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Applied and Environmental Microbiology, December 2005, p. 8706-8713, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8706-8713.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Allelic Variation of Polymorphic Locus lytB, Encoding a Choline-Binding Protein, from Streptococci of the Mitis Group

Miriam Moscoso,{dagger} Virginia Obregón,{dagger},{ddagger} Rubens López, José L. García, and Ernesto García*

Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain

Received 31 May 2005/ Accepted 27 July 2005


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ABSTRACT
 
The choline-binding protein LytB, an N-acetylglucosaminidase of Streptococcus pneumoniae, is the key enzyme for daughter cell separation and is believed to play a critical pathogenic role, facilitating bacterial spreading during infection. Because of these peculiarities LytB is a putative vaccine target. To determine the extent of LytB polymorphism, the lytB alleles from seven typical, clinical pneumococcal isolates of various serotypes and from 13 additional streptococci of the mitis group (12 atypical pneumococci and the Streptococcus mitis type strain) were sequenced. Sequence alignment showed that the main differences among alleles were differences in the number of repeats (range, 12 to 18) characteristic of choline-binding proteins. These differences were located in the region corresponding to repeats 11 to 17. Typical pneumococcal strains contained either 14, 16, or 18 repeats, whereas all of the atypical isolates except strains 1283 and 782 (which had 14 and 16 repeats, respectively) and the S. mitis type strain had only 12 repeats; atypical isolate 10546 turned out to be a {Delta}lytB mutant. We also found that there are two major types of alternating repeats in lytB, which encode 21 and 23 amino acids. Choline-binding proteins are linked to the choline-containing cell wall substrate through choline residues at the interface of two consecutive choline-binding repeats that create a choline-binding site. The observation that all strains contained an even number of repeats suggests that the duplication events that gave rise to the choline-binding repeats of LytB involved two repeats simultaneously, an observation that is in keeping with previous crystallographic data. Typical pneumococcal isolates usually grew as diplococci, indicating that an active LytB enzyme was present. In contrast, most atypical isolates formed long chains of cells that did not disperse after addition of purified LytB, suggesting that in these strains chains were produced through mechanisms unrelated to LytB.


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INTRODUCTION
 
Streptococcus pneumoniae (pneumococcus) is a major human pathogen and a leading cause of pneumonia, bacteremia, and meningitis in adults and otitis media in children. Choline-binding proteins (ChBPs) are among the most well-known surface proteins that have been investigated to date in pneumococcus due to their role in pathogenesis, as well as the fact that they are candidate antigens for improved conjugate vaccines. Choline, which is absolutely required for normal pneumococcal growth, is a structural component of teichoic and lipoteichoic acids and anchors ChBPs to the surface of S. pneumoniae (13, 16). In addition, in the ChBPs that hydrolyze the cell wall, such as the major LytA autolysin, LytB, LytC, and Pce, choline-containing teichoic acids are also needed for enzymatic activity (see references 26 and 27 for recent reviews). ChBPs are also important since they represent a paradigm for supporting the modular theory of protein evolution. These proteins are built by combination or fusion of different functional domains with a peculiar choline-binding domain (ChBD) consisting of up to 18 choline-binding repeats (ChBRs) of about 20 amino acid residues each (27). Crystallographic studies have revealed that the ChBD of LytA is organized as a left-handed superhelix consisting exclusively of ß-hairpins and that linkage of ChBPs to the choline-containing cell wall substrate is carried out through choline residues at the interface of two consecutive ChBRs that create a choline-binding site (11, 12). Similar structures have been reported recently for the ChBD of the Cpl-1 lysozyme, a ChBP encoded by the pneumococcal phage Cp-1 (19), and the Pce phosphocholine esterase (18).

The lytBR6 allele (2,109 bp) codes for an 81.9-kDa protein containing a 23-amino-acid, cleavable signal peptide (predicted Mr of the processed protein, 79,317) (5). The N-terminal moiety of LytBR6, which is responsible for the cell wall binding, contains 18 ChBRs, whereas the C-terminal moiety confers the enzymatic specificity. It has been shown recently that the LytB glucosaminidase is the key enzyme for cell separation at the end of cell division; i.e., lytB pneumococcal mutants grow and form long chains of cells, and reversion to this phenotype can occur upon addition of purified LytB to the culture medium (5, 14). We have suggested that LytB catalyzes a final step in cell division that disperses intact cells.

Gosink and coworkers reported that lytB mutants showed significantly reduced colonization of the nasopharynx but exhibited a behavior similar to that of the parental lytB+ strain when they were tested in a model of pneumococcus-induced sepsis (17). On the other hand, it has been reported that immunization of mice with LytB induced protective antibodies in a mouse sepsis model (37), making this enzyme a good candidate for the development of improved vaccines against S. pneumoniae. In addition to being immunogenic and protective, a reliable vaccine candidate should ideally be structurally stable (to avoid misfolding of the surface-located proteins) and conserved. Unfortunately, only very limited information on LytB variation is currently available. To date, the nucleotide sequence of the lytB gene has been determined for four pneumococcal strains, namely, the unencapsulated laboratory strain R6 (14, 20), the type 19F clinical isolate G54 (8), and the type 4 strains N4 (37) and TIGR4 (36). Although it has been reported that N4 is an ancient designation for the TIGR4 strain (36), unexpectedly, the lengths of the sequences of these strains included in the data banks are clearly different (1,845 and 1,977 bp for N4 and TIGR4, respectively) (see below).

Precise identification of species in the mitis group of streptococci (23) is still a challenging task, as illustrated by the fact that some atypical pneumococci that exhibit some properties similar to those of the atypical isolates studied here have recently been reclassified as a new species, Streptococcus pseudopneumoniae (2). Furthermore, atypical S. pneumoniae strains have been identified as isolates that are bile (deoxycholate)-insoluble streptococci, and some genetic and physiological peculiarities of this group have recently been studied in detail (33).

To extend our studies of ChBPs, we decided to investigate the allelic variation and molecular evolution of LytB, a choline-binding surface protein, in clinical isolates identified as typical and atypical pneumococci, as well as the organization of the lytB gene of the type strain of Streptococcus mitis. We found that the main difference among the LytB alleles involves the number of repeats of the ChBD.


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MATERIALS AND METHODS
 
Strains, growth conditions, and purification of LytB.
The streptococcal strains used in this study are shown in Table 1. Cultures were grown either in Todd-Hewitt broth supplemented with 0.5% yeast extract or in C medium (24) supplemented with 0.08% yeast extract (C+Y medium). Escherichia coli BL21(DE3)(pRGR5) was used as a source of LytB. This enzyme was overproduced and purified by affinity chromatography on DEAE-cellulose as previously described (5).


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  		TABLE 1. Some characteristics of the streptococcal strains used in this study

PCR amplification and nucleotide sequencing.
Chromosomal DNA from typical pneumococcal isolates was prepared as previously described (10). The DNA extraction procedure described by Ezaki et al. (9) was used for all other streptococcal isolates. Routine DNA manipulations were performed as described elsewhere (33). The relevant oligonucleotide primers used were 5'pyrDII (position 857346; 5'-CCCATCTCTTACGTCGTCC-3'), 5'pyrD (position 858134; 5'-GACAACCCTTGAACCACG-3'), lytBN0 (position 858816; 5'-ACCAATCCTTATGCCTGCCC-3'), lytB-23 (position 860399; 5'-GTCGTATGGCTAGATAAGG-3'), lytB-C (position 861138/c; 5'-ccgaattctTACTAATCTTTGCCACCTAGC-3'), lytB-C2 (position 861277/c; 5'-GGCTTTGTCACTTACACAGG-3'), 3'flpA (position 862061/c; 5'-GAATAAGGCTGAGCGCGAC-3'), and 3'hyp (position 863146/c; 5'-CATCTGTCGGACGGTCGG-3') (the position numbers indicate the positions of the first nucleotide of the primer in the sequence reported previously [accession no. AE007317]; c indicates that the sequence corresponds to the complementary strand; lowercase letters indicate nucleotides introduced to construct appropriate restriction sites [indicated by underlining]). Unless indicated otherwise, all the lytB alleles studied were PCR amplified using primers 5'pyrD and 3'flpA. The DNA sequence was determined by the dideoxy chain termination method with an automated ABI Prism 3700 DNA sequencer (Applied Biosystems). Primers for PCR amplification and nucleotide sequencing were synthesized with a Beckman model Oligo 1000 M synthesizer or were purchased from Sigma.

Miscellaneous techniques.
The lytBR6 allele was PCR amplified, labeled with a digoxigenin luminescence detection kit (Boehringer Mannheim), and used as a probe. A fragment of lytBR6 encoding the C-terminal domain of LytB was amplified using oligonucleotide primers lytB-23 and lytB-C2 and also used as a probe. Southern blotting and hybridization were carried out according to the manufacturer's instructions. For phenotypic curing tests (i.e., chain dispersion by exogenous addition of LytB) (5), exponentially growing cultures (3 ml) in C+Y medium (A550, 0.15 to 0.2) received 1.5 µg of a purified LytB preparation and incubation was continued at 37°C. A control culture did not receive LytB. After 1.5 and 4.5 h of incubation, the cultures were examined by phase-contrast microscopy.

Data analysis.
DNA and protein sequences were analyzed with the Genetics Computer Group software package (version 10.0) (6). Pairwise evolutionary distances (PEDs) (estimated number of substitutions per 100 bases) were determined using the Distances program with the correction adequate for each case. Multiple alignments were created with Pileup and, when required, adjusted manually.

Nucleotide sequence accession numbers.
The nucleotide sequences determined in this study have been deposited in the EMBL/GenBank/DDBJ databases. The lytB alleles have been assigned accession numbers AJ870411 to AJ870429 (Table 1).


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RESULTS AND DISCUSSION
 
Allelic variants at the lytB locus.
Globally, 20 different alleles could be found among the 24 strains which we sequenced (Table 1). It should be noted that every typical S. pneumoniae strain studied harbors a different lytB allele, indicating that this gene is relatively polymorphic. It was also noted that the length of the alleles varied from 1,707 nucleotides (for the atypical strain 1078 and the S. mitis type strain) to 2,109 nucleotides (for strains R6, N, 8249, G54, and 436). Pairwise sequence comparison revealed that strains 8224 and 11923 had identical lytB alleles. This was not unexpected since it has been reported that these strains are closely related as determined by sequencing of the lytA and sodA genes, as well as by multilocus sequence typing (33). In addition, the lytB genes of strains 236, 1338, and 1629 were also identical to each other. Surprisingly, no PCR product could be obtained by using DNA prepared from strain 10546 and any combination of the primers mentioned above. Moreover, Southern blot hybridization of chromosomal DNA from this strain with two different lytB probes (see Materials and Methods) failed to reveal any hybridization band (not shown), indicating that strain 10546 is a {Delta}lytB mutant.

In the region encoding the C-terminal catalytic domain (not shown) the only remarkable change was detected in alleles 15 and 20, which contain a 6-bp deletion after nucleotide 882. Because of this characteristic, PEDs were determined out separately for the 5' and 3' ends of the corresponding alleles in the gene regions that appeared to be well conserved (Fig. 1). A perfect match was found between alleles 3 and 6 from position 1 to position 726 and for the last 918 nucleotides. In the 3' region, allele 2 was also identical to alleles 3 and 6, but since the sequence available from the data banks (accession number AF291696) contains an indeterminate nucleotide in the 5' part of allele 2, it was not possible to ascertain whether these three alleles were also identical in this region. However, the fact that these alleles differ markedly in the number of repeats (14, 16, and 18 repeats for alleles 2, 3, and 6, respectively) should be taken into account. Alleles 1 to 11, which correspond to alleles of typical pneumococci, were well conserved (PEDs, <1%), whereas alleles 14 and 16 from atypical strains were slightly divergent (PEDs, 1 to 5%). All other alleles exhibited PEDs ranging from 5 to nearly 10%. Among the latter group, however, allele 15 (from atypical pneumococcal strain 1078) and allele 20 (from the S. mitis type strain) appear to be closely related (PED, ~4%). This result fully agreed with the result previously reported for the informative galU gene of the same two isolates, for which 97% nucleotide identity was found (28). However, the PEDs found when we compared lytB alleles from typical and atypical pneumococci were significantly lower than those reported previously for the lytA gene (PEDs, >20%) (33). It is possible that selection of clinical isolates for deoxycholate insolubility may introduce a bias for greater variability of the lytA gene, whereas there was no selective pressure for LytB among the same set of strains.



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  		FIG. 1. Pairwise comparison of the nucleotide sequences of 20 different lytB alleles. The values above the diagonal are pairwise evolutionary distances for positions 1 to 726. The values below the diagonal are similar values for the most 3'-located 918 nucleotides, corresponding to positions 1192 to 2109 in the lytBR6 allele (allele 1). The values are the estimated numbers of substitutions per 100 bases as determined by using the Jukes-Cantor distance correction (22). A black background indicates PEDs that are less than 1%, a gray background indicates PEDs that are between 1 and 5%, and a white background indicates PEDs that are more than 5%.

Polymorphism outside the ChBR region.
In fact, precisely 50% of the point mutations detected in both the 5' and 3' regions corresponded to nucleotide changes at the third position of the codons. However, the 6-bp deletion in alleles 15 and 20 (Fig. 2), where the sequence of the two alleles matched and completely differed from the sequences of other lytB alleles, might be an indication of the occurrence of a localized recombination event, as previously suggested (33). Additional mutations not involving the third position of the codons were also found in this region in other alleles (see below). Notwithstanding the central part of LytB, where polymorphism in the number of ChBRs was noted (see below), the N- and C-terminal parts of the protein were very well conserved. In fact, nonconserved substitutions of amino acid residues were observed in as few as 17 of 527 positions (Table 2). Most of the changed residues were in atypical strains and the S. mitis type strain, whereas differences among typical LytB proteins (corresponding to alleles 1 to 11) were found at positions 425, 560, and 687 exclusively. It is noteworthy that position 425 was the most polymorphic position, since up to four different residues were observed. Globally, in the DNA region encoding amino acid residues 421 to 431 we observed that mutations coding for nonconserved amino acid substitutions crowded together (Table 2).



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  		FIG. 2. Structure of lytB alleles 15 and 20. Each of the positions at which the sequence of one or more of the lytB alleles differs from the sequence of allele 1 is shown. Colons indicate nucleotides that are identical to those of allele 1. Sites where all the sequences are identical are not shown. Sites 1, 2, and 3 indicate the first, second, and third nucleotides, respectively, in the codon. The numbering of the codons corresponds to that of allele 1 (14).


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  		TABLE 2. Nonconserved amino acid subtitutions in different LytB proteins at the N- and C-terminal endsa

Polymorphism in the number of ChBRs: evolutionary insights.
LytBR6 contains a ChBD that is located N terminally and is made up of 18 ChBRs (14). Sequence conservation among the different ChBRs of LytBR6 was evident not only at the amino acid level but also at the nucleotide level, suggesting that the repeats arose through a duplication process. Sequence homology was most evident from repeat 10 to repeat 17 (Fig. 3), which suggests that these repeats appeared recently in evolutionary terms. Surprisingly, although all ChBRs were very similar, there are two major types of alternating repeats, one encoding 21 amino acid residues (ChBR2, -3, -6, -9, -10, -12, -14, and -16) and the other coding for 23 amino acid residues (ChBR1, -5, -8, -11, -13, -15, and -17). Repeats 4 and 7 appeared to be partially deleted as they encode only 17 amino acids, and repeat 18 is highly degenerate at its 3' end. The most noticeable difference among the different lytB alleles involved the number of repeats found in the region encoding the ChBD, which ranged from a minimum of 12 to a maximum of 18 (Table 1). Typical pneumococcal strains contain 14, 16, or 18 repeats, whereas atypical lytB isolates, including the S. mitis type strain, typically have only 12 repeats, although strains 1283 and 782 have 14 and 16 repeats, respectively.



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  		FIG. 3. Multiple alignment of the ChBRs of the lytBR6 allele. A black background indicates nucleotides that are conserved in at least one-half of the repeats. A gray background indicates other conserved nucleotides. Hyphens indicate gaps introduced to maximize homology. Consensus nucleotide and amino acid sequences are shown at the bottom. The location of the ß-sheets (arrows) and choline-binding residues (triangles) as deduced from the known crystal structures of C-LytA (12) and Cpl-1 (19) are also indicated at the bottom.

The alternating structure of the repeats (see above) together with the observation that there is always an even number of repeats suggested that the duplication events that gave rise to the ChBRs of LytB probably involved two repeats simultaneously, one encoding 21 amino acid residues and the other coding for 23 amino acid residues.

As mentioned above, striking polymorphism among the lytB alleles was found in the central region of the gene. Moreover, multiple-sequence alignments also showed that the differences in the number of ChBRs among strains were exclusively in the region corresponding to repeats 11 to 17 of allele 1. Taking allele 1 as reference, we first constructed a multiple alignment of every repeat of the heterogeneous region using Pileup to obtain the correct assignment of these repeats in the gene. After this, the evolutionary distances were determined using Distances. A schematic representation of the results of this procedure is shown in Fig. 4. In spite of the fact that different alleles from typical pneumococcal strains may contain the same number of repeats, it is evident that the organization of such repeats appears to be very different, although the repeats always assemble as alternating repeats consisting of 63 and 69 bp. Interestingly, this finding correlates with crystallographic data showing that a choline-binding site is formed by two hairpins of consecutive ChBRs (12). Whether an increased number of pairs of choline-binding sites is important for improving and/or fine-tuning the activity of LytB or whether a modified LytB might provide an evolutionary advantage for the cell is not known at present.



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  		FIG. 4. Polymorphism of ChBR11 to ChBR17 of the LytB protein from various streptococcal strains. The upper part is a diagram of LytBR6. The numbers of amino acid residues of each ChBR and the whole protein are indicated in the boxes and on the right, respectively. The cross-hatched and black boxes represent the signal peptide and the C-terminal domain, respectively. ChBR1 to ChBR10 and ChBR18 are indicated by gray boxes. The region corresponding to ChBR11 to ChBR17 is enlarged. For clarity, ChBR11 to ChBR17 are labeled with different colors. ChBRs that have identical nucleotide sequences are indicated by the same color and shading. Nearly identical nucleotide sequences (PEDs, <5%) are indicated by stippled boxes that are the corresponding color. The open boxes represent highly divergent ChBRs (PEDs, 6 to 15%).

Two previous studies revealed noticeable allelic polymorphism in the gene encoding pneumococcal surface protein C (pspC), particularly in the number of ChBRs, which varies between 7 and 13 (4, 21). This observation appears to differ from our results for LytB, in which the duplication events that gave rise to the choline-binding repeats of LytB involved two repeats simultaneously. In addition, the previous authors noted that the ChBD of PspC was followed by a highly conserved, weakly hydrophobic stretch of 18 amino acids (VNTTVDGYGVNANGEWVN) (21). It is conceivable that in spite of no significant sequence similarity with the canonical ChBRs, this protein segment may also follow a superhelical fold. In fact, the conserved C-terminal end of PspC is 77.8% similar (44.4% identical) to the C-terminal "tail" of a recombinant form of the pneumococcal Pce phosphorylcholine esterase (GKTPEGYTVDSSGAWLVD), whose three-dimensional structure has been solved recently (18). Most importantly, it has been shown that the Pce C-terminal tail (residues 517 to 534) fold as an additional ChBR, and this might also be the case for PspC.

Morphology of species of the mitis group containing different lytB alleles.
It is well established that lytA mutants form only short chains of 7 to 10 cells (34, 35), whereas S. pneumoniae lytB mutants grow in long chains (>30 cells) (5, 14). In addition, it has recently been reported that underexpression of PcsB, a protein containing a CHAP domain found in bacterial murein hydrolases, also led to formation of long chains of pneumococcal cells (31). Interestingly, the essential VicRK two-component signal transduction system controls the expression of both pcsB and lytB (32). It is noteworthy that all the typical pneumococcal isolates studied here exhibited a diplococcus morphology (some short chains were also observed), mainly when they were growing exponentially (not shown). This observation supports the hypothesis that the presence of the LytB protein could play a critical role in the survival of pneumococci since growth as diplococci should facilitate bacterial spreading during infection (30). This result also indicated that in spite of the number of ChBRs (range, 14 to 18) (Table 1), all the typical pneumococci synthesize an active LytB enzyme.

In sharp contrast to what was found for typical S. pneumoniae isolates, the majority of atypical strains grew by forming long chains of cells (unpublished observations). Only atypical strain 101 and the type strain of S. mitis grew chiefly by forming short chains (≤10 cells). Diplococci were seldom found in cultures of these two strains. This behavior could be anticipated since the type strain of S. mitis does not have a lytA gene and strain 101 (like every atypical isolate tested so far) synthesizes a LytA amidase having reduced specific activity (33). The type strain of S. mitis, like strain 101, contains lytB alleles with only 12 ChBRs, indicating that even the shortest forms of LytB can exhibit normal enzymatic activity. Chaining was anticipated in the case of strain 10546, which completely lacks lytB, but not in other atypical strains. Although the possibility that one or more of the nonconservative amino acid changes present in the different alleles (Table 2) might result in an inactive (or less active) LytB enzyme cannot be completely ruled out at present, phenotypic curing experiments strongly suggested that chaining in these atypical pneumococci was not related to a LytB deficiency. We have reported that addition of purified LytB glucosaminidase to a pneumococcal mutant defective in this enzyme dispersed the chains of the mutant in a dose-dependent manner until the cells largely took on the appearance of typical diplococci or small chains, as illustrated in Fig. 5A and B (5). However, when a purified preparation of LytB was added to exponentially growing cells of chain-forming strains, chain-dispersing activity was not observed in any atypical strain in this functional "complementation" experiment (Fig. 5C and D). A plausible explanation for this is that the atypical isolates synthesized a modified peptidoglycan refractory to the enzymatic action of LytB from the R6 strain. Actually, it has been shown that LytB acts exclusively at the poles of the cells, where presumably the fine composition or structure of the peptidoglycan differs from that in the rest of the cell wall. Moreover, in remarkable contrast to other ChBPs, LytB is able to bind to the surface of ethanolamine-grown cells but cannot degrade this compound (5). Some strains of S. mitis contain at least two cell wall polysaccharides, one identical to the C polysaccharide of S. pneumoniae and one whose composition is not known (3). Some atypical pneumococci which exhibit some properties similar to those of the atypical isolates studied here have recently been reclassified as a new species, S. pseudopneumoniae (2). It is conceivable that as reported for S. mitis, some strains of this new streptococcal species may have a cell wall composition different in some way from that of S. pneumoniae.



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  		FIG. 5. Phenotypic curing test for typical and atypical pneumococci. (A and B) Phase-contrast microscopy examination of S. pneumoniae R6B, a LytB mutant, that was not treated (A) and was treated (B) with 1.5 µg of purified LytB. (C and D) Atypical strain 1508 that was not treated (C) and was treated (D) with LytB. (E) Cultures of strains R6 and R6B (lytB mutant) growing in the liquid medium (C+Y medium). Note that chains of R6B cells sedimented at the bottom of the tube.

In summary, in spite of exhibiting noticeable polymorphism in the number of repeats encoding the ChBRs, LytB is a well-conserved protein that should be incorporated into the ongoing studies aimed at developing improved vaccines. In particular, several other surface-associated pneumococcal proteins are currently being tested for protein-based antipneumococcal vaccines (1). As lytB mutants are impaired in the capacity to colonize the nasopharynx (17), it is conceivable that immunization with LytB either alone or in combination could reduce pneumococcal carriage, particularly in children.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the Dirección General de Investigación Científica y Técnica (BMC2003-00074) and from Redes Temáticas de Investigación Cooperativa (G03/103 and C03/14).

We are grateful to P. García and D. Llull for helpful comments and critical reading of the manuscript. We also thank E. Cano for skillful technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain. Phone: (34) 91 837 3112. Fax: (34) 91 536 0432. E-mail: e.garcia{at}cib.csic.es. Back

{dagger} M.M. and V.O. contributed equally to this work. Back

{ddagger} Present address: Bioferma Murcia S.A., Research Centre and Production, Parque Industrial Las Salinas, Avenida de Europa s/n, 30840 Alhama de Murcia, Spain. Back


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Applied and Environmental Microbiology, December 2005, p. 8706-8713, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8706-8713.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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