ABSTRACT
The genes encoding the 62-kDa lipoproteins from theMycoplasma mycoides subsp. mycoideslarge-colony type (LC) strain Y-goat and the M. mycoidessubsp. capri strain PG3 were cloned and analyzed by sequencing. These two lipoproteins have been named LppA[MmymyLC] and LppA[Mmyca], and their corresponding genes have been namedlppA[MmymyLC] and lppA[Mmyca], respectively. The nucleotide and deduced amino acid sequences of these two lipoproteins showed a very high degree of similarity between these two mycoplasmas. Given the sequence data, LppA seems to fulfill the same structural functions as the previously described major lipoproteins P72 of M. mycoides subsp. mycoidessmall-colony type and P67 of the Mycoplasma species bovine group 7. Based on lppA gene sequences of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri type strains, a specific PCR assay was developed so that it amplified this gene in all field strains of the two species analyzed in this study but not in the other members of the M. mycoides cluster. Analysis of the PCR-amplifiedlppA genes with frequently cutting restriction enzymes showed a certain degree of genetic variability which, however, did not cluster the two subspecies. This PCR therefore allows a rapid identification of M. mycoides subsp. mycoidesLC and M. mycoides subsp. capri but does not distinguish between these two closely related subspecies. LppA was expressed in Escherichia coli K-12 and used for the production of polyclonal mouse antiserum. Antibodies against recombinant LppA[MmymyLC] reacted with a 62-kDa protein in allM. mycoides subsp. mycoides LC and M. mycoides subsp. capri type strains and field strains tested but not with the other members of the M. mycoidescluster, thus showing the antigenic specificity of LppA and further supporting the concept that a close relationship exists between these two mycoplasmas.
The Mycoplasma mycoidessubsp. mycoides large-colony type (LC) and M. mycoides subsp. capri strains belong to theM. mycoides cluster, a group of six closely related mycoplasmas (13). M. mycoides subsp.mycoides LC causes mastitis, keratoconjunctivitis, polyarthritis, pneumonia, and septicemia in goats (13, 14, 34, 36). It has also been isolated, rarely, from cattle (20, 27) and sheep (25). M. mycoides subsp.capri is reported to cause a pattern of diseases similar to those induced by M. mycoides subsp. mycoidesLC specifically in goats, including mastitis, arthritis, and pulmonary diseases (13, 21, 23, 28, 40, 41). From an epidemiological point of view, differential identification of the members of theM. mycoides cluster is of major importance. An accurate and rapid means of identification of the subspecies or subtypes of mycoplasmas of the M. mycoides cluster is prerequisite for the differentiation, since the different members of this cluster show very strong differences in virulence and epidemiological impact. However, many methods fail in specificity because they are hampered by strong serological cross-reactions between the different members of theM. mycoides cluster (7, 12, 15, 17, 32, 39).
It has been reported that M. mycoides subsp.mycoides LC and M. mycoides subsp.capri are antigenically very similar as assessed by numerical analysis of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein patterns (11, 22). Identical results have been obtained by two-dimensional PAGE protein patterns, which confirm that M. mycoides subsp. mycoides LC strains are more closely related to M. mycoides subsp. capri strains than to M. mycoides subsp. mycoides small-colony type (SC) strains (26, 35).
A DNA probe based on a randomly chosen genomic fragment was developed for the differentiation of the different members of the M. mycoides cluster into four groups. This DNA probe grouped M. mycoides subsp. mycoides LC strains together withM. mycoides subsp. capri and distinguished them from the other members of the M. mycoides cluster. However, it did not allow differentiation between these two mycoplasmas (38). DNA-DNA hybridization studies revealed variable values for DNA homology between M. mycoides subsp.mycoides LC and M. mycoides subsp.capri (75 to 94%), between M. mycoides subsp.mycoides LC and M. mycoides subsp.mycoides SC (88 to 93%), and between M. mycoidessubsp. capri and M. mycoides subsp.mycoides SC (75 and 93%) depending on experimental conditions (2, 9). Phylogenetic studies based on sequence analysis of 16S rRNA genes (rrs) revealed 99.9% similarity between M. mycoides subsp. mycoides LC andM. mycoides subsp. capri. These results suggested that these two mycoplasmas could be grouped into a single subspecies, one distinct from M. mycoides subsp.mycoides SC (29).
With the objective of getting a better insight into the antigenic and genetic differences among the different members of the M. mycoides cluster, the major surface-located lipoprotein antigens of M. mycoides subsp. mycoides SC, P72, and of Mycoplasma sp. bovine group 7, P67, had been characterized (8, 16). Immunoblot analysis of type and field strains of the different species from the M. mycoides cluster by using monospecific polyclonal antibodies against each of these proteins revealed that these two proteins were antigenically species specific.
Southern blot hybridization with a gene probe for P72 revealed that the M. mycoides subsp. capri strain PG3 and the M. mycoides subsp. mycoides LC strain Y-goat as well contained an analogous gene. The aim of the present study was to clone, sequence, and analyze the genes encoding the major lipoproteins from M. mycoides subsp.capri and M. mycoides subsp.mycoides LC.
MATERIALS AND METHODS
Strains and growth conditions.Mycoplasma strains used in this study and their origins are listed in Table1. The Mycoplasma species were cultured in standard mycoplasma medium at 37°C (5). Cells were pelleted by centrifugation at 20,000 × g for 20 min, washed in TES buffer (10 mM Tris, 1 mM EDTA, 0.8% NaCl [pH 8.0]) and resuspended in TES buffer to reach a cell concentration of 109 cells ml−1. Escherichia coliXL1-Blue MRF′ {Δ(mcrA)183Δ(mcrCB-hsdS-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proAB laclqZΔM15Tn10(Tetr)]} and XLOLR {Δ(mcrA)183Δ(mcrCB-hsdS-mrr)173 endA1 thi-1 recA1 gyrA96 relA1 lac [F′ proAB laclqZΔM15Tn10(Tetr)]λr, Su−} (Stratagene, La Jolla, Calif.) were grown on Luria-Bertani broth at 37°C (3). E. coli YN2980 (leuUGAlacZ659UGAtrpA9605UAGhis-29UAGile thyA metB argH rpoB rpsL prfB3[pISM3001]) was used as a suppressor strain for UGATrp and was grown as described (37). Expression vector pBK-CMV phagemid (Stratagene) was propagated in XLOLR strain.
Strains of mycoplasmas used and signals of PCRs for thelppA gene
DNA manipulation, construction and screening of gene library, and PCR.Genomic DNA was extracted by the guanidinium thiocyanate method as indicated (31). DNA from theM. mycoides subsp. mycoides LC strain Y-goat and the M. mycoides subsp. capri strain PG3 was partially digested with SauIIIA and used to construct λ ZAP Express phage banks (Stratagene) as described (8). The gene libraries were screened with a digoxigenin-11-dUTP-labeled DNA probe, and positive clones were transformed to phagemids as indicated (16). All standard techniques of molecular biology have been described previously (3). PCR amplifications were carried out as indicated (16), by using the oligonucleotide primers listed in Table 2.
Primer pairs used in PCR assays and for DNA sequencing
Nucleotide sequencing and sequence analysis.Plasmids pJFFMMLC2 (from M. mycoides subsp. mycoidesLC) and pJFFMMCA4 (from M. mycoides subsp.capri) containing 2.0-kb and 4.5-kb inserts, respectively, were sequenced on both strands. Sequencing was performed with an Applied Biosystems DNA Sequenator AB373 by using the Taq Dye Deoxy Terminator Cycle kit (Applied Biosystems/Perkin Elmer, Norwalk, Conn.), using oligonucleotide primers matching T3 and T7 promoter sequences contained on the cloning vector (Table 2). Full-length sequencing of the insert was obtained by producing an ordered nested set of deletion mutations in the same cloning vector with a Nested Deletion kit according to the directions of the supplier (Pharmacia Biotech, Uppsala, Sweden) and subsequent DNA sequence analysis of the plasmids with the T3 and T7 primers.
The DNA sequences and their deduced amino acid sequences were analyzed with the program PROSITE (4) of the DNA analysis software PC/Gene and with the program PSORT (24) and were aligned with the software GCG (Genetics Computer Group, Madison, Wis.). Sequence comparisons with GenBank/EMBL and NBRF databases were performed with the BLAST programs (1).
Preparation of recombinant LppA[MmymyLC] antigen and production of polyclonal mouse anti-LppA[MmymyLC] serum.ThelppA gene of M. mycoides subsp.mycoides LC cannot be expressed in its full length inE. coli K-12, due to the mycoplasma-specific UGATrp codons (42), which are read in E. coli as stop codons. We have therefore used an expression system which has been developed for efficient suppression of UGATrp for the expression of mycoplasmal proteins inE. coli (37). Strain YN2980 (37) was transformed with plasmid pJFFMMLC2 and grown to the mid-exponential growth phase. Expression of the clonedlppA[MmymyLC] gene from the vector’splac promoter was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside and growth for 4 h further. Cells were then harvested, washed with TES buffer, and resuspended in a 0.1 volume of TES buffer. The resuspended cells were sonicated on ice with a Branson Sonifier 250 (Branson Ultrasonics, Danbury, Conn.) equipped with the Microtip at an output of 3 for 1 min. The protein concentration of the suspension obtained was measured by the method of Bradford (6) and reached 400 μg ml−1.
In order to obtain polyclonal anti-LppA[MmymyLC] serum, 150 μl of the suspension described above containing 60 μg of protein per administration was mixed with an equal volume of Freund’s complete adjuvant (Difco Laboratories, Detroit, Mich.) as described (18) and used to immunize mice. After 3 weeks the mice were booster immunized with the same amount of protein, but Freund’s incomplete adjuvant was used. Blood for serum was taken 2 weeks later. The serum was adsorbed with E. coli YN2980 carrying the empty vector pBK-CMV. The antigen for adsorption was prepared as indicated (8). Immunoblot analysis was done according to standard protocols (3, 8). The mouse serum was diluted 1:2,000, and reaction products were visualized with affinity-purified goat phosphatase-labeled anti-mouse immunoglobulin G (heavy and light chains) (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) diluted 1:2,000.
RESULTS
Cloning of the lppA genes of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri.Southern blot analysis ofHindIII-digested genomic DNA from the type and reference strains of all members of the M. mycoides cluster was performed with a digoxigenin-labeled gene probe for the P72 (lipoprotein) gene of M. mycoides subsp. mycoides SC (8). These hybridization results showed the presence of genes analogous to the P72 gene in all members of the M. mycoides cluster.
Gene libraries of M. mycoides subsp.mycoides LC and M. mycoides subsp.capri containing about 106 phage clones ml−1 were constructed with λ phage ZAP Express vector and screened. From both libraries, plasmids which were shown to contain the entire genes of the lipoproteins namedlppA[MmymyLC] and lppA[Mmyca], as assessed from sequencing data of the extremities of the inserts, were retained. Plasmid pJFFMMLC2 contained a 2.0-kb insert fromM. mycoides subsp. mycoides LC, and plasmid pJFFMMCA4 contained a 4.5-kb insert from M. mycoidessubsp. capri (Fig. 1). The integrity of the inserts of these plasmids was verified by PCR amplification of the corresponding fragments from genomic DNA ofM. mycoides subsp. mycoides LC and fromM. mycoides subsp. capri by using primers matching the sequenced extremities of the inserts (Table 2).
Locations of the lppA gene (black boxes) and surrounding genes on plasmids pJFFMMLC2 containing a 2.0-kb insert cloned from the DNA of the M. mycoides subsp.mycoides LC strain Y-goat (a) and pJFFMMCA4 containing a 4.5-kb insert cloned from the DNA of the M. mycoidessubsp. capri strain PG3 (b). The ORFs are represented by white boxes, and arrowheads indicate the direction of transcription and translation. The locations of the oligonucleotide primers are shown by arrows. Filled triangles indicate the location ofplac promoter of the cloning vector.
DNA sequence analysis of the lipoprotein genes and of flanking genes.The inserts of plasmids pJFFMMLC2 and pJFFMMCA4 were sequenced in both directions. The nucleotide sequence of the insert pJFFMMLC2 contained an open reading frame (ORF) encoding the lipoprotein LppA[MmymyLC] precursor of 526 amino acid (aa) residues with a predicted molecular mass of 60.288 kDa (Fig.2). It is preceded by a consensus sequence for a ribosome binding site (RBS) located five nucleotides (nt) upstream of the initiation codon, AUG. The analogous ORF in plasmid pJFFMMCA4 encoded the lipoprotein LppA[Mmyca] precursor, which shows 523 aa residues and a predicted molecular mass for LppA[Mmyca] of 59.822 kDa (Fig. 2). This ORF is preceded 6 bp upstream by a canonical RBS sequence. The two lppAgenes contain mycoplasma-specific UGATrp codons corresponding to aa 147 and 415 and to aa 144 and 412, respectively, and are terminated by UAA stop codons. Analysis of the amino acid sequences of LppA indicated a consensus sequence for a potential recognition site of a prokaryotic signal peptidase II (19) at aa 21 to 25 and a lipid attachment site at the cysteine residue starting at aa 25 (Fig. 2). A potential transmembrane region is located at aa residues 7 through 23 of the leader sequence. The amino acid sequences of the mature LppA[MmymyLC] and LppA[Mmyca] are virtually the same. They have 94% identical and 95% positive (identical + similar) amino acids. Differences are mainly located at the N terminals of the proteins, where a few deletions and substitutions are located (Fig. 2). The mature LppA proteins of both M. mycoides subsp. mycoidesLC and M. mycoides subsp. capri showed a similarity (42 to 44% identity and 50 to 52% positivity for aa residues) to P72 of M. mycoides subsp.mycoides SC.
Amino acid sequence comparison of LppA precursors from the M. mycoides subsp. mycoides LC strain Y-goat and from the M. mycoides subsp. capristrain PG3. Underlined amino acids correspond to the consensus sequence for the signal peptidase II recognition site. The arrow indicates the potential cleavage site for this peptidase. Vertical bars show identical amino acids, and dots between sequences show similar amino acids.
The sequence analysis of both plasmids pJFFMMLC2 and pJFFMMCA4 revealed a part of an ORF, ORF1, upstream of lppA. Its function is currently unknown. However, ORF1 in both M. mycoidessubsp. mycoides LC and M. mycoides subsp.capri showed a high similarity to the ORF upstream of the gene encoding P72 in M. mycoides subsp.mycoides SC (8). The ORF1 of M. mycoides subsp. mycoides LC showed 52% identical amino acids and 69% identical nucleotides at the DNA level with the ORF1 ofM. mycoides subsp. capri. In both plasmid clones, the lppA gene is followed by a hairpin structure representing a potential transcriptional termination signal (Fig. 1). In pJFFMMCA4, the hairpin structure was followed by two ORFs that sequenced in the opposite direction; one, ORFX, encodes a polypeptide with unknown function, and the second has 63% identical nucleotides with a DNA methylase in Spiroplasma species (accession no.X17195 ) (33) (Fig. 1). Upstream of the potential DNA methylase gene we found a hairpin structure that acts as a transcription stop signal and is part of an ORF showing 93% identical nucleotides to the mtlD (which encodes mannitol-1-phosphate dehydrogenase) gene analogue found in M. mycoidessubsp. mycoides SC (8) (Fig. 1). In pJFFMMLC2, an ORFX was found downstream of the transcription stop signal followinglppA. It showed 89% identical nucleotides to the analogous location in M. mycoides subsp. capri (Fig.1).
Expression and serological specificity of LppA of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri.The expression and the antigenic specificity of LppA in M. mycoides subsp.mycoides LC and M. mycoides subsp.capri were analyzed on immunoblots by using adsorbed polyclonal mouse antibodies directed against recombinant LppA[MmymyLC]. Whole cells of the M. mycoides subsp.mycoides LC and M. mycoides subsp.capri type strains and field strains, as well as the type and reference strains of the other members of the M. mycoides cluster (Table 1), were solubilized in SDS sample buffer and used as antigens on the immunoblots. The mouse anti-LppA[MmymyLC] serum strongly reacted with a 62-kDa band or a 60- and 62-kDa doublet for all strains of M. mycoidessubsp. mycoides LC and M. mycoides subsp.capri tested except strain WK354/80. The apparent molecular mass of LppA on SDS polyacrylamide gels (62 kDa) is slightly higher than the expected molecular mass as calculated from its DNA sequence-deduced amino acid composition (60 kDa). Strain WK354/80, for which the taxonomic classification is not clear, showed weak reactions with a doublet band of 58 and 60 kDa. The other mycoplasmas of the M. mycoides cluster did not react with anti-LppA[MmymyLC] antibodies, thus showing the antigenic specificity of LppA[MmymyLC] to M. mycoides subsp. mycoides LC and M. mycoides subsp. capri (Fig.3).
Expression of lipoprotein LppA[MmymyLC] and LppA[Mmyca] in mycoplasmas of the M. mycoidescluster (a) and in the M. mycoides subsp.mycoides LC and M. mycoides subsp.capri field strains (b and c, respectively). Immunoblots containing total antigens of the different mycoplasmas (Table 1) were probed with anti-LppA[MmymyLC] serum from a mouse immunized with recombinant LppA[MmymyLC]. The positions of the lipoproteins LppA[MmymyLC] and LppA[Mmyca] and of molecular mass standards (broad range) (Bio-Rad Laboratories) are indicated. Certain strains showed a doublet band reacting with anti-LppA[MmymyLC] antibodies (b). We interpret these doublets to be due to posttranslational modification of these lipoproteins. In certain strains, no doublets are visible, which might be due to the fact that the cells were taken in a late stage of cell growth. Secondary bands with molecular masses not within the 60 to 62 kDa range are interpreted as background reactions due to nonspecific serological reaction of the mouse sera. The faint doublet at 58 and 60 kDa seen with strain WK354/80 is supposed to be due to a lipoprotein closely related to, but different from, LppA[MmymyLC] and LppA[Mmyca].
Specificity of lppA to M. mycoidessubsp. mycoides LC and M. mycoides subsp.capri.The presence of lppA[MmymyLC] andlppA[Mmyca] was studied in M. mycoidessubsp. mycoides LC and M. mycoides subsp.capri strains and in the other members of the M. mycoides cluster by PCR amplification with the primer pair MMMLC2-L and MMMLC1-R (Table 2) by using as the templates chromosomal DNA from all mycoplasma strains used in this study (Table 1). All strains of M. mycoides subsp. mycoidesLC and M. mycoides subsp. capri showed the expected DNA fragment of 1.05 kb with the exception of strain WK354/80, which amplified a smaller fragment (0.85 kb). The PCR product obtained from WK354/80 was analyzed further by DNA sequencing. DNA sequencing results showed that the edges of the PCR fragment but not the central part of the segment were identical tolppA[MmymyLC] and lppA[Mmyca]. In total, this 0.85-kb fragment showed 63 to 64% similarity tolppA[MmymyLC] and lppA[Mmyca], 95% similarity to the P67 gene of the Mycoplasma sp. bovine group 7, and 93% similarity to the P72 gene ofM. mycoides subsp. mycoides SC. None of the other mycoplasmas of the M. mycoides cluster analyzed showed any amplification product (Fig. 4and Table 1). These results confirmed that the primer pair MMMLC2-L and MMMLC1-R amplified a specific 1.05-kb fragment from M. mycoides subsp. mycoides LC and M. mycoides subsp. capri. For further analysis, the PCR amplification products of the lppA[MmymyLC] and lppA[Mmyca] genes were digested with the restriction enzyme AluI and examined (Fig.5). Profiles obtained from the different strains showed some variations, indicating a certain heterogeneity of the lppA gene within the different strains. The profiles show that the same differences are found within strains of the same subspecies as well as among strains of different subspecies and hence do not differentiate M. mycoides subsp.mycoides LC strains from M. mycoides subsp.capri strains.
PCR fragments obtained from amplification with the primer pair MMMLC2-L and MMMLC1-R and genomic template DNA from type and reference strains of the M. mycoides cluster and from the M. mycoides subsp. mycoides LC andM. mycoides subsp. capri field strains (Table 1). As size markers, HindIII-digested λ DNA fragments were used. Their sizes are given in kilobases on the left side.
Restriction fragment analysis of PCR-amplifiedlppA genes from the M. mycoides subsp.mycoides LC and M. mycoides subsp.capri strains (Table 1). PCR products of DNA from type strains and field strains were digested with AluI and analyzed by electrophoresis on 8% polyacrylamide gels. The standards used were HinfI-digested pBR322 fragments. Their sizes are indicated in base pairs.
DISCUSSION
Sequence data show that the lipoproteins LppA[MmymyLC] and LppA[Mmyca] form a family together with the major lipoproteins ofM. mycoides subsp. mycoides SC, P72, and ofMycoplasma sp. bovine group 7, P67, and are hence suggested to have a function which is analogous to the two latter lipoproteins. Accordingly, we propose that P72 and P67 be renamed lipoproteins LppA[MmymySC] and LppA[Mbgr7], respectively.
Analysis of LppA from both M. mycoides subsp.mycoides LC and M. mycoides subsp.capri showed that the potential transmembrane region located in the leader sequence of LppA[Mmyca] is identical to that found in LppA[MmymySC] (P72) of M. mycoides subsp.mycoides SC and in LppA[Mbgr7] (P67) ofMycoplasma sp. bovine group 7 (8, 16) and differs only in a single amino acid (residue 11) from the leader sequence of LppA[MmymyLC]. The corresponding gene fragment is also highly conserved and could therefore serve as a valuable probe for the cloning of other mycoplasmal lipoprotein genes.
DNA sequence analysis of the lppA genes of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri revealed a very high degree of similarity which is also reflected antigenically, as shown on immunoblots. Minor variations, which can be observed in thelppA genes of various field strains, are not specific to the two subspecies. The differences seem to have only minor phenotypic impact since they are not reflected antigenically as revealed by immunoblots. Genetic variation among different field isolates from M. mycoides subsp. mycoides LC and M. mycoides subsp. capri has also been detected in other gene loci (40a). Hence, LppA seems to be a common and specific antigen of the two closely related mycoplasmasM. mycoides subsp. mycoides LC andM. mycoides subsp. capri. The chromosomal location of the lppA genes seems to be conserved in the vicinity of the mtlD gene (which encodes mannitol-1-phosphate dehydrogenase).
The specific PCR using the primer pair MMMLC2-L and MMMLC1-R amplified the lppA genes from M. mycoides subsp. mycoides LC and M. mycoides subsp. capri field strains of various geographic origins but not those from other members of theM. mycoides cluster. We therefore propose that this PCR be used for the identification of the phylogenetic and antigenic entityM. mycoides subsp. mycoides LC/M. mycoides subsp. capri.
Interestingly, for strain WK354/80, PCR amplification of thelppA gene resulted in a shorter-than-expected fragment which was shown to be most similar to the lppA[Mbgr7] (P67) gene of Mycoplasma sp. bovine group 7. WK354/80 strain was first described in the literature as Mycoplasma sp. bovine group 7 (10) and was later retyped by us asM. mycoides subsp. capri. Sequence analysis of the rrs gene of strain WK354/80, on the other hand, showed its strong similarity to that of M. mycoidessubsp. mycoides LC. These results reflect the ambiguous taxonomic status of WK354/80, which must be located intermediate between those of Mycoplasma sp. bovine group 7 andM. mycoides subsp. mycoides LC/M. mycoides subsp. capri. It illustrates further the good discriminatory potential of the lppA genes, which encode the major lipoproteins, in the differentiation of the members of theM. mycoides cluster.
ACKNOWLEDGMENTS
We are grateful to Yvonne Schlatter for technical assistance with DNA sequence analysis and PCR and to Margrit Krawinkler for expert help with identification and cultivation of mycoplasmas. We thank Chris Minion, Ames, Iowa, for the kind gift of strain YN2980, which proved to be most helpful for the expression of cloned mycoplasmal genes, and Shmuel Razin, Jerusalem, Israel, Kevin Dybvig, Birmingham, Alabama, and Karl-Erik Johansson, Uppsala, Sweden, for their helpful suggestions in naming lipoproteins.
This study is part of European COST action 826 on ruminants’ mycoplasmoses and was supported by grant C96.0073 of the Swiss Ministry of Education and Science and by the Swiss Federal Veterinary Office.
FOOTNOTES
- Received 29 June 1998.
- Returned for modification 6 November 1998.
- Accepted 4 December 1998.
- Copyright © 1999 American Society for Microbiology
REFERENCES
