and 260nm at 20°C by using a JASCO JA-810spectropolarimeter and quartz cuvettes 0.005,0.02,and 0.01cm in depth.

Analytical ultracentrifugation (sedimentation velocity).Sedimentation veloc-ity experiments were carried out in Beckman XL-A analytical ultracentrifuge equipped with absorbance optics.Proteins were diluted in 50mM sodium phos-phate buffer (pH 8.0),100mM NaCl,and 1%glycerol to a concentration of 0.1mg ml ?1and centrifuged overnight at 129,000?g at 4°C.Scans at an absorbance wavelength of 280nm were taken every 6min.The SEDFIT program was used to analyze 95scans for each protein,which represents the full extent of sedi-

TABLE 3.Plasmids constructed in this study

Plasmid

Relevant features a

pKLB2derivatives

pAGB3.3...................................tacp -parB G2893A (A97T)pAGB3.4...................................tacp -parB C4733T (A158V)

pAGB3.5...................................tacp -parB G6563A/G6673A (R219H/A223T)pAGB3.6...................................tacp -parB C2803T (R94C)pAGB3.7...................................tacp -parB G5933A (G198D)pAGB3.8...................................tacp -parB C7483T (stop249)pAGB3.9...................................tacp -parB G2123A (G71D)pAGB3.10.................................tacp -parB C3193T (P107S)pAGB3.11.................................tacp -parB C2903T (A97V)pAGB3.12.................................tacp -parB C3653T/?758-769

(A122V/?253-256)

pAGB3.13.................................tacp -parB G5703A (M190I)pAGB3.14.................................tacp -parB G1403A (G47D)pAGB3.15.................................tacp -parB G2113A (G71S)pAGB3.16.................................tacp -parB C5003T (T167I)pAGB3.17.................................tacp -parB C4943T (T165I)pMKB3.18.................................tacp -parB C3653T (A122V)pMKB3.19.................................tacp -parB ?758-769(?253-256)

pET28mod derivatives b

pMKB1.3...................................T7p -parB G2893A (A97T)pMKB1.4...................................T7p -parB C4733T (A158V)

pMKB1.5...................................T7p -parB G6563A/G6673A (R219H/A223T)pMKB1.6...................................T7p -parB C2803T (R94C)pMKB1.7...................................T7p -parB G5933A (G198D)pMKB1.8...................................T7p -parB C7483T (stop249)pMKB1.9...................................T7p -parB G2123A (G71D)pMKB1.10.................................T7p -parB C3193T (P107S)pMKB1.11.................................T7p -parB C2903T (A97V)pMKB1.12.................................T7p -parB C3653T/?758-769

(A122V/?253-256)

pMKB1.13.................................T7p -parB G5703A (M190I)pMKB1.14.................................T7p -parB G1403A (G47D)pMKB1.15.................................T7p -parB G2113A (G71S)pMKB1.16.................................T7p -parB C5003T (T167I)pMKB1.17.................................T7p -parB C4943T (T165I)pMKB1.18.................................T7p -parB C3653T (A122V)pMKB1.19.................................T7p -parB ?758-769(?253-256)pBBR1MCS1derivatives c

https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB G2893A (A97T)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB C4733T (A158V)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB G6563A/G6673A

(R219H/A223T)

https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB C2803T (R94C)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB G5933A (G198D)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB C7483T (stop249)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB G2123A (G71D)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB C3193T (P107S)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB C2903T (A97V)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB C3653T/?758-769

(A122V/?253-256)

https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB G5703A (M190I)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB G1403A (G47D)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB G2113A (G71S)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB C5003T (T167I)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB C4943T (T165I)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB C3653T (A122V)https://www.wendangku.net/doc/2a9783145.html,cI q tacp -parB ?758-769(?253-256)pAKE600derivatives

pMKB4......................................BamHI-SalI fragment of pKLB3carrying

parA-parB d

pMKB4.3...................................pMKB4with parB G2893A (A97T)(PCR

mutagenesis with primers 5and 6)*

pMKB4.4...................................pMKB4with parB C4733T (A158V)(PCR

mutagenesis with primers 7and 8)*

pMKB4.6...................................pMKB4with parB C2803T (R94C)(PCR

mutagenesis with primers 9and 10)*

pMKB4.15.................................pMKB4with parB G2113A (G71S)(PCR

mutagenesis with primers 11and 12)*

pMKB4.17.................................pMKB4with parB C4943T (T165I)(PCR

mutagenesis with primers 13and 14)*a

*,The primer sequences are given in Table S1in the supplemental material.b Plasmid derivatives with EcoRI-SalI fragments with parB from relevant pKLB2derivatives.c

Plasmid derivatives with BamHI-SalI fragments with lacI q tacp -parB from relevant pKLB2derivatives.d

The parA allele is truncated at the 5?end.

TABLE 2.Plasmids used in this study

Plasmid Relevant features a

Source or reference

pABB811pGB2with parS 2sequence (orientation I)5pAKE600ori MB1oriT RK2;Ap r sacB

11pBBR1MCS1BHR cloning vector lacZ ?-MCS mob T7p T3p ;Cm r

28pET28mod ori MB1Km r ,T7p ,lacO ,His tag,no BamHI site,T7tag deleted 36pGB2ori SC101;Sp r /Sm r

9pGBT30ori MB1,Ap r ,lacI q tacp expression vector 22pJMB500pBBR1MCS1with tacp-parB Pa 30pKLB2pGBT30with tacp-parB Pa

5pKLB3

pGBT30with tacp-parA-parB Pa

Cm r ,chloramphenicol resistance;Sm r ,streptomycin resistance;Ap r ,ampi-cillin resistance;Sp r ,spectinomycin resistance;Km r ,kanamycin resistance.

3344KUSIAK ET AL.

J.B ACTERIOL .

mentation of the sample.Sedimentation coef?cient distributions were calculated by using the Lamm equation modeling implementing maximum entropy regu-larization(46).

DNA-binding af?nity assay.Puri?ed His6-tagged ParB polypeptides were used in a DNA-binding electrophoretic mobility shift assay(EMSA)as previously described(32).Portions(5.6pmol)of the double-stranded parS oligonucleotide AGCTTGTTGCTTGTTCCACGTGGAACAAGGCCG were incubated with increasing amounts of ParB derivative(10,20,30,and50pmol)in binding buffer (20mM Tris-HCl[pH8.0],1mM dithiothreitol,150mM NaCl)at37°C for15 min.The samples were analyzed on10%polyacrylamide gels run in1?Tris-borate-EDTA(TBE)(45).The gels were stained with ethidium bromide,and DNA was visualized in UV light.

Cross-linking with glutaraldehyde.Puri?ed His-tagged ParB derivatives at concentrations either of0.1or0.3mg ml?1were cross-linked with increasing concentrations of glutaraldehyde(0.0001to0.01%)as described previously(20). Protein complexes were separated on SDS-PAGE gels and analyzed by Western blotting with anti-ParB antibodies.

Western blot technique.The cultures of PAO1161and its mutant derivative strains were grown in L broth to exponential phase.In the“silencing”experi-ments,the cultures of DH5?carrying pKLB2derivatives were induced with0.5 mM IPTG for at least2h.Cells were collected,and serial dilutions were plated on L agar to estimate the CFU.Extracts from approximately the same number of cells were separated by SDS-PAGE.Proteins were transferred onto nitrocel-lulose membranes,and Western blotting with anti-ParB or anti-ParA antibodies was performed as described previously(5).

Introduction of parB mutated alleles into the P.aeruginosa PAO1161chromo-some by homologous https://www.wendangku.net/doc/2a9783145.html,petent E.coli strain S17-1was trans-formed with pAKE600suicide vector derivatives(11).Overnight cultures of the transformants(donor strains)and the recipient P.aeruginosa PAO1161Rif r were conjugated and integrants treated as described before(30).The allele exchange was veri?ed by PCR using chromosomal DNA as a template and the parB1/ parB2pair of primers.The ampli?ed parB orfs were?rst digested with appro-priate restriction enzymes and then sequenced to con?rm the presence of derived mutations.

P.aeruginosa growth experiments.PAO1161Rif,PAO1161parB null,and the various newly constructed PAO1161parB mutant strains were grown overnight and then diluted1:100into fresh L broth.Transformants of PAO1161parB null with pBBR1MCS1vectors containing wild-type(WT)or mutated parB alleles were grown on selective medium overnight and diluted in fresh L broth with antibiotic without or with0.5mM IPTG for tac promoter(tacp)induction.The cultures were incubated at37or30°C with vigorous shaking,and the OD600was measured every hour for the?rst8h of growth.Samples were diluted and spread on L agar to estimate CFU.

DAPI staining.Cells were placed on slides covered with0.01%poly-L-lysine (Sigma).The typical volumes used were5?l for a culture in mid-log phase and 1?l diluted in4?l of LB medium for a culture in stationary phase.The bacteria were allowed to adhere to the poly-L-lysine layer for5min.Unbound bacteria were washed out by rinsing the slide twice with phosphate-buffered saline(10 mM sodium phosphate[pH7.4],15mM KCl,150mM NaCl).Cells were then ?xed using3.7%formaldehyde and stained with DAPI(5?g ml?1)for15min. From this point onward,the slides were kept in the dark.Cells were analyzed by using a Nikon Eclipse EC800microscope.Phase-contrast images were collected using Lucia G software,whereas DNA stained with DAPI was visualized using Lucia G/F software.Overlays of images were obtained with the Lucia G/F software(Nikon).

Immuno?uorescence microscopy.Portions(10?l)of af?nity-puri?ed anti-ParB antibodies(5)were used as primary antibodies(a1:100dilution in2% [wt/vol]bovine serum albumin[BSA]-PBS),followed by1?l of anti-rabbit IgG ?uorescein isothiocyanate(FITC)-conjugate solution(6.9?g ml?1in2%[wt/vol] BSA/PBS)(Sigma).

Molecular modeling.Atomic coordinates of the ParB three-dimensional monomer model were obtained from the Swiss-MODEL server(2,49)on the basis of crystallographic data for KorB of RK2/RP4plasmid(PDB entry1R71) and Spo0J of Thermus thermophilus(PDB entry1VZ0).The structures of KorB and Spo0J monomers are signi?cantly similar,as shown by the FATCAT server (56)with a P value of2.20e?06,although the predicted“dimer”formation and its interaction with DNA seem to be different for the two proteins.Two models for ParB dimers were created:N-terminal P114-T229fragments modeled on the basis of the KorB RK2/RP4structure(26)and N-terminal Q37-L224fragments modeled on the basis of Spo0J from T.thermophilus structure(32).The struc-tures of the WT and mutated ParB proteins were subjected to energy minimi-zation in vacuo using an AmberFF99force?eld as implemented in Sybyl?1.2 (Tripos,Inc.).

RESULTS

Isolation of parB mutants impaired in spreading.It has been shown previously(5)that ParB of P.aeruginosa,when in excess,is able to bind to the centromere-like sequence parS, spread on DNA,and silence the expression of the genes adja-cent to parS.The E.coli strain DH5?carrying pABB811(the stably inherited test plasmid based on the pSC101replicon[9] with parS inserted200bp from the repA promoter[5])was transformed with the high-copy expression vector pKLB2tacp-parB.The frequency of double transformants selected in the presence of IPTG was105-fold lower than the number of transformants selected only for incoming plasmid.Transfor-mation of the same competent cells with empty vector pGBT30 showed no IPTG effect.Even slight overproduction of WT ParB in trans to plasmid with parS(uninduced conditions)gave a10-fold decrease in the number of double transformants compared to DH5?(pABB811)transformed with empty vector pGBT30(5).When parS was absent in the resident plasmid (pGB2),no plasmid loss was observed after transformation with pKLB2in the presence of IPTG(the same frequency of transformation was observed when selecting either for incom-ing plasmid or both incoming and resident plasmid).Since the reduction in numbers of double transformants arising in the presence of excess ParB was such a strong phenotype,we applied it to select for parB mutants unable to spread and transcriptionally silence the repA gene.

Plasmid DNA of pKLB2mutagenized in vitro with hydro-xylamine was used to transform E.coli NR9786strain carrying pABB811.The transformants were plated on MacConkey me-dia with galactose containing penicillin,streptomycin,and IPTG to induce ParB production.The parB gene in pKLB2is inserted between the tac promoter and the galK gene involved in the utilization of galactose as a carbon source.Mutants of pKLB2impaired in the transcriptional signals could then be easily identi?ed as unable to rescue the galactose fermentation in the recipient strain.Approximately10%of transformants formed white colonies on MacConkey plates and were ex-cluded from further analysis.The plasmid pro?les of49puta-tive mutants were also analyzed to eliminate plasmids with visible rearrangements or lowered copy number,but all trans-formants seemed to carry the plasmids identical to pKLB2 (data not shown).Finally,extracts from exponentially growing cultures of transformants induced by the presence of IPTG were checked by SDS-PAGE,followed by Western blotting with anti-ParB(data not shown).The mutants could be divided into three groups:(i)mutants with no detectable ParB(n?11),(ii)mutants with visibly truncated ParBs(n?18),and mutants with ParBs with a molecular mass close to that of WT ParB and produced at levels similar to the WT ParB level(n?20).DNA sequencing revealed that most of the mutants in the ?rst two groups of parB mutants still producing ParB protein had acquired stop codons at different positions and therefore produced truncated ParBs deprived of the C-terminal domain that is responsible for dimerization,which we have shown to be essential for all detectable activities of ParB(J.Mierzejewska, unpublished data).The shortest identi?ed parB deletion lead-ing to a“silencing”de?ciency removed just seven amino acids from the C terminus.Among the third group of mutants we found a number of single-or double-nucleotide substitutions,

V OL.193,2011parB SPREADING MUTANTS OF PSEUDOMONAS AERUGINOSA3345

so 12alleles were chosen for further analysis.One of the alleles,parB40,encoded a ParB derivative with a substitution of A122V (in conserved region 2)and an in-frame deletion of four amino acids in the C-terminal domain close to the variable linker region (visible on a Western blot as a slightly truncated ParB version).The individual mutations were in-troduced by site-directed mutagenesis and,for further analysis,we used the double mutant producing ParB A122V/?253-256and single mutants producing ParB A122V and ParB ?253-256separately (Fig.1).The mutant allele parB14producing ParB without the C-terminal 51amino acids was also included in the analysis as an internal control representing protein de?nitely unable to dimerize.

The mutated plasmids were introduced again into strain DH5?(pABB811)to con?rm the de?ciency in silencing of the repA promoter.Transformants were plated out with selection for the incoming plasmid,for both incoming and resident plasmids and with selection for both plasmids in the pres-ence of IPTG.We observed,after induction with IPTG,a 105-fold decrease in the number of transformants for WT ParB (pKLB2)but no difference in the frequency of transfor-mations for any of the mutants except parB40A (see Fig.S1in the supplemental material).The overproduction of ParB A122V gave a strong silencing effect comparable to that of WT ParB,whereas ParB ?253-256was impaired in the ability to silence the repA gene.Further functional analysis indicated no differences between ParB A122V/?253-256and ParB ?253-256,so for clarity only results for the deletion mutant produc-ing ParB ?253-256are presented.Western blot analysis with anti-ParB antibodies of extracts from transformants grown in the presence of IPTG showed neither signi?cant differences in the level of ParB overproduction between WT ParB and mu-tant derivatives nor increased instability of mutant versions of ParB (data not shown).

The N terminus of ParB is involved in oligomerization.The parB mutant alleles were recloned into pET28mod vector and their products were puri?ed as N-terminally His 6-tagged de-rivatives.Puri?ed ParB mutant derivatives were initially screened by circular dichroism (CD)measurements at wave-lengths between 190and 260nm to check for major perturba-tions in protein structure.Analysis of CD spectra revealed no signi?cant changes in the ?-helical content in the secondary structure of any of the tested proteins in comparison to WT ParB (even the shorter form of ParB 1-249);hence,only CD spectra for representative ParB derivatives are shown in Fig.2.

The ability of ParB derivatives impaired in spreading to form high-molecular-mass structures was tested by two methods:analytical ultracentrifugation and cross-linking with glutaral-dehyde.The results from analytical ultracentrifugation (Fig.3A)demonstrated that WT ParB exists mainly as dimers in solution,and no higher-order complexes (e.g.,tetramers)were detected.The results also showed that all ParB spreading mu-tants were still able to dimerize (data are presented for only two representative derivatives,ParB T167I and ParB M190I).On the other hand,ParB 1-249remained monomeric,whereas the short deletion derivative ParB ?253-256seemed to form less-stable dimers than other tested proteins.

Cross-linking with glutaraldehyde and separation of com-plexes by SDS-PAGE con?rmed the ability of 10derivatives to form dimers (molecular mass of ?66kDa)with the exception of ParB 1-249,ParB G71S,ParB G71D,and ParB ?253-256,for which dimers could not be observed by this method (see Fig.S2in the supplemental material).To better visualize the higher-molecular-mass forms,cross-linked proteins were

sep-

FIG.1.Summary of “silencing”parB mutants of P.aeruginosa used in the present study.A schematic map of ParB (290amino acids)with highly conserved motifs (5)marked in black and the approximate positions of amino acid substitutions in ParB derivatives defective in silencing ability (mutant alleles selected in pKLB2tacp-parB )is shown.The deletion mutants are presented underneath the scheme.The ParB derivative with two regions modi?ed A122V and ?253-256was originally encoded by allele parB40and was also included in the analysis.

FIG.2.Circular dichroism spectra of ParB derivatives.Puri?ed 6?His-tagged versions of mutated ParB were analyzed by CD as de-scribed in Materials and Methods.Since all spectra were similar,only four are shown as representative ParB derivatives for clarity.

3346KUSIAK ET AL.J.B ACTERIOL .

arated by SDS-PAGE,transferred to nitrocellulose ?lters,and analyzed by Western blotting with anti-ParB antibodies.We used two different concentrations of proteins (0.1and 0.3mg/ml,respectively)and treated the proteins with glutaraldehyde in the presence or absence of double-stranded oligonucleotide corresponding to parS .The ability to be cross-linked did not depend on the presence of DNA for any of the analyzed pro-teins.Only ParB 1-249remained strictly monomeric under all conditions.Other ParB proteins were able to form higher-order complexes,although some of them with less ef?ciency than WT ParB at the same concentration of protein and cross-linking agent (Fig.3B).The results for all tested ParB derivatives are shown in Fig.S3in the supplemental mate-rial.On the basis of the ability to form higher-order struc-tures,the proteins were separated into two groups.ParB derivatives G47D,G71S(D),R94C,A97T,P107S,and A158V were all noticeably impaired in oligomerization abil-ity under these conditions.The ParB T165I,T167I,M190I,and G198D mutants and the double mutant R219H/A223T showed no defect in the in vitro oligomerization test.The results from analytical ultracentrifugation and cross-linking experiments are summarized in Fig.3C and Table 4.The results indicated that while the C-terminal dimerization do-main is the major determinant of higher-order complex

for-

FIG.3.Structural analysis of ParB derivatives.(A)Analytical ultracentrifugation of ParB derivatives.Puri?ed His 6-tagged versions of mutated ParB at concentrations 0.1mg ml ?1were centrifuged in the Beckman XL-A ultracentrifuge in sets of eight tubes,with WT ParB and ParB 1-249included in each set.“S”represents the sedimentation coef?https://www.wendangku.net/doc/2a9783145.html,parison of the S values for four ParB mutants are shown,where an S value close to 2corresponds to dimeric form of the protein and an S value close to 1corresponds to the monomeric form of the protein.(B)Cross-linking of ParB derivatives with glutaraldehyde (GA).Puri?ed His 6-tagged proteins at 0.3mg ml ?1were incubated at room temperature for 20min (18)without or with different concentrations of glutaraldehyde:0.001,0.002,0.005,and 0.01%.Proteins were separated by SDS-PAGE on 12%gels and visualized by Western blotting with anti-ParB antibodies.Monomers are indicated as “M,”and higher-order are indicated as complexes as “P.”(C)Graphic summary of the ability of ParB mutants to form higher-order complexes.Localization of analyzed mutations is indicated by arrows.Black solid arrows indicate mutants with the ability to form higher-order complexes comparable to WT ParB.Black V-type arrows indicate mutants impaired in higher-order complex formation.The gray arrow indicates mutant ParB 1-249,which remains in the monomeric state.

V OL .193,2011parB SPREADING MUTANTS OF PSEUDOMONAS AERUGINOSA 3347

mation,several residues in the N-terminal of ParB play an important role in oligomerization.

The H-T-H motif of ParB is required but not suf?cient for effective DNA recognition/binding.EMSAs using mutated ParB proteins were performed to check the ability of these proteins to bind double-stranded oligonucleotides correspond-ing to the parS 2sequence.It was previously demonstrated that WT ParB exhibits the highest af?nity to this perfect palin-drome sequence among tested parS sites (5).The His 6-tagged WT ParB was used as a positive control,whereas an unrelated double-stranded oligonucleotide served as the negative control (nonspeci?c binding [data not shown]).DNA-protein com-plexes were separated by PAGE.The group of ParB mutants unable to bind parS oligonucleotide consisted of ParB T165I,T167I,and G198D;the double-mutant ParB R219H/A223T;and ParB 1-249with the C terminus removed (see Fig.S4

FIG.4.DNA-binding activity of ParB derivatives.(A)EMSA for representative ParB derivatives.Different amounts (10,20,30,and 50pmol)of puri?ed ParB proteins were incubated with 5.6pmol of parS double-stranded oligonucleotide at 37°C for 15min.The samples were analyzed on 10%(wt/vol)nondenaturing polyacrylamide gels run in TBE.After electrophoresis,the gels were stained with ethidium bromide,DNA was visualized in UV light and photographed.(B)Graphic summary of DNA-binding af?nity of ParB mutants.Localization of analyzed mutations is indicated by arrows.Three types of arrow correspond to distinct DNA binding abilities:highly impaired mutants are indicated by gray arrows,partially impaired mutants are indicated by black V-type arrows,and mutants binding to DNA with WT ParB activity are indicated by black solid arrows.For comparison,the results for oligomerization ability of mutant derivatives are shown below the ParB scheme.

TABLE 4.Summary of analysis of parB mutants

No.of parB allele (substitution)

conserved motif a Form in the solution (AUC)

Higher-order complex formation (after GA

cross-linking)

DNA binding (EMSA)

Toxic effect (after overproduction in PAO1161parB )

Wild type Dimer ?????????52(G47D)

Dimer ?????23(G71D)Box I Dimer ???55(G71S)*,Box I Dimer ???9(R94C),Box II Dimer ?????3(A97T),Box II Dimer ???????24(P107S)

Dimer ?????7(A158V),H-T-H Dimer ?????62(T165I),H-T-H Dimer ?????61(T167I),H-T-H Dimer ?????44(M190I)Dimer ?????14(G198D)

Dimer ?????89(R219H/A223T),region III Dimer

?????40B (?253-256)Dimer/monomer ???18(stop249)

Monomer ???

*,The parB alleles introduced into PAO1161chromosome by allele exchange are shaded.

3348KUSIAK ET AL.

J.B ACTERIOL .

the supplemental material).The remaining proteins were still able to bind parS either with af?nity comparable to that of WT ParB(ParB G47D,R94C,A97T,P107S,and A158V)or with lower af?nity,as observed for ParB G71D,G71S,M190I,and ?253-256.Figure4shows representative data and a summary of the EMSA experiments,whereas data for all ParB mutants tested using EMSA with parS

are presented in Fig.S4in the supplemental material.Our previous studies showed that ParB binds to parS as a dimer and that a ParB derivative with the H-T-H motif deleted had lost its DNA-binding activity(5).We expected substitutions in the H-T-H motif(ParB A158V, T165I,and T167I)and ParBs with impaired C-terminal dimerization domain(ParB1-249and?253-256)to be defec-tive in DNA binding,and this was con?rmed except for ParB A158V.Two mutants with amino acid substitutions in the sequence following the H-T-H motif(ParB G198D[between region3and the H-T-H motif]and ParB R219H/A223T[re-gion3])did not bind parS.In addition,mutant ParB M190I, also located between region3and the H-T-H motif,had a lower binding af?nity for parS.Since the ability to dimerize and polymerize is not impaired in these derivatives,the new deter-minants involved in the ParB interactions with DNA have been identi?ed.Two derivatives—ParB G71S and ParB G71D—had lower binding af?nities toward parS,while substitutions of gly-cine at position71in the highly conserved Box I sequence led to a strong polymerization defect(Fig.4B).

De?ciency in spreading on the DNA is not equivalent to the loss of ParB toxicity.As shown previously,ParB,when in excess,is able to inhibit the growth of P.aeruginosa(5),and this effect is independent of ParA(an excess of ParB is toxic in a parA-null mutant)(30).To test the effect of the overproduc-tion of the mutant versions of ParB on P.aeruginosa,the alleles were cloned into broad-host-range vector pBBR1MCS1under the control of the tacp.All pBBR1MCS1derivatives were transformed into a PAO1161parB deletion strain.The cultures of transformants were grown with or without IPTG.The aver-age growth rate in exponential phase was calculated from three independent experiments,while the strains with the empty vector pBBR1MCS1and pJMB500were used as controls.The results showed that the overproduction of ParB derivatives impaired in spreading still demonstrated either a very strong toxic effect(ParB A97T),a weaker but signi?cant toxic effect (ParB G47D,G71D,G71S,R94C,A158V,T165I,M190I, G198D,and R219H/A223T;ParB1-249;and ParB?253-256), or no effect(ParB P107S and T167I).DNA binding and spreading is therefore not the main factor responsible for slow-ing down the growth rate of P.aeruginosa in the presence of a ParB excess since some spreading-de?cient mutants retained their toxicity(Fig.5).

ParBs defective in spreading cause defects in chromosome segregation and changes in cell motilities.To analyze the role of ParB spreading in various cellular functions,?ve parB al-leles—parB55,parB9,parB3,parB7,and parB62(coding for ParB G71S,R94C,A97T,A158V,and T165I,respectively)—were introduced into the PAO1161chromosome.The products

FIG.5.Growth inhibition of P.aeruginosa parB-null mutant by over-

production of ParB mutant derivatives.(A)Growth of PAO1161parB null

(pBBR1MCS1tacp-parB mutants).Overnight cultures were diluted100-

fold into L broth supplemented with chloramphenicol or chloramphen-

icol and0.5mM IPTG.The cultures were incubated with shaking at

37°C,and the OD600was measured at hourly intervals.As controls,

PAO1161parB null(pBBR1MCS1)(vector)and PAO1161parB null

(pJMB500)(WT parB)were used.For clarity,only the results for

IPTG-induced cultures for three representative mutant ParB deriva-

tives are shown.(B)Graphic summary of inhibition effect of ParB

derivatives on PAO1161parB null growth.The ParB derivatives retain-

ing a growth inhibition effect when overproduced are indicated either

by black solid arrows(high toxicity)or black V-type arrows(slightly

lower toxicity),whereas modi?cations abolishing growth inhibition are

indicated by gray solid arrows.

TABLE5.Growth rate and percentage of anucleate cells formation

for P.aeruginosa WT and parB mutants

P.aeruginosa strain a Division time

(min)at30°C

on L broth b

%Anucleate cells

in population c

PAO116142?0.03

PAO1161parB null54 2.30

PAO1161parB55(G71S)44 1.41

PAO1161parB9(R94C)45 1.31

PAO1161parB3(A97T)44 2.3

PAO1161parB7(A158V)41 1.2

PAO1161parB62(T165I)55 2.4

a The amino acid substitution in the ParB derivative is indicated in paren-

theses.

b Cultures were grown at30°on L broth and at hourly intervals appropriate

dilutions were spread on L-agar plates to estimate the CFU.The division time

was calculated from three independent cultures.

c Cells from a logarithmically grown culture were DAPI staine

d and visualized

by microscopy.The percentage of anucleate cells was estimated in three exper-

iments on the sample of at least1,000cells each.

V OL.193,2011parB SPREADING MUTANTS OF PSEUDOMONAS AERUGINOSA3349

of the mutated alleles when analyzed in vitro differed in their ability to bind DNA,form higher-order complexes,and disturb cell growth or divisions when overproduced(Table4).The mutant alleles were cloned into the suicide vector pAKE600 (11)and then introduced into the P.aeruginosa genome by homologous recombination.The allele exchange was con?rmed by sequencing of parB genes ampli?ed by PCR on chromo-somal DNA of the putative mutants.Separation of proteins from mutant strains by SDS-PAGE and analysis by Western blotting con?rmed that the level of ParB production was com-parable to that of WT strain PAO1161(see Fig.S5in the supplemental material).The extracts were also screened for the presence of ParA with anti-ParA antibodies and showed no differences in the level of ParA production between PAO1161 and the various parB mutants(data not shown). Chromosomal mutants were tested for growth rate and colony morphology,two types of motility(swimming and swarming),cell size,chromosome segregation,anucleate cell production(by DAPI staining),and ParB focus forma-tion(by immuno?uorescence microscopy using primary anti-ParB antibodies and FITC-conjugated secondary antibodies). The colony morphology of tested mutants was indistinguish-able from the parB-null mutant:they formed irregular-shaped colonies in contrast to the smooth-edged colonies of the WT strain(data not shown).Growth rate was much slower in mu-

tant PAO1161parB

62(comparable to strain PAO1161parB

null

whereas in four other strains the cell division rate was not signi?cantly different from the WT strain(Table5).All?ve mutants showed defects in chromosome segregation.The per-centage of anucleate cell production by mutants varied be-tween1.2and2.4%,whereas it was?0.03%for the PAO1161 WT and2.3%for the PAO1161parB

null

strains under the same growth conditions(Table5).The number of anucleate cells was estimated in three independent experiments on the sam-ples of1,000cells.Monitoring of ParB localization using the FITC technique showed multiple,randomly distributed,and fuzzy ParB foci in all?ve mutants(Fig.6A).Thus,it appears that formation of two to four condensed and regularly spaced foci observed in the WT strain depends on the ability of ParB to bind to DNA and spread.All mutants also showed defects in swarming and swimming,although two of the?ve mutant strains,

PAO1161parB

7(expressing ParB A158V)and PAO1161parB

(expressing ParB G71S),were signi?cantly less impaired than the parB-null mutant(Fig.6B).

DISCUSSION

Representatives of the ParB family(including chromosomal ParBs)have a main dimerization domain located in the C terminus and centrally located H-T-H motif involved in DNA binding(Fig.7).Conserved N and C domains of ParB protein are separated by a variable linker region(5,6,35).The ability to form dimers is a prerequisite for strong operator binding since monomers either do not bind DNA or bind weakly(5,21, 32).Despite the homology and overall very similar structure ParBs seem to differ signi?cantly in their interactions with DNA.The“transcriptional silencing”function observed for several representatives of the type IA ParB family depends on the ability of the protein to recognize and bind speci?c DNA sequences(centromere-like parS),oligomerize by protein-pro-tein interactions,and spread on DNA starting from parS(5,35, 37).In the present study we analyzed mutant parB alleles from P.aeruginosa defective in spreading on DNA.

The mutations affecting spreading of ParB from P.aerugi-nosa were found dispersed throughout the protein.The most common parB mutants had stop codons producing a range of truncated derivatives,all deprived of the dimerization domain. The shortest deletion from the C terminus,which abolished spreading,had seven amino acids removed,con?rming our previous observation about the importance of the C terminus of the ParB protein in dimerization(5).Among the mutants there was also a parB allele with a12-nucleotide deletion in the linker proximal part of the C-domain(in-phase deletion of four amino acids).ParB?253-256was impaired signi?cantly in the dimerization,demonstrating that other residues in region4(5) are also involved in self-association.

The residues from the N-terminal part of the protein form the oligomerization surface in ParB.In the present study,we isolated12parB point mutants impaired in spreading but not in dimerization,a?nding previously demonstrated by analytical ultracentrifugation and cross-linking experiments(Fig.3and Table4).None of the ParB derivatives seemed to have a greatly disturbed CD spectrum,suggesting no major misfold-ing of the proteins.The ParB derivatives mutated in the N terminus—G47D,G71S/D,R94C,A97T,P107S,and A158V—were impaired in the ability to form higher-order complexes, identifying the N-terminal half of ParB as the major oligomer-ization determinant(Table4).

Identi?cation of new DNA-binding determinants in the ParB sequence outside of the H-T-H motif.Three of the ParBs with substitutions in the predicted H-T-H motif(Fig.1)were thought to be impaired in DNA binding,but only two of them, ParB T165I and T167I,with mutations in the recognition helix, were unable to bind parS.Among other ParB derivatives de-fective in parS binding were ParB M190I,ParB G198D,and the double-mutant ParB R219H/A223T.These three deriva-tives with amino acids substitutions localized outside of the H-T-H motif showed no defect in dimerization or oligomer-ization in vitro,suggesting that their inability to spread may be the result of changes in amino acids involved in speci?c DNA contacts.

Model of ParB-DNA complex formation.For years the at-tempts to crystallize the intact ParB proteins of type IA from different sources have been unsuccessful.The?rst structure solved was that of the C terminus of KorB(residues297to358) from RK2plasmid of IncP-1(10),representing the dimeriza-tion domain.It was followed by crystal structures of part of the N terminus of KorB(residues117to294)crystallized with the O

KorB binding site(26),the N-terminal residues1to222of Spo0J(269amino acids)from Thermus thermophilus(32),and fragment from residues142to333bound to parS of ParB of P1 (333amino acids in total)(47).In a recent report(48),crystals of full-length SopB of the F plasmid(323amino acids)mixed 1:1with18-mer(the sopC consensus site)were obtained and resolved;however,the density was only observed for central residues157to271containing determinants for speci?c bind-ing to DNA.

ParB from P.aeruginosa shares much greater similarity to KorB from the RK2/RP4plasmid and Spo0J(ParB)from T.thermophilus(17and36%sequence identities,respectively,

3350KUSIAK ET AL.J.B ACTERIOL.

Fig.7)than to ParB of P1plasmid and SopB of F plasmid (47,48).From the available crystallographic data,two models for ParB Pa have been created on the basis of KorB RK2/RP4structure (Fig.8A and B)and Spo0J from T.thermophilus (Fig.8C and D),and mutations defective in either DNA binding or oligomerization were localized in the structure.Neither of these models fully accommodates all of our experimental data.P.aeruginosa ParB seems to interact with DNA similarly

FIG.6.Characterization of PAO1161parB mutants defective in spreading.(A)ParB subcellular localization in PAO1161parB spreading mutants.Fixed cells were prepared from exponential phase of culture growth (OD 600?0.4)on L broth at 37°C.Overlaid images of immuno-?uorescence (green)signal and DAPI (blue)staining show ParB foci in the cells of PAO1161(WT ParB),PAO1161parB 3(ParB A97T),PAO1161parB 7(ParB A158V),PAO1161parB 9(ParB R94C),PAO1161parB 55(ParB G71S),and PAO1161parB 62(ParB T165I)mutants.Mag-ni?cations of single cells are shown for clarity.(B)Swimming motility of PAO1161parB mutants defective in spreading.Arrows demonstrate the diameters of the zones of swimming (extending beyond visible growth zones on the surface).(C)Swarming motility of PAO1161parB mutants defective in spreading.

V OL .193,2011parB SPREADING MUTANTS OF PSEUDOMONAS AERUGINOSA 3351

KorB,engul?ng DNA sequence and not“sitting”on it as the Spo0J model predicts(32).The polymerization de?cient mutations in the N terminus of ParB

?t into Spo0J T. thermophilus structure(the adequate part of KorB has not been crystallized[26]),however,on the assumption that the “dimerization”surface described by the authors(32)is in-deed an oligomerization surface(see below).

ParB interacts with DNA in a similar way to KorB.Previous reports on the KorB structure of the RK2/RP4plasmid(26) demonstrated that four helices make contact with the opera-tor:helices?3and?4forming the typical H-T-H motif and also helices?6and?8(Fig.7).While?4(the recognition helix)plays a role in nonspeci?c KorB operator binding,it does not contribute to operator sequence recognition.The recogni-tion speci?city is based mainly on the two side-chain interac-tions(hydrogen bonding)outside the H-T-H motif,T211in?6 and R240in?8,which was con?rmed experimentally by mu-tagenesis(26).Alignment of the amino acid sequences of KorB

and ParB

Pa indicated that M190and G198of ParB

are lo-

cated in the predicted?6helix for P.aeruginosa ParB(based

on the KorB structure;Fig.7and Fig.8A),whereas R219H

corresponds to R240in the KorB structure.On the basis of our

mutant analysis,we conclude that the speci?c DNA binding of

ParB

outside of H-T-H is highly similar to KorB-DNA in-

teractions,with the exception that the recognition helix of

H-T-H(mutant substitutions in T165and T167of ParB)is also

involved in the speci?c recognition of https://www.wendangku.net/doc/2a9783145.html,putational

modeling of the WT and mutated protein structures indicated

that A158of the“stabilization”helix of H-T-H motif is not

buried in the DNA duplex and,hence,the substitution A158T

may not affect the interactions with DNA(data not shown).

The ParB domain responsible for polymerization corre-

sponds to the Spo0J putative dimerization surface.Only N-

terminal domains of KorB and Spo0J proteins were crystal-

lized,both deprived of the main C-terminal dimerization

domains.In the biochemical tests(gel?ltration,in vitro cross-

linking,and analytical ultracentrifugation),both truncated pro-

teins were monomeric in solution(26,32).The fragment

FIG.7.Alignment of ParB from P.aeruginosa PAO1with selected representatives of ParB family.Dark gray shadowing indicates the similar residues in more than three representatives.Light gray shadowing indicates similarities between two proteins.Structural motifs from crystallo-graphic studies on KorB of plasmid RP4/RK2(above the alignment in green)and Spo0J/ParB from T.thermophilus(indicated below in yellow) are drawn with rectangles corresponding to?-helices and arrows corresponding to?https://www.wendangku.net/doc/2a9783145.html,beling of the helices corresponds to the original nomenclature(26,32).H-T-H motifs are striped.Red-shaded residues in ParB P.aeruginosa sequence correspond to substituted residues,and pink-shaded residues correspond to deleted residues in the course of this study.A red arrow indicates the localization of stop codon in the ParB 1-249derivative.Green residues marked in the KorB sequence correspond to DNA-binding determinants outside of the H-T-H sequence(26). Blue residues in Spo0J of B.subtilis correspond to mutations analyzed previously(3).

3352KUSIAK ET AL.J.B ACTERIOL.

KorB-O was crystallized in the presence of the O B operator,and binding to DNA linked two monomers in such a way that the operator was completely engulfed within the “dimer”(26).In the case of T.thermophilus Spo0J,no DNA was present during crystallization,but the authors still obtained N domains interlinked into antiparallel “dimer”with an extensive dimer interface (28%of the surface of one monomer)(32).

Mutants of ParB Pa ,which were able to dimerize but not to polymerize,have been found at positions G47and G71,?ank-ing ?-helix H2;at positions R94and A97located in ?-helix H3;and at P107preceding the ?-sheet S3.This region corresponds to the Spo0J dimer interaction interface (according to the Spo0J model;Fig.7and Fig.8A and B).The glycine at position G71seems to be particularly important,since two spreading-defective mutants were isolated with substitutions G71S and G71D,https://www.wendangku.net/doc/2a9783145.html,putational modeling showed no dras-tic change in folding of mutant proteins (data not shown),although both mutated proteins at position G71are defective in DNA binding and polymerization,suggesting that amino acid substitutions change ?exibility of the protein and di-minish its ability for stable self-interactions and interactions with DNA.

Observed “toxicity”of ParB when overproduced is not the result of ParB spreading.All tested mutant ParBs defective in spreading (with the exception of ParB P107S and ParB T167I)inhibit the growth of P.aeruginosa when in excess (Fig.5).However,one of the substitutions (A97T)increased the “tox-icity”compared to WT https://www.wendangku.net/doc/2a9783145.html,putational modeling dem-onstrated that this substitution introduces structural changes in the region from E61to L103(see Fig.S6in the supplemental material).Our current hypothesis about “toxicity”is that when in excess ParB interacts and sequesters important cell compo-nents and interferes with their normal function.It is then feasible that observed changes in ParB A97T modify the pro-tein-protein interaction surface.

Spreading is necessary for ParB’s role in cell division and precise nucleoid segregation.The pleiotropic character of parB deletion mutants (4)prompted us to introduce ?ve different parB alleles with single amino acid substitutions into the PAO1161chromosome.The growth rate of the four tested parB point mutants did not differ from the growth rate of the WT strain,whereas the PAO1161parB 62mutant (producing ParB T165I,impaired in DNA binding)had a division time similar to the parB deletion mutant.All parB substitution mu-tants were impaired in swarming and swimming (two of three tested motilities),as observed previously for the parB -null mu-tant (4);however,the de?ciency in motilities was less pro-nounced in the presence of ParB A158V and ParB G71S.At present,we cannot explain these differences between particu-lar mutant derivatives.Despite the fact that mutated

ParBs

FIG.8.Model of N-terminal part of ParB from P.aeruginosa interacting with DNA.(A and B)Different projections of two ParB monomers (the N-terminal P114-T229fragment)modeled on the basis of the KorB RK2/RP4structure (26).(C and D)Different projections of two ParB monomers (the N-terminal Q37-L224fragment)modeled on the basis of Spo0J from the T.thermophilus structure (32).Monomer subunits are indicated in green and dark blue,with regions of the H-T-H highlighted in light blue.Residues mutated in the present study are colored red (impaired in DNA binding)and yellow (impaired in polymerization)in both monomers but numbered only in the dark blue one.

V OL .193,2011parB SPREADING MUTANTS OF PSEUDOMONAS AERUGINOSA 3353

were defective either in DNA binding(ParB T165I),oligomer-ization(ParB A97T,R94C,and A158V)or both(ParB G71S), all of them demonstrated aberrant chromosome segregation and formed from1.2to2.4%anucleate cells under logarithmic growth conditions.Delocalized,multiple,and fuzzy ParB foci were also observed in all?ve mutants(Fig.6).Since the com-mon feature of the tested mutant ParB proteins was the in-ability to spread on DNA,we can conclude that any mutation prohibiting spreading yields a phenotype similar to the lack of ParB in relation to the chromosome segregation and the ability to move.

The only previous mutant analysis of chromosomal homo-logue of ParB family was performed on Spo0J from B.subtilis (3).Among sporulation-de?cient spo0J mutants,amino acid substitution R206C was found at a position corresponding to our mutant ParB R219H de?cient in spreading(Fig.7).The R206C substitution in Spo0J leads to an abnormal nucleoid appearance,dispersed Spo0J foci,and loss of DNA binding similarly to ParB R219H.Another spo0J mutant(spo0J13) impaired in focus production and chromosome segregation produced Spo0J R80A corresponding in position to our mu-tant ParB R94C(Fig.7).Both of these mutations in ParB and Spo0J do not decrease the DNA-binding ability,con?rming the signi?cant and conserved roles that these residues play in ParB homologues.

ACKNOWLEDGMENTS

We thank Tim R.Dafforn,University of Birmingham,School of Biosciences,for guidance in the analytical ultracentrifugation(AUC) technique and Joanna Zylinska,Institute of Biochemistry and Biophys-ics,Microbial Biochemistry Department,for assistance in plasmid se-quencing.

This study was funded by Wellcome Trust Collaborative Research Initiative grant067068/Z/02/Z and in part by an EMBO short-term fellowship(ASTF176.00.06)awarded to M.K.and MNiSW grant2913/ B/PO1/2008/34.

REFERENCES

1.Adachi,S.,K.Hori,and S.Hiraga.2006.Subcellular positioning of F plas-

mid mediated by dynamic localization of SopA and SopB.J.Mol.Biol.

356:850–863.

2.Arnold,K.,L.Bordoli,J.Kopp,and T.Schwede.2006.The Swiss-MODEL

workspace:a web-based environment for protein structure homology mod-eling.Bioinformatics22:195–201.

3.Autret,S.,R.Nair,and J.Errington.2001.Genetic analysis of the chromo-

some segregation protein Spo0J of Bacillus subtilis:evidence for separate domains involved in DNA binding and interactions with Soj protein.Mol.

Microbiol.41:743–755.

4.Bartosik,A.A.,J.Mierzejewska,C.M.Thomas,and G.Jagura-Burdzy.

2009.ParB de?ciency in Pseudomonas aeruginosa destabilizes the partner protein ParA and affects a variety of physiological parameters.Microbiology 155:1080–1092.

5.Bartosik,A.A.,https://www.wendangku.net/doc/2a9783145.html,socki,J.Mierzejewska,C.M.Thomas,and G.Jagura-

Burdzy.2004.ParB of Pseudomonas aeruginosa:interactions with its partner ParA and its target parS and speci?c effects on bacterial growth.J.Bacteriol.

186:6983–6998.

6.Bingle,L.E.,D.P.Macartney,A.Fantozzi,S.E.Manzoor,and C.M.

Thomas.2005.Flexibility in repression and cooperativity by KorB of broad host range IncP-1plasmid RK2.J.Mol.Biol.349:302–316.

7.Breier,A.M.,and A.D.Grossman.2007.Whole-genome analysis of the

chromosome partitioning and sporulation protein Spo0J(ParB)reveals spreading and origin-distal sites on the Bacillus subtilis chromosome.Mol.

Microbiol.64:703–718.

8.Cervin,M.A.,et al.1998.A negative regulator linking chromosome segre-

gation to developmental transcription in Bacillus subtilis.Mol.Microbiol.

29:85–95.

9.Churchward,G.,D.Belin,and Y.A.Nagamine.1984.pSC101-derived plas-

mid which shows no sequence homology to other commonly used cloning vectors.Gene31:165–171.

10.Delbru¨ck,H.,G.Ziegelin,https://www.wendangku.net/doc/2a9783145.html,nka,and U.Heinemann.2002.An Src

homology3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4.J.Biol.Chem.

277:4191–4198.

11.El-Sayed,A.K.,J.Hothersall,and C.M.Thomas.2001.Quorum-sensing-

dependent regulation of biosynthesis of the polyketide antibiotic mupirocin in Pseudomonas?uorescens NCIMB10586.Microbiology147:2127–2139.

12.Gerdes,K.,J.Moller-Jensen,and R.Bugge Jensen.2000.Plasmid and

chromosome partitioning:surprises from phylogeny.Mol.Microbiol.37:455–466.

13.Gerdes,K.,M.Howard,and F.Szardenings.2010.Pushing and pulling in

prokaryotic DNA segregation.Cell141:927–942.

14.Godfrin-Estevenon,A.-M.,F.Pasta,and https://www.wendangku.net/doc/2a9783145.html,ne.2002.The parAB gene

products of Pseudomonas putida exhibit partition activity in both P.putida and Escherichia coli.Mol.Microbiol.43:39–49.

15.Hanahan,D.1983.Studies on transformation of Escherichia coli with plas-

mids.J.Mol.Biol.166:557–580.

16.Hanai,R.,et al.1996.Molecular dissection of a protein SopB essential for

Escherichia coli F plasmid partition.J.Biol.Chem.271:17469–17475.

17.Hayes,F.2000.The partition system of multidrug resistance plasmid TP228

includes a novel protein that epitomizes an evolutionarily distinct subgroup of the ParA superfamily.Mol.Microbiol.37:528–541.

18.Irani,V.R.,and J.J.Rowe.1997.Enhancement of transformation in Pseu-

domonas aeruginosa PAO1by Mg2?and heat.Biotechniques22:54–56. 19.Ireton,K.,and A.D.Grossman.1994.A developmental checkpoint couples

the initiation of sporulation to DNA replication in Bacillus subtilis.EMBO J.

13:1566–1573.

20.Jagura-Burdzy,G.,and C.M.Thomas.1995.Puri?cation of KorA protein

from broad host range plasmid RK2:de?nition of a hierarchy of KorA operators.J.Mol.Biol.253:39–50.

21.Jagura-Burdzy,G.,et al.1999.Repression at a distance by the global reg-

ulator KorB of promiscuous IncP plasmids.Mol.Microbiol.32:519–532. 22.Jagura-Burdzy,G.,J.P.Ibbotson,and C.M.Thomas.1991.The korF region

of broad-host-range plasmid RK2encodes two polypeptides with transcrip-tional repressor activity.J.Bacteriol.173:826–833.

23.Jakimowicz,D.,B.Gust,J.Zakrzewska-Czerwinska,and K.F.Chater.2005.

Developmental-stage-speci?c assembly of ParB complexes in Streptomyces coelicolor hyphae.J.Bacteriol.187:3572–3580.

24.Jakimowicz,D.,K.Chater,and J.Zakrzewska-Czerwínska.2002.The ParB

protein of Streptomyces coelicolor A3(2)recognizes a cluster of parS se-quences within the origin-proximal region of the linear chromosome.Mol.

Microbiol.45:1365–1377.

25.Kahn,M.,et al.1979.Plasmid cloning vehicles derived from plasmids ColE1,

F,R6K,and RK2.Methods Enzymol.68:268–280.

26.Khare,D.,G.Ziegelin,https://www.wendangku.net/doc/2a9783145.html,nka,and U.Heinemann.2004.Sequence-

speci?c DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB.Nat.Struct.Mol.Biol.11:656–663. 27.Kois,A.,M.Swiatek,D.Jakimowicz,and J.Zakrzewska-Czerwin′ska.2009.

SMC protein-dependent chromosome condensation during aerial hyphal development in Streptomyces.J.Bacteriol.191:310–319.

28.Kovach,M.E.,et al.1995.Four new derivatives of the broad-host-range

cloning vector pBBR1MCS1,carrying different antibiotic-resistance cas-settes.Gene166:175–176.

https://www.wendangku.net/doc/2a9783145.html,rsen,R.A.,et al.2007.Treadmilling of a prokaryotic tubulin-like protein,

TubZ,required for plasmid stability in Bacillus thuringiensis.Genes Dev.

21:1340–1352.

https://www.wendangku.net/doc/2a9783145.html,socki,K.,A.A.Bartosik,J.Mierzejewska,C.M.Thomas,and G.Jagura-

Burdzy.2007.Deletion of the parA(soj)homologue in Pseudomonas aerugi-nosa causes ParB instability and affects growth rate,chromosome segrega-tion,and motility.J.Bacteriol.189:5762–5772.

31.Lee,P.S.,D.C.-H.Lin,S.Moriya,and A.D.Grossman.2003.Effects of the

chromosome partitioning protein Spo0J(ParB)on oriC positioning and replication initiation in Bacillus subtilis.J.Bacteriol.185:1326–1337.

32.Leonard,T.A.,P.J.Butler,and J.Lo¨we.2004.Structural analysis of the

chromosome segregation protein Spo0J from Thermus thermophilus.Mol.

Microbiol.53:419–432.

33.Lewis,R.A.,C.R.Bignell,W.Zeng,A.C.Jones,and C.M.Thomas.2002.

Chromosome loss from par mutants of Pseudomonas putida depends on growth medium and phase of growth.Microbiology148:537–548.

34.Livny,J.,Y.Yamaichi,and M.K.Waldor.2007.Distribution of centromere-

like parS sites in bacteria:insights from comparative genomics.J.Bacteriol.

189:8693–8703.

35.Lobocka,M.,and M.Yarmolinsky.1996.P1plasmid partition:a mutational

analysis of ParB.J.Mol.Biol.259:366–382.

36.Lukaszewicz,M.,et al.2002.Functional dissection of the ParB homologue

(KorB)from IncP-1plasmid RK2.Nucleic Acids Res.30:1046–1055.

37.Lynch,A.S.,and J.C.Wang.1995.SopB protein-mediated silencing of

genes linked to the sopC locus of Escherichia coli F plasmid.Proc.Natl.

Acad.Sci.U.S.A.92:1896–1900.

38.Mohl,D.A.,and J.W.Gober.1997.Cell-cycle dependent polar localization

of chromosome partitioning proteins in Caulobacter crescentus.Cell88:675–684.

39.Mohl,D.A.,J.Easter,Jr.,and J.W.Gober.2001.The chromosome parti-

3354KUSIAK ET AL.J.B ACTERIOL.

tioning protein,ParB,is required for cytokinesis in Caulobacter crescentus.

Mol.Microbiol.42:741–755.

40.Mori,H.,et al.1989.Puri?cation and characterization of SopA and SopB

proteins essential for F plasmid partitioning.J.Biol.Chem.264:15535–15541.

41.Murray,H.,and J.Errington.2008.Dynamic control of the DNA replication

initiation protein DnaA by Soj/ParA.Cell135:74–84.

42.Pansegrau,W.,et https://www.wendangku.net/doc/2a9783145.html,plete nucleotide sequence of Birmingham

IncP alpha https://www.wendangku.net/doc/2a9783145.html,pilation and comparative analysis.J.Mol.Biol.

239:623–663.

43.Rashid,M.H.,and A.Kornberg.2000.Inorganic polyphosphate is needed

for swimming,swarming,and twitching motilities of Pseudomonas aerugi-nosa.Proc.Natl.Acad.Sci.U.S.A.97:4885–4890.

44.Rodionov,O.,M.Lobocka,and M.Yarmolinsky.1999.Silencing of genes

?anking the P1plasmid centromere.Science283:546–549.

45.Sambrook,J.,E.F.Fritsch,and T.Maniatis.1989.Molecular cloning:a

laboratory manual,2nd ed.Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY.

46.Schuck,P.2000.Size-distribution analysis of macromolecules by sedimen-

tation velocity ultracentrifugation and Lamm equation modeling.Biophys.J.

78:1606–1919.

47.Schumacher,M.A.,and B.E.Funnell.2005.Structures of ParB bound to

DNA reveal mechanism of partition complex formation.Nature438:516–519.

48.Schumacher,M.A.,K.M.Piro,and W.Xu.2010.Insight into F plasmid

DNA segregation revealed by structures of SopB and SopB-DNA complexes.

Nucleic Acids Res.38:4514–4526.

49.Schwede,T.,J.Kopp,N.Guex,and M.C.Peitsch.2003.SWISS-MODEL:an

automated protein homology-modeling server.Nucleic Acids Res.31:3381–3385.

50.Simon,R.,M.O’Connell,https://www.wendangku.net/doc/2a9783145.html,bes,and A.Puhler.1986.Plasmid vectors for

the genetic analysis and manipulation of Rhizobia and other gram-negative bacteria.Methods Enzymol.118:640–659.

51.Simpson,A.E.,R.A.Skurray,and N.Firth.2003.A single gene on the

staphylococcal multiresistance plasmid pSK1encodes a novel partitioning system.J.Bacteriol.185:2143–2152.

52.Surtees,J.A.,and B.E.Funnell.1999.P1ParB domain structure includes

two independent multimerization domains.J.Bacteriol.181:5898–5908. 53.Surtees,J.A.,and B.E.Funnell.2001.The DNA binding domains of P1

ParB and the architecture of the P1plasmid partition complex.J.Biol.

Chem.276:12385–12394.

54.Yamaichi,Y.,and H.Niki.2000.Active segregation by the Bacillus subtilis

partitioning system in Escherichia coli.Proc.Natl.Acad.Sci.U.S.A.97: 14656–14661.

55.Yamaichi,Y.,M.A.Fogel,S.M.McLeod,M.P.Hui,and M.K.Waldor.

2007.Distinct centromere-like parS sites on the two chromosomes of Vibrio spp.J.Bacteriol.189:5314–5324.

56.Ye,Y.,and A.Godzik.2004.FATCAT:a web server for?exible structure

comparison and structure similarity searching.Nucleic Acids Res.32(web server issue):W582–W585.

V OL.193,2011parB SPREADING MUTANTS OF PSEUDOMONAS AERUGINOSA3355

管径公制英制对照表及常用尺寸转换.doc

1. 以公制 (mm)为基准，称 DN (metric unit) 公制单位 2. 以英制 (inch) 为基准，称 NB(inch unit) 英寸单位 3. DN(nominal diameter 公称内径 ) ，NB(nominal bore 公称内径 ) ，OD(outside diameter 外径 ) 长度单位换算： 1 千米（公里） = 2 市里英里 =海里 1米 =3 市尺 =英尺 1 海里 =千米（公里） =市里 =英里 1市尺 =米=英尺 1 英里 =千米（公里） =市里 1英尺 =12 英寸 =市尺 1 平方千米（平方公里） =100 公顷 =4 平方市里 .= 平方海里 1公亩 =100 平方米 =市亩 1公顷 =10000平方米 =100 公亩 =15 市亩一千克（公斤） =2 市斤 =英磅 1市斤 =千克（公斤） =英镑 1英磅 =16 盎司 =千克（公斤） =市斤 1升（公制） =1 公斤 =1 六升 =1 市升 =加仑（英制） 1毫升 =1 西西 =升（公制） 1加仑（英制） =升=市升管径公制及英制对照表 DN(mm)公称内径NB(inch) 公称内径OD(mm)外径对应欧标内螺纹俗称DN6 1/8 10 1 分管DN8 1/4 2 分管DN10 3/8 17 3 分管DN15 1/2 RP1/2 4 分管DN20 3/4 RP3/4 6 分管DN25 1 RP1 1 寸管DN32 1 1/4 RP1 1/4 1 寸 2 DN40 1 1/2 RP1 1/2 1 寸半DN50 2 RP2 2 寸DN65 2 1/2 RP2 1/2 2 寸半DN80 3 RP3 3 寸DN100 4 RP4 4 寸DN125 5 RP5 5 寸DN150 6 RP6 6 寸DN200 8 RP8 8 寸DN250 10 273 DN300 12 325 DN350 14 377 DN300 16 426 DN450 18 478 DN500 20 529 DN600 24 630 规范不同，外径会有些许差异，请注意。 1 英寸 =8 英分 =，4 分==半寸， 6 分==3/4 寸。

英制与公制对照表公称管子尺寸和公称直径

英制与公制对照表公称管子尺寸和公称直径管道尺寸（英制与公制对照表) 英寸是长度单位。1 英寸= 2.539999918 厘米(公分)。英寸或[吋]是使用于联合王国(UK，即英国(英联邦)、其前殖民地的长度单位。美国等国家也使用它。在台湾与香港，“英寸”通常写作“吋”。英寸的常用简写为[in]或["]“吋”是近代新造的字，念作“英寸”，属汉字中一字念两音的字，其他如“浬”念作“海里”等，借用中国传统的长度单位“寸”，并加口旁以示区别。一、尺寸： DN15（4分管）、DN20（6分管）、DN25（1寸管）、DN32（1寸2管）、DN40（1寸半管）、DN50（2寸管）、DN65（2寸半管）、DN80（3寸管）、DN100（4寸管）、DN125（5寸管）、DN150（6寸管）、DN200（8寸管）、DN250（10寸管）等。二、把1英寸分成8等分: 1/8 1/4 3/8 1/2 5/8 3/4 7/8 英寸。相当于通常说的1分管到7分管,更小的尺寸用1/16、1/32、1/64来表示，单位还是英寸。如果分母和分子能够约分（如分子是2、4、8、16、32）就应该约分。英寸的表示是在右上角打上两撇，如1/2" 如DN25（25mm，下同）的水管就是英制1"的水管，也是以前的8分水管。 DN15的水管就是英制1/2"的水管，也是以前的4分水管。如DN20的水管就是英制3/4"的水管，也是以前的6分水管外径与DN，NB的关系如下：公称管子尺寸(NPS)/in 0. 25 0. 5 0. 75 1. 1. 25 1. 5 2. 2. 5 3. 4. 6.

管径对照表

管径对照表 Company number：【0089WT-8898YT-W8CCB-BUUT-202108】

多大管径配多大阀门多大管径（外径）与多大阀门（通径）DN尺寸对照表工程管径对照表（常用）： 1 英寸=毫米 =8英分？ 1/2 是四分(4英分) DN15？ 3/4 是六分(6英分) DN20？ 2分管 DN8 4分管 DN15？ 6分管 DN20？ 1′ DN25 ′ DN32 ′ DN40 2′ DN50 ′ DN65？ 3′ DN80 4′ DN100 5′ DN125 6′ DN150 8′ DN200？ 10′ DN250 12′ DN300？ GB/T50106-2001？管径？ 1 水煤气输送钢管（镀锌或非镀锌）、铸铁管等管材，管径宜以公称直径DN表示；？ 2 无缝钢管、焊接钢管（直缝或螺旋缝）、铜管、不锈钢管等管材，管径宜以外径×壁厚表示；？ 3 钢筋混凝土（或混凝土）管、陶土管、耐酸陶瓷管、缸瓦管等管材，管径宜以内径d表示；？ 4 塑料管材，管径宜按产品标准的方法表示；？ 5 当设计均用公称直径DN表示管径时，应有公称直径DN与相应产品规格对照表。？建筑排水用硬聚氯乙烯管材规格用de（公称外径）×e（公称壁厚）表示（GB ）？给水用聚丙烯（PP）管材规格用de×e表示（公称外径×壁厚）. 关于DN与De的区别：？ 1、DN是指管道的公称直径，注意：这既不是外径也不是内径；应该与管道工程发展初期与英制单位有关；通常用来描述镀锌钢管，它与英制单位的对应关系如下：？ 4分管：4/8英寸：DN15；？ 6分管：6/8英寸：DN20；？ 1寸管：1英寸：DN25；？寸二管：1又1/4英寸：DN32；？

管道直径尺寸规格

管道直径尺寸规格五金手册中可以查一般来说，管子的直径可分为外径、内径、公称直径。管材为无缝钢管的管子的外径用字母D来表示，其后附加外直径的尺寸和壁厚，例如外径为108的无缝钢管，壁厚为5MM，用D108*5表示，塑料管也用外径表示，如De63,其他如钢筋混凝土管、铸铁管、镀锌钢管等采用DN表示，在设计图纸中一般采用公称直径来表示，公称直径是为了设计制造和维修的方便人为地规定的一种标准，也较公称通径，是管子（或者管件）的规格名称。管子的公称直径和其内径、外径都不相等，例如：公称直径为100MM的无缝钢管邮102*5、108*5等好几种，108为管子的外径，5表示管子的壁厚，因此，该钢管的内径为（108*5-5）＝98MM，但是它不完全等于钢管外径减两倍壁厚之差，也可以说，公称直径是接近于内径，但是又不等于内径的一种管子直径的规格名称，在设计图纸中所以要用公称直径，目的是为了根据公称直径可以确定管子、管件、阀门、法兰、垫片等结构尺寸与连接尺寸，公称直径采用符号DN表示，如果在设计图纸中采用外径表示，也应该作出管道规格对照表，表明某种管道的公称直径，壁厚。 . 管子系列标准压力管道设计及施工，首先考虑压力管道及其元件标准系列的选用。世界各国应用的标准体系虽然多，大体可分成两大类。压力管道标准见表3。法兰标准见表4。表3 压力管道标准分类大外径系列小外径系列规格 DN-公称直径 Ф－外径 DN15-ф22mm，DN20-ф27mm DN25-ф34mm，DN32-ф42mm DN40-ф48mm，DN50-ф60mm DN65-ф76(73)mm，DN80-ф89mm DN100-ф114mm，DN125-ф140mm DN150-ф168mm，DN200-ф219mm

钢管尺寸对照表

压力管道标准分类，大外径系列，规格，DN-公称直径，Ф－外径， DN10—Ф14mm，DN15—Ф18mm，DN20—Ф25mm，DN25—Ф32mm，DN32—Ф38mm，DN40—Ф45mm， DN50—Ф57mm，DN65—Ф76mm，DN80—Ф95mm，DN100—Ф114mm，DN125—Ф140(146)mm， DN150—Ф168mm，DN175—Ф194mm，DN200—Ф219mm，DN225—Ф245mm，DN250—Ф299mm， DN300—Ф325mm，DN350—Ф377mm，DN400—Ф426mm，DN450—Ф480mm，DN500—Ф530(529)mm， DN550—Ф560(559)mm；DN600—Ф630mm；DN650—Ф666mm；DN700-Ф720； DN750-Ф762；DN800-Ф820；DN850-Ф870；DN900-Ф920；DN950-Ф965； DN1000-Ф1020；DN1050-Ф1090；DN1100-Ф1120；DN1200-Ф1220；DN1300-Ф1320；DN1400-Ф1420；DN1500-Ф1520；DN1600-Ф1620；DN1700-Ф1720； DN1800-Ф1820；DN1900-Ф1920；DN2000-Ф2020；DN2200-Ф2220；DN2400-Ф2420 小外径系列，规格，DN-公称通径或称平均外径，Ф－外径， DN15—Ф22mm，DN20—Ф27mm，DN25—Ф34mm，DN32—Ф42mm，DN40—Ф48mm，DN50—Ф60mm， DN65—Ф73mm，DN80—Ф89mm，DN100—Ф108mm，DN125—Ф133mm，DN150—Ф159(152)mm ，DN175—Ф180mm， DN200—Ф203mm，DN225—Ф245mm，DN250—Ф273mm，DN300—Ф324mm，DN350—Ф356(351)mm， DN400—Ф406mm，DN450—Ф457mm，DN500—Ф508mm，DN550—Ф560(559)mm，DN600—Ф610mm； DN650—Ф660mm；DN700-Ф711；DN750-Ф762；DN800-Ф813；DN850-Ф864；DN900-Ф914； DN950-Ф965；DN1000-Ф1016；DN1100-Ф1118；DN1200-Ф1219； DN1300-Ф1321；DN1400-Ф1422；DN1500-Ф1524；DN1600-Ф1626； DN1700-Ф1727；DN1800-Ф1829；DN1900-Ф1930；DN2000-Ф2032；DN2200-Ф2235；DN2400-Ф 2438 公称直径对应(DN A/B系列英制)钢管外径 A 系列为国际通用系列(俗称英制管) B 系列为国内沿用系列(俗称公制管)(mm) 公称直径钢管外径 A系列(英制管) B系列(公制管) DN10 17.2 14 DN 15 21.3 18 DN 20 26.9 25 DN 25 33.7 32 DN 32 42.4 38 DN 40 48.3 45 DN 50 60.3 57 DN 65 76.1 76 DN 80 88.9 89

钢管尺寸对照表

钢管尺寸对照表 Document number：BGCG-0857-BTDO-0089-2022

镀锌管标准尺寸表

查看文章镀锌管尺寸规格表 2010-10-29 10:00 镀锌管基本知识一般来说，管子的直径可分为外径、内径、公称直径。管材为无缝钢管的管子的外径用字母 D 来表示，其后附加外直径的尺寸和壁厚，例如外径为 108 的无缝钢管，壁厚为 5MM，用 D108*5 表示，塑料管也用外径表示，如 De63，其他如钢筋混凝土管、铸铁管、镀锌钢管等采用 DN 表示，在设计图纸中一般采用公称直径来表示，公称直径是为了设计制造和维修的方便人为地规定的一种标准，也较公称通径，是管子（或者管件）的规格名称。管子的公称直径和其内径、外径都不相等，例如：公称直径为 100MM 的无缝钢管邮 102*5、108*5 等好几种，108 为管子的外径，5 表示管子的壁厚，因此，该钢管的内径为（108*5 -5）＝98MM，但是它不完全等于钢管外径减两倍壁厚之差，也可以说，公称直径是接近于内径，但是又不等于内径的一种管子直径的规格名称，在设计图纸中所以要用公称直

径，目的是为了根据公称直径可以确定管子、管件、阀门、法兰、垫片等结构尺寸与连

接尺寸，公称直径采用符号 DN 表示，如果在设计图纸中采用外径表示，也应该作出管道规格对照表，表明某种管道的公称直径，壁厚。? .?管子系列标准? 压力管道设计及施工，首先考虑压力管道及其元件标准系列的选用。世界各国应用的标准体系虽然多，大体可分成两大类。压力管道标准见表 3。法兰标准见表 4。? 表 3 压力管道标准分?类大外径系列小外径系列规格 DN-公称直径 Ф－外径 DN15-ф22mm，DN20-ф27mm DN25-ф34mm，DN32-ф42mm DN40-ф48mm，DN50-ф60mm DN65-ф76(73)mm，DN80-ф89mm DN100-ф114mm，DN125-ф140mm DN150-ф168mm，DN200-ф219mm DN250-ф273mm，DN300-ф324mm DN350-ф360mm，DN400-ф406mm DN450-ф457mm，DN500-ф508mm DN600-ф610mm，

公称管子尺寸和公称直径对照表

公称管子尺寸和公称直径令狐采学管道尺寸（英制与公制对照表）英寸是长度单位。1 英寸= 2.539999918 厘米(公分)。英寸或[吋]是使用于联合王国(UK，即英国(英联邦)、其前殖民地的长度单位。美国等国家也使用它。在台湾与香港，“英寸”通常写作“吋”。英寸的常用简写为[in]或["]“吋”是近代新造的字，念作“英寸”，属汉字中一字念两音的字，其他如“浬”念作“海里”等，借用中国传统的长度单位“寸”，并加口旁以示区别。一、尺寸： DN15（4分管）、DN20（6分管）、DN25（1寸管）、DN32（1寸2管）、DN40（1寸半管）、DN50（2寸管）、DN65（2寸半管）、DN80（3寸管）、DN100（4寸管）、DN125（5寸管）、DN150（6寸管）、DN200（8寸管）、DN250（10寸管）等。二、把1英寸分成8等分: 1/8 1/4 3/8 1/2 5/8 3/4 7/8 英寸。相当于通常说的1分管到7分管,更小的尺寸用1/16、1/32、1/64来表示，单位还是英寸。如果分母和分子能够约分（如分子是2、4、8、16、32）就应该约分。英寸的表示是在右上角打上两撇，如1/2" 如DN25（25mm，下同）的水管就是英制1"的水管，也是以前的8分水管。

如DN15的水管就是英制1/2"的水管，也是以前的4分水管。如DN20的水管就是英制3/4"的水管，也是以前的6分水管外径与DN，NB的关系如下：管子规格及有关数据

国际管道尺寸对照表 DN公称直径不锈钢无缝管尺寸对照表

一般来说，管子的直径可分为外径、内径、公称直径。管材为无缝钢管的管子的外径用字母D来表示，其后附加外直径的尺寸和壁厚，例如外径为108的无缝钢管，壁厚为5MM，用D108*5表示。塑料管也用外径表示，如De63。其他如钢筋混凝土管、铸铁管、镀锌钢管等采用DN表示。在设计图纸中一般采用公称直径来表示，公称直径是为了设计制造和维修的方便人为地规定的一种标准，也较公称通径，是管子（或者管件）的规格名称。管子的公称直径和其内径、外径都不相等，例如：公称直径为100MM的无缝钢管有102*5、108*5等好几种，108为管子的外径，5表示管子的壁厚，因此，该钢管的内径为（108-5-5）＝98MM，但是它不完全等于钢管外径减两倍壁厚之差，也可以说，公称直径是接近于内径，但是又不等于内径的一种管子直径的规格名称，在设计图纸中所以要用公称直径，目的是为了根据公称直径可以确定管子、管件、阀门、法兰、垫片等结构尺寸与连接尺寸，公称直径采用符号DN表示，如果在设计图纸中采用外径表示，也应该作出管道规格对照表，表明某种管道的公称直径，壁厚。 (其实在工程领域并没有一个完全的管道公称直径与外径的对照，外径与公称直径的换算基本要靠经验。大致是公称直径大约等于内径（外径减两个皮厚）

管道英制与公制对照表

管道尺寸（英制与公制对照表) 英寸是长度单位。1 英寸= 2.539999918 厘米(公分)。英寸或[吋]是使用于联合王国(UK，即英国(英联邦)的长度单位。美国等国家也使用它。在台湾与香港，“英寸”通常写作“吋”。英寸的常用简写为[in]或["]“吋”是近代新造的字，念作“英寸”，属汉字中一字念两音的字，其他如“浬”念作“海里”等，借用中国传统的长度单位“寸”，并加口旁以示区别。一、尺寸： DN15（4分管）、DN20（6分管）、DN25（1寸管）、DN32（1寸2管）、DN40（1寸半管）、DN50（2寸管）、DN65（2寸半管）、DN80（3寸管）、DN100（4寸管）、DN125（5寸管）、DN150（6寸管）、DN200（8寸管）、DN250（10寸管）等。二、把1英寸分成8等分: 1/8 1/4 3/8 1/2 5/8 3/4 7/8 英寸。相当于通常说的1分管到7分管,更小的尺寸用1/16、1/32、1/64来表示，单位还是英寸。如果分母和分子能够约分（如分子是2、4、8、16、32）就应该约分。英寸的表示是在右上角打上两撇，如1/2" 如DN25（25mm，下同）的水管就是英制1"的水管，也是以前的8分水管。 DN15的水管就是英制1/2"的水管，也是以前的4分水管。如DN20的水管就是英制3/4"的水管，也是以前的6分水管外径与DN，NB的关系如下：公称管子尺寸(NPS)/in 0.25 0.5 0.75 1.0 1.25 1.5 2.0 2.5 3.0 4.0 6.0 公称直径(DN)/mm 6 15 20 25 32 40 50 65 80 100 150 公称管子尺寸(NPS)/in 8.0 10.0 12.0 14.0 16.0 18.0 20.0 24.0 36.0 42.0 48.0 公称直径(DN)/mm 200 250 300 350 400 450 500 600 900 1000 1200 管子规格及有关数据公称直径英寸外径近似内径壁厚相当于无缝管普厚/加厚 mm ＂mm mm mm mm 15 4分21.25 15 2.75/3.25 22 20 6分26.75 20 2.75/3.5 25 25 1寸33.5 25 3.25/4 32 32 1.2寸42.25 32 3.25/4 38 40 1.5寸48 40 3.5/4.25 45 50 2寸60 50 3.5/4.5 57 70 2.5寸75.5 70 3.75/4.5 76 80 3 88.5 80 4/4.75 89 100 4 114 106 4/5.0 108 125 5 140 131 5/5.5 133 150 6 165 156 5/5.5 159 200 8 219 207 6 219 250 10 273 259 7 273 300 12 325 309 8 435 350 14 377 9 485

钢管DN尺寸对照表

钢管DN尺寸换算表：钢管尺寸对应DN 计算值对应管道的通称直径 4分DN15 φ21.3 φ18 6分DN20 φ26.9 φ25 1寸DN25 φ33.7 φ32 1.2寸DN32 φ4 2.4 φ38 1.5寸DN40 φ48.3 φ45 2寸DN50 φ60.3 φ57 2.5寸DN65 φ76.1 φ76 3寸DN80 φ88.9 φ89 4寸DN100 φ114.3 φ108 5寸DN125 φ139.7 φ140 6寸DN150 φ168.3 φ159 8寸DN200 φ219.1 φ219 10寸DN250 φ273 φ273 12寸DN300 φ323.9 φ325 14寸DN350 φ355.6 φ377 16寸DN400 φ406.4 φ426 18寸DN450 φ457 φ480 20寸DN500 φ508 φ530 24寸DN600 φ610 φ630 28寸DN700 φ711 φ720 32寸DN800 φ813 φ820 36寸DN900 φ914 φ920 40寸DN1000 φ1016 φ1020 吋=英寸(inch,缩写为in)1分＝1/8寸＝DN6 2分＝1/4寸＝DN8 3分＝3/8寸＝DN10 4分＝1/2寸＝DN15 6分＝3/4寸＝DN20 8分＝1寸＝DN25 4分管,是以英制尺寸来表示的，也就是4英分管。英制尺寸的表示方法为：1英尺(1’)=12英寸(12’’)；1英寸=8英分。英分用1/8’’，1/4’’，3/8’’，1/2’’，5/8’’，3/4’’，7/8’’表示。1英分=125英丝，1英寸=1000英丝。 (三) 英制尺寸换成“米制”尺寸1英寸=25.4毫米,即是”米制”尺寸。如：5/8’’×25.4=15.875毫米；5/16’’×25.4=7.938毫米。 4分管也是1/2’’×25.4=12.7mm 、 6分管3\4"---管道公称直径近似19mm、 1寸管1"---管道公称直径近似25.4mm。大外径系列，规格，DN-公称直径，Ф－外径， DN10—Ф14mm，DN15—Ф18mm，DN20—Ф25mm，DN25—Ф32mm，DN32—Ф38mm，DN40—Ф45mm，DN50—Ф57mm，DN65—Ф76mm，DN80—Ф89mm，DN100—Ф108mm，DN125—Ф133mm，DN150—Ф159mm，DN175—Ф194mm，DN200—Ф219mm，DN225—Ф245mm，DN250—Ф273mm，DN300—Ф325mm，DN350—Ф377mm，DN400—Ф426mm，DN450—Ф480mm，

水管对照尺寸参数表

称直径外径壁厚、重量 Sch.5S Sch.10S Sch.20S Sch.40S DN NPS in. mm in. mm kg/m in. mm kg/m in. mm kg/m in. mm kg/m 15 1/2 0.840 21.34 0.065 1.65 0.809 0.083 2.11 1.01 20 3/4 1.050 26.67 0.065 1.65 1.03 0.083 2.11 1.29 25 1 1.315 33.40 0.065 1.65 1.30 0.109 2.77 2.11 0.120 3.05 2.31 0.133 3.38 2.53 32 11/4 1.660 42.16 0.065 1.65 1.67 0.109 2.77 2.72 0.120 3.05 2.97 0.140 3.56 3.42 40 11/2 1.900 48.26 0.065 1.65 1.92 0.109 2.77 3.14 0.120 3.05 3.43 0.145 3.68 4.09 50 2 2.375 60.33 0.065 1.65 2.41 0.109 2.77 3.97 0.120 3.05 4.35 0.145 3.91 5.50 65 21/2 2.875 73.03 0.083 2.11 3.73 0.120 3.05 5.32 0.156 3.96 6.81 0.203 5.16 8.72 80 3 3.500 88.90 0.083 2.11 4.56 0.120 3.05 6.52 0.156 3.96 8.38 0.216 5.49 11.4 90 31/2 4.000 101.60 0.083 2.11 5.23 0.120 3.05 7.49 0.156 3.96 9.63 0.226 5.74 13.7 100 4 4.500 114.30 0.083 2.11 5.90 0.120 3.05 8.45 0.203 5.16 14.0 0.237 6.02 16.2 125 5 5.563 141.30 0.109 2.77 9.56 0.134 3.40 11.7 0.203 5.16 17.5 0.258 6.55 22.0 150 6 6.625 168.28 0.109 2.77 11.4 0.134 3.40 14.0 0.216 5.49 22.3 0.280 7.11 28.5 200 8 8.625 219.08 0.109 2.77 14.9 0.148 3.76 20.2 0.237 6.02 32.0 0.322 8.18 42.9 250 10 10.750 273.05 0.134 3.40 22.8 0.165 4.19 28.1 0.237 6.02 40.0 0.365 9.27 60.9 300 12 12.750 323.85 0.156 3.96 31.6 0.180 4.57 36.3 0.237 6.02 47.7 0.375 9.53 74.6 350 14 14.000 355.60 0.156 3.96 34.7 0.188 4.78 41.8 0.258 6.55 57.0 0.437 11.1 95.3

公称管子尺寸与直径对照表

公称管子尺寸与直径数值对照表一般来说，管子的直径可分为外径、内径、公称直径。管材为无缝钢管的管子的外径用字母D来表示，其后附加外直径的尺寸和壁厚，例如外径为108的无缝钢管，壁厚为5MM，用D108*5表示。塑料管也用外径表示，如De63。其他如钢筋混凝土管、铸铁管、镀锌钢管等采用DN表示。在设计图纸中一般采用公称直径来表示，公称直径是为了设计制造和维修的方便人为地规定的一种标准，也较公称通径，是管子（或者管件）的规格名称。管子的公称直径和其内径、外径都不相等，例如：公称直径为100MM的无缝钢管有102*5、108*5等好几种，108为管子的外径，5表示管子的壁厚，因此，该钢管的内径为（108-5-5）＝98MM，但是它不完全等于钢管外径减两倍壁厚之差，也可以说，公称直径是接近于内径，但是又不等于内径的一种管子直径的规格名称，在设计图纸中所以要用公称直径，目的是为了根据公称直径可以确定管子、管件、阀门、法兰、垫片等结构尺寸与连接尺寸，公称直径采用符号DN表示，如果在设计图纸中采用外径表示，也应该作出管道规格对照表，表明某种管道的公称直径，壁厚。 (其实在工程领域并没有一个完全的管道公称直径与外径的对照，外径与公称直径的换算基本要靠经验。大致是公称直径大约等于内径（外径减两个皮厚）但也不完全相等，大致差不多，取整就行。比如φ108*7的外径108，它的公称直径是100；再比如φ32*4.5，外径32，公称直径应该是25。以此类推，就能得到外径与公称直径的对应关系了。) 国家标准架子管每米的重量为0.00384吨，全管的实际重量：0.00384*钢管长度外径与DN，NB的关系如下：管子规格及有关数据

管径规格尺寸对照表

管径规格尺寸对照表管径规格尺寸对照表1 英寸=25.4毫米 =8英分 1/2" 是四分(4英分) DN15 3/4" 是六分(6英分) DN20 2分管 DN8 4分管 DN15 6分管 DN20 1′ DN25 1.2′ DN32 1.5′ DN40 2′ DN50 2.5′ DN65 3′ DN80 4′ DN100 5′ DN125 6′ DN150 8′ DN200 10′ DN250 12′ DN300 GB/T50106-2001 管径----管径应以mm为单位。管径的表达方式应符合下列规定： 1 水煤气输送钢管（镀锌或非镀锌）、铸铁管等管材，管径宜以公称直径DN表示；

2 无缝钢管、焊接钢管（直缝或螺旋缝）、铜管、不锈钢管等管材，管径宜以外径×壁厚表示； 3 钢筋混凝土（或混凝土）管、陶土管、耐酸陶瓷管、缸瓦管等管材，管径宜以内径d表示； 4 塑料管材，管径宜按产品标准的方法表示； 5 当设计均用公称直径DN表示管径时，应有公称直径DN与相应产品规格对照表。建筑排水用硬聚氯乙烯管材规格用de（公称外径）×e（公称壁厚）表示（GB 5836.1-92）给水用聚丙烯（PP）管材规格用de×e表示（公称外径×壁厚）关于DN与De的区别：。 1、DN是指管道的公称直径，注意：这既不是外径也不是内径；应该与管道工程发展初期与英制单位有关；通常用来描述镀锌钢管，它与英制单位的对应关系如下：。 4分管：4/8英寸：DN15；。 6分管：6/8英寸：DN20；。 1寸管：1英寸：DN25；。寸二管：1又1/4英寸：DN32；。寸半管：1又1/2英寸：DN40；。两寸管：2英寸：DN50；。三寸管：3英寸：DN80（很多地方也标为DN75）；。四寸管：4英寸：DN100；。 De主要是指管道外径，一般采用De标注的，均需要标注成外径X壁厚的形式；。主要用于描述：无缝钢管、PVC等塑料管道、和其他需要明确壁厚的管材。拿镀锌焊接钢管为例，用DN、De两种标注方法如下：。 DN20De25X2.5mm。 DN25De32X3mm。 DN32De40X4mm。

钢管尺寸对照表

钢管尺寸对照表 Document number：PBGCG-0857-BTDO-0089-PTT1998

钢管尺寸对照表

钢管尺寸对照表内部编号：（YUUT-TBBY-MMUT-URRUY-UOOY-DBUYI-0128）

管径对照表

多大管径配多大阀门多大管径（外径）与多大阀门（通径）DN尺寸对照表工程管径对照表（常用）： 1 英寸=25.4毫米=8英分 1/2 是四分(4英分) DN15 3/4 是六分(6英分) DN20 2分管 DN8 4分管 DN15 6分管 DN20 1′DN25 1.2′DN32 1.5′ DN40 2′DN50 2.5′DN65 3′DN80 4′ DN100 5′ DN125 6′DN150 8′DN200 10′ DN250 12′DN300 GB/T50106-2001 2.4管径 2.4.1管径应以mm为单位。 2.4.2管径的表达方式应符合下列规定： 1 水煤气输送钢管（镀锌或非镀锌）、铸铁管等管材，管径宜以公称直径DN表示； 2 无缝钢管、焊接钢管（直缝或螺旋缝）、铜管、不锈钢管等管材，管径宜以外径×壁厚表示； 3 钢筋混凝土（或混凝土）管、陶土管、耐酸陶瓷管、缸瓦管等管材，管径宜以内径d 表示； 4 塑料管材，管径宜按产品标准的方法表示； 5 当设计均用公称直径DN表示管径时，应有公称直径DN与相应产品规格对照表。建筑排水用硬聚氯乙烯管材规格用de（公称外径）×e（公称壁厚）表示（GB 5836.1-92）给水用聚丙烯（PP）管材规格用de×e表示（公称外径×壁厚）. 关于DN与De的区别： 1、DN是指管道的公称直径，注意：这既不是外径也不是内径；应该与管道工程发展初期与英制单位有关；通常用来描述镀锌钢管，它与英制单位的对应关系如下： 4分管：4/8英寸：DN15； 6分管：6/8英寸：DN20； 1寸管：1英寸：DN25；寸二管：1又1/4英寸：DN32；寸半管：1又1/2英寸：DN40；

管径大小对应表

DN15,DN20,DN25是外径。四分管和六分管的直径 1 英寸=25.4毫米=8英分 1/2 是四分(4英分) DN15 3/4 是六分(6英分) DN20 2分管DN8 4分管DN15 6分管DN20 1′DN25 1.2′DN32 1.5′DN40 2′DN50 2.5′DN65 3′DN80 4′DN100 5′DN125 6′DN150 8′DN200 10′DN250 12′DN300 GB/T50106-2001 2.4管径 2.4.1管径应以mm为单位。 2.4.2管径的表达方式应符合下列规定： 1 水煤气输送钢管（镀锌或非镀锌）、铸铁管等管材，管径宜以公称直径DN表示； 2 无缝钢管、焊接钢管（直缝或螺旋缝）、铜管、不锈钢管等管材，管径宜以外径×壁厚表示； 3 钢筋混凝土（或混凝土）管、陶土管、耐酸陶瓷管、缸瓦管等管材，管径宜以内径d 表示； 4 塑料管材，管径宜按产品标准的方法表示； 5 当设计均用公称直径DN表示管径时，应有公称直径DN与相应产品规格对照表。建筑排水用硬聚氯乙烯管材规格用de（公称外径）×e（公称壁厚）表示（GB 5836.1-92）给水用聚丙烯（PP）管材规格用de×e表示（公称外径×壁厚）随着人们生活水平、环保意识的提高以及对健康的关注，在给排水领域掀起了一场建材行业的绿色革命。据大量水质监测数据表明：采用冷镀锌钢管后，一般使用寿命不到5年就锈蚀，铁腥味严重。居民纷纷向政府部门投诉，造成一种社会问题。塑料管材与传统金属管道相比，具有自重轻、耐腐蚀、耐压强度高、卫生安全、水流阻力小、节约能源、节省金属、改善生活环境、使用寿命长、安装方便等特点，受到了管道工程界的青睐并占据了相当重要的位置，形成一种势不可当的发展趋势。塑料管特点及应用

钢管公制英制换算表

1、公称直径就是公称口径或叫公称通径或叫公称尺寸，哪里来的区别啊？ 2、直径与口径在管道工程上也没有区别的呀。 3、公称直径就是各种管子与管路附件的通用口径。同一公称直径的管子与管路附件均能相互连接，具有互换性.它不是实际意义上的管道外径或内径，虽然其数值跟管道内径较为接近或相等，是供参考用的一个方便的圆整数，与加工尺寸仅呈不严格的关系。公称直径不是外径，也不是内径，而是近似普通钢管内径的一个名义尺寸。为了使管子、管件连接尺寸统一，每一公称直径，对应一个外径，其内径数值随厚度不同而不同。 3、符号是DN，单位是毫米，但省略不写。也可用英制in表示。 4、通常公称直径是对有缝钢管、铸铁管、混凝土管等管子的标称，无缝钢管不用此表示法（用外径*壁厚）不过就算你用了，大家也能看明白，个人觉得无所谓。管路附件也用公称直径表示，意义同有缝管。 5、钢管安装时无需换算，因为在同一标准里同一公称直径同一压力等级的连接尺寸相同，并且同一材料的外径也相同，实行公称直径的目的就是为了方便连接性，要换算的话岂不是白实行了吗。 6、强调：公称直径即不是管的外径，也不是管的内径，还不是内外径的平均值(设备的公称直径是指外径) 。举实际列子：DN20的焊管外径26.75壁厚2.75 ；DN25的焊管外径33.50壁厚3.25 ；DN32的焊管外径42.25壁厚3.25 ；DN150的焊管外径165壁厚4.5。一、1吋=25.4 二、吋是公称直径的英制叫法换算成毫米也只能是公称直径的米制叫法，不能对应外径，因为同一公称直径的管子还有大小口径之分三、在管道的规格上没有绝对的换算公式，对于小于100只能以1吋=25 来模糊计算，取其相近的数，对于大于100的管子用1吋=25可以完全匹配计算。常用的如下：公称直径DN6 对应的俗称1分公称直径DN8 对应的俗称2分公称直径DN10 对应的俗称3分公称直径DN15 对应的俗称4分公称直径DN20 对应的俗称6分公称直径DN25 对应的俗称1吋公称直径DN32 对应的俗称1吋2分公称直径DN40 对应的俗称1吋半公称直径DN50 对应的俗称2吋公称直径DN65 对应的俗称2吋半公称直径DN80 对应的俗称3吋公称直径DN100 对应的俗称4吋公称直径DN125 对应的俗称5吋公称直径DN150 对应的俗称6吋公称直径DN200 对应的俗称8吋公称直径DN250 对应的俗称10吋公称直径DN300 对应的俗称12吋公称直径DN350 对应的俗称14吋

管道直径尺寸规格

。管道直径尺寸规格五金手册中可以查一般来说，管子的直径可分为外径、内径、公称直径。管材为无缝钢管的管子的外径用字母D来表示，其后附加外直径的尺寸和壁厚，例如外径为108的无缝钢管，壁厚为5MM，用D108*5表示，塑料管也用外径表示，如De63,其他如钢筋混凝土管、铸铁管、镀锌钢管等采用DN 表示，在设计图纸中一般采用公称直径来表示，公称直径是为了设计制造和维修的方便人为地规定的一种标准，也较公称通径，是管子（或者管件）的规格名称。管子的公称直径和其内径、外径都不相等，例如：公称直径为100MM的无缝钢管邮102*5、108*5等好几种，108为管子的外径，5表示管子的壁厚，因此，该钢管的内径为（108*5-5）＝98MM，但是它不完全等于钢管外径减两倍壁厚之差，也可以说，公称直径是接近于内径，但是又不等于内径的一种管子直径的规格名称，在设计图纸中所以要用公称直径，目的是为了根据公称直径可以确定管子、管件、阀门、法兰、垫片等结构尺寸与连接尺寸，公称直径采用符号DN表示，如果在设计图纸中采用外径表示，也应该作出管道规格对照表，表明某种管道的公称直径，壁厚。 . 管子系列标准压力管道设计及施工，首先考虑压力管道及其元件标准系列的选用。世界各国应用的标准体系虽然多，大体可分成两大类。压力管道标准见表3。法兰标准见表4。表3 压力管道标准分类大外径系列小外径系列规格 DN-公称直径 Ф－外径 DN15-ф22mm，DN20-ф27mm DN25-ф34mm，DN32-ф42mm DN40-ф48mm，DN50-ф60mm DN65-ф76(73)mm，DN80-ф89mm DN100-ф114mm，DN125-ф140mm DN150-ф168mm，DN200-ф219mm

英制钢管尺寸对照表

外径壁厚重量英寸毫米.单位毫米.英寸.公斤/米.磅/英尺. 1/8”10.310S 1.240.0490.280.19 1/8”10.3STD-40 1.730.0680.370.25 1/8”10.3XS-80 2.410.0950.480.32 1/4”13.710S 1.650.0650.500.34 1/4”13.7STD-40 2.240.0880.640.43 1/4”13.7XS-80 3.020.1190.810.55 3/8”17.110S 1.650.0650.640.43 3/8”17.1STD-40 2.310.0910.850.57 3/8”17.1XS-80 3.200.126 1.110.75 1/2”21.35S 1.050.0420.530.36 1/2”21.310S 2.110.083 1.010.68 1/2”21.3STD-40 2.770.109 1.280.86 1/2”21.3XS-80 3.730.147 1.63 1.10 1/2”21.3160 4.780.188 1.97 1.33 1/2”21.3XXS7.470.294 2.57 1.73 3/4”26.75S 1.650.065 1.030.69 3/4”26.710S 2.110.083 1.290.87 3/4”26.7STD-40 2.870.113 1.70 1.14 3/4”26.7XS-80 3.910.154 2.22 1.49 3/4”26.7160 5.560.219 2.93 1.97 3/4”26.7XXS7.820.308 3.68 2.48 1”33.45S 1.650.065 1.310.88 1”33.410S 2.770.109 2.12 1.42 1”33.4STD-40 3.380.133 2.53 1.70 1”33.4XS-80 4.550.179 3.27 2.18 1”33.4160 6.350.250 4.28 2.88 1”33.4XXS9.090.358 5.51 3.71 1 1/4”42.25S 1.65 0.065 1.67 1.12 1 1/4”42.210S 2.770.109 2.72 1.83 1 1/4”42.2STD-40 3.560.140 3.43 2.31 1 1/4”42.2XS-80 4.850.191 4.51 3.03 1 1/4”42.2160 6.350.250 5.67 3.81