Proc. Natl. Acad. Sci. USAVol. 89, pp. 10252-10256, November 1992Microbiology

A periplasmic protein disulfide oxidoreductase is required fortransformation of Haemophilus influenzae RdJEAN-FRANCOIS TOMB*Johns Hopkins University School of Medicine, Department of Molecular Biology and Genetics, Baltimore, MD 21205

Communicated by Hamilton 0. Smith, June 30, 1992

ABSTRACT The mutated gene in JG16, a Haemophilusinfluenzae strain deficient in competence-induced DNA bindingand uptake, was cloned and the wild-type allele was sequenced.The gene was shown by Northern analysis to be constitutivelyexpressed on a 1.7-kilobase transcript. The gene product wasidentified as a 20.6-kDa protein targeted to the periplasm. Theprotein contains the sequence Cys-Pro-His-Cys (CPHC) and ishighly similar to two other periplasmic CPHC motif-containingproteins: DsbA, anEscherichia col protein (45% identity, 87%homology) and TcpG, a Vibrio cholrae protein (32% identity,74% homology). Both DsbA and TcpG promote disulfide bondformation in periplasmic proteins, are required for pilusbiogenesis, and, like thioredoxin, are capable of reducinginsulin in vitro. The Haemophilus protein was shown to com-plement an E. coli mutation in DsbA and was named Por(periplasmic oxidoreductase). In JG16 the competence-dependent redistribution of inner membrane proteins did notoccur. These rmdings suggest that Por is required for thecorrect assembly and/or folding of one or more disulfide-containing cell envelope proteins involved either in competencedevelopment or in the DNA-binding and -uptake machinery.

In Haemophilus influenzae Rd, development of competencefor genetic transformation is an inducible phenomenon (1, 2).Transformation involves sequence-specific binding of donorDNA (3-5), its uptake into a DNase-resistant membranecompartment (transformasome) (6), translocation into thecytoplasm, and integration into the cell chromosome (7). Thetransformasome was tentatively identified by electron mi-croscopy as a vesicular surface structure appearing on com-petent cells (8).To elucidate the protein components involved in the as-

sembly and structure of the transformasome, a number oftransformation-deficient (Tfo-) mutants produced by mini-TnlOkan insertional mutagenesis have been isolated and thecorresponding genes cloned (9). One mutant strain, JG16,that is deficient in competence-induced binding and uptakeofDNA is analyzed in this paper. The gene corresponding tothe mutation in JG16 was cloned on an 8.7-kilobase (kb)Pst I DNA fragment, identified by additional mutagenesis,sequenced,t and shown to be constitutively expressed. Thegene encodes a 20.6-kDa periplasmic protein which is ho-mologous to DsbA (also called PpfA) (10, 11) and TcpG (12),two periplasmic protein disulfide oxidoreductases found inEscherichia coli and Vibrio cholerae. Based on this homologyand its ability to transcomplement aDsbA mutation in E. coli,it is concluded that this Haemophilus protein, named Por, isa member of a family of periplasmic protein disulfide oxi-doreductases. The role of Por in transformation is explored.

MATERIALS AND METHODSBacteria Strains and Plasmids. H. influenzae strains KW20,

MAP7 (Strr), and JG16 (KW20::mini-Tnl0kan Kanr Tfo-)

have been described (9). JFH400 and JFH403 are KW20strains containing a 1.3-kb kan insertion at the Dra I site ofopen reading frame 1 (ORF1) (JFH400) and at the Mlu I siteofORF3, respectively (Figs. 1 and 2). The E. coli strains usedwere JCB474 [F'(traD30 proAB 1aclq lacZAM15)], JCB477(JCB474 dsbAl zih-12::TnlO) (10), CC118 (AlacX74phoAA2OrecAl), CC202 (CC118/F42 lacI3 zzf-2::TnphoA) (13, 14),DH5a [hsdRl7 recAl 480dA(lacZ)MJS] (BRL), BL21-(DE3pcn) (hsdS pcnB zad::TnlO) (15), and MC1060 [A(lacI-lacY) relAl rpsL150 spoTi hsdRl] (16).

Plasmids used in this work and previously described werepEUPi {pBR322 [EcoRI::15-mer containing an 11-base-pair(bp) uptake signal sequence (USS)]} (17); pBluescript M13+(Stratagene), named here pBS1 for convenience; pSU2718and pSU2719, pl5A-derived plasmids, capable of replicationin Haemophilus (18); pUC19 and M13mpl8 (19); andpUCKSAC (20). Plasmids constructed during the course ofthis work were pBBO [pUC19 (Sac I::L-mer Bgi II linker)];pBB1 [pBBO (Sma I::237-bp Ssp 1-HindIII from pEUPI)];pBB36 [pBBO (Bgl II-BamHI::36 repeats of 256-bp Bgl II-BamHI from pBB1)]; pJF450 [pBS1 (Pst I::8.7-kb Pst I flae-mophilus DNA)]; pJF500 [pBS1 (Xba I-Pst I::2-kb Xba I-PstI from pJF450)]; pJF501 (pJF500::mini-TnlOkan insertion no.16); pJF510, pJF511, and pJFS12 (pJF500::TnphoA at posi-tions I, II, and III, respectively; see Figs. 1 and 2); pJF539[pSU2718 (Xba I-Pst I::2-kb Xba I-Pst I from pJF500)];pJF540 {pJF539 [Dra I(372)::8-mer BamHI linker]}; pJF542{JpJF539 (Dra I(852)::8-mer BamHI linker]}; and pJF601[pSU2719 (BamHI: :BamHI fragment amplified by PCR usingprimers G16L and G16R); see Fig. 2].Growth and Competence Development. E. coli and H.

influenzae cells were grown aerobically at 37°C (21, 22). Theefficiency of transformation of H. influenzae strains madecompetent in MIV medium (22) was determined as described(23). Competence mutants were transformed with plasmidDNA by electroporation (24). Concentrations of antibioticswere as described (22).

Insertional Mutagenesis. Mini-TnlOkan mutagenesis ofpJF450 was done in MC1060 according to Way et al. (25).TnphoA mutagenesis of pJF450 was done in CC202 (26). Thepositions of mini-TnlOkan and TnphoA insertions in thetarget DNA were determined by restriction analysis. Eachinsertion was crossed back into wild-type H. influenzae andits effect on transformation was determined (9, 27).DNA Sequencing. The 2-kb Xba I-Pst I fragment containing

the por gene was subcloned between the Xba I and Pst I sitesof M13mpl8 and M13mpl9 and sequenced by the dideoxymethod (r7Sequencing kit; Pharmacia). The positions of theTnphoA insertions in pJF510, pJF511, and pJF512 were de-termined using the primer 5'-GCCGGGTGCAGTAATATCG-

Abbreviations: ORF, open reading frame; Tfo-, transformation-deficient.*To whom reprint requests should be addressed at: Johns HopkinsSchool of Medicine, Department of Molecular Biology, 725 NorthWolfe Street, PCTB 505, Baltimore, MD 21205.tThe sequence reported in this paper has been deposited in theGenBank data base (accession no. M94205).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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tIRV? ?WH

I

-Vi-IV?T Vi hI IIN N 416

a*-- 2.0kb -~--

FIG. 1. Map of the 8.7-kb insert in pJF45O showing restriction sites, positions of mini-TnlOkan and TnphoA insertions, and ORFs found inthe sequenced 2.0-kb Xba I-Pst I fragment. Black lollipops indicate Tfo- mini-TnlOkan insertions. The inverted lollipop (16) is the originalinsertion present in JG16. The other loilipops indicate Tfo+ mini-TnlOkan insertions. Shaded triangles indicate TnphoA fusions producing activealkaline phosphatase. Those labeled I, II, and III are in-frame fusions to the transformation gene por (also called OR.F2 in the text). Unshadedtriangles represent TnphoA insertions producing no alkaline phosphatase activity.

3', which is complementary to the 5' end of the codingsequence of the alkaline phosphatase (phoA) gene. The posi-tion of the original mini-TnlOkan insertion was determined onthe plasmid pJF501 by using the primer 5'-CCACCTTAACT-TAATGATTTTTACC-3', complementary to the ends of themini-TnlOkan (23).Bam1H Linker Insertions, Kanamycin Camette Mutagene-

sis, and PCR Amplification. BamHI linker insertion at Dra Isites of pJF500 was done using dephosphorylated 5'-CGGATCCG-3'. A 1.3-kb Pst I fragment containing theTnO3 Kanr gene was isolated from pUCKSAC and used incassette mutagenesis (20). PCR ampliffication was done ac-cording to the standard protocol recommended by Perkin-Elmer/Cetus. A fragment containing a truncated ORMi andintact ORF2 (Por) was amplified by using primers G16L andG16R (Fig. 2). To facilitate cloning, a BamHI restriction sitewas added to the 5' end of both primers.

Preparation of Pepl6B Antibodies. Pepl6B (amino acidsequence VNPEGLNYDDFVK), corresponding to a regionin ORF2 (Fig. 2) of high antigenicity index (28), was synthe-

sized and conjugated to the carrier protein hemocyanin (29).Antibodies were raised in rabbits (Hazleton Research Prod-ucts, Vienna, VA) (29).RNA Preparation and Northern Analysis. RNA was isolated

by hot-phenol extraction (30). For Northern analysis, 20 ,&gof total RNA was electrophoresed in a 1.5% formalde-hyde/1% agarose gel and transferred to nylon membrane(Schleicher & Schuell). The probe was prepared by PCRamplification of an internal por gene fragment with theprimers G16C and G16R (Fig. 2) in the presence of[a-32P]dCT'P and [a-32P]dATP (50,gCi each; 1IpCi = 37 kBq).Membrane and Periplasmic Protein Preparation. Prepara-

tion of total membrane proteins and separation of inner andouter membrane fractions by sucrose density gradients weredone as described (31). Fractions ofinner or outer membranewere collected using an ISCO fractionator model 640, recon-centrated by centrifugation, and resuspended in 0.1% theoriginal volume of cells. Succinic dehydrogenase activity ofmembrane fr-actions was used to measure inner membranecontent (31). Periplasmic proteins were prepared by osmotic

Xbal

G16L ----

0111 -4 N K C K R L N Z V L Z L L Q S Y N S K D S D L S L N

GAATTCAAATCATATAGTCAAACTAAGRTATAGATATATACGTAAGAGTCGTATTACATCTGCCAAAATCAK I L IQ K I A N I S G F Q K P L N I L 1' D I V I I Y Q L K X D G T D K Y I P I P G L K K D Y I

Dral G16C -F 1------4 4,I D F K T A L L R A R G I I K-o 4NKKVLL 'LN S VNSFAADLQIVI ~~(0112)

G K Q Y V QV S Q Q AS

Q Q K I V I1FFSFP8 Y C P H C Y AFIZNI YKX I P Q Q V VD A L P K

Dral A ID V K K Q YNV NF L G Q SIN L T RA WA L A NA L G Al S K VK S P L AA Q D A

TTAATCATGAGAATCGGCATTMTAC AATGAACGCAGATIAGIGCTI - TTCAT~LGTTGT~LAAAGGJAGACGAA~MAkqa~L KS N D D I RA IFP L8SN G I T AB Q FD G GI NS0 FA VNG L V NK Q VNA A Q K V R

G V P D F Y V N G R V N P 0 L N Y D D F V D Y V Q T V G L L Q K * G1SR ----------

Pepl6BTTTTGTAAATATTAATGAAAGAA"G&"ATATTAWGCTGCAAGINPMAGCGTAACGCGMGTMTTTQACGACAAAAACTACCCA~~~~~~~~~~~~~~~~~~~~~~~~cavaa*4'***k'-e'-In -IL IL- . -

0113 N A A 9 V T R D D N Y P G R G D T I Q V L

Q Y G QAF KXA L D L G Z RI P AT KINKZ D F VAFP C R G I R A AI TF F BKZ T N N K TART

R I N T K K R VY T LBS D V S IAA S G GBZ D Y P ARK I R

1548

1689

0114 -4 N Q L P I S Q Y N I L L Q K K L I K

L T A LL H P F NA P D I Q V F D SP! 8 Y AK R A IFP RI WHI Q D D F YI INK F D Q1966

FIG. 2. Sequence of the 2.0-kb Xba I-Pst I por gene fr-agment. Putative ribosome binding sites and ATG codons are in boldface andunderlined. Dashed arrows indicate primers used in this work. The down arrow at position 688 indicates the leader peptidase cleavage site, andthe preceding underlined sequence is the leader peptide. The doubly underlined sequence is the synthetic peptide Pepl6B used to generateantibodies against Por. Black triangles indicate the positions of the active alklmine phosphatase fusions I, II, and III. The inverted lollipop,between bases 737 and 738, indicates the position of mini-TnlOkan insertion 16.

H4Jto

140

280

421

562

701

842

983

1124

1265

.. . . . . . .. .. -- i0

GATGTGAAATMAACAiLTATCATGTAAATTTCTTAGGTCATCAATCTGAAAACTTAACACGTGCTTGGGCGTTAGCAATGGCATTAGGTGCAGAAAGTAAAGTAAAATCACCATTATTTGAAGCGGCTCAA6AAGGATGCC

iT

LASAX-r-r47KAI'U-rAAATWUAAAATTUCUTGTAAACCCTOAAGGGTTAAATTATQATQATTWGTOAAAGATTATGTIGCAAACCGTAAAAGGTTTATTGCAAAAATAACQAAAAATTGGTTTJLATGCCAGCCCTA

A1407

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Table 1. Transformation phenotype and complementation of thetransformation deficiency of JG16 with plasmids containing por

RelativeStrain* transformations

KW20 (wild type) 1JG16 1.1 x 10-5JFH400 (ORFl-) 0.66JFH403 (ORF3-) 0.1JG16/pSU2718 1.6 x 10-5JG16/pJF539(pSU2718::2-kb Xba I-Pst I) 0.15JG16/pJF540(ORF1::BamHI linker) 0.13JG16/pJF542(por::BamHI linker) 1.4 x 10-5JG16/pJF601('0RF1 + por) 0.13*The genotype ofeach strain is described in Materials and Methods.tTransformation frequency (TF) is the ratio of Ste' colonies to totalcolony-forming units. Relative transformation is (TF of a strain)/(TF of KW20). TF of KW20 was 0.35%.

shock (32). Freshly prepared phenylmethanesulfonyl fluoridewas added (0.1 mM) to all fractions.Western and Southwestern Analysis. Western immunoblot

analysis was done according to the ECL protocol (Amer-sham) on 10 ,ug ofmembrane proteins after SDS/15% PAGE.Probing of Western blots with DNA ("Southwestern" anal-ysis) was done as described (33). Filters were probed with 0.5Ag of 32P-labeled (5 x 106 cpm) double-stranded or single-stranded, heat denatured, pBB36 DNA.

RESULTSMapping the Tfo Gene in JG16 by Insertional Mutageness.

The mutated gene in JG16 was previously cloned and thewild-type allele was recovered on an 8.7-kb DNA fragmentby in vivo recombination (23). To map Tfo- loci on thefragment, mini-TnlOkan and TnphoA insertional mutagenesiswas performed. Ofthe 80 mini-TnlOkan insertions examined,20 were unique and were mapped by restriction analysis (Fig.1). Only 1 produced a Tfo- phenotype, and it mapped to thesame position as the original mini-TnlOkan insertion (Figs. 1and 2). Of the 32 independent TnphoA insertions examined,half of which were chosen as active alkaline phophatasefusions (blue colonies), 12 were unique. Three of thesefusions (shaded triangles in Fig. 1) were near the Tfo-mini-TnlOkan insertions, suggesting that the product of thetransformation gene is targeted to the cell envelope. Attemptsto cross these TnphoA insertions, as well as other, non-activeinsertions (from white colonies), back into H. influenzaewere unsuccessful.

Sequencing and Identification of the ITo Gene. Since all ofthe Tfo- insertions were within the Xba I-Pst I region ofpJF540, this fragment was subcloned into pSU2718 (18). Thenew plasmid, pJF539, complemented the Tfo- defect in JG16(Table 1). To identify the gene(s) coding for the complement-ing activity, the 2-kb fragment and thejunction regions of themini-TnlOkan insertion and the three active TnphoA inser-

tions were sequenced. This analysis identified three tandemORFs (Figs. 1 and 2) plus part of a fourth ORF (Fig. 2). Thethree active alkaline phosphatase fusions were in frame withORF2 (por), into which the Tfo- mini-TnlOkan had inserted.To determine whether ORF1 and ORF3 had functions relatedto transformation, a Kanr cassette was inserted at the Dra Isite within ORF1 and at the Mlu I site within ORF3. Theinsertions were crossed back into the chromosome of Hae-mophilus and were found to have only a minimal effect ontransformation efficiency (Table 1). To ascertain the involve-ment ofORF2 in transformation, aBamHI linker (8-mer) wasintroduced into the Dra I site at sequence position 852 (Fig.2). The complementation activity of ORF2 was abolished bythis insertion (Table 1). In addition, a region containing ORF2was amplified by PCR using the primers G16L and G16R andthen subcloned in pSU2719. The resulting plasmid, pJF601,restored transforming ability to JG16 (Table 1).Comparison of the predicted amino acid sequences of

ORFs 1, 3, and 4 with the translated GenBank data base(Release 62) revealed that ORF3 is 58% identical with anuntranslated ORF in the intergenic region between ORFI andORFII of the ilvG-rnnC loci of E. coli (34), and the 62 aminoacids of the partial ORF4 are 54.8% identical with trnA, thetRNA (m5U54) methyltransferase gene of E. coli (35).For ORF2, sequence analysis predicted two possible ATG

start codons, one at base 528 and the other at base 621 (Fig.2). Position 621 was determined to be correct for two reasons.First, it was followed by a typical signal sequence fortargeting to the cell membrane, in agreement with the alkalinephosphatase fusion result. Second, the predicted sizes of theprecursor protein and the mature processed protein were 22.9kDa and 20.6 kDa, in agreement with results in the nextsection. Comparison with GenBank sequences revealed thatthe ORF2 protein was 45% identical and 87% homologous tothe E. coli protein DsbA, and 32% identical and 74% homol-ogous to TcpG, a homolog of DsbA from V. cholerae (12)(Fig. 3). To test whether the ORF2 protein could functionallysubstitute for DsbA, E. coli JCB477, which is deficient inDsbA production and consequently does not plaque M13(10), was transformed with pJF539, pJF542, pJF601, andpSU2718. Ability to plaque M13 was restored when a plasmidcarrying an intact ORF2 was introduced into JCB477 (Table2). Based on these findings, the ORF2 protein was namedPor, for periplasmic oxidoreductase.

Characterization and Ceflular Lcalization of Por. In vivolabeling of plasmid-encoded proteins with the T7 RNA poly-merase expression system (15) showed that pJF500 encodedtwo polypeptides of apparent molecular mass 21.7 kDa and20.5 kDa (Fig. 4A), in agreement with the predicted sizes ofthe precursor and mature forms obtained by sequence anal-ysis. Plasmids pJF510 and pJF512, each carrying an activephoA fusion within por (I and III of Fig. 2), did not encodethe 20.5- and 21.7-kDa polypeptides, but instead encoded 49-and 55-kDa polypeptides, respectively. Western blots (Fig.4B) probed with a polyclonal serum against Pep16B showed

110I V

Por MKKVLLALGL GVSTLMSVNS FAADLQEGKQ YVQVSQQASQ QKEVIEFFSF YCPHCYAFEM EYKIPQQVVD ALPKDVKFKQ YHVNFLGHQ SENLTRAWAL AMALGAESKV

DsbA MKXIWLALA GLVLAFSA SAAQYEDGKQ YTTLEKPVAG APQVLEFFSF FCPHCYQFEE VLHISDNVKK KLPEGVKMTK YHVNFMGGDL GKDLTQAWAV AMALGVEDKV

TcpG MKKLF AL VATLMLSVSA YAAQFKEGEH YQVLKTPASS SPVVSEFFSF YCPHCNTFE PIIAQLKQ QLPEGAKFQK NHVSFMGGNM GQAMSKAYAT MIALEVEDKM*** ~~** * * * ***** **** ** * ** ** * * * * ** * *

111

Por KSPLFEAAQKDsbA TVPLFEGVQK

TcpG VPVMFNRIHT

160 212

DALKSMDD IRAIFLSNGI TAEQFDGGIN SFAVNGLVNK QVNAAEQFKV RGVPDFYVNG KFRVNPEGL NYDDFVKD YVQTVKGLLQ K.

TQTIRSASD IRDVFINAGI KGEEYDAAWN SFVVKSLVAQ QEKAAADVQL RGVPAMFVNG KYQLNPQGMD TSNMDVFVQQ YADTVKYLSE KK

LRKPPKDEQE LRQIFLDEGI DAAKFDAAYN GFAVDSMVRR FDKQFQDSGL TGVPAVVVNN RYLVQGQSVK S LDE YFDLVNYLLT LK* * ** * * * * * *** ** * * *

FIG. 3. Sequence alignment of Por, DsbA, and TcpG. The CXXC motif of thioredoxin and protein disulfide isomerase is underlined. Starsindicate identical amino acids in the three proteins. Arrowhead points to the leader peptidase cleavage site. Overall identity among the threeproteins is 24%. Alignment was done using the program PILEUP (University of Wisconsin Genetics Computer Group).

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Table 2. Complementation of the dsbAl mutation in E. coliwith por

M13mpl8 plaqueStrain* formation

JCB474 +JCB477JCB477/pSU2718 -JCB477/pJF601 +JCB477/pJF539 +JCB477/pJF542 -

*See Materials and Methods for description.

that the antibodies recognized a 22-kDa periplasmic proteinthat was present in the wild-type strain of Haemophilus,absent from the mutant JG16, and present in higher amountsin JFH539 (JG16 containing the cloned 2-kb Xba I-Pst Ifragment). The protein was undetectable in osmoticallyshocked cells. These results show that the only detectableprotein product from por starts at the ATG codon at 621 andconfirm that the product ofpor is targeted to the periplasm ofboth E. coli and H. influenzae.

Alterations in Membrane Protein Composition Associatedwith Competence Development. Proteins from total mem-branes and from outer and inner membranes of competentand noncompetent cells were compared on Coomassie-stained gels (Fig. SA). Three polypeptides of 120, 78, and 31kDa that were barely detectable, if at all, in total membranesfrom logarithmic-phase wild-type KW20 cells were visible intotal membrane proteins from competent cells (arrows in Fig.SA). The two larger species partitioned to the inner mem-brane fraction, whereas the 31-kDa polypeptide partitionedto the outer membrane fraction. In addition to these newspecies, a striking alteration was seen in the outer membraneprotein composition as a result of competence development.At least 11 new polypeptide bands (dots in Fig. SA) appeared,but these were apparently derived from inner membranepolypeptides, since the staining intensities of correspondingbands in the inner membrane samples were diminished. InJG16, the three new polypeptides appeared with competencedevelopment, but the competence-related change in the out-er-membrane protein profile was not observed.

Southwestern analysis showed that several of the mem-brane proteins had affinity for double-stranded DNA, whileonly one major signal was seen, at 32 kDa, with single-stranded DNA (Fig. 5 B and C). The pattern with double-

KW2 0A NC c

68-

41*

FIG. 4. SDS/15% PAGE and Western blot analysis of plasmid-encoded proteins from osmotically shocked cells and periplasmic,"6shockates." (A) In ViVo [35S]methionine-labeled proteins fromBL21(DE3pcn) cells containing plasmid pBS1 Qlane 1), pJF500 (lane2), pJF510 (lane 3), and pJF512 (lane 4). (B) Western blot analysis,using antibodies against Pep16B, of proteins (20 p~g per lane) fromosmotically shocked cells (lanes 1-3) and from periplasmic prepa-rations (lanes 4-6). Proteins were from KW20 Qlanes 1 and 4),JG16/pSU2718 (lanes 2 and 5), and JG16/pJF500 (lanes 3 and 6).Molecular size standards were from BRL.

stranded and single-stranded DNA confirmed that the pro-teins involved were derived from the inner membrane frac-tion. Since the blot was probed under relatively low-stringency conditions (100 mM NaCl) and in the absence ofcompeting nonspecific DNA or heparin, it was concludedthat most of the observed signals were nonspecific.

Transcription ofper. Fig. 6 shows that por is constitutivelyexpressed on a 1.7-kb transcript. Transcription is transientlydecreased during competence induction and is undetectableafter 150 min in the MIV competence-inducing medium.

DISCUSSION

The absence of a 20.6-kDa periplasmic protein was deter-mined to be the cause of the mutant phenotype in JG16, a

B I;

4 3 e ~ ~ a

4 ,W -294= N-

i4

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p

*

FIG. 5. SDS/15% PAGE and Southwestern analysis of membrane proteins from KW20 and JG16. (A) Coomassie-stained total membrane(T), inner membrane (I), and outer membrane (0) proteins (10 pg per lane) from noncompetent logarithmic-phase cells (NC) and MIV-competentcells (C). Arrowheads point to three competence-specific proteins produced in both KW20 and JG16 (120, 78, and 31 kDa). (B and C)Autoradiograms of gels similar to A after electroblotting to a nylon membrane and probing with double-stranded (B) heat-denatured or (C)32P-labeled pBB36 DNA. The pBB36 DNA contains 36 repeats of the uptake signal sequence (USS) (see ref. 36).

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7%;7

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FIG. 6. Northern analysis ofthepor transcript. RNA (20 1Lg) fromlog-phase cells (log) and at various intervals in MIV competence-inducing medium was electrophoresed in a 1.5% formaldehyde/1%agarose gel. Transcript sizes were determined by comparison withRNA standards (BRL).

Haemophilus strain deficient in competence-induced DNAbinding and uptake. The transformation gene was shown tobe constitutively expressed on a 1.7-kb transcript, suggestingthat it is expressed as part of an operon. The observedtransient decrease in transcription of this gene during com-petence development and the absence of any expression at150 and 200 min in MIV medium are not unique to thistranscript. These changes have been observed for otherconstitutively expressed genes not related to transformation(unpublished data). The transient decrease in transcriptioncoincides with the increased transcription of competence-induced genes (unpublished results and ref. 37) and might bedue to the appearance of a competence-specific o factor thatsequesters the RNA polymerase complex. Depletion ofRNAprecursors in the growth-limiting MIV medium is probablythe cause of the observed late shutdown in transcription.

Por is homologous to DsbA and TcpG, two periplasmicproteins required for the formation of disulfide bonds andessential for the biogenesis of pili. Por also restores M13sensitivity to an E. coli strain deficient in the production ofDsbA. Based on these findings, it was concluded that Por isa functional homolog of DsbA. Since mutations in dsbA andtcpG seem to only affect disulfide-forming proteins targetedto the periplasm, a mutation in por should have little effect onthe protein composition of the cell envelope ofHaemophilus.This was confirmed by the observed identity of proteinprofiles of total membranes (Fig. 5A) and of periplasmicpreparations from JG16 and KW20 (data not shown). More-over, the three newly synthesized competence-specific pro-teins seen in the inner and outer membrane samples of KW20were also present in the por mutant, suggesting that Pordeficiency has no effect on regulatory steps in competencedevelopment. However, a major difference was observedwhen inner and outer membranes from Por+ and Por- strainswere compared. In wild type, at least 11 proteins associatedwith inner membrane fractions cofractionated with outermembrane vesicles in response to competence development,but this phenomenon was not observed in the Por- mutant,nor has it been previously reported by other workers in thefield. No outer membrane protein was observed to shift to theinner membrane fractions, and the percentage of succinicdehydrogenase (a marker for the inner membrane compart-ment) that partitioned with the outer membrane remainedunchanged during competence development. Thus, this re-distribution of specific inner membrane proteins (SIMPs)requires Por, presumably in its capacity as a periplasmicprotein disulfide oxidoreductase.

It is possible that some or all of these proteins are part ofthe transformasome structure. One of them, a 32-kDa poly-peptide which binds to double-stranded DNA and has thehighest affinity for single-stranded DNA, might possibly playa role in the competence-specific DNA binding (38, 39). If thisis true and if other SIMPs are also part of the binding anduptake machinery, the formation of the transformasomerequires the recruitment and assembly of constitutively ex-pressed inner membrane proteins. However, it is also plau-sible that these SIMPs constitute a signal transduction com-

plex that mediates the induction ofcompetence development.Whatever their role is, elucidation of the mechanism bywhich these SIMPs redistribute will undoubtedly contributeto our understanding of membrane morphogenesis.

I am grateful to Hamilton Smith, in whose laboratory the work wasdone, for critical review of the manuscript. I thank Jon Beckwith,Cohn Manoil, and James Bardwell for sending strains; Ron Taylorforsending a preprint of his manuscript and for communicating unpub-lished results; and Mark Chandler and Bill Bishai for their review ofthe manuscript. Special thanks go to Gerry Barcak for his help in theinitial phase of the work. This work was supported by Grants5-PO1-CA16519 and 1-RO1-AI27783 from the National Institutes ofHealth and by Grant MV-517 from the American Cancer Society.

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