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FEMS Microbiology Letters 146 (1997) 181^188

Isolation and characterization of the integration host factor genes of

Pasteurella haemolytica

Sarah K. Highlander a; *, Orlando Garza 1;a , Billie Jo Brown 2;a , Simi Koby b , Amos B. Oppenheim b

b

a Department of Microbiology and Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Department of Molecular Genetics, The Hebrew University, Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel

Received 26 September 1996 ; revised 10 November 1996 ; accepted 10 November 1996

Abstract Using a bacteriophage lambda complementation system in

Escherichia coli,

we cloned genes encoding subunits of the

Pasteurella haemolytica. ihfA and ihfB mutations in E. coli demonstrated that the P. haemolytica gene products form functional heterodimers. The ihfA and ihfB genes encode polypeptides predicted to be 99 and 93 amino acids long,

heterodimeric DNA binding/bending protein, integration host factor, from the bovine pathogen, Complementation of heterologous

respectively, and are very similar to integration host factor subunits from other Gram-negative bacteria, although phylogenetic analysis indicated that the

P. haemolytica sequences are distantly related to those from other bacteria. Most significant amino

acid differences were restricted to the amino-terminal domains of the predicted peptides.

Keywords:

Integration host factor ;

Pasteurella haemolytica ;

DNA-binding protein

1. Introduction

replication, and transcriptional control of gene expression [1,2]. IHF binds to speci¢c DNA sequences

Integration host factor (IHF) is a heterodimeric

creating a 140³ bend in the DNA [3]. In the case of

DNA binding/bending protein involved in a variety

genes positively regulated by IHF, this bending can

of cellular processes including bacteriophage lambda

enhance the interaction of an upstream activator

integration into the bacterial chromosome, phage

protein with core RNA polymerase and the alternate

replication and packaging, transposition, plasmid

sigma factor,

c54 ,

bound downstream at the promo-

ter. Examples include nitrogen ¢xation operons in * Corresponding author. Tel. : +1 (713) 798 6311 ; fax : +1 (713) 798 7375 ; e-mail : [email protected] 1

Present address : Edcouch-Elsa High School, P.O. Box 127, Edcouch, TX 78538, USA.

2

Present address : Department of Biochemistry and Molecular Biology, The Mayo Clinic, Rochester, MN 55905, USA.

Klebsiella pneumoniae [4], £agellar genes in Caulobacter crescentus [5], and Pseudomonas putida operons involved in hydrocarbon utilization [6]. In other systems, IHF acts as a negative regulator and it can repress its own transcription [7]. We have been studying regulation of leukotoxin expression in the bovine respiratory pathogen,

0378-1097 / 97 / $17.00 Copyright ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

PII S 0 3 7 8 - 1 0 9 7 ( 9 6 ) 0 0 4 6 9 - 7

Pas-

S.K. Highlander et al. / FEMS Microbiology Letters 146 (1997) 181^188

182

VZAP-HLP1 VZAP-A1175 (ihfA‡ ), respectively, using

teurella haemolytica. A model for leukotoxin tran-

pHLP2 were excised from the phages,

scriptional regulation, that involves DNA bending

(ihfB

and

the

a

requirement

for

upstream

activation,

has

‡)

and

ExAssist

plasmid excision system

(Stratagene).

for

Deletion derivatives of pOG2067 and pHLP2 were

IHF was found 240 bp upstream of the transcrip-

created by restriction digestion, followed by recircu-

tional start-site and gel mobility shift assays demon-

larization of the plasmid using T4 DNA ligase. DNA

E. coli IHF binds to the promoter region

sequences were determined by the dideoxy chain ter-

been

proposed.

strated that

A

near-consensus

binding

site

[8], yet its role in leukotoxin regulation has not yet

mination

been established. As a ¢rst step toward examining

plates,

P. haemolytica,

the role of IHF in gene expression in

it was necessary to clone the genes encoding both

method

using

quencing

on

double-stranded

Sequenase

Kit,

as

Version

2.0

recommended

by

DNA

T7

tem-

DNA

the

Se-

supplier

(Amersham).

subunits of IHF from this organism. Here we describe

this

cloning, genetic complementation in

E.

2.3. Complementation of IHF function in E. coli

coli, and features of IHF from P. haemolytica. IHF

function

was

assessed

plaque formation using phage

2. Materials and methods

described

[12].

genes from

2.1. Bacterial strains, phage and plasmids

strain

Plasmids

by

quantitation

VD69,

expressing

of

as previously

ihfA

or

ihfB

P. haemolytica were electroporated into

vihfA82 : : Tn10)

A5427

(

or

strain

A5179

(ihfB157 : : Tn10) and the resulting strains were used

P. haemolytica strain PHL101 was the source of chromosomal DNA for library construction and was grown

at

(Difco).

gyrA96

37³C

E.

coli

endA1

q

in

v

Brain

strain

Heart

JM109

hsdR17

relA1

Infusion

of phage plating.

broth

v(lac-proAB)

(

supE44,

as plating bacteria to determine the relative e¤ciency

thi

[FPtraD36

3. Results and discussion

proAB lacI Z M15]) was used for routine cloning and strains A5427 (N99

vihfA82 : : Tn10),

hupA16 : : kan hupB11 : : cat

A5179

hupB11 : : cat

ihfB157 : : Tn10)

hupA16 : :kan

hupB11 : :cat

were used to analyze

and

vihfA82

3.1. Library construction and isolation of ihfA and

hupA16 : : kan

(N99

A5477

ihfB complementing clones

(N99

ihfB-157)

[9]

ihf complementation. E. coli

We used the strategy of Haluzi et al. [12] to identify

ihf-encoding clones within a

V

phage library of

strains were grown at 37³C in LB medium supple-

P. haemolytica DNA. In the absence of the histone-

mented with ampicillin (100 mg l

like

31 ) or tetracycline 3 1 ), where appropriate. Bacteriophage VD69 (10 mg l (imm21

nin5) [10] was used to evaluate phage plating

e¤ciencies on recombinant strains and bacteriophage

VZAP

VcI857 vnin5

II (

red

‡

‡

gam , Stratagene) was

used as the vector for library construction.

protein,

tion. Using HU

bacteriophage

lambda

requires

3

(hupA

hupB) strains carrying either

ihfA (previously termed himA) or ihfB (previously known as

hip or himD) mutations [13], phage clones

encoding

ihfA or ihfB function, respectively, were

identi¢ed.

2.2. DNA manipulations and sequencing

HU,

IHF protein for DNA packing and plaque forma-

Two

unique

plasmids,

one

expressing

ihfA (pOG2067) and one expressing ihfB (pHLP2) were recovered and their restriction maps determined

Standard recombinant DNA techniques were used, as described in [11]. A

(Fig. 1).

P. haemolytica EcoRI-XhoI

recombinant library was created using the

VZAP

vector and DNA was packaged in vitro. Putative

II

ihf

3.2. Deletion mapping and complementation of IHF function in E. coli

clones were identi¢ed as those that could produce

hupAB ihfA or hupAB ihfB strains,

A series of deletions of pOG2067 and pHLP2 was

as previously described [12]. Plasmids pOG2067 and

created to identify minimal regions that expressed

plaques on either

S.K. Highlander et al. / FEMS Microbiology Letters 146 (1997) 181^188

183

Fig. 1. Physical maps of P. haemolytica IHF subclones and complementation of IHF function in E. coli (indicated by a + and assayed as described in Table 1). Only P. haemolytica DNA sequences, cloned between the EcoRI and XhoI sites of pBluescript SK II vector (Stratagene, La Jolla, CA), are illustrated. The locations of open reading frames, obtained from DNA sequence of the region (see Fig. 2), are represented by the arrows above the maps; solid lines indicate regions where sequence was determined and dashed lines denote the expected length of £anking open reading frames, based on comparison to similar sequences in E. coli or H. in£uenzae. Speci¢c digests for creation of deletions were as follows: pBA2068, BamHI+BglII; pBA2069, SmaI; pBA2072, SspI partial digestion; pBA2084, PstI partial digestion; pOG2061, HindIII; pOG2062, HindII; pOG2063, StyI; pOG2064, BamHI+BglII; pOG2065, PstI-NsiI. N.T., not tested.

or ihfB, respectively (Fig. 1). Each deletion derivative was tested for its ability to complement ihfA or ihfB mutations by quantitating V plaque formation in A5427 or A5179 (Fig. 1 and Table 1). For pOG2067, this allowed us to place tentatively the ihfA gene within a 0.9 kb fragment between the BglII and PstI sites. For pHLP2, the ihfB gene was tentatively limited to region that included the central 0.4 kb HindIII-BglII fragment. ihfA

3.3. DNA sequence of ihfA and ihfB

We determined the nucleotide sequence of the ihfencoding regions on pBA2068 and pOG2065 (Fig. 2). The mol% G+C of the coding regions is 40 and 39% for ihfA and ihfB, respectively, consistent with other P. haemolytica genes. On pBA2068 (Fig. 2a), we observed a 99 amino acid ihfA ORF that is 67% identical and 82% similar (including identical and

184

S.K. Highlander et al. / FEMS Microbiology Letters 146 (1997) 181^188

S.K. Highlander et al. / FEMS Microbiology Letters 146 (1997) 181^188 Fig. 2. DNA sequence of

ihfA

(a) and

ihfB

(b) genes from

P. haemolytica.

185

Amino acid sequences, predicted from the DNA sequence, are

indicated. Partial peptide sequences for putative upstream and downstream ORFs are also shown. Potential translational regulatory sequences (purine-rich ribosome binding sites, start and stop codons) are indicated in boldface.

E. coli-like c70

promoter

310 and 335 sites

ihfA and ihfB are underlined. Inverted repeat sequences are indicated by the dashed arrows, and a consensus binding site for IHF, upstream of ihfB, is indicated by the carets (^). Sequences have been assigned GenBank accession numbers U56138 (ihfA) and U56139 (ihfB). upstream of

6

chemically related amino acids) to E. coli IhfA. As in E. coli, a sequence similar to pheT lies upstream of ihfA. A partial orf1, similar to a sequence found 3P to the putative Haemophilus in£uenzae ihfA gene, initiates downstream of the P. haemolytica gene. A potential c70 -like promoter sequence is located immediately upstream of the ihfA start codon and the putative 10 region is £anked by a perfect dyad repeat. This organization of potential expression signals is unlike that in the E. coli pheT-ihfA intergenic region, where only 4 bp separate the two genes and ihfA transcription initiates within the upstream pheST region [7]. For pOG2065 (Fig. 2b), a 93 amino acid ihfB ORF, that is 63% identical and 80% similar to E. coli IhfB, was detected. Again, the map location of ihfB, downstream of rpsA, is similar to that in E. coli, and rpsA is followed by a region of dyad symmetry, similar to the transcription terminator found

3

Table 1

E. coli molytica ihfA and ihfB-like genes

IHF complementation in

by plasmids expressing

P. hae-

eopa

Plasmid

ihfA complementation in A5427 (hupA hupB ihfA) pOG2067

0.1

pBA2068

NTb

1035

pBA2069

1032

pBA2072

1034

pBA2084

1034

pBluescript II SK

ihfB complementation in A5179 (hupA hupB ihfB) pHLP2

0.1

1036

pOG2061 pOG2062

1036

pOG2063

0.8

1036

pOG2064 pOG2065

0.1

1035

pBluescript II SK a

eop=e¤ciency of

VD69

plating with respect to strain A5196 (N99

hupA16 : :kan hupB11 : :cat).

b

NT=not tested. Strain could not be established.

3P to rpsA in E. coli. Sequences similar to a c70 promoter lie between rpsA and ihfA ; a putative IHF-binding site overlaps the 10 region of the putative promoter. The presence of this binding site suggests that ihfB expression could be repressed by IHF binding, as it is in E. coli. A second partial reading frame of unknown function, orf2, follows ihfB.

3

3.4. Phylogenetic relationships of IHF subunits Sequences of IHF subunits from eight Gramnegative organisms have been reported and mutations and peptide alignments have been used to illustrate genetic relationships and conservation of key residues [14]. We chose ¢rst to compare the peptides by generating phylogenetic trees using a distancematrix method (Fig. 3). Relative genetic distances (shown as numbers along each branch) were calculated for pairwise amino acid alignments using the PAM-250 amino acid substitution matrix [15]. On the unrooted trees, P. haemolytica sequences are clustered on branches with those deduced from H. in£uenzae ; this was anticipated since both organisms are members of a distinct bacterial family called HAP (Haemophilus-Actinobacillus-Pasteurella). Nevertheless, these sequences are not as closely related to one another as are subunits from enteric bacteria or the pseudomonads. Overall, subunit sequence similarities ranged from approx. 67^84% and identities from 40^67%, although Rhodobacter capsulatus sequences were the least similar. With respect to the E. coli sequences, IhfA shares 66 identical and an additional 14 similar amino acids, while IhfB has 59 identical and 15 similar residues. In comparison, the IhfA sequence from H. in£uenzae has 59 identical and 13 similar amino acids and IhfB has 62 identical and 13 similar residues, when compared to the P. haemolytica sequences. Alignments of predicted IhfA and IhfB subunits, showing conserved and non-conserved amino acids, are shown in Fig. 4.

186

S.K. Highlander et al. / FEMS Microbiology Letters 146 (1997) 181^188

Fig. 3. Phylogenetic trees of predicted IhfA (a) and IhfB (b) peptides from Gram-negative bacteria. Branch lengths (numbers between branch points) indicate the estimated phylogenetic distances between sequences, and the ¢lled circles denote the weighted centroid of each tree. Note that the Rhodobacter capsulatus branches have been truncated and their lengths are not drawn to scale. Trees were generated by the TreeGen algorithm on the Computational Biochemistry Research Group Server at the Swiss Federal Institute of Technology, Zuërich. Eco, E. coli; Ech, Erwinia chrysanthemi; Hin, Haemophilus in£uenzae; Pau, Pseudomonas aeruginosa; Ppu, Pseudomonas putida; Pha, Pasteurella haemolytica; Rca, Rhodobacter capsulatus; Sma, Serratia marcesens; Sty, Salmonella typhimurium.

Fig. 4. Alignment of predicted amino acid sequences for IhfA (a) and IhfB (b) subunits from Gram-negative bacteria. The alignments were generated using the GCG GAP program and the output manipulated using the BOXSHADE server at the Swiss Institute for Experimental Cancer Research, Lausanne. Identical residues are indicated by the black boxes and functionally similar residues by the shaded boxes. A consensus for each alignment appears below the sequence, where upper-case letters represent identities for all sequences and lower-case letters indicate amino acid similarity for at least 50% of the sequences. Eco, E. coli; Ech, Erwinia chrysanthemi; Hin, Haemophilus in£uenzae; Pau, Pseudomonas aeruginosa; Ppu, Pseudomonas putida; Pha, Pasteurella haemolytica; Rca, Rhodobacter capsulatus; Sma, Serratia marcesens; Sty, Salmonella typhimurium.

S.K. Highlander et al. / FEMS Microbiology Letters 146 (1997) 181^188

A model for IHF structure has been proposed based on the crystal structure of the similar heterodimeric histone-like protein, HU [16]. The new sequences presented here, along with the predicted sequences from H. in£uenzae, extend and con¢rm previous reports concerning sequence requirements for IHF function. According to the proposed IHF structure, residues 53^78 are predicted to form the `arms' of the heterodimer that are required for DNA binding [17]. Key arginine and lysine residues within the arms (IhfA R55, R60, R63, R74, K66; IhfB R56, R59, R62, K65, K74) are believed to make primary contact with the minor groove; these are completely conserved within the P. haemolytica and H. in£uenzae sequences. Mutations that changed amino acids P65 and K66 in IhfA and E44 in IhfB altered DNA binding speci¢city [17]; these are also conserved. Several site-directed changes (E5K, E8K, S50T, H54A, R59A, K65Q, H79A, K81A, and triple mutant R73A G74A K75A) in IhfB were shown to have no e¡ect on IHF function. Most of the original residues examined are the same in the P. haemolytica and H. in£uenzae sequences, but it is of interest to note that one of these (H79Y) was observed in H. in£uenzae IhfB and changes at residues 73 and 74 were observed in both the P. haemolytica (E73G G74A) and H. in£uenzae (E73S G74A) subunits; thus it appears that substitutions at positions 73, 74 and 79 can be tolerated. Residues within the amino-terminus of the protein are thought to be involved in the protein-protein interactions that permit dimer formation between IhfA and IhfB. The largest sequence variations were observed within this region, especially within the IhfB subunit. While some of the changes within this region are conservative, many are not. These changes may de¢ne critical residues that are involved in dimerization and may, in part, be the cause of the weak complementation that we observed using P. haemolytica genes in the heterologous E. coli system. The alignments in Fig. 4 also reveal the location of several positions that are not conserved. Within IhfA, no sequence conservation was observed at amino acids 11, 58, 89, 93, 96, 98 or 99. For IhfB, non-conserved residues occur at positions 15, 24, 35, 39, 71 and 93. Finally, the predicted sequence of IhfB from P. haemolytica includes a single cysteine residue at position 25 within the variable amino-terminal domain. The relevance of

187

this is not known, though the cysteine substitution is probably tolerated as a conservative replacement for the small alanine amino acid that occurs in other sequences. Acknowledgments

This work was supported by USDA grant 9303427 and a grant from the German-Israeli Foundation for Scienti¢c Research and Development. References [1] Friedman, D.I. (1988) Integration host factor: a protein for all reasons. Cell 55, 545^554. [2] Goosen, N. and Van de Putte, P. (1995) The regulation of transcription initiation by integration host factor. Mol. Microbiol. 16, 1^7. [3] Thompson, J.F. and Landy, A. (1988) Empirical estimation of protein-induced DNA bending angles: applications to V sitespeci¢c recombination complexes. Nucleic Acids Res. 16, 9687^9705. [4] Hoover, T.R., Santero, E., Porter, S. and Kustu, S. (1990) The integration host factor stimulates interaction of RNA polymerase with NIFA, the transcriptional activator for nitrogen ¢xation operons. Cell 63, 11^22. [5] Gober, J.W. and Shapiro, L. (1990) Integration host factor is required for the activation of developmentally regulated genes in Caulobacter. Genes Dev. 4, 1494^1504. [6] de Lorenzo, V., Herrero, M., Metzke, M. and Timmis, K.N. (1991) An upstream XylR- and IHF-induced nucleoprotein complex regulates the c54 -dependent Pu promoter of the TOL plasmid. EMBO J. 10, 1159^1167. [7] Mechulam, Y., Blanquet, S. and Fayat, G. (1987) Dual level control of the Escherichia coli pheST-himA operon expression. tRNAPhe -dependent attenuation and transcriptional operatorrepressor control by himA and the SOS network. J. Mol. Biol. 197, 453^470. [8] Highlander, S.K. and Weinstock, G.M. (1994) Static DNA bending and protein interactions within the Pasteurella haemolytica leukotoxin promoter region: development of an activation model for leukotoxin transcriptional control. DNA Cell Biol. 13, 171^181. [9] Mendelson, I., Gottesman, M. and Oppenheim, A.B. (1991) HU and integration host factor function as auxiliary proteins in cleavage of phage lambda cohesive ends by terminase. J. Bacteriol. 173, 1670^1676. [10] Miszuswa, S. and Ward, D.F. (1982) A bacteriophage lambda vector for cloning with BamHI and Sau3A. Gene 20, 317^322. [11] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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