Biogenesis of E. coli Pap pili: PapH, a minor pilin subunit involved in cell anchoring and length modulation

Cell, Vol. 49, 241-251,

April 24, 1987, Copyright

0 1987 by Cell Press

Biogenesis of E. coli Pap Pili: PapH, a Minor Pilin Subunit Involved in Cell Anchoring and Length Modulation Monica Bgiga, Mari Notgren, Department of Microbiology University of Umei S-901 87 UmeH, Sweden

and Staffan

Normark

Summary The biogenesis of Escherichia co5 Pap pili, encoded by the pap gene cluster, was studied. A novel gene, pap/+, was identified and found to encode a weakly expressed pilin-like protein. PapH was dispensable for digalactoside-specific binding and for formation of Pap pili. However, in papH deletion mutants 50%-70% of total pilus antigen was found free of the cells. We present evidence showing coregulation of papff and the adjacent gene, papA, which encodes the major pilin subunit. A decrease in the PapA to PapH ratio resulted in a large fraction of cells producing shortened pili, whereas overproduction of PapA relative to PapH resulted in cells with lengthened pili. The data show that PapH has roles in anchoring the pilus to the cell and in modulating pilus length. Introduction Filamentous protein polymers are formed in both eukaryotic and prokaryotic cells. The coordinated polymerization and depolymerization of actin fibers are required for movement and phagocytosis by eukaryotic cells (Pollard and Cooper, 1988). Pili and flagella are classes of bacterial surface polymers involved in attachment (Korhonen et al., 1980a) and cell motility (reviewed by Doetsch and Sjoblad, 1980), respectively. Both prokaryotic organelle types are easily amenable to structural and functional analyses, and are therefore attractive models to study the formation of linear protein polymers and to investigate how the lengths of such fibers are regulated. Pili are 4-10 nm in width and roughly 1 urn in length, and are found on many gram-negative bacteria (Duguid and Old, 1980). A number of pili are known to mediate the specific binding of bacteria to host-ceil glycoconjugate receptors and are therefore important for bacterial attachment to epithelial tissue (Leffler and Svanborg-Eden, 1986). Pili organelles also play a fundamental role in bacterial conjugation and act as receptors for several phages (Manning and Achtman, 1979; Bradley, 1974). All pili gene clusters of Escherichia coli characterized to date contain a number of genes in addition to those coding for the major pilin subunit (for review, see Normark et al., 1986). Each of these gene clusters expresses a high molecular weight protein that is thought to act as a channel and polymerization center for the pilin subunits, and a periplasmic protein that appears to be involved in the transport of the processed pilin subunit (Norgren et al., 1984; Mooi and de Graaf, 1985; Norgren et al., unpublished data). Besides coding for these two gene products,

which have central roles in pilin export and pilus formation, each pilus gene cluster contains additional genes, some of which have been sequenced and found to code for proteins that bear a striking homology to pilins (Mooi et al., 1984; Lindberg et al., 1986). More than 90% of E. coli strains isolated from the urine of uncompromised children with acute pyelonephritis carry one or more chromosomal pap gene clusters, which encode the so-called Pap pili or P-fimbriae that bind to (a-o-Galp-[1+4)-8-DGal&containing glycolipids (Leffler and Svanborg-Eden, 1986). The specific adhesion function was recently shown to be exerted by minor pilin-like proteins and not by the major PapA pilin subunit (Lindberg et al., 1986). In this paper we investigated the cell association and length modulation of Pap pili. We have identified in the pap gene cluster a novel gene, pa@-/, that encodes a pilinlike protein. Data are presented that suggest that this protein anchors the pilus to the bacterial cell and that the stoichiometric relationship between PapH and the major pilin subunit, PapA, determines pilus length. Results Nucleotide Sequence of the papH Gene and Identification of Its Gene Product Plasmid pPAP5 in the E. coli strain HBlOl expresses digalactoside-binding Pap pili of serotype F13 (Lund et al., 1985). The pap gene cluster responsible for regulation and biogenesis of these pili is diagrammed in Figure 1. Previous mapping experiments revealed a region of approximately 700 bp between papA, the structural gene for the major pilin subunit (Bdga et al., 1984) and papC, a gene specifying export and assembly functions (Norgren et al., 1984; Norgren et al., unpublished data). The nucleotide sequence of this intervening region was determined, and found to contain a 627 nucleotide long open reading frame with the same polarity as pap4 (Figure 2). A potential initiation codon (ATG), preceded by a sequence showing homology to ribosome binding sites (Shine and Dalgarno, 1974), was found at position 2384. A protein initiated here and ending at the TGA triplet at position 2969 would contain 195 amino acid residues and exhibit a calculated molecular size of 21.8 kd. The NHrterminal amino acid sequence of the open reading frame has all the features of a signal peptide sequence (Inouye and Halegoua, 1980). The suggested cleavage site between Ala -1 and Gly +l conforms to the rules for prokaryotic signal peptidase cleavage sites as described by von Heijne (1984). Deletions were constructed within the open reading frame to assess if it encoded a gene product. Plasmid pPAP207 is deleted for 0.1 kb of DNA around the Smal site located at position 2626, whereas pPAP265 was constructed by deleting 0.35 kb of DNA (Figure 1). The expression of Pap proteins from the intact plasmid pPAP5 as well as from the deletion derivative pPAP207 was analyzed in E. coli minicells (Figure 3). The wild-type pPAP5 ex-

Cell 242

1 kb

E ,

A Ev H S HpHpA Ev I L-000-II Pad Pam pap*papH WPC

EvEvH

Figure 1. Genetic and Physical Plasmid pPAP5 and Its Derivative

SS

X A

E I E I E I

pPAP 5

001 PaPEPaPFWPG

Paw

B A X

I B

E I E

B

E

Abbreviations for restriction endonuclease sites are as follows: A, Apal; 6, SamHI; Ev, EcoRV; H, Hindlll; Hp. Hpal; S, Smal; and X, Xhol. Filled bars indicate pSR322 DNA, open bars show pACYC184 sequences, and the /acZ vector pRZ5202 is represented by hatched bars. Physical locations of the deletions (A) within the papN and the pa@ genes, and the insertion(V) within the paps gene, are shown.

pPAP253 pPAP 207 pPAP 265

H -

H

H pSNlO0

‘d

pPAP212

-

I

pPAP259

I

pPAP 261

I

pPAP 269

:“W”

pPAP 267 -

E { E >

Map of the Plasmids

S

E

S

E

pHMG9 pPAP249 S 4

pPAP264

pressed, at very low levels, a 20 kd protein that was not produced by the mutant. Instead, a unique 14 kd polypeptide species was expressed from pPAP207; this most likely represents a truncated derivative of the 20 kd protein. Thus, the identified open reading frame represents a novel pap gene, designated pa@, and its mutant alleles are denoted pap/f7 (plasmid pPAP207) and pap/+2 (plasmid pPAP265). The deduced mature PapH protein of 173 amino acid residues shares many structural features with known E. coli pilin proteins (Figure 4; Normark et al., 1966). PapH containstwo cysteine residues 36 amino acids apart in the NHTterminal half of the protein, a tyrosine residue as the penultimate amino acid, and shows an overall sequence similarity to other E. coli pilins, especially in the regions from Gly 23 to Gly 50 and in the COOH-terminal region. It was recently demonstrated that the PapE and PapF proteins encoded by the pap gene cluster (Figure 1) are minor pilin-like proteins present in a pilus preparation

(Lindberg et al., 1966). The PapH protein shares the pilinlike features of both these proteins. However, no band migrating at the position of the PapH protein was detected when purified Pap pili were analyzed on silver-stained SDS-polyacrylamide gels (data not shown). Hence, if PapH is part of the purified Pap pilus it must be present in a significantly lower amount than PapE, which is clearly detected under the same experimental conditions (Lindberg et al., 1966). papH Mutations Affect the Cell Association of Pap Pili Derivatives of E. coli strain HBlOl harboring pPAP5 or its papH deletion mutants, pPAP207 and pPAP265, were compared with respect to piliation and hemagglutination. Both strains expressed pili as detected by electron microscopy, both agglutinated human Pl erythrocytes, and purified pili prepared from these strains agglutinated erythrocytes to the same extent (data not shown). Electron microscopy revealed piliated cells both in the Start

Figure

2. Nucleotide

Sequence

of the pep/-/ Gene

and the Deduced

Amino

papH

*a”?

Acid Sequence

The numbers above the DNA sequence show base positions relative to the left EcoRl site of the plasmid pPAP5 (see Figure 1). The vertical arrow indicates the postulated cleavage site for the signal peptidase. The region of dyad symmetry, which may form a hairpin structure at the RNA level, is indicated by horizontal arrows. The putative Shine-Dalgarno sequence and the restriction site for endonuclease Smal are shown by horizontal lines.

Biogenesis 243

of Pap Pili

release of Pap pili, showing that the observed phenotype was specific for mutations in papH (data not shown). - 94

PapC -)

papH+ Is Dominant over papHf and papH2 Complementation studies on the papH mutations were performed using either the papH allele (pPAP259) or the truncated variant papH7 (pPAP261) (Figure 5 and Figure 6). E. coli cells harboring pPAP267 (papH7) or pPAP265 @apH2), when complemented with pPAP259 @apH+), reieased the same low amount of pili into the supernatant as cells carrying pPAP5. Protein expression from pPAP261 or from the vector plasmid pACYC184 could not complement pPAP207, showing that it must be the PapH protein expressed from pPAP259 that corrects the defective phenotype of the papH7 mutant. Introducing the papH7 mutant pPAP261 into cells harboring pPAP5 had no effect on the cell association of pili, thereby implying that the truncated polypeptide expressed from the papH7 allele could not compete with the wild-type PapH protein.

- 67

PapG+ PapD *

1 30 1

PapH + PapA * 1 PapE -+ 1 PapF * 1

20.1

14.4

PapB -. Pap1 -) Figure 3. Autoradiograph of [35S]Methionine-Labeled pressed in Minicells Isolated from the Strain ORN103

Proteins

Ex-

Analyzed here are minicells harboring the plasmid pPAP5 (lane I), pPAP207 (lane 2). or pBR322 (lane 3). The asterisk indicates the location of the truncated form of the PapH protein. The molecular sizes of standard proteins are marked in kd at the right.

wild type and in the papH mutants (Figure 5). In contrast to wild-type cells, the mutants demonstrated a large proportion of free pili. To quantify the amounts of cellbound and free pilus antigen, immunoblot experiments were performed using antiserum raised against purified Pap pili (Figure 6). Bacteria were grown on solid medium and were gently resuspended in saline buffer. The amounts of pilus antigen remaining in the supernatant fluids after centrifugation were compared between the wild type and thepapH mutants. As shown in Figure 6, significantly more Pap pilus antigen was found in the supernatant from cells carrying a mutant papH allele. However, the total amount of pilus antigen was not significantly altered by mutations in papH. The pilus antigen detected in the supernatants from the mutants ran as a ladder on a nondenaturing polyacrylamide gel, in the same way as did purified wild-type Pap pili (data not shown). This showed that the free antigen represented polymerized Pap pili, as suggested by the electron microscopy. To determine whether the papH mutations had any effect on cell lysis or on the release of periplasmic proteins, the amount of periplasmic f3-lactamase in the supernatants of the mutants and the wild type was compared. In each case, only 3% of the total 8-lactamase activity was found in the supernatant, implying that the pilus antigen was not released through spontaneous lysis of the papH mutants. Mutations abolishing expression of the other minor Pap proteins (i.e., PapE, PapF, or PapG) had no effect on the

Transcriptional Coregulation of papA and papH The functional data described above strongly suggested that PapH has a role in the biogenesis of Pap pili. We therefore asked whether papH and the major pilin subunit gene pap4 are coregulated. The intercistronic region between papA and papH consists of only 63 bp. Within this region we found no sequences typical of the conserved -10 and -35 regions of E. coli promoters (Hawley and McClure, 1983). Instead, this region contains a sequence of dyad symmetry that we recently showed to be the papA transcriptional terminator (Bbga et al., 1985). Therefore, transcription of papH could depend on transcriptional read-through of the papA terminator. Transcription of pap4 is stimulated by the regulatory PapB protein. To determine if papH was also under PapB control, two papH-lacZ transcriptional fusions were examined: pHMG9 is wild-type for paps, whereas pPAP249 carries a mutant pap6 allele (Figure 1). The P-galactosidase activity in HBlOl cells harboring pPAP249 was significantly lower than that in cells harboring pHMG9 (Table l), but could be complemented to the level of the latter by introducing plasmid pPAP221, which expresses the PapB protein. To investigate the reason for the residual j3-galactosidase activity of pPAP249, the pap6 promoter and the upstream region coding for the Papl regulatory protein were deleted. The P-galactosidase activity level of the derivative plasmid pPAP264 was reduced to that of the vector control (pRZ5202), and could not be increased by complementation with papB alone (pPAP221) or pap6 plus pap/ (pHMG72) (BBga et al., 1985) expressed by coresident plasmids. These data showed that the transcription initiated and regulated from the paps promoter is responsible for the expression of papH. It was recently reported that in vivo transcription initiated from the pap!3 promoter was required for the papA gene to be primarily represented as an 800 base long mRNA with its 5’ end located between papB and papA (BBga et al., 1985; Figure 7). One hypothesis that accounts for the length of this transcript is that it is a product

Cell 244

1 I

20 I

40 I .

PapH

v

60 I T

T

v

v

T

VI0

TPVRDL-QNGFSGPERKFSLRLRNCRFNSQ----GGNLFSDSR~RVTFDGV

PapA

LSKSFLEAGGVSKP-NDLDIELVNCDITAFK---GGNGAKKGTVKLAFTGP

v

T'j

v

v

vvvv

VRTASLAQEGATSSAVGFNIQLNDCDTNVASKAA--VASKAA--VAFLGTAlDAGHTNV

v

v

120 I v v

v

vv

PapH

RGETPDKFNLSGQAKGINLQIADVRGNIARAGKVMPAl-----PLTGN~~A

PapA

IVNCHSDELDT---NGGTGTAlVVQGA-GKNVVFDG-SEGDANTLKDGENV

F7

PAENADDMLQT---VGDTNTAlVVTDSSGKRVKFDGATETGASNLINGDNT

v

VVVV

.

140 I vvv

PSGPQSDMLQT---VGATNTAIVVTDPHGKRVKFDGATATGVSYLVDGDNT LA------LQSSAAGSATNVGVQILDRTGAALTLDGATFSSSTTLNNGTNT

V

vvv

v

v

T

V

PapH

LDYTLRIVRN---GKKLEAGNYEAVLGFRVDYR

PapA

L HYT

F7

IHFTAFVKKDNS-GKNVAEGAFSAVANFNLTYQ

Figure

vv

VSKLFLENDGESQP-KSFDIKLINCDITNFKKAAGGGGAKTGTVSLTFSGV

v

Type1

T

ISKSFLQEGGETQP-KDLNIKLVNCDITNLKQLQ-GGAAKKGTVSLTFSGV

100 I

1

vv

80 I v

PapH

F72

v

AATTVNGGTVHFKGEVVNAACAVDAGSVDQTVQLGQ

P

F72

I

APTIPQGQGKVTFNGTVVDAPCGIDAQSADQSIDFGQ

TYPO 1

Type1

v

AASIPQGQGEVSFKGTVVDAPCGIETQSAKQEIDFGQ

=72

1

. .

APTIPQGQGKVTFNCTVVDAPCSISQKSADQSIDFGQ

F71

F72 Type1

.

GPFPPPGMSLPEYWGEEH"WWDGRAAFHGEVVRPACTLAME"A~QIIDUG~

Pap.4

F71

.

TVV

,vv

T

v

,v

AVVKKSSAVGAAVTEGAFSAVANFNLTYQ

IHFTAAVRKDGS-GNPVTEGAFSAVANFNLTYQ IPFQARYFAT---GAA-TPGAANADATFKVQYQ 4. Comparison

of the Deduced

Amino

Acid Sequences

of the PapH Protein

and Other

Pilin Proteins

Sequences for the pilin proteins PapA (Biga et al., 1994) F7, (Rhen et al., 1995) F7, (van Die and Bergmans, 1994). and Type 1 (Orndorff and Falkow, 1985) are aligned with the PapH sequence. The amino acids are given in the one-letter code and are numbered according to the PapH protein. Gaps have been inserted into the sequences to improve the alignment. A filled triangle indicates identical amino acids in all sequences. Positions of identical or conservative replacements of amino acids in four out of five sequences are indicated by open triangles. Conservative replacements are defined as being within the groups (0, E), (K, R), (S, T), (F, Y, W), and (I, L, V, M).

of the larger, 1300 base long pap&papA transcript. To monitor the transcriptional activity over pa@ directly, Northern blot hybridization experiments were performed using strand-specific papH, papA, and pap8 probes. A weakly expressed 1300 base long transcript was detected when the pap/f probe was hybridized with RNA prepared from cells carrying pPAP5 (Figure 7). The intensity of this band was markedly reduced when RNA from cells harboring the paps3 mutant pPAP253 was used. A transcript similar in size and abundance, and comigrating with the paps-papA mRNA of pPAP5, could also be detected when RNA from the mutant was hybridized with a pap4 probe. These data show that transcription of pap/f is represented by a weak, 1300 base long pap4pap/-i mRNA, which like

the heavily expressed 800 base pap4 transcript is dependent on the transcription from the pap8 promoter. Changes in the Stoichiometric Ratio of PapA to PapH Affect Pilus Length In wild-type cells PapA, the major pilin subunit, is produced in much larger amounts than PapH. From minicell analysis we estimated that the difference is at least lOOfold, taking into account four methionine residues in PapH and only one in PapA. Plasmid derivatives were constructed that selectively overproduce PapA or PapH, and the effects on pilus expression were studied by electron microscopy and immunoblot analysis of PapA antigen. Plasmid pPAP267, which overproduces the PapA pilin

Wyis

of Pap Pili

a

b

C

d

f

Figure

5. Electron

Micrographs

of HE101 Derivatives

(a) HBlOl harboring pPAP5 and pACYC164. (b) HBlOl harboring pPAP207 and pACYC164. (e) HBlOl harboring

harboring pPAP207

pPAP265 and pACYC164. (c) HBlOl harboring pPAP265 and pPAP259. (d) HBlOl and pPAP259. (f) HBlOl harboring pPAP207 and pPAP261. The bar indicates 1 w.

subunit, was introduced into HBlOl cells harboring either the wild-type pap gene cluster on pPAP5 or thepapH2 mutant pPAP265. In both cases, expression of PapA was lofold higher in comparison with the controls, i.e., HBlOl cells harboring pPAP5 or pPAP265 together with the vector pACYC184 (Table 2). When PapA was overproduced in

the presence of pPAP5, pili remained ceil-associated, but became longer (Figure 8 and Figure 9). Thus, 22% of the cells harboring pPAP5 plus pPAP267 expressed more than 10 pili per cell that were longer than 2.0 pm, compared with 4% of control cells (harboring pPAP5 plus pACYC184) (Table 2). In the case of HE101 cells that over-

Cell 246

Figure

6. lmmunoblot

Analysis

of the PapA Antigen

in the Wild Type and in pap/+ Mutants

Shown is an analysis of supernatant (s) and pellet(p) fractions of strain HBIOI harboring plasmids having the wild-type allele (H+) or apap/f mutant allele (H-). (a) HBlOl harboring pPAP5 and pACYC184. (b) HBlOl harboring pPAP5 and pPAP259. (c) HBlOl harboring pPAP5 and pPAP261. (d) HBlOl harboring pPAP207 and pACYC184. (e) HBlOl harboring pPAP207 and pPAP259. (1) HBlOl harboring pPAP207 and pPAP261. (g) HBlOl harboring pPAP5 and pACYC184. (h) HBlOl harboring pPAP265 and pACYC184. (i) HBlOl harboring pPAP265 and pPAP259.

Table 1. Expression of 8Galactosidase Activity with Plasmids Encoding PapB and Papl. papH-/acZ

Fusion

Introduced

pRW202 (/acZ vector) pHMG9 (pep/+, SC, A+, H-la@ pPAP249 (papl+, 137, A+, H-lad) ‘I pPAP249 ‘I pPAP249 pPAP264 @ap82, A+, H-lad) pPAP264 ” Y pPAP284 pPAP284 ” Cells were

grown

in MC1029

Cells

Carrying

Various

papH-/ad

Plasmid

Transcriptional

5-Galactosidase

-

Specific

Fusions

Activity

Complemented

(units)

4 100 23 18 100 3 5 4 6

pACYCl84 (vector) pPAP221 (paps+) pACYC184 (vector) pPAP221 @epB+) pHMG72 @apTc, B+)

in CASA medium.

produced PapA in the absence of F’apH, 500/b-70% of the total pilus antigen was in the form of free pili. Despite overproduction of PapA, only 4% of the cells had more than 10 pili longer than 2.0 pm (Figure 8 and Table 2). However, the majority of free pili appeared long in the electron microscope. Unfortunately, because of the high degree of aggregation of free pili, it was not possible to determine accurately the length distribution of these structures. To increase expression from pap/-/, DNA corresponding to the pap4 terminator was deleted, and papH transcription was placed under the control of the tetracycline promoter. This construct, pPAP289, showed an 8-fold increase in PapH production compared with pPAP5 when expression was monitored in minicells. HBlOl harboring pPAP289 together with pPAP5 or pPAP267 produced the same amount of pilus antigen as the controls (harboring pPAP5 or pPAP265 plus pACYC184). Moreover, the pilus antigen remained cell-associated. When examined in the electron microscope, cells with unusually short pili were observed (Figure 9). Only 10% and 8%, respectively, of cells that overproduced PapH carried pili longer than 1.4 pm (Table 2). Plasmid pPAP269 was also introduced into cells, with thepapA mutation on pPAP23 (Lindberg et al., 1964). Despite the overproduction of PapH, no pili were detected,

supporting our view that PapH cannot be polymerized a pilus dominated by PapH subunits.

into

Discussion Despite the medical importance of bacterial pili as attachment factors, little is known about their biogenesis. We show here for digalactoside-binding Pap pili of uropathogenie E. coli that membrane anchorage and pilus length are governed by the presence of the PapH protein. More specifically, mutations in the pap/-/ gene resulted in 50%-70% of the total pilus antigen to be found free of the cell in the form of polymerized structures. The fact that this phenotype could be complemented by having an intact pap/-I gene on a coresident plasmid suggests that the PapH protein is directly involved in the cell anchoring of Pap pili. Overproduction of PapH relative to PapA resulted in shorter pili, whereas overproduction of PapA gave rise to very long pili. Recently, we demonstrated that Pap pili encoded by the pap gene cluster contain minor pilin-like proteins that are required for pili to bind to glycoconjugates containing U-DGalp(l-4)-8-o-Galp (Lindberg et al., 1986). The PapH protein was not required for intact cells or purified Pap pili to have digalactoside-specific binding properties. Never-

E$enesis

of Pap Pili

Figure 7. Northern Blot Analysis of Transcription of the papH, pap4, and paps Genes Samples of total RNA isolated from HBlOl cells carrying pBR322, pPAP5. or pPAP253 were blotted as described in Experimental Procedures. A blot of 30 pg RNA samples was hy bridized with the papH probe, and blots of 10 ug RNA samples were analyzed with the papA and pap6 probes. Autoradiography was for 1 day (pep4 probe, lanes 1-4; pap6 probe, lanes l-3) 2 days @apH probe), and 5 days @apA probe, lane 5; pap8 probe, lane 4). The 16s and 23s ribosomal RNAs are indicated. The location of the 1300 base papA-papH transcript is shown by an asterisk. Denatured, end-labeled Hindlll fragments of 1, DNA were used as size markers.

Table 2. Effect

Plasmids

of PapA to PapH

in HE101

pPAP5, pACYC184 pPAP5, pPAP267 pPAP5, pPAP269 pPAP265, pACYC184 pPAP265, pPAP267 pPAP265, pPAP269

Cells

Stoichiometry

Relative Amount PapA Antigena 1 10 1 1 10 1

on Pilus Length

of

Relative Amount PapH Proteinb 1 1 8 0 0 8

of

% Free Pilus AntigenC 1O-20 1O-20 1O-20 50-70 50-70 1O-20

% Ceils with More Than 10 Pilid >1.4

pm

31 78 10 54 41 8

>2.0

urn

4 22 1 13 4 1

a The amount of PapA antigen in cell extracts was determined by the immunoblotting technique. The values given are relative to cells harboring pPAP5 and pACYC184. b The amount of PapH protein was determined in minicells, and the values presented are relative to cells harboring pPAP5 and pACYC184. c The fraction of free pilus antigen was estimated from immunoblot experiments, d Pilus lengths were measured on 200 cells on electron micrographs at 5000 x magnification.

theless, the deduced amino acid sequence of F’apH showed all the features characteristic of known E. coli pilins. However, unlike other major and minor pilins, PapH carries a 14 residue long proline-rich NHrterminal extension. Proline-rich regions in staphylococcal protein A (Guss et al., 1984) and streptococcal M protein (Hollingshead et al., 1986) have been suggested to link these proteins to the cell wall. Likewise, a compilation of sequences from several proteins involved in transport show that their membrane-buried regions almost always contain one or more proline residues (Brand1 and Deber, 1986). Therefore, we speculate that the unique NH2 terminus of PapH is membrane-associated. In minicells, taking into consideration the number of methionine residues in the two proteins, PapA is at least 100-fold more abundant than PapH. Our data suggest that the activity of papH is dependent on transcriptional readthrough of a terminator structure positioned between pap4 and papH. A read-through product 1300 bases in length was detected, and was about lOO-fold less abun-

a

S-P

Figure 8. lmmunoblot Overproduce PapA

Analysis

b S-P

of PapA Antigen

in HBlOl

Cells That

Shown is an analysis of supernatant (s) and pellet (p) fractions of HBlOl cells overproducing PapA in the presence of PapH (pPAP5 and pPAP267) (a) or in the absence of PapH (pPAP265 and pPAP267) (b).

dant than the terminated 800 base long pap4 transcript. Furthermore, we provide evidence to indicate that papA and papH are coregulated. Transcription of both these genes depends on the activity of the papB promoter and is positively affected by the PapB protein. We believe that

a

b

C

Figure 9. Electron PapA or PapH

Micrographs

(a) HBlOl harboring pPAP5 and pPAP267. bar indicates 1 urn.

of HE101 Derivatives

Overproducing

pPAP5 and pACYC164. (b) HBlOl harboring (c) HBlOl harboring pPAP5 and pPAP269. The

the difference in the transcriptional activity over papA and papH is an important factor governing the stoichiometry between the PapA and PapH proteins. One can envisage several modes by which the length of a polymeric structure could be determined. The length

of a pilus could simply be the result of spontaneous breakage of the fiber, the likelihood of which would increase with increasing length of the pilus. We believe spontaneous breakage of pili does occur, but at a low level since only small amounts of free pilus antigen were found in supernatants of cells harboring the wild-type pap gene cluster. Furthermore, a ItMold increase in the expression of PapA, which led to longer pili, did not result in any increased amount of free pilus antigen. An alternative mechanism may be that pilus length is determined by an equilibrium between polymerization and depolymerization, similar to that described for actin (Pollard and Cooper, 1988). Furthermore, Pseudomonas pili and F pili have been shown to be retractable (Bradley, 1972; Novotny and Fives-Taylor, 1974). However, we have no evidence for retraction of pili in the pap system. We favor that pilus length is governed by specific terminator proteins, for which PapH is one candidate. The pilinlike nature of PapH suggests that it may be polymerized into the pilus fiber. However, papH mutants do not negatively affect the total amount of surface-exposed pilus antigen, showing that PapH is not required in the secretion or assembly processes of pilin subunits. We therefore believe that the entrance of PapH into the Pap pilus occurs late in biogenesis. Because of the low cellular concentration of PapH, its polymerization with other pilin subunits would be a rare event. We suggest that the growing Pap pilus can be detached because of unstable interaction between PapA and the cell envelope. Once PapH enters the pilus structure, we believe that its presence allows no further polymerization. The low amount of free pilus antigen found outside cells harboring the wild-type gene cluster could represent pili released in the growing phase. Both length modulation and membrane anchoring would thereby depend on the same protein. Furthermore, these data are consistent with a model in which Pap pilus growth is accomplished by the addition of subunits to the base of the organelle. This has recently been shown to be the mechanism of elongation for E. coli type 1 pili (Lowe et al., 1987) and filamentous phages (Armstrong et al., 1983), whereas flagella grow by polymerization of subunits onto the tip (Emerson et al., 1970). Length of the bacteriophage li tail is determined by a specific protein, gpH, which acts as template during polymerization (Katsura and Hendrix, 1984). In tobacco mosaic virus and filamentous bacteriophages, length is governed in a similar way by the nucleic acid molecule and specific terminator proteins (Griffith and Kornberg, 1974). In these systems the interplay with DNA requires an exact mechanism for length determination. In our model, the length of Pap pili depends on the ratio between PapA and PapH and on the affinity these proteins have for the export (PapD) and polymerization (PapC) proteins. Such a mechanism would result in pili with individual variations in length. We feel that Pap pili can tolerate a certain flexibility with respect to length and still maintain biological function. It is probably more important for uropathogenic E. coli to ensure a firm association of Pap pili to cells, since released pilus adhesin would compete with the

Biogenesis 249

of Pap Pili

piliated tempts termine pilus

bacteria

for

are now

under

whether

the

as our

Experimental Bacterial

model

binding way protein

to the mucosal

to purify

the

is located

PapH at the

lining.

At-

protein

to de-

base

of the

suggests.

Procedures Stralns

and Growth

Conditions

Strains used in this study were the E. coli K-12 derivatives HB101 (Boyer and Roulland-Dussoix, 1989), MC1029 (Casadaban and Cohen, 1980). JM103 (Messing et al., 1981), and ORN103 (Orndorff et al., 1985). Cells were grown in L broth (Bertani, 1951) supplemented with medium E (Vogel and Bonner, 1958) or in CASA medium, which consists of 1.5% Casamino acids, medium E, and 1 &ml thiamine. The antibiotics carbenicillin (100 &ml), chloramphenicol (30 pg/ml), and tetracycline (10 pg/ml) were used for selection of plasmid-containing strains.

Recombinant

DNA Techniques

Restriction endonucleases, T4 DNA ligase, Xhol linkers, Bai 31 nuclease, Sl nuclease, exonuclease Ill, the Klenow fragment of polymerase I, T4 polymerase, and RNAase-free DNAase I were used according to conditions recommended by the commercial suppliers (New England Biolabs, Boehringer Mannheim, and Pharmacia PL). isolation of plasmid DNA, agarose gel electrophoresis, and transformation procedures were essentially as described by Maniatis et al. (1982). DNA fragments were isolated from low-melting agarose gels according to the manufacturer (Bethesda Research Laboratories).

Plasmld

Constructlons

To create deletions within the pep/+ gene, plasmid pSNlO0 (Normark et al., 1983; Figure 1) was linearized at the Smal site, digested with exonuclease III, and then treated with nuclease Sl. Digestion products were ligated and transformed into strain HBiOl, and clones obtained were analyzed by restriction enzyme mapping. It should be noted that Xhol linkers had been added to one portion of the digestion products before ligation was initiated. Two deletion derivatives of pSNlO0 containing deletions within the pepH gene were used for isolation of the Hindlll pap DNA fragments, which were subsequently cloned into pPAP208 (B%ga et al., 1985). Piasmid pPAP208 is a pPAP5 derivative lacking the central 4.1 kb Hindill fragment of pap DNA. The resulting plasmids, designated pPAP207 and pPAP285, contain a complete pap gene cluster except for mutations, denoted papHI (pPAP207) or papH2 (pPAP285), within the papH gene (Figure 1). In the mutation papHI, 0.1 kb of thepapH gene around the Smal site is deleted, whereas in papH2 0.35 kb of DNA is substituted by a Xhol linker. Construction of plasmids that exhibited low-level expression of the PapH protein or its truncated variant PapHl was undertaken in the following way. Plasmids pSNlO0 and pPAP207 were restricted with HindIll, and the protruding ends were filled in using the Klenow fragment of polymerase I. After restriction with the endonuclease Hpal, the smaller Hindlll-Hpal DNA fragments were isolated and then inserted by blunt-end ligation into the EcoRV site of the plasmid vector pACYC184 (Chang and Cohen, 1978). ThepapH (pPAP259) and papH7 (pPAP281) alleles are transcribed in the same orientation as the tetracycline genes in both of these constructs. To obtain high transcriptional activity through the papH gene, pPAP289 (Figure 1) was constructed, in which DNA covering the pap4 terminator was deleted. This plasmid was made as follows. Plasmid pPAP212 (Figure 1) was linearized with Hindlli, and treated with Bal31 nuclease. After addition to the DNA of Xhoi linkers and restriction with Smal endonuclease. Xhol-Smal fragments approximately 485 bp in size were isolated and then ligated with Sali- and Smai-digested M13mp8. DNA sequencing confirmed that one recombinant plasmid carried pap DNA covering the region from position 2357 (see Figure 2) to the Smal site at position 2828. In this construct, the pap DNA is flanked by Hindlll and EcoRl recognition sites. After cleavage at these sites, the protruding ends were filled in, and the 0.50 kb blunt-ended fragment was cloned into the EcoRV site of pACYC184. In the resulting construct, the small Smai-BamHI fragment was replaced with the 0.59 kb Smal-BamHI fragment isolated from pPAP259, to create a com-

pletepapH gene. In the final construct, pPAP289, the papH gene is under the control of the tetracycline promoter. To construct a plasmid that carries only the pap.4 gene, plasmid pPAP285 was restricted with Xhol, and the protruding ends were filled in. After cleavage of the DNA with EcoRV, the 0.79 kb fragment spanning the pap4 gene was cloned into the EcoRV site of pACYC184, resulting in pPAP287 (Figure I), in which the pap4 gene is transcribed from the tetracycline promoter. Plasmid pPAP253 was constructed as follows. The sticky ends of the 0.78 kb Taqi fragment (positions 857-1834) from pPAP5 were filled in and inserted at the Smal site of piasmid pHMGl1 (Bfa et al., 1985). The construct obtained was digested wiih EcoRl and Apal, and ligated with the 1.22 kb EcoRI-Apal fragment (position 1-1219) of pPAP5. In the resulting plasmid, pPAP251, a partial duplication of the pap6 gene, encompassing a BamHl site, was created. Plasmid pPAP251 was linearized with BamHI, treated with Bal31 nuclease, and ligated after addition of Xhol linkers to the ends. In one of the resultant deletion derivatives, pPAP24-102, thepapSgene had been deleted for 0.15 kb of DNA located at the 3’ end of the gene. This pap6 allele was called pap/33 The 1.8 kb EcoRI-Hindlll fragment from pPAP24-102 was cloned into the corresponding sites of pPAP208; the subsequent insertion of the central 4.1 kb Hindlll pap DNA fragment created pPAP253. This construct contains a complete pap gene cluster except for the deletion within the paps gene (Figure 1). Transcriptional fusions between pap DNA and the lad gene were made by cloning into the vector pRZ5202 (kindly provided by L. Munson and W. S. Reznikoff) followed by transformation into the Lacstrain MC1029. Piasmid pHMG9 (Bgga et al., 1985; Figure 1) has the 2.8 kb EcoRI-Smal fragment of pap DNA cloned between the EcoRl and Smal sites of pRZ5202. Fusion pPAP249 (Figure 1) was constructed in the same way as pHMG9, but contains a 200 bp insertion of DNA within the pap13 gene that gives rise to a papB mutation denoted papBl (Biga et al., 1985). To obtain a papH-/acZ fusion lacking the transcriptional activity of the pap6 promoter region, plasmid pPAP5 was first restricted with Apal and then treated with T4 polymerase to render the fragments blunt-ended. Following digestion of the fragments with Smal, the 1.8 kb Apal-Smal fragment was isolated and then ligated with Smal-restricted pRZ5202 DNA. The pap genes in the resulting construct, pPAP284 (Figure 1). are orientated in the same direction as in pHMG9, and the mutated paps allele is denoted pap82.

Nucleotide

Sequence

Analysis

Overlapping DNA fragments covering the region from the Hindlll site at position 1.95 kb to the Hpal site at position 3.22 kb (Figure 1) were isolated from the plasmid pSN100. After restriction of the DNA with appropriate restriction enzymes, digestion products were cloned into M13mp8 and M13mp9 vectors (Messing and Vieira, 1982) and transformed into the strain JM103. The DNA sequence on both strands of the papH gene was determined using the dideoxynucleotide chain, termination method of Sanger et al. (1977).

Expmsslon

of Protelns

In Minicells

Proteins encoded by the plasmids pPAP5, pPAP207, and pBR322 were analyzed in minicells prepared from strain ORN103 and labeled with [35S]mathionine (Amenham) as described by Thompson and Achtman (1978). The radioactive polypeptide products were separated on a linear 15% (w/v) SDS-polyacrylamide gel (Laemmli, 1970), and detected by autoradiography. Protein molecular weight standards employed ranged in size from 14.4 kd to 94 kd.

Hemagglutlnation

Assay

Binding properties of strains were determined by slide agglutination using human Pl erythrocytes as described previously (Normark et al., 1983). The agglutination of purified pili was performed as described by Lindberg et al. (19&Q), using Z-fold serial dilutions starting with 500 pg/ml of protein. A positive reaction was determined macroscopically.

Pill Preparation After 48 hr incubation on solid selective CASA medium, bacteria were harvested, and pili were purified essentially according to the method of Korhonen et al. (1980b). The pili preparations were analyzed on an SDS-polyacrylamide gel by silver staining (Oakley et al., 1980).

Cell 250

Quantification

of Piius Antigen

Cell extracts of strains to be analyzed were prepared by resuspending the cells gently in saline, followed by centrifugation at 12,000 x g for 15 min at 4OC. The supernatant and pellet from 1 x 16 bacteria were evaporated, and the residues were suspended and boiled in sample buffer consisting of 62.5 mM Tris-HCI (pH 6.6), 1% SDS, 0.5% pmercaptoethanol, and 10% glycerol. The proteins were separated by electrophoresis on 15% poiyacrylamide slab gels containing 0.1% SDS. immunoblot transfer experiments were carried out essentially as described by Swanson et al. (1962), using antiserum (1 :I000 dilution) raised against Pap pili. The amounts of pilus protein attached and released from the bacteria were estimated from autoradiograms after immunoblot transfer.

Electron

Mlcroscopy

Electron microscopy was performed using a JEOL 1008 microscope with 150-mesh copper grids coated with thin films of 2% Formvar. The bacterial colonies were overlayed with buffer (IO mM Tris-HCI [pH 7.51, 10 mM magnesium chloride), and the cell suspension was allowed to sediment for 15 min on a grid. Grids were washed with the buffer, negatively stained for 5 set with 3.55% ammonium molybdate, and then washed with redistilled water.

Determination

of BLactamase

Activity

The B-lactamase activity in cell extracts and supernatants of 1.6 x lOa bacteria was determined spectrophotometrically at 462 nm, using the chromogenic cephalosporin nitrocefin (Oxoid) as a substrate (O’Callaghan et al., 1972). Cell extracts were prepared by disrupting the cells with four 10 set bursts from a Sonifier B-12 sonicator at setting 3. Extracts and supernatants were clarified by centrifugation at 12,000 x g for 15 min at 4OC.

Determination

of pGalactosidase

Activity

The specific activity of P-galactosidase was determined as described by Miller (1972). Results presented in Table 1 represent mean values of three independent experiments in which p-galactosidase activity was assayed in duplicate.

Hybridization

Probes

and Labellng

Procedures

Ml3 clones containing DNA of the paps, papA, or papH gene were used for preparation of single-stranded templates (Messing and Vieira, 1962). Strand-specific probes used in Northern blot hybridization experiments were obtained by labeling the templates by primer extension as described by Hu and Messing (1962), using [a-=P]dATP (Amersham) and a hybridization probe primer from New England Biolabs. The specific activity obtained was 1 x 108 cpmlrg DNA. Hindllldigested ?. DNA was end-labeled with [a-zP]dATP by a fill-in reaction using the Klenow fragment of DNA polymerase I.

isoiatlon of RNA and Northern Blot Hybridization E. coli HBlOl cells carrying one of the plasmids pPAP5, pPAP253,

or pBR322 were grown at 37“C in CASA medium to ~2 x 108 cells per ml. Cells were lysed as described by von Gabain et al. (1963), and total RNA was extracted by the hot-phenol method (Salser et al., 1967). The preparations were treated with RNAase-free DNase I, extracted with phenol and chloroform (l:i), and stored at -20% as an ethanol precipitate. The RNA sample was dissolved in 20 pl of sample buffer consisting of 50% formamide, 2.2 M formaldehyde, and electrophoresis buffer (5 mM sodium acetate, 20 mM HEPES [pH 7.01). The RNA was heated for 5 min at 65OC, quickly cooled on ice, and 2 VI of dye mix (50% glycerol, 1 mM EDTA, 0.4% xylene cyanol, 0.4% bromophenol blue, 10% ethanol) was added. RNA species were fractionated by electrophoresis on a 1.O% agarose, 2.2 M formaldehyde gel. Denatured, ond-labeled Hindiil X DNA fragments were run in parallel. Following electrophoresis the gel was soaked in electrophoresis buffer for 15 min, and the nucleic acids were blotted onto nitrocellulose filter (Schleicher and Schiill, BA85) using the procedure of Thomas (1960). Filters were rinsed in 3x SSC (lx SSC = 150 mM sodium chloride, 15 mM sodium citrate [pH 7.01) and then baked at 60°C in vacua for 2 hr. Prehybridization was performed at 44OC for 6 hr in a solution of 50% formamide, 2x Denhardt’s, 5x SSC, 10 mM EDTA, and 0.2% SDS containing 500 pglml of denatured calf thymus DNA; hybridization with 1 x Iti cpmlml probe proceeded at 42oC for 24 hr in a simi-

lar solution, but prepared with only lx Denhardt’s. The blots were washed four times for 5 min at room temperature in 2x SSC, 0.5% SDS, followed by twice for 15 min at 45OC in 0.1x SSC, 0.5% SDS. Filters were dried and autoradiographed.

Acknowledgments We thank E. Skogman for skilled technical assistance, and L. Johansson for excellent technical assistance with the electron microscopy. We also thank Drs. J. Tennent and C. Korch for critically reading the manuscript, and B.-i. Str6mberg for typing. This work was supported by the Swedish Medical Research Council (Grants B66-13X-04768IlC and B66-16P-O6693-03A), the Swedish Natural Science Research Council (Grant B-BU 3373112), and the Swedish Board for Technological Development (Grant 613364). M. 8. was supported in part by a grant from the Lennander Foundation, and M. N. was supported in part by the Swedish Medical Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘adverfisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received

October

29, 1966; revised

January

6, 1967.

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