Investigation of the translation-initiation factor IF2 gene, infB, as a tool to study the population structure of Streptococcus agalactiae

Jakob Hedegaard1, Majbritt Hauge2, Jeppe Fage-Larsen1, Kim Kusk Mortensen1, Mogens Kilian2, Hans Uffe Sperling-Petersen1 and Knud Poulsen2

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The sequence of infB, encoding the prokaryotic translation-initiation factor 2 (IF2), was determined in eight strains of Streptococcus agalactiae (group B streptococcus) and an alignment revealed limited intraspecies diversity within S. agalactiae. The amino acid sequence of IF2 from S. agalactiae and from related species were aligned and revealed an interspecies conserved central and C-terminal part, and an N-terminal part that is highly variable in length and amino acid sequence. The diversity and relationships in a collection of 58 genetically distinct strains of S. agalactiae were evaluated by comparing a partial sequence of infB. A total of six alleles were detected for the region of infB analysed. The alleles correlated with the separation of the same strains of S. agalactiae into major evolutionary lineages, as shown in previous work. The partial sequences of infB were furthermore used in phylogenetic analyses of species closely related to S. agalactiae, yielding an evolutionary tree which had a topology similar to a tree constructed using 16S rRNA sequences from the same species.

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
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RESULTS AND DISCUSSION
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In the past 25 years Streptococcus agalactiae, also known as group B streptococci, has emerged as the leading cause of illness and death among newborn infants (Schuchat, 1998 ). An estimated 7500 episodes of neonatal sepsis and meningitis occurred in 1998 in the USA (information provided by the Centers for Disease Control and Prevention, the National Center for Infectious Diseases and the Division of Bacterial and Mycotic Diseases). S. agalactiae is part of the normal microflora in the vagina in many women from where it may colonize the newborn during delivery. It is not completely understood why some infants develop disease upon colonization. At present, no vaccine preventing invasive diseases caused by S. agalactiae is available and intrapartum prophylactic treatment with antibiotics is used routinely in some countries (Schuchat, 1999 ). The polysaccharide capsule is recognized as a major virulence factor in S. agalactiae and, in addition, the bacterium may express a number of proteins including C5a peptidase, hyaluronidase, alpha-antigen or RIP, and beta-antigen that are potential virulence factors (Cleat & Timmis, 1987 ; Li et al., 1997 ; Pritchard & Lin, 1993 ; Stlhammar-Carlemalm et al., 1993 ; Wexler et al., 1985 ). Differences in pathogenic potential among subpopulations of S. agalactiae have been disputed. Musser et al. (1989) identified a high-virulence clonal type of S. agalactiae serotype III in a collection of strains from the USA, Quentin et al . (1995) found that among French isolates three clonal families were responsible for the majority of invasive disease in neonates and Granlund et al. (1998) described an association between strains characterized by the presence of an insertion element within the hyaluronidase gene and endocarditis. We found that a population of S. agalactiae strains isolated in Denmark was predominantly clonal and there was no evidence of association between specific evolutionary lines and neonatal disease (Hauge et al., 1996 ). Additional molecular markers with a suitable variation within the species would be valuable tools for epidemiological and evolutionary studies on S. agalactiae to evaluate the discrepancies described above. Sequence variation in infB, encoding the prokaryotic translation-initiation factor 2 (IF2), has been studied in different isolates of Escherichia coli (Steffensen et al., 1997 ) and the part of infB encoding the GTP-binding domain of IF2 has been found to contain adequate variation for use as a phylogenetic marker within the family Enterobacteriaceae (Hedegaard et al., 1999 ).

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{alpha}, IF2ß and IF2{gamma} 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Bacterial strains and cloning vectors.
The S. agalactiae strains used in this study for complete sequencing of the infB gene were 3164, 941, 3114, 3076, 949, 3074, 3163 and 3098 of serotypes III, Ia, Ia, Ib, II, III, III and Ia, respectively, representing the clonal types 4, 13, 15, 25, 37, 43, 52 and 58, respectively, described by Hauge et al. (1996) . Strains 3164 and 3163 represented the so-called low and high virulent types, respectively, isolated in the USA (Musser et al., 1989 ), whereas the remaining strains were human isolates from Denmark. In addition, 50 strains representing each of the remaining clonal types described by Hauge et al. (1996) were included for partial sequencing of the infB gene. The strains were grown at 37 °C in atmospheric air plus 5% CO2 on blood agar or in Todd–Hewitt broth. Bacteriophage {lambda}L47.1 (Loenen & Brammar, 1980 ) was used as a BamHI substitution vector and recombinant phages were plated on E. coli K802 (Wood, 1966 ) as described by Sambrook et al. (1989) . E. coli UT5600 (Elish et al., 1988 ) was used as host for the thermo-inducible runaway-replication plasmid pCP40 in combination with the regulatory plasmid pcI857 (Remaut et al., 1983 ). The expression of genes inserted in pCP40 is under control of the {lambda}pL promoter and the temperature- sensitive cI repressor. The gene inserted must contain its own translational regulatory elements.

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 {lambda}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.
{lambda} 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 infB{Delta}991–2784. 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 {Delta}991–2784), 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 infB{Delta}991–2784)] 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{Delta}331–927, 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{Delta}331–927 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{Delta}331–927 was sequenced at the N terminus by automated Edman degradation essentially as described by Nyengaard et al. (1991) .


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Sequencing and characterization of infB from S. agalactiae strain 3076
We have previously cloned and characterized the infB gene encoding IF2 from Lactococcus lactis (accession no. AJ005118). In the present study we have used a DNA probe encoding the GTP-binding domain of L. lactis IF2, i.e. Ile396 to Val584 , to screen for an infB homologue in S. agalactiae . Southern blot experiments using low stringency conditions for hybridization showed that the probe recognized a single EcoRI restriction fragment of the same size in the genome of all 58 strains analysed (data not shown). Thus, the probe appeared to be highly specific under the conditions used. Subsequently, the same probe and conditions were used to identify a positive clone, {lambda}Sa-IF2-4.4, in a genomic lambda phage library of S. agalactiae strain 3076. Recognition sites for the restriction enzymes EcoRI and HindIII were mapped within the DNA of {lambda}Sa-IF2-4.4. The results showed that this recombinant phage contained an insert of 12·5 kb including an EcoRI fragment of 10·6 kb with homology to the infB probe from L. lactis as revealed by Southern blot analyses.

A fragment of infB encoding the central and conserved part of IF2 was amplified by PCR using degenerate oligonucleotide primers and DNA of phage {lambda}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 {Delta}991–2784), was transformed into competent E. coli UT5600(pcI857) followed by overexpression and purification of S. agalactiae IF2{Delta}331–927 (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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Fig. 1. Coomassie brilliant blue R250 stained SDS-PAGE summarizing the purification of S. agalactiae IF2{Delta}(331–927). MW: molecular mass marker (Amersham Pharmacia Biotech); S30: sample from supernatant after centrifugation of cell extract at 30000 g; S-FF: sample of pooled fractions after chromatography on S-Sepharose-FF; S-HP: sample of pooled and concentrated fractions after chromatography on S-Sepharose- HP; AcA: sample of pooled and concentrated fractions after chromatography on AcA54. The fragment has a calculated molecular mass of 32·9 kDa but migrates as an ~40 kDa protein due to the very basic pI of the fragment.

 
Homology between IF2 sequences from S. agalactiae and related species
IF2 sequences from several bacterial species are available from public databases. A comparison of the sequences shows that this initiation factor varies considerably in size among different species from 571 aa (63·2 kDa) in Thermus thermophilus (accession no. Z48001) to 1054 aa (111·3 kDa) in the myxobacterium Stigmatella aurentica (accession no. X87940; Bremaud et al., 1997 ). With 927 aa (102·4 kDa), IF2 from Sre. agalactiae is among the larger IF2 proteins. A general feature observed when comparing IF2 sequences from different bacteria is a conserved C terminus and an N terminus that is highly variable in length and amino acid sequence. The amino acid sequences of IF2 from S. agalactiae and four closely related species were aligned (Fig. 2). A strong homology of the central and C-terminal part is observed, whereas the N-terminal part is more variable except for a homologous stretch including the first 50 aa. A segment, Ile185–Glu237 in S. agalactiae IF2, was also found to be conserved between S. agalactiae and S. pyogenes IF2.



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Fig. 2. Alignment of selected sequences of IF2. B. subt , Bacillus subtilis (accession no. Z18631; Shazand et al., 1990 ); E. faec, Enterococcus faecium (accession no. M36878; Friedrich et al., 1988 ); S. agal, Streptococcus agalactiae 3076 (accession no. AJ003164; this work); S. pyog, Streptococcus pyogenes (The Streptococcal Genome Sequencing Project); and L. lact , Lactococcus lactis (accession no. AJ005118). The proposed domain borders are shown by >|< and the five essential regions, G1 to G5, of the GTP-binding motif are indicated by a ~. The polymorphic amino acid positions in S. agalactiae IF2 are indicated by asterisks.

 
A structural model has been proposed for IF2 from E. coli, dividing it into six structural domains (Mortensen et al., 1998 ). No function has been assigned to domain I (aa 1–157); domain II (aa 158–289) interacts with the ribosomal 30S and 50S subunits (Moreno et al., 1998 , 1999 ); domain III (aa 290–389) is the link between domain II and IV; domain IV (aa 390–559) is responsible for the binding of GTP and exhibits GTPase activity together with the 50S ribosomal subunit; domain V (aa 560–671) interacts with the T arm of (Yusupova et al., 1996 ) and may, together with IF1, mimic the structure of elongation factor G at the ribosomal A site (Brock et al., 1998 ; Ævarsson et al., 1994 ); domain VI (aa 671–890) is believed to contain the recognition site of IF2 (Gualerzi et al., 1991 ). Domain III and/or V may interact with the initiation factor IF1 during translation initiation (Moreno et al. , 1999 ). Based on the strong homology of the central and C-terminal part, the six-domain model of E. coli IF2 can be adapted to IF2 from other bacteria regarding domains IV, V and VI, while the N-terminal domains can not be assigned due to lack of homology. Consequently, S. agalactiae IF2 can be divided into an N-terminal region (aa 1–427) and three domains, Glu428 –Ala598, Asp599–Arg710 and Val711–Lys927, being homologous to domains IV, V and VI, respectively, in E. coli IF2 (Fig. 2). The GTP-binding motif in domain IV of IF2 is well characterized and five regions, G1 to G5, essential for the activity have been described (Bourne et al., 1991 ). As indicated in Fig. 2 the S. agalactiae IF2 contains the consensus sequence for each of the five regions.

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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Fig. 3. Dendrograms based on sequence data from infB and 16S rRNA using the distance method. For both infB and 16S rRNA sequences, phylogenetic analyses using the parsimony method resulted in one most parsimonious tree, having a topology identical to the one obtained by the distance method. The numbers at branch sites are the percentages of bootstrap replications supporting the branch. The infB tree is based on data from S. agalactiae, accession no. AJ003164 (this work); S. pyogenes (the streptococcal genome sequencing group); L. lactis, accession no. AJ005118; Ent. faecium, accession no. M36878 (Friedrich et al., 1988 ); B. subtilis, accession no. Z18631 (Shazand et al., 1990 ); B. stearothermophilus, accession no. X04399 (Brombach et al. , 1986 ); M. genitalium, accession no. U39695 (Fraser et al., 1995 ); M. pneumoniae, accession no. AE000062 (Himmelreich et al., 1996 ) and Esc. coli, accession no. K01175 (Sacerdot et al., 1984 ). The central part of infB (bp 1285–1794 in S. agalactiae infB) was used for phylogenetic analyses. The parsimony analyses showed that of 519 characters, 259 were parsimony informative (202 were identical and 58 were parsimony uninformative). The 16S rRNA tree is based on data from S. agalactiae, accession no. AB002479; S. pyogenes, accession no. AB002521; L. lactis, accession no. M58837; Ent. faecium, accession no. Y18294; B. subtilis, accession no. AF008220 (Green et al., 1985 ); B. stearothermophilus, accession no. X60640 (Ash et al., 1991 ); M. genitalium, accession no. X77334; M. pneumoniae, accession no. M29061 (Weisburg et al., 1989 ) and Esc. coli, accession no. J01695 (Brosius et al., 1978 ). A segment of the 16S rRNA gene corresponding to nucleotide positions 43–1374 with reference to the S. agalactiae 16S rRNA gene was used for the analyses. Positions containing ambiguously determined nucleotides were deleted in all 16S rRNA sequences. The parsimony analyses showed that of 1343 characters, 416 were parsimony informative (760 were identical and 167 were parsimony uninformative).

 
Diversity in the sequence of infB among S. agalactiae strains
The infB sequence obtained for S. agalactiae strain 3076 was used to design oligonucleotide primers for amplification by PCR and sequencing of infB in an additional seven human isolates of S. agalactiae. The results divide the eight isolates into four alleles of infB sequences termed A to D (Fig. 4). Within infB, no deletions or insertions were observed and 14 polymorphic nucleotide positions were found, leading to three polymorphic amino acid positions in IF2, each with two possible amino acids. Residue 94 is Pro in allele A, B and C but Ser in allele D; residue 169 is His in allele A and B, and Tyr in allele C and D; and residue 744 is Ala in allele A, C and D, and Val in allele B (Fig. 4). The number of polymorphic nucleotide positions is lower than previously found for infB in E. coli, where a total of 66 polymorphic positions were detected in the sequence of infB among 10 human isolates (Steffensen et al., 1997 ). The polymorphic nucleotide positions were unevenly distributed within infB, as the majority of the positions were located in the part encoding the GTP- binding domain and the C-terminal region of IF2. This resembles the distribution of variations found in infB from E. coli . In contrast, the observed number of polymorphic amino acid positions was higher than that reported among E. coli, where only a single amino acid variation was found (Steffensen et al., 1997 ). The sequence results confirm our previous finding in E. coli that the sequence of infB/IF2 is extremely conserved within species.



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Fig. 4. Polymorphic positions detected for infB (top) and IF2 (bottom) in eight strains of S. agalactiae. Domain borders, as defined by homology to E. coli IF2, and functional regions are indicated. The borders between domains I, II and III are dashed because their exact position can not be determined due to lack of homology to E. coli IF2. The four allele groups (A–D) are indicated.

 
To further assay for variation in the infB gene among S. agalactiae strains, we included an additional 50 strains for sequencing of the central variable region of infB encoding Lys 361–Gln517. To obtain a diverse collection of S. agalactiae, the strains, together with the eight strains described above, were selected to represent each of the 58 clonal types described by Hauge et al. (1996) . A total of six alleles were detected for the region of infB analysed. In addition to the four alleles shown in Fig. 4, which we will refer to as A to D as indicated, one allele termed E, represented by two closely related strains of clonal types 55 and 56, was identical to allele A except for a silent-site mutation of an A instead of T at position 1104, and one allele termed F, represented by a single strain of clonal type 28, was identical to allele A except for a T instead of G at position 1170 resulting in a conservative substitution of Glu 390 to Asp. We observed a correlation between alleles of the region of infB analysed and the division into six major lineages, termed I through VI, based on our previous clonal typing of S. agalactiae (Hauge et al., 1996 ) (Fig. 5). Thus, all 16 strains representing lineage V had the infB allele C, a cluster of six closely related strains within lineage II had allele B, whereas the remaining two distantly related strains of lineage II had infB allele A like the 11 strains of lineage I, the two strains of lineage IV, and 16 out of 17 strains of lineage III analysed. The exceptional strain of lineage III had the unique infB allele F as described above. Two of four strains of lineage VI had infB allele E, strain 3098 belonging to clonal type 58 of this lineage had the unique allele D, and the strain included of clonal type 57, lineage VI had infB allele A. This association between certain infB alleles and distinct clonal lineages strongly supports our previous suggestion of dividing this population of S. agalactiae into distinct evolutionary lineages. Notably, division of serotype III strains into two genetically distinct subpopulations, lineage I and V, is confirmed by differences in their infB alleles. The fact that some lineages shared the same infB allele may indicate that these are more closely related than suggested by the topology of the dendrogram.



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Fig. 5. Association between phylogenetic lineages and specific alleles of the central variable part of infB among 58 different clonal types of S. agalactiae. The dendrogram is adapted from Hauge et al. (1996) and was originally constructed on the basis of 23 different characteristics. The serotype of the strains is indicated. The dagger symbol indicates that the full length sequence of infB was determined in these strains.

 
In conclusion, our results, based on variation in a partial sequence of infB, support the previously proposed clonal population structure of S. agalactiae. Though less discriminative, the central variable segment of infB proved to be useful as a molecular marker for phylogenetic studies of the species. The prokaryotic IF2 has been studied in several bacteria and the C-terminal part is found to be conserved between different species while the N- terminal part is characterized by its variability in both length and composition. Several different steps in the protein-synthesis mechanism can be affected by antimicrobial drugs and the species-specific N terminus of IF2 may be useful as a target for species-specific selective drugs.


   ACKNOWLEDGEMENTS
 
The authors are grateful to Jens Jacob Hansen for providing the probe used for Southern blotting and to Katja Adolf for providing the sequence of infB from Lactococcus lactis. The sequence of infB from Streptococcus pyogenes was provided by B. A. Roe, S. P. Linn, L. Song, X. Yuan, S. Clifton, R. E. McLaughlin, M. McShan and J. Ferretti from the Streptococcal Genome Sequencing Project at the University of Oklahoma funded by USPHS/NIH grant AI38406.

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.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 24 December 1999; revised 30 March 2000; accepted 10 April 2000.