Genetic diversity of three lgt loci for biosynthesis of lipooligosaccharide (LOS) in Neisseria species

Peixuan Zhu1, Michael J. Klutch2, Margaret C. Bash1, Raymond S. W. Tsang3, Lai-King Ng3 and Chao-Ming Tsai1

Division of Bacterial, Parasitic and Allergenic Products1 and Division of Viral Products2, Center for Biologics Evaluation and Research, FDA, 8800 Rockville Pike, Bethesda, MD 20892, USA
National Microbiology Laboratory, Population and Public Health Branch, Health Canada, Canada3

Author for correspondence: Peixuan Zhu. Tel: +1 301 496 4177. Fax: +1 301 402 2776. e-mail: Zhu{at}cber.fda.gov


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic Neisseria. Nine lgt genes at three chromosomal loci (lgt-1, 2, 3) encoding the glycosyltransferases responsible for the biosynthesis of LOS oligosaccharide chains were examined in 26 Neisseria meningitidis, 51 Neisseria gonorrhoeae and 18 commensal Neisseria strains. DNA hybridization, PCR and nucleotide sequence data were compared to previously reported lgt genes. Analysis of the genetic organization of the lgt loci revealed that in N. meningitidis, the lgt-1 and lgt-3 loci were hypervariable genomic regions, whereas the lgt-2 locus was conserved. In N. gonorrhoeae, no variability in the composition or organization of the three lgt loci was observed. lgt genes were detected only in some commensal Neisseria species. The genetic organization of the lgt-1 locus was classified into eight types and the lgt-3 locus was classified into four types. Two types of arrangement at lgt-1 (II and IV) and one type of arrangement at lgt-3 (IV) were novel genetic organizations reported in this study. Based on the three lgt loci, 10 LOS genotypes of N. meningitidis were distinguished. Phylogenetic analysis revealed a gene cluster, lgtH, which separated from the homologous genes lgtB and lgtE. The lgtH and lgtE genes were mutually exclusive and were located at the same position in lgt-1. The data demonstrated that pathogenic and commensal Neisseria share a common lgt gene pool and horizontal gene transfer appears to contribute to the genetic diversity of the lgt loci in Neisseria.

Keywords: Neisseria meningitidis, Neisseria gonorrhoeae, glycosyltransferase, oligosaccharide structure, antigenic variation

Abbreviations: LOS, lipooligosaccharide

The GenBank accession numbers for the sequences reported in this paper are AF470655AF470685.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Lipooligosaccharide (LOS) is a major virulence factor of two pathogenic Neisseria species, Neisseria meningitidis and Neisseria gonorrhoeae (Kahler & Stephens, 1998 ; Preston et al., 1996 ). In contrast to the lipopolysaccharide (LPS) in many enteric bacteria, N. meningitidis and N. gonorrhoeae LOS lack repeating O-chains and possesses variable oligosaccharide chains. These oligosaccharide chains are branched and designated {alpha}, ß and {gamma} chains (Kahler & Stephens, 1998 ; Verheul et al., 1993 ; Zhu et al., 2001 ) (Fig. 1). The Neisseria LOS is heterogeneous and the expression of LOS is subject to phase variation (Burch et al., 1997 ; Danaher et al., 1995 ; Jennings et al., 1995 ; Yang & Gotschlich, 1996 ). N. meningitidis LOS is divided into 12 immunotypes (L1–12) on the basis of specific antibody reactions (Mandrell & Zollinger, 1977 ; Zollinger et al., 1977 , 1980 ). Immunotypes L1–L8 are primarily associated with N. meningitidis serogroups B and C while immunotypes L9–L12 are mainly associated with serogroup A.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1. General structure of Neisseria LOS and functional relationship of the lgt genes. The {alpha} chain of LOS immunotype L8 is limited to Galß1–4Glc attached to the heptose I of the inner core. The {alpha} chain of LOS immunotype L3,7 contains a lacto-N-neotetraose structure of Galß1–4GlcNAcß1–3Galß1–4Glc. The alternative {alpha} and ß chain of oligosaccharides are indicated with dotted lines. PEA and Ac refer to phosphoethanolamine and acetyl residue, respectively. PEA replaces the ß chain at the 3' position of HepII for some N. meningitidis LOSs. The terminal residue present in N. gonorrhoeae is indicated with (Ng). The lgt genes at the three chromosomal loci are indicated as lgt-1 (lgtA, lgtB, lgtC, lgtD and lgtE), lgt-2 (lgtF and rfaK) and lgt-3 (lgtG).

 
Three genetic loci (lgt-1, 2 and 3) were reported to encode the glycosyltransferases responsible for biosynthesis of the LOS oligosaccharide chains (Fig. 1). The lgt-1 locus consists of 7 ORFs designated lgtA, lgtB, lgtC, lgtD, lgtE, lgtH and lgtZ (Gotschlich, 1994 ; Zhu et al., 2001 ). LgtA, LgtB and LgtE proteins are responsible for the biosynthesis of the lacto-N-neotetraose structure (Galß1–4GlcNAcß1–3Galß1–4Glc) of the {alpha} chain. LgtC is an {alpha}1,4-galactosyltransferase and is responsible for adding galactose to an alternative {alpha} chain. LgtD is an N-acetylgalactosaminyl-transferase and is responsible for adding an N-acetylgalactosamine to the terminus of the {alpha} chain in N. gonorrhoeae. lgtH and lgtZ at the lgt-1 locus in N. meningitidis are homologous to other lgt genes; however, their biological function remains to be determined (Zhu et al., 2001 ).

The lgt-2 locus includes lgtF and rfaK that are involved in the biosynthesis of {alpha} and {gamma} chains, respectively (Fig. 1) (Kahler et al., 1996a , b ; van der Ley et al., 1997 ). lgtF encodes a ß1,4 glucosyltransferase that is responsible for adding Glc to HepI in the inner core ({alpha} chain). rfaK encodes the {alpha}1,2-N-acetylglucosaminyltransferase that adds GlcNAc to HepII in the inner core ({gamma} chain). The lgt-3 locus has an lgtG gene encoding {alpha}1,3 glucosyltransferase for biosynthesis of the ß chain (Fig. 1) (Banerjee et al., 1998 ). Four genes at the lgt-1 and lgt-3 loci, lgtA, lgtC, lgtD and lgtG, have a homopolymeric tract that is thought to be involved in LOS phase variation by the slipped strand mispairing mechanism (Danaher et al., 1995 ; Jennings et al., 1995 ; Yang & Gotschlich, 1996 ). Therefore, both the composition and expression of lgt will regulate the biosynthesis of LOS.

In N. gonorrhoeae, the lgt-1 locus has been sequenced in three strains (F62, FA1090 and 1291) and analysed in an additional four strains (15253, MS11, M94 and R10), by DNA hybridization (Erwin et al., 1996 ; Gotschlich, 1994 ; Harvey et al., 2000 ). The composition and organization of lgt-1 for N. gonorrhoeae is the same for all sequenced strains; only the hybridization data from strain 15253 suggests that there may be some heterogeneity (Erwin et al., 1996 ). In N. meningitidis, lgt-1 has been sequenced in five strains [126E (L1), MC58 (L3), Z2491 (L9), A1 (L8) and M978 (L8)] and analysed for 10 representative strains of 10 LOS immunotypes (L1, L2, L3, L4, L5, L6, L8, L10, L11 and L12) by DNA hybridization (Jennings et al., 1995 , 1999 ; Parkhill et al., 2000 ; Tettelin et al., 2000 ; Zhu et al., 2001 ). These studies indicate that N. meningitidis has a small and variable repertoire at the lgt-1 locus. Both the composition and organization of lgt-1 differ among the N. meningitidis strains examined (Jennings et al., 1999 ). The lgt-1 locus in two immunotypes, L7 and L9, has not been determined, and the lgt genetic organization and DNA sequences of 8 LOS immunotypes of N. meningitidis (L2, L4, L5, L6, L7, L9, L10, L11 and L12) have not been reported. In commensal Neisseria, only one lgt-1 sequence from strain 44 of Neisseria subflava has been reported (Arking et al., 2001 ).

To understand the genetic basis of LOS heterogeneity and antigenic variation in Neisseria, nine lgt genes at three chromosomal loci were examined in 95 strains of N. meningitidis, N. gonorrhoeae and commensal Neisseria by DNA hybridization, PCR, restriction analysis and nucleotide sequencing.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, growth conditions and DNA isolation.
Twelve LOS prototype strains [126E (L1), 35E (L2), 6275 (L3), 89I (L4), M981 (L5), M992 (L6), 6155 (L7), M978 (L8), M120 (L9), 7880 (L10), 7889 (L11), 7897 (L12)] and four N. meningitidis strains [A1 (L8), M986 (L3), BB305 (L3) and 44/76] were obtained from the culture collection of Dr C. E. Frasch of the Center for Biologics Evaluation and Research, FDA, Bethesda, MD, USA. Eight N. meningitidis strains (2001083, 2001130, 2001113, 2001117, 2001110, 2001111, 2001147 and 2001148), representing strains of the serogroup C ET-15 variant endemic in Canada since the 1980s and strains associated with the 2001 outbreak in Canada, are from the authors’ collection. N. gonorrhoeae strains 920905–920911, 920913–920920, 920921–920925, 921001–921010 are clinical isolates from individuals with urethritis and were obtained from Dr J. Zenilman, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Disseminated gonococcal infection strains no. 3, 4, 5, 8, 10, 18, 19, 26, 27 and 29 have been previously described (O’Brien et al., 1983 ). Nine N. gonorrhoeae strains (880140, 880250, 880288, 880447, 881051, 881096, 882602, 882916, 883021) associated with pelvic inflammatory disease, N. gonorrhoeae reference strain F62 and 18 commensal Neisseria strains were from the authors’ culture collection. The bacterial strains were grown on Brain–Heart Infusion (BHI) agar at 37 °C in 5% CO2 for 16 h. Chromosomal DNA was isolated as described (Olyhoek et al., 1991 ; Sarkari et al., 1994 ). The DNA samples of N. meningitidis serogroup A, subgroup IV-1 strain Z2491 and N. gonorrhoeae strain FA1090 were obtained from Dr M. Achtman of the Max-Planck-Institut für molekulare Genetik in Berlin, Germany. The DNA sample of N. meningitidis serogroup B, ET-5 complex strain MC58 (ATCC accession no. BAA-335) was obtained from Dr H. Tettelin of the Institute for Genomic Research (TIGR), Rockville, MD, USA.

PCR amplification of the lgt genes.
Three lgt loci were examined by PCR using external primers at the flanking region and internal primers shown in Table 1. Primers for lgt-1, lgt-2 and lgt-3 were designed from the conserved regions identified by multiple alignment of eight [strain 126E, GenBank accession no. U65788; MC58, U25839; Z2491, AL162753.2; M978, AF355193; A1, AF355194; F62, U14554; 1291, AF121135; and FA1090, AE004969], four [strain CDC8201085, GenBank no. NMU58765; MC58, AE002520; Z2491, AL162757; and FA1090, AE004969] and four [strain 15253, GenBank no. AF076919; MC58, AE002553; FA1090, AE004969; and 44, AF241526] available lgt gene sequences, respectively.


View this table:
[in this window]
[in a new window]
 
Table 1. Primers used in this study

 
The size of each locus in the bacterial strains was examined by PCR using the external primers in the flanking regions. The presence or absence of each lgt gene was detected by PCR using internal primers (Table 1) using both the chromosomal DNA and the PCR product of the whole locus as templates for parallel analysis. The gene order was mapped using multiple combinations of external and internal primers and also by comparison to the control strains, whose lgt organization were known. The whole lgt region was sequenced when the size or order of lgt was different from the control strains.

The PCR reaction mixtures contained 1 µl 10 mM dNTPs, 10 pmol each primer, 0·1 µg chromosomal DNA, 5 µl 10x PCR buffer, 1·5 U Taq DNA polymerase (Perkin-Elmer) and sterile redistilled H2O in a final volume of 50 µl. PCR amplification was performed on a GeneAmp 9700 cycler (Perkin-Elmer) using the following protocol: denaturation at 94 °C for 2 min, 30 cycles of amplification at 94 °C for 30 s, 56 °C for 30 s and 72 °C for 2 min, and a final extension at 72 °C for 4 min. The PCR products were analysed by electrophoresis on 1% agarose gels and stained with ethidium bromide.

Restriction analysis.
Amplified DNA fragments from the three lgt loci were subjected to digestion with restriction endonucleases AluI, HaeIII, RsaI and MspI (Roche Molecular Biochemicals). The digestion was performed using 8 µl PCR product, 1·0 µl 10xrestriction endonuclease buffer, 10U restriction endonuclease and sterile distilled water to a final volume of 10 µl. The mixture was incubated at 37 °C for 1 h and then separated on a 1·5% agarose gel and stained with ethidium bromide.

DNA dot blot hybridization.
Bacterial genomic DNA was boiled for 2 min and blotted to Hybond-N+ membranes (Amersham). The DNA probes for lgtA, lgtB, lgtC, lgtD, lgtE, lgtH, lgtF and lgtG were labelled with DIG-11-dUTP (PCR-DIG labelling mix, Roche Molecular Biochemicals) during PCR amplification using the primers in Table 1 and the genomic DNAs from strains F62 (lgtA, lgtB, lgtC, lgtD and lgtE probes), MC58 (lgtF and lgtG) and Z2491 (lgtH) as the templates. DNA hybridization and detection were performed as described by the manufacturer (DIG Nucleic Acid Detection Kit, Roche Molecular Biochemicals).

DNA sequencing.
The PCR products were purified by QIAquick spin columns (Qiagen). DNA sequences were determined from both strands of three independent PCR products for each strain using the primers in Table 1 and 28 additional internal primers (presented in a supplementary table at http://mic.sgmjournals.org). The sequences of homopolymeric regions in lgtA, lgtC and lgtG were determined again from cloned templates. The PCR products were cloned into pCRT7 vector (Invitrogen) and three clones for each strain were used for sequencing. DNA sequencing was performed using the ABI PRISM Dye Terminator Sequencing Kit with AmpliTaq DNA polymerase FS (Perkin Elmer) on a model 377 automated sequencer (Applied Biosystems). DNA sequences were analysed with the Genetics Computer Group package (GCG10.2-Unix, University of Wisconsin) and the Molecular Evolutionary Genetics Analysis software (MEGA2.1, Arizona State University) (Devereux et al., 1984 ; Kumar et al., 1994 ). The sequence data from this study have been submitted to the GenBank database with accession numbers AF470655AF470685.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Distribution of the lgt genes in Neisseria
The distribution of nine lgt genes at the three chromosomal loci, lgt-1, lgt-2, lgt-3, was examined by both DNA hybridization and PCR. Ninety-five strains of Neisseria, including 26 N. meningitidis strains, 51 N. gonorrhoeae strains and 18 strains of commensal Neisseria were examined. The lgt loci have been partially or fully described previously for five of the N. meningitidis strains and two of the N. gonorrhoeae strains studied. The results obtained were in agreement with previous reports (Gotschlich, 1994 ; Jennings et al., 1995 , 1999 ; Parkhill et al., 2000 ; Zhu et al., 2001 ); these seven strains served as controls for subsequent comparisons.

The distributions of lgt genes at the three loci in different Neisseria species are shown in Table 2. At the lgt-1 locus, all 49 strains of N. gonorrhoeae had the same lgt genes as the reference strain FA1090: lgtA, lgtB, lgtC, lgtD and lgtE. The lgt genes for N. meningitidis were more variable. lgtD was present only in N. meningitidis strain 126E (L1). lgtE and lgtH in N. meningitidis were mutually exclusive. lgtA and lgtB were present in all but one and three strains of N. meningitidis, respectively. At the lgt-2 locus, all N. gonorrhoeae strains and all 25 N. meningitidis strains tested in this study had lgtF. However, only three commensal Neisseria strains had lgtF: Neisseria cinerea (strain 81176), Neisseria lactamica (81186) and Neisseria polysaccharea (87043). At the lgt-3 locus, 18 N. meningitidis strains, four N. lactamica strains, one N. subflava strain and all N. gonorrhoeae strains had lgtG. The lgt genes were not detected in two strains of N. subflava (81187 and 81201) and one strain each of Neisseria canis, Neisseria caviae, Neisseria flavescens, Neisseria mucosa, Neisseria ovis, Neisseria sicca and Neisseria weaveri.


View this table:
[in this window]
[in a new window]
 
Table 2. Distribution of nine lgt genes in 95 strains of Neisseria

 
The results from DNA hybridization and PCR analysis were consistent except for two strains, 81176 of N. cinerea and N9 of Neisseria elongata. In strain 81176, the lgtC signal was detected by hybridization but the size of the PCR product (4·8 kb) for lgtC using primer pair P13/P18 differed from the size expected (747 bp). Further analysis showed that the size difference resulted from a DNA fragment of ~4 kb inserted into the 5' region of lgtC. In strain N9, hybridization signals of lgtA, lgtH and lgtG were also observed but the genes were not detected by PCR amplification. The details of the genetic variation in the lgt genes in these two strains have not been analysed further.

Genetic organization of the lgt loci
The lgt-1 locus of 12 N. meningitidis strains (35E, 6275, M986, 89I, M981, M992, 6155, M120, 7880, 7889, 7897 and BB305) was sequenced. For the other 66 strains that yielded positive signals in the hybridization and PCR, the lgt gene order was mapped by PCR using multiple combinations of flanking and internal primers. The data were compared to the reported lgt-1 from five strains of N. meningitidis (strains Z2491, MC58, 126E, A1 and M978) (Jennings et al., 1995 , 1999 ; Parkhill et al., 2000 ; Zhu et al., 2001 ), seven strains of N. gonorrhoeae (FA1090, F62, 15253, 1291, MS11, M94 and R10) (Erwin et al., 1996 ; Gotschlich 1994 ; Harvey et al., 2000 ) and one strain of N. subflava (44) (Arking et al., 2001 ). Based on the genetic organization, the lgt-1 locus in these strains was classified into eight genetic types (Types I–VIII; Fig. 2). The organization of all N. gonorrhoeae strains was identical and designated Type I. This type was not observed in any other Neisseria species. Type II was found in both N. lactamica and N. subflava. Types III–VIII were found in N. meningitidis as well as in commensal Neisseria (one N. subflava, VI; two N. polysaccharea, VII).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2. Schematic representation of genetic organizations of the lgt-1 locus in Neisseria. Letters on the left indicate the genetic types (Types I–VIII) of lgt-1. Letters on the right indicate the Neisseria species as follows: Ng, N. gonorrhoeae; Nl, N. lactamica; Nm, N. meningitidis; Ns, N. subflava; Np, N. polysaccharea. Numbers in parentheses indicate the number of strains for each type. Arrows indicate the ORFs of the lgt genes. For comparison, the arrows for the ORF of lgtA correspond to previous reports (Gotschlich, 1994 ; Jennings et al., 1995 ). Two primers (P1/P2) in the flanking region of lgt-1 are indicated by small arrows. The shaded boxes indicate partial lgt genes including: a, 78 bp of lgtA; b, 17 bp of lgtC; c, 120 bp of lgtA.

 
Only two types of lgt-2 could be distinguished: with lgtF (Type I) or without lgtF (Type II). In contrast to lgt-2, differences between N. gonorrhoeae and N. meningitidis were seen in the gene distribution at lgt-3. The lgtG gene was found in all N. gonorrhoeae strains but only in 68% of N. meningitidis strains. To understand the genetic basis for the absence of lgtG in the N. meningitidis strains, PCR was performed for 24 N. meningitidis strains with the lgt-3 flanking primers (P47/P48) and internal primers. Three reference strains whose lgt-3 sequences were known (FA1090, MC58 and Z2491) were used as controls. Comparative analysis divided the genetic organization of lgt-3 into four types, Types I–IV (Fig. 3). Types I, II and III were represented by three control strains (FA1090, MC58 and Z2491, respectively). Type IV was a new organization and therefore the sequence of this locus was determined for a representative strain (126E).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Schematic representation of genetic organizations of the lgt-3 locus in Neisseria. Labelling is as in Fig. 2. Deletions are shown as dotted lines.

 
At the lgt-3 locus, the lgtG gene is flanked with ORFs encoding hypothetical proteins (Parkhill et al., 2000 ; Tettelin et al., 2000 ). These ORFs are indicated as orf1 (NMA0409, NMB2031), orf2 (NMA0408) and orf3 (NMA0405, NMB2033) in Fig. 3. The basic arrangement of orf1 and orf3 is similar in the four types at lgt-3; however, the composition between orf1 and orf3 is variable (Fig. 3). Fifteen N. meningitidis strains (35E, 6275, M986, 89I, M992, A1, BB305, 2001083, 2001130, 2001113, 2001117, 2001110, 2001111, 2001147 and 2001148) and FA1090 have Type I organization with lgtG and orf2 between orf1 and orf3. Two strains (MC58 and M981) have Type II organization (without orf2). Five strains (Z2491, 7880, 7889, 7897 and M120) have Type III organization (without lgtG but with an insertion between orf2 and orf3 of a 569 bp pseudogene; NMA0407, putative acetyltransferase) (Parkhill et al., 2000 ). Three strains (126E, 6155 and M978) have type IV organization (without lgtG or orf2) (Fig. 3). In addition, two lgt-3 types were observed in commensal Neisseria strains. N. subflava strain 85071, N. cinerea strain 81176 and four N. lactamica strains (81186, 89375, 89421 and 93302) had Type II organization. Two N. polysaccharea strains (85323 and 87043) had Type IV organization.

Size variation and sequence polymorphism of the lgt genes
In addition to the lgt-1 sequences from 12 N. meningitidis strains, the sequences of lgtF were determined from 14 N. meningitidis strains, which included all 12 of the LOS prototype strains and two additional N. meningitidis strains (M986 and A1). The sequences of lgtG were determined from seven N. meningitidis strains (35E, 6275, M986, 89I, M981, M992 and A1) as well. lgtF and lgtG from N. gonorrhoeae and commensal Neisseria were analysed by digestion of PCR products with four restriction endonucleases (AluI, HaeIII, MspI and RsaI). Based on the distinct restriction patterns, five lgtF genes and six lgtG genes were also sequenced from N. gonorrhoeae strain 880140, and commensal Neisseria strains 81186, 93302, 81176 and 85071. The sequences obtained in this study were compared to the lgt sequences reported in the databases (Arking et al., 2001 ; Gotschlich, 1994 ; Harvey et al., 2000 ; Jennings et al., 1995 , 1999 ; Kahler et al., 1996a , b ; Parkhill et al., 2000 ; Tettelin et al., 2000 ; Zhu et al., 2001 ). The size variation and sequence polymorphisms of the lgt genes are shown in Table 3. The mean G+C content for each lgt gene varied from 45·09 mol% to 56·51 mol% (Table 3). Six genes (lgtA, lgtB, lgtC, lgtD, lgtE and lgtG) showed size variations whereas three genes (lgtF, lgtH and lgtZ) had a conserved length (Table 3). The size variations resulted from variations in the homopolymeric region, insertions and deletions, or presence of a premature stop codon. The genetic organization of the three lgt loci for 25 N. meningitidis strains and homopolymeric tract length for 17 N. meningitidis strains are summarized in Table 4.


View this table:
[in this window]
[in a new window]
 
Table 3. Size variation and sequence polymorphism of the lgt genes in Neisseria

 

View this table:
[in this window]
[in a new window]
 
Table 4. Genotypes and homopolymeric tracts of the lgt genes in N. meningitidis

 
Each distinctive nucleotide sequence at the coding region was given an arbitrary allele designation for the lgt genes. The lgt genes in Neisseria were highly diverse as evidenced by allelic variation. Among the 20 lgtA genes sequenced, there were 18 unique lgtA alleles (lgtA1–18). A total of 116 (11·08%) polymorphic sites were identified for the aligned lgtA nucleotide sequences. Of the 18 lgtB gene sequences, 16 distinct lgtB alleles (lgtB1–16) were observed. One hundred and fifty-four (18·33%) polymorphic sites were identified in the aligned lgtB sequences. Of the 12 lgtE sequences, 11 distinct lgtE alleles were observed. One hundred and twenty-one (14·35%) polymorphic sites were identified among the aligned lgtE sequences. Sequence variations were also observed for six lgtC genes (lgtC1–6) and four lgtD genes (lgtD1–4). Sixty-two (6·62%) and 95 (9·32%) polymorphic sites were identified in lgtC and lgtD, respectively. Twenty lgtF sequences showed 13 alleles with 56 polymorphic sites (7·38%). Fifteen lgtG sequences showed 14 alleles with 109 polymorphic sites (10·29%) (Table 3).

The number of synonymous substitutions per synonymous site (Ks) and nonsynonymous substitutions per nonsynonymous site (Ka) were estimated for all pairwise comparisons by the Li method using the DIVERGE in the GCG 10.2 program package (Devereux et al., 1984 ; Li et al., 1985 ; Li, 1993 ). Ks varied from 1·94% to 11·52% and Ka from 0·73% to 4·71%. Based on the amino acid substitution frequencies, lgtF, lgtH and lgtZ were the most conserved genes. The Ka/Ks ratio, which is a measure of the selective constraints on a gene, varied from 0·14 to 0·83. In the lgtA, lgtB, lgtC, lgtD and lgtE genes at the lgt-1 locus, the levels of synonymous and nonsynonymous site variation were elevated, and similarly, the Ka/Ks ratios (0·38 to 0·83) were extremely high (Table 3).

Phylogenetic analysis of the lgt genes
A phylogenetic tree was constructed using the neighbour-joining method by program MEGA 2.1 from the nucleotide sequences for each distinct allele of the lgtB, lgtE and lgtH genes (Kumar et al., 1994 ; Saitou & Nei, 1987 )(Fig. 4A). The phylogenetic tree reveals that lgtH was related to but separated from lgtB and lgtE. The genetic distances among lgtH alleles are shorter than that among lgtB and lgtE in the phylogenetic tree, suggesting that lgtH might be a new paralogue resulting from a recent gene duplication event or imported from other bacterial sources. It was also noted that lgtB and lgtE from three strains of N. gonorrhoeae (F62, 1291 and FA1090) clustered into a group, similar to their relationship in a tree of lgtA genes (not shown).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4. Phylogenetic trees of the lgtB, lgtE, lgtH, lgtF and lgtG genes encoding glycosyltransferases in Neisseria. Trees were constructed from the representative strains for each distinct lgt gene allele using the neighbour-joining method as implemented by the Molecular Evolutionary Genetics Analysis software (MEGA 2.1, Arizona State University) (Kumar et al., 1994 ; Saitou & Nei, 1987 ). (A) The sequence relationships among the distinct alleles of the three homologous genes lgtB, lgtE and lgtH. A cluster of eight alleles of the lgtH gene from N. meningitidis strains is indicated by a box of broken lines. ’.b’ indicates the lgtB sequence to distinguish the same strain name for the lgtE or lgtH sequences. (B, C) The sequence relationships among the 13 alleles of lgtF and the 14 alleles of lgtG. Cn, Ng and Nm indicate commensal Neisseria, N. gonorrhoeae and N. meningitidis, respectively.

 
Phylogenetic trees were constructed for the 13 distinct lgtF alleles and for the 14 distinct lgtG alleles at the other two loci (Fig. 4B, C). The phylogenetic tree shows the lgtF genes from three species are highly related (Fig. 4B). Sequence relationships could be observed between N. lactamica strain 88186 and N. meningitidis strain M120, as well as N. cinerea strain 81176 and N. gonorrhoeae strain FA1090. In contrast, the lgtG genes formed three distinct groups (Fig. 4C). The first group consisted of three lgtG sequences from the commensal Neisseria strains 85071, 81186 and 93302. The second group consisted of three sequences from N. gonorrhoeae strains FA1090, 15253 and 880140. The third included five alleles from six lgtG sequences of N. meningitidis strains A1, M986, 6275, M981, M992 and 35E (Fig. 4C). The 3' 51 bp of lgtG in the commensal Neisseria was very different from that in the other two groups of lgtG, with only ~50% homology.

The lgtG gene from strain MC58 was an intermediate branch between the N. meningitidis group and the commensal Neisseria. The 5' end of lgtG from MC58 was similar to other N. meningitidis strains but the 3' 51 bp was identical to the lgtG genes from the commensal Neisseria, suggesting intragenic recombination between commensal Neisseria and N. meningitidis. The reported lgtG from N. subflava strain 44 (Arking et al., 2001 ) was closely related to N. meningitidis strain 89I (Fig. 4C). These two strains formed an intermediate branch between the N. gonorrhoeae and N. meningitidis groups. The 5' and 3' ends of lgtG from these two strains were similar to N. meningitidis but five polymorphic sites in the internal region were identical to lgtG from N. gonorrhoeae. These data implied an independent evolutionary history for the genes at three lgt loci involved in LOS biosynthesis.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this report, we described the genetic diversity of the three lgt loci encoding glycosyltransferases responsible for biosynthesis of the oligosaccharide chains of LOS in N. meningitidis, N. gonorrhoeae and commensal Neisseria.

In 51 N. gonorrhoeae strains examined in this study, only one genetic type of lgt-1, Type I, was found. This type contained five genes: lgtA, lgtB, lgtC, lgtD and lgtE. These results agree with all reports in the literature except for strain 15253 (Erwin et al., 1996 ). In this study we have examined a variety of clinical isolates including urethritis, disseminated gonococcal infection and reference strains. Therefore, it appears that the lgt-1 organization of 15253 is not common and lgt-1 of N. gonorrhoeae strains is not diverse. At the lgt-2 and lgt-3 loci, all 51 strains of N. gonorrhoeae contain lgtF and lgtG, respectively. N. gonorrhoeae strains showed only Type I organization at the lgt-3 locus. These results revealed that most N. gonorrhoeae strains have a common lgt composition and arrangement at three chromosomal loci for biosynthesis of LOS. The LOS heterogeneity and antigenic variation in N. gonorrhoeae may be controlled at the transcriptional or the translational level.

Six genetic types of lgt-1 from Type III to Type VIII were recognized in 25 N. meningitidis strains, representing 12 LOS phenotypes. The common lgt-1 types in N. meningitidis were Types VI and VII, which contained three genes, lgtABE or lgtABH. At the lgt-3 locus, N. meningitidis strains had a variable organization from Type I to IV. This suggests that the lgt composition and arrangement in N. meningitidis are important factors in LOS heterogeneity and antigenic variation. In two N. meningitidis strains, 7880 and 7889, the genes at the lgt-1 locus detected in this study differ from the previous report (Jennings et al., 1999 ). The PCR, DNA hybridization and nucleotide sequencing have been repeated to verify the data. The reproducible results showed that these two strains contain lgtABH and lgtAH at the lgt-1 locus, respectively.

The N. meningitidis LOS is classified into 12 immunotypes (Mandrell & Zollinger, 1977 ; Zollinger et al., 1977 , 1980 ). However, N. meningitidis LOS is heterogeneous and its expression is subject to antigenic variation (Burch et al., 1997 ; Danaher et al., 1995 ; Jennings et al., 1995 ; Yang & Gotschlich, 1996 ), which complicate the LOS phenotype as a marker for epidemiological purposes. On the basis of genetic organization of the three lgt loci, 25 N. meningitidis strains examined in this study can be divided into 10 LOS genotypes (Table 4). The eight Canadian strains all had LOS genotype 3. Furthermore, the sequence comparison showed that N. meningitidis strains with the same LOS genotype had variable combinations of the lgt gene alleles. Therefore, they could be further divided into LOS genetic subtypes.

No absolute correlation was observed in the relationships between the LOS genotypes and LOS phenotypes (Table 4). LOS phenotyping detects the antigenic variation of LOS products whereas the LOS genotyping detects the genetic basis for the LOS biosynthesis. Many N. meningitidis strains express a LOS mixture shown as multiple bands on SDS-PAGE gels, while some strains express only a single LOS band. LOS phenotyping classifies N. meningitidis strains into a major LOS immunotype plus one or more minor LOS immunotypes. LOS genotyping could distinguish N. meningitidis strains with the potential for variation in LOS expression. For example, strain A1 has the same L8 major immunotype as M978 but lacks expression of L3,7 (Zhu et al., 2001 ).

In some cases, differences in expression of lgt genes may result in different phenotypes among strains with the same genotype. For example the homopolymeric tract of lgtG is in-frame in M981 (C11) but out-of-frame in MC58 (C12). Extension of the LOS ß chain controlled by lgtG might be one of the reasons for the phenotype difference. Strain 7889 (L11) has the same organization at the lgt-1 locus as A1 (L8) but expresses a different LOS immunotype. LOS from strain 7889 does not contain the terminal Gal (unpublished data), indicating that lgtH is not expressed or non-functional. Comparison of the LgtH protein sequences showed three amino acids difference between the two strains. At the lgt-3 locus, strain A1 has lgtG for extension of the ß chain but strain 7889 does not. This suggests that the antigenic difference between L8 and L11 LOS may be influenced by both the {alpha} and ß chains.

Therefore, while LOS genotyping cannot replace LOS phenotyping, it provides complementary information that will contribute to our understanding of the mechanisms of LOS antigenic variation. Also, since LOS genotype subdivided several common LOS phenotypes and is not phase variable, it may serve as a useful epidemiologic marker.

Interspecies gene transfer and frequent DNA recombination in pathogenic and commensal Neisseria species has been observed at several chromosomal loci, e.g. the penA and tbpB genes (Linz et al., 2000 ; Spratt et al., 1992 ). In this study, the genes lgtA and H were detected in six species, lgtB and F in five species, lgtC and G in four species and lgtD in three species of Neisseria (Table 2). The distribution of the lgt genes in multiple species provides evidence that pathogenic Neisseria and some commensal Neisseria share a common lgt gene pool for LOS biosynthesis. Some genes seem specific for certain species, e.g. lgtE was detected almost exclusively in the pathogenic species N. gonorrhoeae and N. meningitidis, and the mosaic lgtZ was detected only in N. meningitidis. No lgt genes were detected in seven species: N. canis, N. caviae, N. flavescens, N. mucosa, N. ovis, N. sicca and N. weaveri. Only the hybridization signals of lgtA, H and G were observed in N. elongata and no specific products were obtained in the PCR amplification of this strain. These commensal Neisseria strains do express LOS and therefore must have lgt genes with low homology in the primer-binding regions or differ in other ways from those examined in this paper.

Two ORFs, lgtH and lgtZ (Zhu et al., 2001 ), are homologues to the lgt genes. The lgtH gene reported in strain Z2491 and strain A1 is closely related to two known genes, lgtB and lgtE. This ORF was originally described as lgtB2 in the genome of N. meningitidis strain Z2491 (Parkhill et al., 2000 ) based on its close relationship to lgtB. However, its 3' end differs from that of both lgtB and lgtE. In this study, the lgtH gene was found in seven additional N. meningitidis strains and in three species of commensal Neisseria. Eight distinct lgtH alleles were identified from nine lgtH sequences from N. meningitidis. Phylogenetic analysis revealed that lgtH is a gene cluster separated from lgtB and lgtE (Fig. 4A). The location of lgtH and its mutual exclusivity with lgtE suggests that it may be an allele of lgtE, so further discussion regarding the nomenclature of this gene may be warranted. lgtZ is a mosaic structure consisting of parts of lgtA and lgtB. It was observed in three N. meningitidis strains, 126E (Jennings et al., 1999 ), M978 (Zhu et al., 2001 ) and BB305. The biological function of lgtH and lgtZ is currently being evaluated.

Both the genetic organization and DNA sequence of the lgt-2 locus were more conserved than in other lgt loci. The G+C content of lgtF (45 mol%) was remarkably lower than the mean G+C content of two N. meningitidis genomes (52 mol%) (Parkhill et al., 2000 ; Tettelin et al., 2000 ). Furthermore, all strains of N. gonorrhoeae and N. meningitidis contained lgtF at the lgt-2 locus and the Ka/Ks ratio of lgtF (0·14) was the lowest of the lgt genes (Table 4). This suggests that there are functional constraints on the LgtF protein in these two pathogenic species. It has been reported that the proximal glucose residue of the {alpha} chain catalysed by LgtF protein is required for efficient invasion of gonococci into the host mucosa (Minor et al., 2000 ). The conservation of lgtF in N. meningitidis and N. gonorrhoeae may reflect the LOS structural requirement of the proximal glucose for interaction with the human host.

Horizontal gene transfer through interspecies and intraspecies DNA recombination appears to be one of the factors responsible for diverse genetic arrangements of the lgt locus. It is interesting to observe in the phylogenetic tree of lgtG that N. meningitidis strain MC58 was separated from other N. meningitidis strains. The 3' end of lgtG from MC58 was identical to that of lgtG from three strains of commensal Neisseria (Cn group, Fig. 4C). This suggests that lgtG in MC58 is a hybrid gene that resulted from horizontal gene transfer via DNA recombination. In addition, the genetic organizations of regions surrounding lgtG are also similar between N. meningitidis strain MC58 and four N. lactamica strains (Type II of lgt-3, Fig. 2), consistent with interspecies gene transfer at the MC58 lgt-3 locus.

On the basis of comparisons of genetic organizations, sequence variations, G+C content and patterns of synonymous and nonsynonymous substitution, we conclude that Neisseria shares a common lgt gene pool for biosynthesis of LOS. The lgt-1 and lgt-3 loci are hypervariable genomic regions in N. meningitidis, whereas lgt-2 is conserved in both pathogenic Neisseria species. The typical polymorphisms of lgt appear to have arisen through horizontal gene transfer and homologous recombination in addition to mutation events. This study of the genetic diversity of the lgt loci provides fundamental information for understanding the heterogeneity and antigenic variation of Neisseria LOS.


   ACKNOWLEDGEMENTS
 
P. Zhu was supported by a NIH Fogarty Postdoctoral Fellowship (369VFFD018551). We thank Carl E. Frasch, Mark Achtman and Hervé Tettelin for contributing bacterial strains and DNA samples.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Arking, D., Tong, Y. & Stein, D. C. (2001). Analysis of lipooligosaccharide biosynthesis in the neisseriaceae. J Bacteriol 183, 934-941.[Abstract/Free Full Text]

Banerjee, A., Wang, R., Uljon, S. N., Rice, P. A., Gotschlich, E. C. & Stein, D. C. (1998). Identification of the gene (lgtG) encoding the lipooligosaccharide ß chain synthesizing glucosyl transferase from Neisseria gonorrhoeae. Proc Natl Acad Sci USA 95, 10872-10877.[Abstract/Free Full Text]

Burch, C. L., Danaher, R. J. & Stein, D. C. (1997). Antigenic variation in Neisseria gonorrhoeae: production of multiple lipooligosaccharides. J Bacteriol 179, 982-986.[Abstract]

Danaher, R. J., Levin, J. C., Arking, D., Burch, C. L., Sandlin, R. & Stein, D. C. (1995). Genetic basis of Neisseria gonorrhoeae lipooligosaccharide antigenic variation. J Bacteriol 177, 7275-7279.[Abstract]

Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395.[Abstract]

Erwin, A. L., Haynes, P. A., Rice, P. A. & Gotschlich, E. C. (1996). Conservation of the lipooligosaccharide synthesis locus lgt among strains of Neisseria gonorrhoeae: requirement for lgtE in synthesis of the 2C7 epitope and of the ß chain of strain 15253. J Exp Med 184, 1233-1241.[Abstract]

Gotschlich, E. C. (1994). Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide. J Exp Med 180, 2181-2190.[Abstract]

Harvey, H. A., Porat, N., Campbell, C. A., Jennings, M., Gibson, B. W., Phillips, N. J., Apicella, M. A. & Balke, M. S. (2000). Gonococcal lipooligosaccharide is a ligand for the asialoglycoprotein receptor on human sperm. Mol Microbiol 36, 1059-1070.[Medline]

Jennings, M. P., Hood, D. W., Peak, I. R., Virji, M. & Moxon, E. R. (1995). Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. Mol Microbiol 18, 729-740.[Medline]

Jennings, M. P., Srikhanta, Y. N., Moxon, E. R., Kramer, M., Poolman, J. T., Kuipers, B. & van der Ley, P. (1999). The genetic basis of the phase variation repertoire of lipopolysaccharide immunotypes in Neisseria meningitidis. Microbiology 145, 3013-3021.[Abstract/Free Full Text]

Kahler, C. M. & Stephens, D. S. (1998). Genetic bases for biosynthesis, structure and function of meningococcal lipooligosaccharide. Crit Rev Microbiol 24, 281-334.[Medline]

Kahler, C. M., Carlson, R. W., Rahman, M. M., Martin, L. E. & Stephens, D. S. (1996a). Two glycosyltransferase genes, lgtF and rfaK, constitute the lipooligosaccharide ice (inner core extension) biosynthesis operon of Neisseria meningitidis. J Bacteriol 178, 6677-6684.[Abstract]

Kahler, C. M., Carlson, R. W., Rahman, M. M., Martin, L. E. & Stephens, D. S. (1996b). Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the {alpha}1,2 N-acetylglucosamine transferase (RfaK). J Bacteriol 178, 1265-1273.[Abstract]

Kumar, S., Tamura, K. & Nei, M. (1994). MEGA: Molecular Evolutionary Genetics Analysis software for microcomputers. Comput Appl Biosci 10, 189-191.[Abstract]

Li, W. H. (1993). Unbiased estimation of the rates of synonymous and nonsynonymous substitution. J Mol Evol 36, 96-99.[Medline]

Li, W. H., Wu, C. I. & Luo, C. C. (1985). A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes. Mol Biol Evol 2, 150-174.[Abstract]

Linz, B., Schenker, M., Zhu, P. & Achtman, M. (2000). Frequent interspecific genetic exchange between commensal neisseriae and Neisseria meningitidis. Mol Microbiol 36, 1049-1058.[Medline]

Mandrell, R. E. & Zollinger, W. D. (1977). Lipopolysaccharide serotyping of Neisseria meningitidis by hemagglutination inhibition. Infect Immun 16, 471-475.[Medline]

Minor, S. Y., Banerjee, A. & Gotschlich, E. C. (2000). Effect of {alpha}-oligosaccharide phenotype of Neisseria gonorrhoeae strain MS11 on invasion of Chang conjunctival, HEC-1-B endometrial, and ME-180 cervical cells. Infect Immun 68, 6526-6534.[Abstract/Free Full Text]

O’Brien, J. P., Goldenberg, D. L. & Rice, P. A. (1983). Disseminated gonococcal infection: a prospective analysis of 49 patients and a review of pathophysiology and immune mechanisms. Medicine 62, 395-406.[Medline]

Olyhoek, A. J. M., Sarkari, J., Bopp, M., Morelli, G. & Achtman, M. (1991). Cloning and expression in Escherichia coli of opc, the gene for an unusual class 5 outer membrane protein from Neisseria meningitidis (meningococci/surface antigen). Microb Pathog 11, 249-257.[Medline]

Parkhill, J., Achtman, M., James, K. D. & 25 other authors (2000). Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 404, 502–506.[Medline]

Preston, A., Mandrell, R. E., Gibson, B. W. & Apicella, M. A. (1996). The lipooligosaccharides of pathogenic Gram-negative bacteria. Crit Rev Microbiol 22, 139-180.[Medline]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.[Abstract]

Sarkari, J., Pandit, N., Moxon, E. R. & Achtman, M. (1994). Variable expression of the Opc outer membrane protein in Neisseria meningitidis is caused by size variation of promoter containing poly-cytidine. Mol Microbiol 13, 207-217.[Medline]

Spratt, B. G., Bowler, L. D., Zhang, Q. Y., Zhou, J. & Smith, J. M. (1992). Role of interspecies transfer of chromosomal genes in the evolution of penicillin resistance in pathogenic and commensal Neisseria species. J Mol Evol 34, 115-125.[Medline]

Tettelin, H., Saunders, N. J., Heidelberg, J. & 39 other authors (2000). Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 278, 1809–1815.[Abstract/Free Full Text]

van der Ley, P., Kramer, M., Martin, A., Richards, J. C. & Poolman, J. T. (1997). Analysis of the icsBA locus required for biosynthesis of the inner core region from Neisseria meningitidis lipopolysaccharide. FEMS Microbiol 146, 247-253.

Verheul, A. F. M., Snippe, H. & Poolman, J. T. (1993). Meningococcal lipopolysaccharides: virulence factor and potential vaccine component. Microbiol Rev 57, 34-39.[Abstract]

Wakarchuk, W., Martin, A., Jennings, M. P., Moxon, E. R. & Richards, J. C. (1996). Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. J Biol Chem 271, 19166-19173.[Abstract/Free Full Text]

Yang, Q. L. & Gotschlich, E. C. (1996). Variation of gonococcal lipooligosaccharide structure is due to alterations in poly-G tracts in lgt genes encoding glycosyltransferases. J Exp Med 183, 323-327.[Abstract]

Zhu, P., Klutch, M. J. & Tsai, C. M. (2001). Genetic analysis of conservation and variation of lipooligosaccharide expression in two L8-immunotype strains of Neisseria meningitidis. FEMS Microbiol Lett 203, 173-177.[Medline]

Zollinger, W. D. & Mandrell, R. E. (1977). Outer-membrane protein and lipopolysaccharide serotyping of Neisseria meningitidis by inhibition of a solid-phase radioimmunoassay. Infect Immun 18, 424-433.[Medline]

Zollinger, W. D. & Mandrell, R. E. (1980). Type-specific antigens of group A Neisseria meningitidis: lipopolysaccharide and heat-modifiable outer membrane proteins. Infect Immun 28, 451-458.[Medline]

Received 6 November 2001; revised 11 February 2002; accepted 21 February 2002.