Department of Biostructural Chemistry, Institute of Molecular and Structural Biology, Aarhus University, Gustav Wiedsvej 10C, DK-8000 Aarhus C, Denmark1
Department of Medical Microbiology and Immunology, The Bartholin Building, Aarhus University, DK-8000 Aarhus C, Denmark2
Author for correspondence: Knud Poulsen. Tel: +45 89421736. Fax: +45 86196128. e-mail: kp{at}microbiology.au.dk
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ABSTRACT |
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Keywords: Streptococcus agalactiae, infB , initiation factor 2, clonal population structure
The EMBL accession numbers for the sequences reported in this paper are AJ003164 and AJ251493 to AJ251499.
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INTRODUCTION |
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IF2 interacts with , GTP, IF1, IF3 and both 30S and 50S ribosomal subunits. Through these interactions, IF2 promotes the binding of the initiator tRNA to the 30S ribosomal subunit and catalyses the hydrolysis of GTP following 70S initiation-complex formation. Initiation-complex formation has been extensively reviewed (Gold, 1988
; Gualerzi & Pon, 1990
; Hershey, 1987
; Maitra et al., 1982
). IF2 from E. coli exists in three forms: IF2-1 (97·3 kDa), IF2-2 (79·7 kDa) and IF2-3 (78·8 kDa). A new nomenclature for translation factors has been proposed by International Union of Biochemistry and Molecular Biology (1996)
. According to this new nomenclature, IF2
, IF2ß and IF2
are named IF2-1, IF2-2 and IF2-3, respectively. The IF2-2 and IF2-3 forms are both translated from in- frame translation-initiation codons in the single-copy gene infB . Outside the family Enterobacteriaceae, internal translation initiation within infB has been found in Bacillus subtilis, where two forms of IF2 were detected: IF2-1 (78·6 kDa) and IF2-2 (68·2 kDa) (Hubert et al., 1992
; Shazand et al., 1990
).
In this study, we confirm the separation of 58 strains of S. agalactiae into separate evolutionary lineages as previously reported by Hauge et al. (1996) . Furthermore, we found that a partial sequence of infB was useful as a molecular marker for phylogenetic analysis of S. agalactiae and closely related species. The amino acid sequence deduced from the full-length sequence of infB from eight strains of S. agalactiae verified the high interspecies variation in the N- terminal sequence of IF2 and the conserved C-terminal region. The predicted start codon of infB in S. agalactiae was verified by subcloning and overexpression in E. coli followed by purification and N-terminal sequencing of an N-terminal fragment of IF2 from S. agalactiae.
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METHODS |
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Southern blot analysis.
The Southern blots were prepared in a previous study on the population structure of S. agalactiae (Hauge et al., 1996 ) and reused for hybridization at low stringency including a stepwise decline in temperature as described by Poulsen et al. (1996)
.
Construction and screening of the S. agalactiae 3076 genomic library.
Whole-cell DNA from S. agalactiae strain 3076 was prepared as described by Hauge et al. (1996) and a partial Sau3AI digest was fractionated by agarose gel electrophoresis. Fragments in the size range from 10 to 20 kb were extracted from the gel by electroelution and used to prepare a genomic library using
L47.1 as a BamHI substitution vector (Sambrook et al., 1989
). The recombinant phages were packaged in vitro with Gigapack II Packaging Extracts (Stratagene) and plated on E. coli K802. Positive plaques were identified by in situ hybridization using a DNA fragment labelled with [32P]dATP as probe. The low-stringency conditions used in the hybridization were as described previously (Poulsen et al., 1996
). A single positive plaque was purified by replating on E. coli K802 and phage DNA was isolated from a 20 ml phage lysate (Mikkelsen et al., 1985
).
DNA sequencing.
phage DNA and different segments of the infB gene amplified by PCR served as template in the sequencing reactions. The PCRs were done using ThermoPrime Plus DNA Polymerase (Advanced Biotechnologies) or ReadyToGo PCR beads (Amersham Pharmacia Biotech) following the protocols supplied with the enzymes. Degenerate oligonucleotide primers for PCR and sequencing were designed by back- translation of selected conserved stretches in an alignment of IF2 sequences from species closely related to S. agalactiae. Specific primers for PCR and sequencing were made as the sequence of infB from S. agalactiae was obtained. Primers for amplifying and sequencing the central variable part of infB were 5'-TACTGAGGGCATGACCGTTGC-3' and 5'- GACACCCGCAGCTTTAGAGTGAT-3'. All oligonucleotide primers were purchased from DNA Technology. The amplicons were purified for sequencing as described by Hedegaard et al. (1999)
or using the Wizard Minicolumns purification system (Promega). The sequencing was performed using the Thermo Sequenase fluorescent labelled primer cycle sequencing kit (Amersham Pharmacia Biotech) and analysed on an ALF DNA sequencer (Amersham Pharmacia Biotech) or using Thermo Sequenase dye terminator cycle sequencing kit (Amersham Pharmacia Biotech) and analysed on an ABI 377 DNA sequencer (Perkin Elmer).
Sequence analyses.
Sequences obtained were analysed and edited using programs contained in the GCG software package (Genetics Computer Group, University of Wisconsin, Madison, WI, USA). Phylogenetic trees were constructed using the distance-matrix method and parsimony analysis. The distances were corrected using the Kimura two-parameter method (Kimura, 1980 ). All trees were generated by heuristic searches with tree-bisection branch-swapping and resampled with 100 bootstrap replications to test the robustness of the data. A tree based on 16S rRNA gene sequences was constructed and compared to the one obtained from infB sequence data. All phylogenetic analyses were performed with PAUP 4.0.0d55 for UNIX (Distributed by D. L. Swofford & S. Olson, Laboratory of Molecular Systematics, Smithsonian Institution).
Expression in E. coli, purification and characterization of an N-terminal fragment of IF2 from S. agalactiae .
An 1108 bp fragment containing the translational-regulatory region and the part of infB encoding the first 330 aa of IF2 from S. agalactiae strain 3076 was termed S. agalactiae infB9912784. This fragment was ligated into pCP40 as an EcoRI/BamHI-digested PCR product made using the primers EcoRI-Forward (5 '-GGCAGGGAATTCAAGAAAAGTGGTTGCTGTCGC-3') and BamHI-Reverse (5'-GGGACGTGGATCCTGTTACTGGTTATGGAG-3'). The obtained plasmid, pCP40(S. agalactiae infB
9912784), was transformed into competent E. coli UT5600(pcI857) cells, prepared as described by Chung et al. (1989)
; plated on 2xTY agar containing 100 µg ampicillin ml-1 and 50 µg kanamycin ml -1 and incubated overnight at 30 °C. Colonies were screened for the correct insert by PCR using the primers pCP40-Forward (5'-ACTGGCGGTGATACTGAGC-3') and Saga819-Reverse (5'- TTTACGACGACTATCTGCTGT-3'). An overexpression test was performed by inoculating 3 ml 2xTY containing 100 µg ampicillin ml-1 and 50 µg kanamycin ml -1 with selected colonies and incubating at 30 °C for 7 h, followed by 42 °C for 1 h and analysing for overexpression by SDS-PAGE. DNA sequencing of plasmid DNA using the pCP40-Forward primer furthermore verified the presence of the correct insert.
Colonies of E. coli UT5600[pcI857, pCP40(S. agalactiae infB9912784)] were inoculated into 2xTY containing 100 µg ampicillin ml-1 and 50 µg kanamycin ml-1, and grown overnight at 30 °C. The overnight culture was diluted 1:100 into 2xTY containing 100 µg ampicillin ml-1 and 50 µg kanamycin ml-1 and grown at 30 °C to an OD550 of 1. Overexpression of the N- terminal fragment, termed S. agalactiae IF2
331927, was induced by diluting the cultures with an equal volume of 2xTY medium containing 100 µg ampicillin ml-1 and 50 µg kanamycin ml-1 at 56 °C, followed by growth for 1·5 h at 42 °C. The cells were harvested by centrifugation at 2200 g for 20 min at 4 °C and the cell pellet was washed with 0·9% NaCl. Samples were analysed for overexpression by SDS-PAGE. The harvested cells were resuspended in buffer H(300) [Buffer H is 50 mM HEPES, pH 7·6, 1 mM DTT, 0·1 mM PMSF, 15 mM NaN3 and NaCl as indicated in parentheses, e.g. H(300) has an NaCl concn of 300 mM) and disrupted by passing through an Aminco French pressure cell at 1500 p.s.i. (10350 kPa). The cell extract was clarified by 1 h centrifugation at 30000 g at 4 °C. S. agalactiae IF2
331927 was captured on an S-Sepharose Fast Flow 50/3 column (Amersham Pharmacia Biotech), moderately purified on an S-Sepharose High Performance 16/10 column (Amersham Pharmacia Biotech), concentrated in Centricon30 (Amicon), highly purified in an AcA54 16/92 column (Biosepra) and finally concentrated in Centricon30. All chromatographic steps were performed at 4 °C using an ÄKTAexplorer system (Amersham Pharmacia Biotech). The S-Sepharose FF and S-Sepharose HP columns were equilibrated with H(300) before loading the sample. The FF column was used at a flow rate of 7 ml min-1 and bound protein was eluted in a linear gradient from H(300) to H(800) in 3·5 column vols. The HP column was used at a flow rate of 2 ml min-1 and bound protein was eluted in a step gradient from H(300) to H(430) in 4 column vols, to H(530) in 0·3 column vols and to H(1000) in 4 column vols. Isocratic elution with H(100) from the AcA54 column was performed at 0·2 ml min-1. During the purification, absorption at 280, 270 and 254 nm were measured on- line and fractions were analysed by SDS-PAGE. The final protein concentration was determined by measuring A280. Purified S. agalactiae IF2
331927 was sequenced at the N terminus by automated Edman degradation essentially as described by Nyengaard et al. (1991)
.
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RESULTS AND DISCUSSION |
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A fragment of infB encoding the central and conserved part of IF2 was amplified by PCR using degenerate oligonucleotide primers and DNA of phage Sa-IF2-4.4 as the template. The amplicons were purified and subsequently sequenced. The terminal parts of infB and flanking regions were sequenced using phage DNA as the template. A sequence of 3391 bp was obtained covering, in addition to infB , the gene downstream, rbfA, encoding ribosome-binding factor A. The identity of both genes was verified by comparison with known sequences of infB and rbfA from other species. In S. agalactiae strain 3076, infB was found to constitute an ORF of 2784 bp starting with a UUG start codon and with the potential of encoding a 102·4 kDa protein of 927 aa with a calculated pI of 9·0. Due to the strongly basic N-terminal region this pI value is exceptionally high, compared to IF2 in other prokaryotes. The proposed translation-start codon was confirmed by heterologous overexpression in E. coli, purification and N-terminal sequencing of an N-terminal fragment of S. agalactiae IF2. A 1108 bp fragment including sequences upstream of the start codon and encoding the first 330 aa (32·9 kDa, pI 9·9), was subcloned into pCP40 and the obtained plasmid, pCP40(S. agalactiae infB
9912784), was transformed into competent E. coli UT5600(pcI857) followed by overexpression and purification of S. agalactiae IF2
331927 (Fig. 1
). The first 5 aa of the purified recombinant protein were determined as Ser-Lys-Lys-Arg-Leu, which is in agreement with the deduced amino acid sequence, assuming that the N- terminal fMet is removed by methionyl-aminopeptidase (Hirel et al. , 1989
).
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The sequence of infB encoding the GTP-binding domain of IF2, which is homologous among species, is expected to be useful as a phylogenetic marker since infB is present in all bacteria as a single copy gene and has been shown to contain sufficient variation (Hedegaard et al., 1999 ; Steffensen et al. , 1997
). The DNA sequences of infB from Gram- positive species related to S. agalactiae and from Mycoplasma species supposed to originate from Gram-positive bacteria and, as outgroup, the Gram-negative E. coli, were aligned and the central part of the gene was used for phylogenetic analyses. A distance tree based on 16S rRNA sequences from the same species was constructed and compared to the topology obtained from infB sequence data (Fig. 3
). The topology of the 16S rRNA tree obtained resembles the topology of 16S rRNA trees from the literature except for a difference in the position of Enterococcus. This is presumably because different parts of the 16S rRNA sequences and different methods for constructing the trees have been used. The genus Enterococcus groups with Bacillus, as in the work of Stackebrandt & Teuber (1988)
, whereas it groups with Streptococcus and Lactococcus in the work of Schleifer & Kilpper-Bälz (1987)
and Olsen et al. (1994)
. The overall topology of the infB tree is equivalent to the 16S rRNA tree but supports the assumption that Enterococcus is closely related to the two other genera of Streptococci, Streptococcus and Lactococcus, rather than to Bacillus. Consequently, our results indicate that the partial sequence of infB is useful as a molecular marker for phylogenetic studies within the Gram-positive bacteria.
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ACKNOWLEDGEMENTS |
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This work was funded by grants from the Familien Hede Nielsens Fund and the Biotechnology Programme of the Danish Natural Science Research Council (28807-9502036, 9602401) to H. U. Sperling-Petersen and by grants from the Danish Medical Research Council (9702265) to M. Kilian.
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Received 24 December 1999;
revised 30 March 2000;
accepted 10 April 2000.