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
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