tRNA gene clusters at the 3' end of rRNA operons are specific to virulent subgroups of Streptococcus agalactiae strains, as demonstrated by molecular differential analysis at the population level

Karine Rolland1, Laurent Mereghetti1, Stephane Watt1, Sonia Chatellier1 and Roland Quentin1

Département de Microbiologie Médicale et Moléculaire, EA 3250, Unité de Bactériologie, Centre Hospitalier Universitaire Bretonneau, 37044 Tours Cedex 01, France1

Author for correspondence: Roland Quentin. Tel: +33 2 47 47 80 56. Fax: +33 2 47 47 38 12. e-mail: quentin{at}med.univ-tours.fr


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The aim of this work was to characterize a 2·4 kb randomly amplified polymorphic DNA (RAPD) fragment described as a marker for a phylogenetic group of Streptococcus agalactiae strains significantly associated with neonatal meningitis. This fragment was analysed by cloning and sequencing, and showed that two types of tRNA gene cluster flank the 3' end of the rRNA operons in S. agalactiae strains. Both types of tRNA gene cluster act as markers for phylogenetic subgroups of strains within the species. One type could be used to distinguish two of the three virulent intraspecies subgroups to which most of the S. agalactiae strains able to invade the central nervous system of neonates belong. This raises the possibility that there is a link between these tRNA genes and the virulence of the bacterium. Based on this analysis, PCR primers were designed to determine whether S. agalactiae strains are likely to belong to lineages of organisms in which most of the highly virulent strains isolated from cerebrospinal fluid cluster. In addition, this work demonstrated that RAPD can be used to detect novel particularities within intraspecies variants of pathogens.

Keywords: neonatal meningitis, molecular analysis, RAPD

Abbreviations: CNS, central nervous system; MEE, multilocus enzyme electrophoresis; RAPD, randomly amplified polymorphic DNA

The GenBank accession numbers for the sequences reported in this paper are AF302130 (for the 2·4 kb amplified fragment) and AF302131 (for the 2·6 kb amplified fragment).


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptococcus agalactiae often colonizes the genital and gastrointestinal tracts of adults without causing symptoms, but it is also a major cause of neonatal meningitis and septicaemia (Schuchat, 1999 ). Indeed, two-thirds of infections due to S. agalactiae occur in pregnant women and neonates. The newborn becomes colonized with S. agalactiae by aspiration of colonized amniotic fluid or during passage through the birth canal. Therefore, the risks of neonatal infection are directly correlated with maternal carriage of the bacterium (Baker, 1997 ).

Serotyping and molecular characterization studies of S. agalactiae isolates from various anatomical sites have suggested that only a limited number of strains from the vagina can cause neonatal meningitis. In the United States, over two-thirds of the cases of S.-agalactiae-related neonatal diseases are caused by strains that produce the type III antigen (Baker & Edwards, 1995 ). In some countries, other serotypes are frequently isolated from patients with early-onset disease (Geslin et al., 1992 ). Molecular genetic studies have demonstrated that there are two major phylogenetic lineages of S. agalactiae (Helmig et al., 1993 ; Musser et al., 1989 ; Quentin et al., 1995 ). In addition, most of the strains responsible for neonatal meningitis are non-randomly distributed in these two phylogenetic groups (Musser et al., 1989 ; Quentin et al., 1995 ). Therefore, the different clones of S. agalactiae strains must have different virulence attributes. However, no molecular data are currently available to support this conclusion.

An insertion sequence (IS1548) has been discovered in the hylB gene, which encodes hyaluronate lyase, of a group of S. agalactiae strains isolated from patients with endocarditis (Granlund et al., 1998 ) and in a homogeneous subgroup of isolates identified by multilocus enzyme electrophoresis (MEE). This sequence identified strains able to invade the central nervous system (CNS) of neonates (Rolland et al., 1999 ). Nevertheless, many other strains isolated from the cerebrospinal fluid of neonates with meningitis do not possess this insertion sequence.

Many of the current strategies used to prevent neonates from becoming infected with S. agalactiae at birth are based on treating all women who provide positive lower vaginal or anorectal cultures with intrapartum chemoprophylaxis by intravenous administration of penicillin G or ampicillin (Baker, 1997 ). These approaches may have adverse impacts; for example, they may lead to the emergence of resistant pathogens and some patients have serious allergic reactions to penicillin. Therefore, it is important to seek genomic particularities that characterize invasive groups of strains. This will enable us to clarify the molecular mechanism of invasion of this subgroup of S. agalactiae strains and to recognize strains in the genital flora that are likely to cause invasive diseases. This may also help obstetricians to limit the use of prophylaxis and, therefore, its adverse effects.

We previously carried out randomly amplified polymorphic DNA (RAPD) experiments to assess the ability of 29 different oligonucleotide primers to characterize 114 S. agalactiae strains of various anatomical origin. One primer (5'-AgggggTTCC-3') generated a 2·4 kb fragment significantly associated with strains belonging to one of the two major lineages of the S. agalactiae species (Chatellier et al., 1997 ). Therefore, this fragment may have specific genetic features that account for its significant association with the phylogenetic distribution of strains within the S. agalactiae species. In this study, we used the same collection of strains as above to characterize this 2·4 kb fragment and to determine whether its composition contributes to this specific association. This approach identified two tRNA gene clusters flanking the 3' end of the rRNA operons in S. agalactiae. Specific combinations of tRNA genes were found to be associated with the two major lineages of S. agalactiae, and these could be used to identify two of the three virulent intraspecies subgroups of strains able to cause neonatal meningitis. Based on this analysis, we designed PCR primers to determine whether given S. agalactiae strains have a high probability of belonging to a virulent subgroup of organisms. In addition, this work illustrated that differential population analysis of bacterial genomes characterized by molecular epidemiological methods may be used to identify new genetic particularities within intraspecies variants of pathogens.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains.
We studied the 114 strains that have been used previously to establish the phylogenetic structure of the S. agalactiae species (Quentin et al., 1995 ; Chatellier et al., 1996 , 1997 ; Rolland et al., 1999 ). A molecular population genetic study, using MEE, showed that this population is divided into two lineages, phylogenetic divisions I and II. Three virulent families of clones contain most of the strains that can invade the CNS of neonates (phylogenetic division I and two electrophoretypes of phylogenetic division II, ET11 and ET12) (Quentin et al., 1995 ). Forty-one of the 114 strains belonged to phylogenetic division I, and 73 belonged to phylogenetic division II. Six strains from phylogenetic division II belonged to the invasive ET11, and 15 belonged to the invasive ET12. Ten of the ET12 strains possess an insertion sequence (IS1548) in the hylB gene (Rolland et al., 1999 ). RAPD analysis carried out with primer 5'-AgggggTTCC-3' showed that the 2·4 kb RAPD fragment was generated for 37 of the 41 phylogenetic division I strains, but for only 1 of the 73 phylogenetic division II strains (Chatellier et al., 1997 ).

Isolation and cloning of the 2·4 kb RAPD fragment.
The 2·4 kb fragment, amplified by RAPD using the 5'-AgggggTTCC-3' primer (Chatellier et al., 1997 ), was subjected to electrophoresis in a 1·4% low-melting-point-agarose (FMC) gel containing ethidium bromide (1 mg l-1). The fragment was purified from the agarose by incubating the gel at 65 °C for 15 min, followed by two phenol extractions (v/v) and a chloroform extraction (v/v). DNA was precipitated with 0·1 M ammonium acetate and absolute ethanol. The precipitated DNA was air-dried and resuspended in 10 µl double-distilled water. The DNA fragment was inserted into the pCRII vector (TA cloning kit, Invitrogen). The fragment was partially sequenced at the 5' and 3' ends with universal primers SP6 and T7 (both from Eurogentec).

PCR.
A number of different primers were designed, based on known sequences (Table 1). A 20 µl PCR mixture was prepared as follows: PCR buffer (10 mM Tris/HCl, 50 mM KCl, 1·5 mM MgCl2; pH 8·3); 0·1 mM of each dNTP (Boerhinger Mannheim); 0·5 µM primers (Eurogentec) (Table 1); 0·5 U Taq polymerase (Perkin Elmer); 25 ng of template DNA, prepared as previously described (Chatellier et al., 1997 ). The cycling conditions were as follows: initial denaturation at 94 °C for 30 s, followed by 30 cycles at 94 °C for 15 s, 55 °C for 15 s and 72 °C for 15 s (additional 15 s at 72 °C for the last extension). The products were separated in a 1% agarose gel in TBE buffer (8·9 mM Tris, 8·9 mM boric acid, 0·25 mM EDTA; pH 8·0). Amplified products contained within the gel were stained with ethidium bromide and visualized by UV transillumination. A 1 kb ladder (Life Technologies) was used as a molecular size standard. The negative control consisted of a reaction in which the DNA template was replaced with double-distilled water. The DNA fragments were subsequently sequenced.


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Table 1. Primers used for sequencing and for PCR analysis

 
Sequencing.
Nucleotide sequences were determined by cycle sequencing, based on the chain-termination method of Sanger (1975) . Cloned products or PCR products were sequenced with the ThermoSequenase dye terminator cycle sequencing pre-mix kit, following the manufacturer’s recommendations (Amersham Life Sciences). Sequenced products were purified with Microcon concentrators (Amicon), to eliminate unused reagents. Products were resolved in a 4·2% acrylamide:bis-acrylamide (19:1) gel (Appligène) containing 6 mM urea in TBE buffer (8·9 mM Tris, 8·9 mM boric acid, 0·25 mM EDTA; pH 8·0) using an ABI Prism 377 DNA sequencer (Perkin Elmer), according to the manufacturer’s instructions.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and sequencing of the 2·4 kb RAPD fragment
We sequenced the extremities of the 2·4 kb RAPD fragment that had been inserted into the pCRII vector. The sequences obtained were used to design two primers, RAPDSP6 and RAPDT7 (Table 1), which were used to test all 114 strains. Three results were obtained: (i) amplification of a 2·4 kb fragment; (ii) amplification of a 2·6 kb fragment; or, (iii) no amplification (Fig. 1a). The correlation between these data and the phylogenetic position of the strains are reported in Table 2. The 2·4 kb fragment was significantly associated with the strains in phylogenetic division I (P<10-4). Moreover, amplification of this 2·4 kb fragment or the absence of amplification was significantly associated with strains belonging to phylogenetic group I and ET11, two of the three clone families of strains able to invade the CNS of neonates (P<10-4).



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Fig. 1. Amplified products whose patterns correlate with the phylogenetic positions of S. agalactiae strains. (a) Results of PCR with primers RAPDSP6 and RAPDT7 (Table 2). A 2·4 kb fragment was observed (lanes 3 and 4), a 2·6 kb fragment was observed (lanes 5 and 6), or no amplified product was observed (lane 2). Lane 1 contains a DNA molecular mass marker. (b) Results of PCR with primers SP6-B2 and T7-B2 (Table 2). Pattern P1, composed of a 1·2 kb fragment (lanes 2 and 4), was observed and pattern P2, composed of two fragments of 1·2 and 1·4 kb in size (lanes 3 and 5), was observed. Lane 1 contains a DNA molecular mass marker.

 

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Table 2. Distribution of amplified products obtained with primers defined at the 5' and 3' extremities of a 2·4 kb RAPD fragment with respect to the phylogenetic position and to the invasive groups of S. agalactiae strains

 
The 2·4 and 2·6 kb DNA phylogenetic markers reflect differences in the genetic composition of the tRNA gene clusters flanking rRNA operons
To determine the genetic differences between the 2·4 and 2·6 kb DNA fragments, the two fragments were completely sequenced using the primers described in Table 1. All six strains examined gave similar results. The nucleotide sequences of the two fragments were 94% similar. The sequences of the 2·4 and 2·6 kb DNA fragments were compared with sequences registered in the GenBank database. They were 70–95% similar to sequences from Streptococcus oralis, Streptococcus uberis, Streptococcus parauberis, Streptococcus cremoris, Streptococcus thermophilus, Lactococcus lactis and Bacillus subtilis (Fig. 2). The 5' ends of these sequences mapped to the ribosomal operon of S. agalactiae. The sequence from nucleotide 9 to nucleotide 740 (2·4 kb fragment) or to nucleotide 741 (2·6 kb fragment) encodes the 23S RNA, followed by a promoter sequence and the 5S rRNA gene. The sequence downstream from the rRNA operon, between nucleotides 756 and 862 (2·4 kb DNA fragment) or between nucleotides 756 and 1105 (2·6 kb DNA fragment), encoded two types of tRNA gene cluster (tRNA gene clusters 1 and 2, Fig. 2). The region corresponding to tRNA gene cluster 1 consisted of 107 bp encoding a tRNAGly. The region corresponding to tRNA gene cluster 2 encoded three different tRNAs: a tRNAVal, a tRNALeu and a tRNALys. Subsequent sequences of the 2·4 kb DNA fragment (nucleotides 896–1680) and the 2·6 kb DNA fragment (nucleotides 1141–1925) were identical. These sequences encoded successively a tRNAAsp, a tRNAGlu, a tRNASer, a tRNAMet, a tRNAPhe, a partial tRNATyr, a tRNATrp, a tRNAHis, a partial tRNAGln and a partial tRNALeu. The remaining 714 (2·4 kb fragment) or 716 nt (2·6 kb fragment) at the 3' ends of the fragments were 87% similar. These sequences did not match any of the sequences registered in the GenBank database. Overall, the 2·4 and 2·6 kb DNA regions associated with the various genetic groups of strains making up the S. agalactiae species differed in terms of the tRNA gene clusters at the 3' ends of the rRNA operons.



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Fig. 2. Genetic features of the 2·4 and 2·6 kb DNA fragments of S. agalactiae strains obtained by PCR with primers RAPDSP6 and RAPDT7 (Table 2) that are associated with the intraspecies phylogenetic distribution of strains. Only one nucleotide region differs between these two fragments, corresponding to two types of tRNA gene cluster (tRNA clusters 1 and 2) located downstream of the ribosomal operon of S. agalactiae strains. Minus symbols indicate the absence of a base; dots indicate identical bases; asterisks above and below the sequences indicate different bases. *This tRNA composition is similar in tRNA gene clusters 1 and 2; 91 and 84% identity with tRNAGly of B. subtilis and L. lactis, respectively; 73% identity with tRNAVal of B. subtilis; §76% identity with tRNALeu of B. subtilis; {int}79% identity with tRNALys of B. subtilis.

 
Use of tRNA gene differences to optimize PCR determination of the phylogenetic position of S. agalactiae strains
As described above, the two fragments (2·4 and 2·6 kb) amplified with RAPDSP6 and RAPDT7 were associated with the phylogenetic clustering of S. agalactiae strains (Table 2). However, it is difficult to distinguish between the two fragments on a 1% agarose gel, due to their high molecular mass and the small difference in their sizes. In addition, no amplification product was obtained for 15 of the 114 strains (Table 2). We therefore tested all of the primers used for sequencing (Table 1) to determine whether a pair of these primers could be used to establish the phylogenetic position of S. agalactiae strains more easily and successfully. All of the primers tested, except the SP6-B2 and T7-B2 primer pair, generated two fragments, both of which were over 1·5 kb long and which differed in size by no more than 200 nt, requiring the use of low-concentration agarose gels for their separation by electrophoresis. In contrast, the SP6-B2 and T7-B2 primer pair generated two patterns that were easy to differentiate. The first (pattern P1, Fig. 1b) consisted of a 1·2 kb DNA fragment and the second (pattern P2, Fig. 1b) consisted of a 1·2 and 1·4 kb fragment. Sequencing showed that the 1·2 kb DNA fragments of patterns P1 and P2 were identical. The 1·4 kb DNA fragment of pattern P2 was 75% similar to the 1·2 kb DNA fragments. The differences between these two nucleotide sequences were similar to those observed between the 2·4 and 2·6 kb fragments, consisting of two different tRNA gene clusters (Fig. 2).

Pattern P1 was significantly associated with the position of S. agalactiae strains in MEE phylogenetic division I (38 P1 strains versus three P2 strains in division I, Table 3), and pattern P2 was significantly associated with division II strains (21 P1 strains versus 52 P2 strains in division II, Table 3) (P<10-4). In addition, pattern P1 distinguished five of the six ET11 strains, but it distinguished only 16 of the 67 strains belonging to ET12 or non-invasive subgroups. Therefore, pattern P1 appears to be a good marker for two of the three subgroups (division I and ET11) of isolates able to invade the CNS of neonates (P<10-4) (Table 3).


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Table 3. Distribution of the P1 and P2 patterns obtained by PCR with primers SP6-B2 and T7-B2 with respect to the phylogenetic position and to the invasive groups of the S. agalactiae strains

 
It is rare that two primers can amplify two fragments under specific conditions, as in the case of pattern P2. Given the positions at which SP6-B2 and T7-B2 prime (Table 1), and that six ribosomal operons have been identified for S. agalactiae species (Dimitriev et al., 1998 ), the double PCR amplification observed in pattern P2 probably indicates that some of the rRNA operons of the strains exhibiting this pattern are flanked by tRNA gene cluster 1 and that some are flanked by tRNA gene cluster 2.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
By characterizing an RAPD fragment that acts as a marker for some phylogenetic and anatomical subgroups of S. agalactiae isolates, we identified new genetic particularities within intraspecies variants of S. agalactiae strains, consisting of two types of tRNA gene cluster that flank the 3' end of the ribosomal operons of S. agalactiae strains. These two clusters correspond to the phylogenetic groups of strains within the S. agalactiae species, including subgroups that can invade the CNS of neonates. This raises questions concerning the possible link between tRNAs and the virulence of the bacterium. It is well known that virulence genes organized in pathogenicity islands are flanked by tRNA genes (Hou, 1999 ). This link could not be established in our strains because no virulence genes were detected in the sequence downstream from tRNA gene clusters 1 and 2. In addition, no specific junction sites similar to the tRNA loci flanking pathogenicity islands in Gram-negative bacteria have yet been identified in Gram-positive bacteria (Hacker et al., 1997 ). Another link between tRNA and bacterial virulence has been established in Shigella flexneri; in this organism, it has been demonstrated that a modified tRNA nucleoside is involved in the expression of the virF gene and in maintaining the correct reading frame (Durand et al., 1997 ; Björk et al., 1999 ).

Our results link tRNA genes to some virulent phylogenetic subgroups of S. agalactiae. This is related to the composition of the tRNA gene clusters located at the 3' end of rRNA operons rather than to tRNA modification. Indeed, the rRNA operons of the two virulent clones of S. agalactiae strains (phylogenetic division 1 and ET11) always seem to be flanked by tRNA gene cluster 1. In contrast, rRNA operons of the other genetic groups of strains were flanked by either of the two types of tRNA gene clusters. As tRNAs are a central interface between the information stored in DNA and its expression in proteins, and as tRNA gene cluster 1 was the only cluster associated with two of the three virulent clones of S. agalactiae strains, it is possible that this tRNA gene cluster is involved in the synthesis of proteins responsible for bacterial pathogenicity. Nevertheless, tRNA gene cluster 1 is also present in the genome of non-invasive subgroups of bacteria but, in these strains, some of the rRNA operons are flanked by tRNA gene cluster 2. Therefore, if tRNA gene cluster 1 is involved in the expression of virulence factors, the smaller number of copies of this tRNA gene cluster in strains that also contain tRNA gene cluster 2 may result in there being fewer proteins involved in virulence.

Whatever the link between tRNA gene cluster composition and virulence in S. agalactiae strains, the specificities described here may be used to identify (by PCR with primers SP6-B2 and T7-B2, Table 1) isolates belonging to two of the three genetic groups of strains that can invade the CNS of neonates (division I and ET11). In addition, as we have shown previously, strains belonging to ET12, the third genetic subgroup of strains able to invade the CNS of neonates, are significantly marked by IS1548 in the hylB gene (Rolland et al., 1999 ). Therefore, there is a high probability that strains that exhibit pattern P1 or contain IS1548 belong to one of these three subgroups (55 of 62 strains) rather than to the non-invasive subgroups (16 of 52 strains) (P<10-4) (Table 3). These two characteristics may be detected by PCR with primers SP6-B2 and T7-B2, as described here, or with primers Hyal1 (5'-CAgCCACTCATAgCACAATgAAACAAg-3') and Hyal2 (5'-gCTAgTTAgATAgCTAATTgTCTgTT-3'), as described previously (Rolland et al., 1999 ). These two pairs of primers may provide a valuable basis for the development of a multiplex PCR method for identifying virulent isolates among the strains present in the genital tract of pregnant women. PCR appears to be a more suitable method than RAPD for developing tools, as the interlaboratory reproducibility of RAPD remains unclear (Saunders et al., 2001 ). In addition, PCR with the primer pair SP6-B2 and T7-B2, as performed in this work, identifies five of the six strains belonging to the virulent clone ET11; these strains were not identified with the 2·4 kb RAPD fragment.

By contrast, differential display of DNA following RAPD seems to be a suitable method for detecting some new genetic specificities within some subgroups of bacteria, as has been suggested previously (Hacker et al., 1997 ). The identified sequences may be phylogenetic markers, pathogenicity islands or genetic elements that play a role in cell physiology or in the expression of virulence factors. RAPD is a quick and inexpensive method, making it possible to use many primers to explore many parts of the genome for bacterial populations composed of numerous strains. This method may be as effective as subtraction hybridization for the identification of new genetic elements involved in the physiology or pathogenicity of bacteria.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Région Centre to K.R. and from the Centre Hospitalier Universitaire de Tours to R.Q.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Baker, C. J. (1997). Group B streptococcal infections. Clin Perinatol 24, 59-70.[Medline]

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Björk, G. R., Durand, M. D., Hagervall, T. G., Leipuviene, R., Lundgren, H. K., Nilsson, K., Chen, P., Qian, Q. & Urbonavicius, J. (1999). Transfer RNA modification: influence on translational frameshifting and metabolism. FEBS Lett 452, 47-51.[Medline]

Chatellier, S., Huet, H., Kenzi, S., Rosenau, A., Geslin, P. & Quentin, R. (1996). Genetic diversity of rRNA operons of unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid of neonates suffering from meningitis. J Clin Microbiol 34, 2741-2747.[Abstract]

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Received 21 September 2001; revised 16 January 2002; accepted 21 January 2002.



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