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Journal of Bacteriology, January 1999, p. 418-425, Vol. 181, No. 2
Department of Microbiology, University of
Hawaii, Honolulu, Hawaii 968221;
Institute for Genetics and Microbiology, University of
Munich, Munich, Germany2; and
Pasteur
Institute, Paris, France3
Received 6 July 1998/Accepted 8 October 1998
We have identified a novel gene, gtcA, involved in the
decoration of cell wall teichoic acid of Listeria
monocytogenes serotype 4b with galactose and glucose.
Insertional inactivation of gtcA brought about loss of
reactivity with the serotype 4b-specific monoclonal antibody c74.22 and
was accompanied by a complete lack of galactose and a marked reduction
in the amounts of glucose on teichoic acid. Interestingly, the
composition of membrane-associated lipoteichoic acid was not
affected. Complementation of the mutants with the cloned
gtcA in trans restored galactose and glucose on teichoic acid to wild-type levels. The complemented strains also recovered reactivity with c74.22. Within L. monocytogenes,
sequences homologous to gtcA were found in all
serogroup 4 isolates but not in strains of any other serotypes. In
serotype 4b, gtcA appears to be the first member of a
bicistronic operon which includes a gene with homology to
Bacillus subtilis rpmE, encoding ribosomal protein L31. In
contrast to gtcA, the latter gene appears conserved among
all screened serotypes of L. monocytogenes.
Listeria monocytogenes is
a gram-positive, facultative intracellular pathogen that is widespread
in the environment and capable of causing severe infections in humans
and animals. Populations at risk include pregnant women, newborns, and
immunocompromised individuals, and infections are
commonly food-borne. Although several serotypes of
L. monocytogenes are present in the environment and in
foods, the majority of infections are caused by strains of serogroup
1/2 (mostly serotypes 1/2a and 1/2b) and 4 (almost always serotype 4b)
(6, 35). Both flagellar and somatic (mostly carbohydrate)
surface antigens contribute to the serotypic designation of the
organism (36).
As for many other gram-positive bacteria, the cell wall of
L. monocytogenes contains large amounts of the anionic
polymer teichoic acid (TA), covalently linked to peptidoglycan. In
Bacillus subtilis, TAs have been shown to be essential for
viability (22, 28). The TA of B. subtilis
168 consists of polyglycerol phosphate, in contrast to polyribitol
phosphate in B. subtilis W23 (1, 5, 28). In
L. monocytogenes, a polyribitol phosphate type of TA
appears to be the prevalent accessory cell wall polymer (7, 14,
40). Pronounced diversity in TA structure and antigenicity is
conferred by glycosidic substitution(s) of the ribitol phosphate units
(7, 10, 40). TA of strains of serogroup 1/2 and 3 consists
of polyribitol phosphate, with N-acetylglucosamine (Glc-NAc) and rhamnose (in the case of serogroup 1/2) substituents on ribitol (Fig. 1). In contrast, in serogroup 4 strains, Glc-NAc is incorporated in the TA chains, and, depending on
the serotype (4a, 4b, etc.), bears galactose and/or glucose
substituents. Serotype 4b strains are unique in bearing both galactose
and glucose substituents on the Glc-NAc of TA (Fig. 1).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cell Wall Teichoic Acid Glycosylation in
Listeria monocytogenes Serotype 4b Requires gtcA,
a Novel, Serogroup-Specific Gene
ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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FIG. 1.
Diagrammatic representation of TA composition in
different L. monocytogenes serotypes. (A) Type 1/2
(serotypes 1/2a, 1/2b, and 1/2c); (B) type 3 (serotypes 3a, 3b, and
3c); (C) serotype 4b. Modified from the work of Uchikawa et al.
(40).
Genetic studies of TA biosynthesis in gram-positive bacteria have mostly involved B. subtilis (19, 28). Although several structural and immunochemical studies of TA in cell walls of different L. monocytogenes serotypes have been performed (7, 10, 14, 39), the molecular genetic mechanisms underlying TA biosynthesis and diversity in Listeria remain to be elucidated. In this communication, we describe gtcA, a novel, serogroup 4-specific gene, which is essential for the presence of normal amounts of galactose and glucose on the TA of L. monocytogenes serotype 4b.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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Bacterial strains and growth media.
Bacteria were grown in
brain heart infusion broth (BHI; Difco) at the indicated temperature as
previously described (16). Growth on agar was on tryptic soy
agar (Difco) supplemented with yeast extract (0.7%) (TSA-YE). All
Listeria strains were preserved in BHI at 
70°C. When
appropriate, antibiotics used for Listeria were streptomycin
(1,200 µg/ml), erythromycin (10 µg/ml), tetracycline (10 µg/ml),
and chloramphenicol (7 µg/ml). Antibiotics used for Escherichia
coli were ampicillin (50 µg/ml) and kanamycin (50 µg/ml).
Antibiotics were purchased from Sigma.
Generation and screening of mutants.
Transposon mutants of
the serotype 4b strains 4b1 and 2381L were produced as described
previously (15) by using filter-mating conjugations
with Enterococcus faecalis CG110 or E. faecalis
RH110, donors of Tn916 and Tn916
E,
respectively. Strains 4b1 and 2381L were streptomycin-resistant
derivatives of NCTC 10527 (sporadic case isolate) and of strain F2381
(implicated in the Jalisco cheese outbreak), respectively
(44). The mutants were grown in 96-well microtiter plates
with the appropriate antibiotics (streptomycin-tetracycline and
streptomycin-erythromycin for Tn916 and Tn916E
mutants, respectively). The mutant banks were kept frozen at 70°C.
E mutant bank of strain 4b1. Its lack of reactivity
with c74.22 but reactivity with c74.33 and c74.180 have been described
(16).
Biochemical analysis of cell wall composition. Cell wall composition was determined as described by Fiedler et al. (7). Preparation and analysis of TA and lipoteichoic acid (LTA) from Listeria were done as previously described (7, 8, 14).
Isolation of transposon-flanking sequences in the mutants. Procedures for extraction of plasmid DNA from E. coli and genomic DNA from listeriae were previously described (20). Unless otherwise indicated, restriction enzymes and other molecular biologic reagents were purchased from Promega (Madison, Wis.).
The thermostable polymerase used was Tfl (Epicenter, Madison, Wis.). The transposon copy number in the mutants was determined by using Southern blots as previously described (15, 20) with the Genius digoxigenin labeling and detection system (Boehringer-Mannheim). The single-copy transposon mutant M44 was used to amplify a transposon-flanking fragment by means of the single specific primer PCR (SSP-PCR) (37) using EcoRI-digested M44 DNA and pUC18, which was digested by EcoRI and dephosphorylated (Pharmacia) as described previously (43). A 500-bp product was amplified, cloned in pCR1000 (Invitrogen), and sequenced. Primers internal to this fragment (PALN, 5' TGG GTT ACT ACA AGA AGA G 3', and PALT, 5' AGT ACT GAT GCG ATA AAA GCA 3') were used to amplify a 300-bp fragment which was cloned in pCR1000, resulting in plasmid pNP1. Transposon-flanking fragments from the other mutants were obtained by PCR using primer OTL (5'-CGG AAT TCC GTG AAG TAT CTT CCT ACA G-3') derived from one of the Tn916 terminal sequences and primer PLNEC (5' CGG AAT TCC TGA GTT ACT ACA AGA AGA G 3'). Both primers contained EcoRI sites (underlined) at their 5' end.Plasmid constructions (pNP500, pNP21, and pNP95). A 4b1 genomic library (5,000 clones; average insert size, 4 to 10 kb) was constructed as described previously (43). This library was screened with pNP1 as a probe, but no positive clones were identified. Another 4b1 library (2,000 clones) was constructed similarly, except that inserts were in the 2- to 4-kb range. One positive clone, pC11A, was identified in this library following screening with pNP1. Plasmid pNP500 was constructed by digesting pC11A with EcoRI (one EcoRI site was in the multiple cloning site of the vector, and the other was in the cloned insert) and subcloning the EcoRI fragment into EcoRI-digested and dephosphorylated pUC18. The locations of the fragments cloned in pNP1, pNP500, and pC11A are indicated in Fig. 3A (see below).
To construct plasmid pNP21, we used primers RhoBam (5' GCG CGG ATC CAA AGG GAC AGG CAA CA 3') and SmeBam (5' GCG CGG ATC CAC TTG AAA ACC TCC T 3') corresponding to nucleotides (nt) 6 to 22 and 1302 to 1319, respectively, and containing BamHI recognition sites (underlined) at their 5' ends. The PCR fragment was digested with BamHI and ligated to the L. monocytogenes-E. coli shuttle vector pKSV7 (38), which had been digested with BamHI and dephosphorylated. To construct pNP95, pNP21 was digested with EcoRI, and the 860-bp EcoRI fragment internal to the cloned insert was cloned into EcoRI-digested and dephosphorylated pKSV7. The fragments cloned in pNP21 and pNP95 are shown in Fig. 3A. To amplify the gtcA gene from strain 2381L, we used primers RhoBam and SmeBam (see above) and genomic DNA from 2381L as the template. The PCR fragment was purified and sequenced directly.Preparation of L. monocytogenes electrocompetent
cells and electroporation.
To prepare electrocompetent cells of
L. monocytogenes, we used the penicillin treatment
protocol of Park and Stewart (24). Electrocompetent cells
were used immediately or frozen at 70°C. For electroporation, 100 µl of cells (thawed on ice if kept frozen) was mixed gently with 1 µg of plasmid DNA (Wizard minipreps; Promega). The cells were then
transferred into 1-mm prechilled electroporation cuvettes (Eppendorf,
Madison, Wis.). The electroporator (Eppendorf) was set to 1.5 kV to
obtain the field strength of 12.5 kV/cm, as suggested by the vendor.
After application of the electrical pulse, 1 ml of BHI with 0.5 M
sucrose was added, and the cuvette was incubated at 30°C for 1 h. The cells (100 µl) were then plated on TSA-YE plates containing
chloramphenicol (7 µg/ml) and incubated at 30°C for 2 to 3 days.
Northern blotting and reverse transcriptase PCR (RT-PCR). RNA for Northern blots was prepared from mid-logarithmic-phase cultures. Following treatment with lysozyme and pronase as for DNA extraction, a 10× volume of Tri Reagent (Molecular Research Center, Inc.) was added and the mixture was incubated at room temperature for 5 min to allow complete dissociation of nucleoprotein complexes. Extraction and precipitation of RNA were done in accordance with the suggestions of the vendor, and Northern transfer was done as described before (2) by using positively charged membranes (Magnacharge Micron Separations Inc.). Radioactive probe labeling and signal detection were done as described previously (2).
4b1 RNA for RT-PCR was prepared by the procedures of Sato (34) and Roels and Losick (32). RNA was resuspended in 100 µl of diethylpyrocarbonate-treated water and stored at70°C. Genomic DNA that might be contaminating the RNA
preparation was destroyed by DNase I (Promega) (1 U/µl, 37°C, for 2 to 3 h), and the DNase was then inactivated by boiling for 10 min.
The absence of contaminating DNA was confirmed by PCR using the
DNase-treated RNA as the template.
For RT-PCR, 15 µl of the RNA was used to synthesize cDNA in a 33-µl
reaction mixture, including 11 µl of first-strand reaction mix
(Pharmacia Biotech), 1 mM dithiothreitol, 1 µl of antisense primer,
and 5 µl of RNase-free water at 37°C for 1 h. The samples were
then heated (95°C, 5 min) to destroy the RT, and 2 to 3 µl of the
cDNA was used as the template in a subsequent 25-µl PCR (94°C, 1 min; 50°C, 1 min; 70°C, 2 min; for a total of 30 cycles in a
Perkin-Elmer thermocycler). Antisense primers used for cDNA synthesis
were P1 (5' CTG ATT AAC AGA ATC ATG AC 3', nt 621 to 640) and P2 (5'
TGT TTA CCA GTA TAG AAC GG 3', nt 1013 to 1032). The subsequent PCRs
included one of these primers as well as one of the following primers,
as indicated in Fig. 7: P3 (5' AAA GGG ACA GGC AAC A 3', nt 6 to 21),
P4 (3' CGC ATC AGT ACT TTT TGC C 3', nt 484 to 503), and P5 (5' GCT TGT
TGC CTT CAA CAT TC 3', nt 751 to 770).
DNA sequencing and sequence analysis. DNA was sequenced on both strands either manually (Silver Sequence; Promega) or by automated sequencing at the Biotechnology Core Facility (University of Hawaii).
For DNA and protein database searches and analyses, we used FASTA (University of Wisconsin GCG package; Genetics Computer Group, Madison, Wis.) and the BLAST protein database (http://www.ncbi.nlm.nih.gov/BLAST/).Nucleotide sequence accession number. The nucleotide sequence data determined in this study have been deposited in the GenBank database under accession no. AFO 72894.
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RESULTS |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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Mutants negative for serotype-specific MAb c74.22 have
impaired TA glycosylation.
Within L. monocytogenes, MAbs c74.22, c74.33, and c74.180 have been
shown to react exclusively with strains of serotypes 4b, 4d, and 4e
(16). We have identified three serotype 4b transposon mutants, M44, B6S (also termed XL1), and 27E8 (also termed XL3), which
were completely negative for c74.22 but retained reactivity with the
other two MAbs (c74.33 and c74.180) on the basis of ELISA and colony
immunoblots (16, 20). Mutant M44 has been shown to harbor a
single copy of Tn916E (20). Western blots of
total cellular and extracellular proteins of M44 and its parental
strain 4b1 with MAb c74.22 did not identify any specific protein
band(s) reactive with this MAb (data not shown). Biochemical analysis of cell wall composition was then undertaken to determine whether the
mutant was altered in components of TA, which, on the basis of
biochemical and immunological studies, have been shown to vary among
different serotypes of L. monocytogenes (7, 10,
40, 41). It was found that cell wall TA from M44 lacked galactose and had only trace amounts of glucose (Fig.
2B). In contrast, cell wall TA from a
wild-type isogenic strain (4WT) contained both galactose and glucose
(Fig. 2A), as is typical of serotype 4b strains. Other cell wall TA
components (phosphate, ribitol, Glc-Nac) remained normal (Fig. 2A and
B), as did the amount and composition of peptidoglycan (data not
shown). Interestingly, LTA composition was also completely
normal, with both galactose and glucose being present at wild-type
levels (data not shown). Results identical to those obtained with M44
were also obtained with the independently derived c74.22-negative
mutants B6S and 27E8 (data not shown).
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Cloning and molecular characterization of gtcA. SSP-PCR was used as described in Materials and Methods to amplify a transposon-adjacent DNA fragment from mutant M44. The amplified flanking fragment (500 bp) was cloned in pCR1000, and the resulting plasmid was used as a probe in Southern blots to determine the sizes of homologous fragments in the wild type (strain 4b1) and in mutant M44. Such blots confirmed that the amplified fragment was indeed from the transposon-flanking region of M44 and localized the transposon insertion in a ca. 0.85-kb EcoRI and a 3.5-kb HindIII genomic DNA fragment. Identical Southern blot results were obtained with mutants B6S and 27E8 (data not shown), suggesting that these independently derived mutants harbored transposon insertions in the same EcoRI and HindIII fragments.
The cloned transposon-flanking fragment was used to screen a genomic library of strain 4b1 (4- to 10-kb fragments) with no success. A single positive clone was identified from the screening of another 4b1 library that contained smaller (2- to 4-kb) inserts. Southern blot analysis suggested that this clone (pC11A) contained 1.2 kb of DNA within the 3.5-kb HindIII fragment which harbored the transposon in M44 (Fig. 3). DNA sequencing of the original SSP-PCR fragment and of the fragment cloned in pC11A revealed that in M44 the transposon was inserted within an open reading frame (ORF), termed gtcA (for galactose-glucose in TA). Nucleotide sequences of transposon-flanking fragments of mutants B6S and 27E8 (amplified as described in Materials and Methods) showed that the transposon had inserted in a common target site within gtcA. In M44, Tn916E was inserted between nt 512 and 513, whereas Tn916E in B6S and Tn916 in 27E8 were
both inserted between nt 514 and 515. The transposon target site in
these mutants, TTTTCGAATAAAAA, was in close agreement with
the Tn916 preferred target sites reported for other
gram-positive bacteria (21).
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ORF analysis.
Sequence analysis revealed two complete ORFs
(gtcA and ORFD) and one incomplete ORF (ORFU) in this
region, all transcribed in the same direction (Fig. 3). An inverted
repeat followed by a series of T's after the end of ORFD may represent
a rho-independent transcription terminator (estimated free energy of
formation, 25 kcal/mol). The transposon-harboring ORF
(gtcA) was preceded by an incomplete ORF (ORFU) with 60 to
74% identity to genes encoding the transcription termination factor
rho in B. subtilis, E. coli, Salmonella typhimurium, Haemophilus influenzae,
Neisseria gonorrhoeae, and other bacteria. The available
sequence of this ORF would be expected to correspond to the 275 bp in
the 3' portion of the putative rho gene in L. monocytogenes, a portion of the coding sequence known to be
especially conserved among rho sequences (23).
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Determination of gtcA sequence in epidemic-associated strain F2381. Strain F2381 is a representative of a clonal lineage implicated in numerous outbreaks of listeriosis and known to be genetically distinct from other serotype 4b strains (3, 25, 44). Sequencing of the gtcA region (nt 289 to 1045) showed that the coding sequence of gtcA was identical for strains 4b1 and F2381. The two sequence differences identified in this region were both in the gtcA-rpmE intergenic region (at nt 756, strain F2381 had an insertion of a T; at nt 819, strain 4b1 had a G that was absent in F2381) (Fig. 3B). The sequenced portion of the coding sequence of rpmE (up to and including nt 1045) was identical for strains 4b1 and F2381.
Transcriptional analysis of gtcA. Northern blots using the gtcA-internal fragment cloned in pNP1 as probe suggested the presence of a ca. 700-nt transcript (data not shown). Signal intensity was quite low in Northern blots, suggesting a low abundance of the message. RT-PCR was therefore used to determine whether gtcA was cotranscribed with its flanking ORFs. The results of a typical RT-PCR experiment are shown in Fig. 5. cDNA synthesis was done by using primers P1 (nt 621 to 640, complementary to the expected gtcA message) and P2 (nt 1013 to 1032, complementary to the expected rpmE message). The RT-PCR results suggest that gtcA was cotranscribed with ORFD (Fig. 5, lane 4) but not with ORFU (lanes 1 and 3). Since only 32 bp separated the gtcA start codon from the ochre codon of ORFU, the gtcA promoter may be located in the 3'-end portion of ORFU. RT-PCR data suggest that the transcript initiation site was within the last 30 to 40 nt of ORFU (16).
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TA defects of gtcA mutants are complemented by wild-type gtcA in trans. Plasmids pNP21 and pNP95, harboring gtcA with or without ORFD (putative rpmE), respectively (Fig. 3), were electroporated into mutant M44. Both plasmids include the ORFU-gtcA intergenic region and the 3'-end region of ORFU, since the gtcA promoter may be contained within this region. The resulting strains were grown in the presence of chloramphenicol at 30°C, a temperature which permits both replication of the temperature-sensitive plasmid (38) and optimal expression of the serotype 4b-specific surface antigens (16). Reactivity of the mutant with c74.22 was restored by both plasmids. Reactivity was relatively low when pNP21 was used but stronger (albeit still somewhat lower than that of the wild type) with pN95 (Fig. 6). Identical results were obtained with mutant 27E8 (data not shown). Interestingly, introduction of either plasmid completely restored TA composition. The TA of M44 complemented with pNP95 contained both galactose and glucose, at levels indistinguishable from those of the wild type (Fig. 2C); identical results were obtained with M44 complemented with pNP21 (data not shown).
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Within L. monocytogenes, only serogroup 4 strains harbor gtcA sequences whereas ORFD appears to be conserved among Listeria spp. Recently, we showed that the region adjacent to the transposon insertion in mutant M44 was present in serogroup 4 (serotypes 4a, 4b, 4c, 4d, and 4e) and in certain strains of Listeria innocua (which, in contrast to all other screened L. innocua strains, were also positive for MAbs c74.22, c74.33, and c74.180 [16]) but not in other serotypes of L. monocytogenes or other Listeria species (20). Southern blots using pNP1 as the probe confirmed these findings (data not shown). In addition, Southern blots with pC11A, which includes gtcA as well as ORFD, suggested that the latter gene (putative rpmE) was present in other serotypes of L. monocytogenes as well as in all other Listeria species (hybridizing signals were weak in Listeria grayi and Listeria seeligeri) (Fig. 7). Such Southern blots also revealed HindIII restriction fragment length polymorphisms (RFLPs) which could differentiate L. monocytogenes strains of serotype 4b (Fig. 7, lanes 2 to 5) from those of serotypes 1/2a and 1/2b (lanes 1 and 6 to 9). Similar differentiation could be obtained with EcoRI RFLPs (data not shown). Southern blots of EcoRI-cut DNA with probe pNP500, which includes gtcA and only the gtcA-ORFD intergenic region, furthermore indicated that the intergenic region was present in strains of other serotypes. In serogroup 4 (serotypes 4a, 4b, 4c, 4d, and 4e), a hybridizing band of 0.8 kb was observed, whereas a 2-kb hybridizing band was observed with serogroup 1/2 (serotypes 1/2a, 1/2b, and 1/2c), serogroup 3 (serotypes 3a, 3b, and 3c), and serotype 7 (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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The gene described in this report, gtcA, appears to be essential for presentation of galactose and glucose substituents on serotype 4b TA. Support for this was provided by the findings that (i) insertional inactivation of gtcA in several independently obtained mutants abolished galactose and markedly reduced glucose levels in TA and (ii) the amounts of both sugars were restored to wild-type levels by the wild-type gtcA gene in trans. These results suggest that the phenotype of gtcA insertion mutants was due to the transposon insertion and not to an unrelated (e.g., spontaneous) mutation. In addition, complementation by the wild-type gtcA in trans strongly suggested that the impact of the gtcA mutations on TA composition was indeed due to inactivation of gtcA itself and not to a possible polar effect of the transposon insertion on a downstream gene(s).
Cell wall compositional analysis of the gtcA mutants led to two unexpected observations: in contrast to its impact on TA, the mutation was found to have no effect on LTA composition, suggesting genetic and enzymatic independence of glycosylation between TA and LTA. Furthermore, TA of gtcA mutants was found not only to lack galactose but also to have markedly reduced glucose. A possible explanation for this may be that the enzymatic step(s) mediating incorporation of glucose onto TA may require the prior presence of galactose. Although the precise function of the gtcA gene product remains to be determined, our genetic, immunological, and biochemical data suggest that it is involved in glycosylation of TA domains and that such domains serve as serotype-specific surface antigens on bacteria of serotype 4b. Key supporting data include the impaired TA glycosylation of the gtcA mutants and their lack of reactivity with MAb c74.22, as well as the finding that normal TA glycosylation and reactivity were restored when gtcA was provided in trans. The presence of hydrophobic, putative membrane-spanning domains in the deduced gtcA product would be in agreement with its involvement in TA biosynthesis. In B. subtilis, several TA biosynthesis gene products have been shown to have membrane-spanning and/or membrane-anchoring domains (18, 19), and enzymes involved in TA biosynthesis have been shown to be membrane associated in Staphylococcus aureus and Staphylococcus xylosus (8, 9). Additional insight into the function of the gtcA product will be provided by biochemical characterization of the protein and, perhaps, by characterization of homologous genes or gene products in other bacteria. In this respect, it is of interest that the gtcA homologue of B. subtilis (ipa34-d) appears to be in an operon dedicated to galactose metabolism (11). However, the precise function of ipa34-d has not yet been determined, and B. subtilis is not known to have galactose in its cell wall TA. In B. subtilis, two glucosyl transferases involved in the incorporation of glucose onto TA have been identified, the products of the gtaB and tagE (rodD), respectively (4, 12, 26, 27, 39). B. subtilis, however, uses glycerol as the glucose acceptor in its TA, whereas in L. monocytogenes serotype 4b, N-acetylglucosamine serves as the galactose and glucose acceptor. It is possible that the gtcA product may have a glycosyl transferase activity, although no homologies with similar transferases were detected by the protein database searches. Another possibility that cannot be excluded at this point is that gtcA may encode a regulatory protein, essential for expression of a gene(s) involved in glycosylation of TA in L. monocytogenes serotype 4b. The potential of gtcA to confer c74.22 reactivity to heterologous serotypes (e.g., serogroup 1/2 or 3) remains to be determined. It must be kept in mind, however, that these strains (e.g., serogroup 1/2 or 3) differ in their TA backbone structure and in TA glycosylation sites from serotype 4b strains (Fig. 1). A more likely candidate for heterologous expression would be c74.22-negative strains of L. innocua, which lack galactose and glucose but have a TA backbone structure identical to that of serotype 4b strains. We were, however, unable to introduce gtcA (cloned in pKSV7) into such an L. innocua strain (serotype 6a), perhaps because of a restriction barrier (data not shown).
It is of interest that in serogroup 4 strains of L. monocytogenes, the gtcA gene was positioned between ORFs with homology to rho and rpmE genes, whereas rho and rpmE were directly adjacent to each other in B. subtilis. Our data suggested that gtcA was the first gene of a bicistronic operon which included the putative rpmE. This was surprising since no apparent commonality in the function of these two genes was readily evident. Our data also indicated that of the two genes, only gtcA was unique to serogroup 4, whereas the putative rpmE (as well as the gtcA-rpmE intergenic region) was conserved among listeriae. It is tempting to speculate that the rho-rpmE genomic arrangement has been preserved in L. monocytogenes strains other than those of serogroup 4 (e.g., 1/2 and 3, etc.) which lack gtcA and that, in the course of evolution of serogroup 4 strains, gtcA was introduced in its present location, conferring serogroup-specific expression of a TA-associated, serotype-specific surface antigen(s). The gene may have been transferred between L. monocytogenes serogroup 4 and a lineage of L. innocua, a species which is nonpathogenic but otherwise closely related genetically and antigenically to L. monocytogenes. The gtcA gene may have originated in an organism other than Listeria, and its unusually low G+C content (30%, in contrast to 38% for L. monocytogenes and other Listeria spp.) may be indicative of this. Introduction of gtcA could have been mediated by a phage or transposon, although the currently available sequence data do not reveal any discernible traces or evidence of such elements (inverted repeats, tRNA sequences, or sequences with possible integrase or transposase functions). An alternative possibility is that the gtcA sequences were present in an ancestral L. monocytogenes-L. innocua lineage but were preserved only in serogroup 4 strains and in certain L. innocua strains. We are currently in the process of further characterizing the genomic region that harbors the putative rho and rpmE genes in strains of L. monocytogenes other than those of serogroup 4, in order to gain further insight on this issue.
Extensive molecular studies of virulence determinants have focused on a select group of extracellular and surface-associated proteins of L. monocytogenes (reviewed in references 13 and 29). In contrast, the possible involvement of carbohydrate-based surface antigens in pathogenesis remains poorly understood. Earlier studies suggested that glycosylated TA components were important antigenic determinants in L. monocytogenes (14, 41). We have obtained data, to be described in a separate presentation, which strongly suggest that galactose on TA of L. monocytogenes serotype 4b was essential as a receptor for serotype-specific phages and, in addition, for invasion of several mammalian cell lines by the bacteria (30). Such data are in agreement with findings from other investigations which showed that in serotype 1/2, rhamnose and Glc-NAc on TA served as receptors for serotype-specific phages (42). However, genes involved in the decoration of serotype 1/2 TA with rhamnose and Glc-NAc have not yet been identified, and information regarding their possible serotype-specific distribution in L. monocytogenes is lacking. Further investigations are needed to enhance our understanding of the evolution of genetic systems dedicated to TA glycosylation and of the possible function(s) of the different types of TA glycosylation in the adaptive physiology and pathogenesis of the microorganism.
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ACKNOWLEDGMENTS |
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This research was partially supported by U.S. Department of
Agriculture Competitive Research Initiative AAFS grant 92-37 201-8095, by ILSI
North America, and by a seed grant from the University of Hawaii.
We thank Vladimir Lazarevic for helpful discussions and exchange of information related to TA biosynthesis. We also thank Nikhat Parveen for assistance with Northern blots, and we are grateful for the valuable participation of Timothy Rodwell, Thong Truong, Xiang-He Lei, and Wei Zheng in certain aspects of this work. We thank the members of our laboratories for valuable feedback and support throughout the course of this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, University of Hawaii, 2538 The Mall, Snyder 207, Honolulu, HI 96822. Phone: (808) 956-8015. Fax: (808) 956-5339. E-mail: ksophia{at}hawaii.edu.
Present address: Centers for Disease Control and Prevention,
Atlanta, Ga.
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REFERENCES |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Journal of Bacteriology, January 1999, p. 418-425, Vol. 181, No. 2
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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