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Journal of Bacteriology, July 2000, p. 3761-3766, Vol. 182, No. 13
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
andSchool of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
Received 3 February 2000/Accepted 17 April 2000
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ABSTRACT |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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The Escherichia coli K5 capsular polysaccharide
[-4)-
GlcA-(1,4)-
GlcNAc-(1-] is a receptor for the
capsule-specific bacteriophage K5A. Associated with the structure of
bacteriophage K5A is a polysaccharide lyase which degrades the K5
capsule to expose the underlying bacterial cell surface. The
bacteriophage K5A lyase gene (kflA) was cloned and
sequenced. The kflA gene encodes a polypeptide with a
predicted molecular mass of 66.9 kDa and which exhibits amino acid
homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E. coli SEBR 3282. There was only limited nucleotide
homology between the kflA and elmA genes,
suggesting that these two genes are distinct and either have been
derived from separate progenitors or have diverged from a common
progenitor for a considerable length of time. Southern blot analysis
revealed that kflA was not present on the chromosome of the
E. coli strains examined. In contrast, elmA was
present in a subset of E. coli strains. Homology was observed between DNA flanking the kflA gene of
bacteriophage K5A and DNA flanking a small open reading frame
(ORFL) located 5' of the endosialidase gene of the E. coli K1 capsule-specific bacteriophage K1E. The DNA homology
between these noncoding sequences indicated that bacteriophages K5A and
K1E were related. The deduced polypeptide sequence of ORFL
in bacteriophage K1E exhibited homology to the N terminus of KflA from
bacteriophage K5A, suggesting that ORFL is a truncated
remnant of KflA. The presence of this truncated kflA gene
implies that bacteriophage K1E has evolved from bacteriophage K5A by
acquisition of the endosialidase gene and subsequent loss of functional
kflA. A (His)6-KflA fusion protein was
overexpressed in E. coli and purified to homogeneity with a
yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme
was active over a broad pH range and NaCl concentration and was capable
of degrading K5 polysaccharide into a low-molecular-weight product.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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At least 80 distinct capsular polysaccharides (K antigens) have been described previously for Escherichia coli (26). Expression of a polysaccharide capsule on the cell surface confers resistance to host immune defenses and other adverse environmental conditions (3, 29). In addition, many of these structurally distinct capsules act as receptors for bacteriophages, and variation of capsular structure may be a mechanism for evasion of bacteriophage infection. Due to their specificity, capsule-specific bacteriophages can be used for identification of numerous E. coli K serotypes. Integral to the tail structure of many capsule-specific bacteriophages are enzymes which degrade the bacterial polysaccharide and provide the bacteriophages access to receptors on the cell surface (5, 32-35).
The E. coli K5 capsular polysaccharide is a polymer of
-4)-GlcA-(1,4)-GlcNAc-(1- (40). This structure is
identical to N-acetylheparosan, the precursor polymer of
heparin and heparan sulfate (17). Coliphage K5A, a
bacteriophage specific for E. coli K5 capsule-expressing
strains, has been previously described (9). This
bacteriophage contains a lyase which degrades the K5 polysaccharide
randomly throughout the polymer by a elimination reaction. The
final reaction products of this bacteriophage lyase consist of hexa-,
octa-, and decasaccharides (8, 12). In addition to the
bacteriophage-borne K5 lyase, a chromosomally encoded K5 lyase enzyme
has been described for E. coli SEBR 3282 (16).
The chromosomal gene (elmA) has been cloned and expressed in
E. coli K-12 and has been shown to produce final reaction
products with higher molecular weights than those of products observed for the bacteriophage lyase (12, 16).
In this communication, we report the cloning and analysis of the bacteriophage K5A lyase gene and describe the expression, purification, and characterization of the recombinant enzyme. We show that the bacteriophage K5A lyase gene is not found on the chromosome of E. coli K5 strains, whereas elmA is present on the chromosome of some strains. The difference in the distribution of the bacteriophage K5A lyase gene and elmA and lack of any shared nucleotide homology indicate that these two genes are distinct variants. These genes either have diverged extensively throughout the evolution of E. coli or have convergently evolved from separate ancestral genes. Additionally, we provide evidence indicating that bacteriophage K5A is likely to be the progenitor to the E. coli K1 capsule-specific bacteriophage K1E.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Bacterial strains, plasmids, and culture conditions.
E.
coli K-12 SURE {e14
mcrA
(mcrCB-hsdSMR-mrr)171 endA1 supE44 thi-1 gyrA96
relA1 lac recB recJ sbcC umuC::Tn5 uvrC
[F' proAB lacIq ZM15
Tn10]} was obtained from Stratagene, and E. coli K-12 TOP10 ([F mcrA
(mcrCB-hsdSMR-mrr) 
80lacZM15
lacX74 deoR recA1 araD139 (araA-leu)7697 galU galK rpsL endA1 nupG])
was obtained from Invitrogen. E. coli K5 wild-type strains
were obtained from K. Jann, Max Planck Institut für
Immunbiologie, Freiburg, Germany. Plasmids used in this study were
pTTQ18 (31) and pBAD/HisB (Invitrogen). Bacteria were
routinely grown at 37°C in Luria-Bertani (LB) medium supplemented
with 100 µg of ampicillin per ml when required. Bacteriophage K5A was
propagated using E. coli Bi8337-41 as a host in LB medium containing 0.2% D-glucose and 10 mM MgCl2.
DNA methods. Bacteriophage DNA was purified by the procedure described for bacteriophage lambda (30). Recombinant DNA techniques were performed according to standard procedures (30). Restriction and modifying enzymes were purchased from Boehringer, Mannheim, Germany. Double-stranded DNA sequencing was performed with custom oligonucleotide primers using the ABI PRISM BigDye Terminator cycle sequencing kit together with an Applied Biosystems automated DNA sequencer.
To construct the (His)6-KflA fusion protein, the kflA gene was amplified from pBL4 by PCR using Pfu DNA polymerase. Two primers, 5'AGGAAGATCTATGGCTAAATTAACCAAACCT3' and 5'TGCCGAATTCTTACTTAGGAAGGGCAGCTAG3', were constructed incorporating BglII and EcoRI restriction sites into the 5' and 3' ends of the kflA gene, respectively. The amplification product was ligated into BglII-EcoRI-digested pBAD/HisB to position kflA in frame with the N-terminal (His)6 fusion tag of the vector. The nucleotide sequences of the kflA and elmA genes were compared using the BLAST 2 program, which aligns two given nucleotide sequences (37).Small-scale recombinant protein expression and preparation of
cell lysates.
The bacteriophage genomic library was screened for
lyase activity by assaying lysates from pools of 10 recombinant clones in E. coli SURE. Individual clones were then screened from
the pools showing lyase activity. E. coli SURE cells (10 ml)
containing the genomic fragments in pTTQ18 were grown to an optical
density at 600 nm (OD600) of 0.6 at 37°C with shaking at
200 rpm and then induced by addition of 1 mM IPTG
(isopropyl--D-thiogalactopyranoside). Incubation was
then continued for 1 h, and the cells were harvested at
5,000 × g for 10 min. The cells were washed once in
ice-cold 100 mM Tris-Cl (pH 7.5), resuspended in a 1/10 volume of the
same buffer, and then sonicated with five 30-s bursts separated by 1 min of cooling in ice water. The sonicates were clarified by centrifugation at 10,000 × g for 20 min.
-mercaptoethanol, 0.05% [wt/vol] bromophenol
blue, pH 6.8) and boiled for 5 min. These lysates (10 µl) were used
directly for SDS-polyacrylamide gel electrophoresis (PAGE).
Purification of K5 polysaccharide. Bi8337-41 cells were grown overnight in 500 ml of LB broth and harvested at 5,000 × g for 10 min. The pellet was washed once in 50 ml of phosphate-buffered saline (pH 7.2), resuspended in 50 ml of extraction buffer (50 mM Tris-Cl, 5 mM EDTA, pH 7.3), and incubated at 37°C for 30 min. The cells were pelleted by centrifugation and treated three times further with extraction buffer. Polysaccharide was precipitated from the pooled supernatants by addition of cetyl-3-ethyl ammonium bromide (Na salt) to a final concentration of 0.1% (wt/vol) followed by incubation at room temperature for 16 h. The precipitate was recovered by centrifugation at 10,000 × g for 20 min at 20°C, dissolved in 1 M NaCl, and precipitated again by addition of ethanol to 80% (vol/vol). The precipitate was dissolved in 5 ml of distilled water and dialyzed against distilled water. The solution was centrifuged at 100,000 × g, and the supernatant containing K5 polysaccharide was freeze-dried.
K5 polysaccharide lyase assays. Polyacrylamide gel assays were performed as previously described (27). Briefly, lysate or purified fusion protein was incubated in 25 µl of 25 mM Tris-acetate (pH 8.5) containing 20 µg of K5 polysaccharide for 1 h at 37°C. A 1/10 volume of 2 M sucrose-0.02% (wt/vol) bromophenol blue in electrophoresis buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3) was added, and 20 µl of each reaction mixture was loaded onto a polyacrylamide gel (25% acrylamide, 0.82% bisacrylamide). Gels were pre-electrophoresed for 1 h at 12 V/cm, and samples were electrophoresed at 6 V/cm. Gels were stained by the combined alcian blue-silver staining method (24).
The spectrophotometric assay for lyase activity took advantage of the4,5 bond formed due to cleavage of the K5 polysaccharide
by elimination (2, 12). The A232
was monitored as a function of time using saturating concentrations of
K5 polysaccharide as substrate. The reaction rate was expressed in
absorbance units (AU) per minute. Typical reactions were performed at
37°C and contained 1 µg of purified KflA fusion protein and 800 µg of K5 polysaccharide in a 1-ml reaction mixture. The rate of
product formation was monitored over 5 min. Buffer conditions were
varied according to the nature of the specific experiment.
Purification of the (His)6-KflA fusion protein. E. coli TOP10(pLYA100) (500 ml) was grown in LB broth supplemented with ampicillin at 37°C at 200 rpm. When the OD600 reached 0.5, L-arabinose was added to a concentration of 0.02% (wt/vol) and the culture was incubated for a further 2 h. The bacteria were harvested at 4,000 × g for 10 min at 4°C. The pellet was washed in 200 ml of buffer A (50 mM Tris-Cl, pH 8.5) and resuspended in 40 ml of ice-cold buffer A. The cell suspension was passed once through a French pressure cell operated at 20,000 lb/in2. The resulting lysate was then centrifuged at 100,000 × g for 1 h at 10°C.
The entire lysate was loaded on a 5-ml Pharmacia HiTrap Q column (pre-equilibrated in buffer A) at 2 ml/min. This was followed by washing with 100 ml of buffer A at 5 ml/min. Elution was performed over 150 ml with a linear 0 to 0.5 M NaCl gradient (in buffer A). Fractions (2 ml) were collected, and those containing the most fusion protein, as determined by SDS-PAGE and Western blotting with-Xpress antibody,
were pooled.
The pooled solution from the anion-exchange chromatography was adjusted
to 500 mM NaCl and 0.1% (vol/vol) Triton X-100. This was then loaded
at 3 ml/min onto a 5-ml Pharmacia HiTrap chelating column (charged with
Ni2+), and the column was washed with 100 ml of buffer B
(50 mM Tris-Cl, 500 mM NaCl, 0.1% [vol/vol] Triton X-100, pH 8.5).
Proteins were eluted into 2-ml fractions at 5 ml/min with a linear 0 to
500 mM imidazole gradient (in buffer B) over 150 ml. Fractions judged to contain the KflA fusion protein by SDS-PAGE and Western blotting were pooled and concentrated in a Centriprep 30 concentrator (Amicon) according to the manufacturer's instructions. The concentrated eluant
was desalted in buffer C (50 mM Tris-Cl, 0.1% [vol/vol] Triton
X-100, pH 8.0) using four 5-ml Pharmacia HiTrap desalting columns
linked in a series. The desalted eluant was concentrated as described
above to 5 ml and stored at 4°C.
Additional analytical methods. SDS-PAGE was performed according to the method of Laemmli (13), and Western blotting was performed as described by Towbin et al. (38) using the ECL detection kit (Amersham). Protein concentration was estimated with the Bio-Rad protein assay kit using bovine gammaglobulin as a protein standard.
Nucleotide sequence accession number. The kflA nucleotide sequence has been submitted to the GenBank database under the accession no. Y10025.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Cloning and nucleotide sequencing of the coliphage K5 lyase gene. A library of bacteriophage K5A DNA was constructed in the expression vector pTTQ18 and transformed into E. coli SURE. Lysates from 250 recombinants were screened for lyase activity by overnight incubation with purified K5 polysaccharide followed by PAGE of the reaction products. One clone, pBL4, exhibited detectable lyase activity (data not shown). Plasmid pBL4 contained a cloned fragment of 2.8 kb, and Southern blot analysis confirmed that the cloned DNA was derived from the genome of bacteriophage K5A (data not shown).
The nucleotide sequence of the cloned DNA in pBL4 was determined. The insert comprised 2,872 bp with a G+C content of 48%. Two open reading frames (ORFs) were identified in the opposite orientation to the tac promoter of pTTQ18 (Fig. 1). The largest ORF (ORF635) was 1,905 bp and encoded a putative protein of 635 amino acids with a predicted molecular mass of 66.9 kDa. A putative ribosomal binding site was identified 10 bp upstream of the ORF635 translational initiation codon. A BLAST search (1) of the GenBank peptide database revealed 53% identity over 619 amino acids (68% similarity) between the ORF635 polypeptide and ElmA, a chromosomally encoded K5 polysaccharide lyase from E. coli SEBR 3282 (O10:K5:H4) (data not shown) (16). This amino acid homology indicates that ORF635 encodes the bacteriophage K5A lyase, and therefore, this gene was designated kflA (K5 lyase). Located 3' to kflA was a partial ORF (ORFP) encoding 147 amino acids (Fig. 1). ORFP was also preceded by a putative ribosomal binding site. A BLAST search of the GenBank database failed to find any proteins with significant homology to the polypeptide encoded by ORFP. The noncoding region of the cloned bacteriophage K5A DNA 5' to kflA contained homology to the consensus for SP6 bacteriophage promoters as described previously for the K1E bacteriophage (6, 22, 23). This implies that kflA is transcribed by a bacteriophage RNA polymerase.
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The kflA gene is distinct from elmA.
Comparison of the nucleotide sequences of kflA and
elmA using a pairwise BLAST2 alignment (36)
identified a short stretch of homology of 77% over 69 bases (data not
shown). The lack of extensive homology suggests that these two genes
are distinct and either have been derived from separate progenitors or
have diverged from a common progenitor for a considerable length of time. To determine if a chromosomal variant of kflA exists,
E. coli K5 wild-type isolates were analyzed by Southern blot
analysis using the kflA gene as a probe. No hybridization
was detected at low stringency among the strains examined (data not
shown). Therefore, it is apparent that KflA is not encoded on the
chromosome of E. coli strains and has evolved solely as a
phage-encoded enzyme distinct from elmA. Southern
hybridization of genomic DNA from the E. coli K5 wild-type
strains using the elmA gene as a probe at high stringency
revealed hybridization to a fragment of approximately 11.5 kb in 4 out
of 11 of these strains (Fig. 2).
Therefore, elmA is not present on the chromosome of all
E. coli K5 strains. Three of the E. coli strains
hybridized by the elmA probe (20026, 21786, and 21834) (Fig.
2, lanes 2, 5, and 9) have been shown to contain K5 lyase activity in
cell lysates and, in addition, produce shorter polymer on their
surfaces than those of other K5 strains (11). It is not
known whether the O15:K5 strain, which also hybridized to
elmA (Fig. 2, lane 8), exhibits intracellular lyase activity or low-molecular-weight polysaccharide. The correlation between K5
polymer size and the presence of elmA suggests that the
chromosomally encoded lyase may have a regulatory role in capsule
synthesis. The relationship between the presence of elmA in
E. coli and expression of low-molecular-weight K5 capsule
with the ecology and pathogenicity of such strains is unclear. It is
possible that ElmA may be encoded by a cryptic K5-specific
bacteriophage (distinct from K5A) which is present only in a subset of
K5 strains. A role for a chromosomally encoded ElmA lyase in control of
capsule size would be different from the role of the KflA enzyme, which
is utilized solely by bacteriophage K5A for recognition of E. coli cells expressing K5 capsule and subsequently to gain access
to the cell surface.
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Coliphage K5 is the progenitor of bacteriophage K1E. Analysis of the 5' noncoding region of the cloned bacteriophage K5A nucleotide sequence revealed that the first 437 nucleotides, up to the kflA translation initiation codon, exhibited 88% identity with sequence 5' to a small ORF (ORFL) encoded by the E. coli K1 capsule-specific bacteriophage K1E (Fig. 1A) (6, 22). In bacteriophage K1E, ORFL is located immediately 5' to an ORF encoding the endosialidase which degrades the polysialic acid capsule of E. coli K1 (22). In addition, the nucleotide sequence spanning 88 bp between the 3' end of kflA and ORFP of bacteriophage K5A exhibited 95% identity with the intergenic region between ORFL and the endosialidase gene (Fig. 1A). This nucleotide sequence homology between noncoding regions of bacteriophage K5A and K1E indicates that these two bacteriophages are related. It was also observed that the first nine consecutive amino acids of the KflA and the ORFL polypeptides were identical (Fig. 1B). The homology between these polypeptides could be extended to 41% identity (60% similarity) over the first 46 amino acids (Fig. 1B), although no homology was detected between the remaining 66 C-terminal amino acids of the ORFL polypeptide and sequences in KflA. Translation of ORFL has not been demonstrated, and the function of this ORF is unknown (22). However, the homology between the N termini of the ORFL polypeptide and KflA suggests that the ORFL may be a truncated remnant of kflA. Thus, it appears that bacteriophage K1E is a derivative of bacteriophage K5A in which the endosialidase gene was acquired by lateral transfer followed by the subsequent loss of the kflA gene. No homology was detected between the noncoding DNA 3' of the K1E endosialidase gene and bacteriophage K5A sequence, and therefore, it is not known if the endosialidase gene was inserted between kflA and ORFP or whether the gene represented by ORFP was replaced by the endosialidase gene. In addition, it cannot be determined whether the endosialidase gene was acquired by K1E before or after loss of the kflA gene.
Expression and purification of a (His)6-KflA fusion
protein.
The KflA lyase was overexpressed in E. coli
K-12 as a fusion protein containing an N-terminal (His)6
tag. The entire kflA gene was amplified by PCR and cloned
downstream of the araBAD promoter in the expression vector
pBAD/His. The resulting plasmid was designated pLYA100. Transcription
of kflA from the araBAD promoter is induced by
the presence of L-arabinose in a dose-dependent manner
(14, 15). Optimum expression of soluble KflA fusion protein
was obtained by induction at an L-arabinose concentration of 0.02% (wt/vol) (Fig. 3).
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Analysis of (His)6-KflA activity.
Cleavage of K5
polysaccharide with recombinant KflA was visualized by analyzing the
products of the lyase reaction by PAGE. Untreated K5 polysaccharide
preparations exhibit a wide range of molecular weights (Fig.
4, lane 1) as a result of varying levels of polymerization during biosynthesis. Incubation of K5 polysaccharide (800 µg/ml) with a dilution series of purified KflA fusion protein showed that at a high enzyme concentration (36 µg/ml) the
polysaccharide was degraded into a single low-molecular-weight product
(Fig. 4, lane 2). As the enzyme concentration was decreased from 9 to 2.25 µg/ml, the polysaccharide was not degraded to completion and
products of successively higher molecular weight were accumulated (Fig.
4, lanes 3 to 6). Since it has been previously shown that incubation of
bacteriophage K5A with K5 polysaccharide results in final reaction
products of hexa-, oct-, and decasaccharides (12), the
lowest-molecular-weight product observed following degradation with 36 µg of KflA per ml (Fig. 4, lane 2) is likely a hexasaccharide. Based
on the elimination reaction, the two sequentially
higher-molecular-molecular weight bands (Fig. 4, lanes 4 to 7)
represent octa- and decasaccharides, respectively. It has been
suggested that cleavage of the K5 polysaccharide by the KflA lyase into
oligosaccharides smaller than three repeating units may not be possible
due to a minimum chain length required for substrate recognition and
cleavage (12).
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GlcA-(1,4)-GlcNAc-(1-]n polymers
followed by epimerization of GlcA residues to iduronic acid and O
sulfation at various positions (17). These modifications are
not stoichiometric, and therefore, heterogeneity exists due to the
degree of epimerization and sulfation. Generally, heparin is more
extensively modified than heparan sulfate. Heparin lyases produced by
Flavobacterium heparinum are enzymes that degrade heparin
and heparan sulfate (21). Three forms of heparin lyase have
been purified and exhibit different specificities for heparin and
heparan sulfate. Applications of these enzymes include structural studies of heparin-like polymers (18, 19), synthesis of
low-molecular-weight therapeutic agents (20), and
neutralization of heparin in blood. These enzymes do not have amino
acid homology to KflA although they exhibit high pH optima similar to
the bacteriophage enzyme (21). KflA recognizes heparan
sulfate as a substrate, cleaving the polymer at regions devoid of
iduronic acid and sulfation (K. Lidholt, personal communication). Thus,
it is possible that the recombinant KflA lyase may be of use as a tool
for structural studies of heparin and heparan sulfate and production of
low-molecular-weight, highly modified heparin-like oligosaccharides.
In conclusion, the gene encoding a bacteriophage lyase
(kflA) specific for the E. coli K5 capsular
polysaccharide has been cloned and sequenced, and a His-tagged KflA
fusion protein has been overexpressed and purified to homogeneity in an
active form. The KflA lyase is not encoded on the E. coli
chromosome, and kflA is distinct from the elmA
polysaccharide lyase gene which is present on the chromosome of a
subset of E. coli serotypes. Therefore, these two genes are
distinct and either have been derived from separate progenitors or have
diverged from a common progenitor for a considerable length of time. In
addition, evidence has been provided which suggests that bacteriophage
K5A is a progenitor to the E. coli K1 capsule-specific
bacteriophage K1E.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Cell Factories Initiative of Framework IV of the European Commission and from the B.B.S.R.C. of the United Kingdom. I.S.R. gratefully acknowledges the support of the Lister Institute of Preventive Medicine.
We thank M. Rolinni for her help with this project.
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FOOTNOTES |
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* Corresponding author. Mailing address: School of Biological Sciences, 1.800 Stopford Building, University of Manchester, Manchester M13 9PT, United Kingdom. Phone: 44 161 275 5601. Fax: 44 161 275 5656. E-mail: ISRobert{at}fs1.scg.man.ac.uk.
Present address: Department of Microbiology, Edo State University,
Ekpoma, Nigeria.
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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