Division of Biological Sciences and
Department of Molecular Microbiology and Immunology, School of
Medicine, University of Missouri
Columbia, Columbia,
Missouri,1 and The Genome Center,
University of Washington, Seattle, Washington2
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INTRODUCTION |
Horizontal acquisition of virulence-determining
genes has accelerated the evolution of bacterial pathogens
(35). The recent availability of complete microbial genome
sequences facilitates investigation of the history of virulence
genes. Of particular interest are contingency loci, which encode
phase-variable surface molecules involved in host tissue interactions
(32). Contingency gene expression is commonly modulated by
slipped-strand mispairing acting on simple sequence repeats within a
control region. Although distantly related, the respiratory commensals
and pathogens Haemophilus influenzae and Neisseria
meningitidis each use tetranucleotide repeat sequences in
translational phase variation of surface proteins (23, 37,
40). Such similarity prompts the question of whether repeat-regulated genes were present in a common ancestor, evolved independently, or had a recent common origin in laterally transferred DNA.
Several important human pathogens (Streptococcus pneumoniae, H. influenzae, and N. meningitidis) coexist in the
respiratory flora of healthy individuals. Kroll et al., who first
reported natural transfer of H. influenzae sequences to
N. meningitidis, have suggested that genetic exchange
between respiratory pathogens may spark the emergence of new invasive
strains (25). They have identified three sequences,
originally from Haemophilus and flagged by
Haemophilus-specific uptake sequences (hUSs), which now
reside in the meningococcal genome.
H. influenzae, a small, gram-negative bacterium, causes
otitis media and bronchitis as well as invasive disease (meningitis and
septicemia) (50). Invasive H. influenzae
disease is usually caused by encapsulated strains of H. influenzae serotype b (Hib); near-universal immunization with
conjugate Hib vaccine has largely eliminated Haemophilus
meningitis from developed countries (3). Nonencapsulated
(nontypeable) H. influenzae (NTHi), against which the Hib
vaccine provides no protection, remains an important cause of
respiratory infections (18). Although NTHi rarely causes invasive infection in immunocompetent hosts, sporadic exceptions warn
of potential virulence. During the 1980s an outbreak of highly lethal
septicemia among children in rural Brazil, termed Brazilian purpuric
fever (BPF), was traced to a single NTHi clone related to H. influenzae biotype aegyptius strains previously associated only
with conjunctivitis (7). In 1994, an NTHi strain (Int1; also called R2866) was isolated in the United States from the blood of
a previously healthy, Hib-immunized child who developed meningitis
(34).
H. influenzae is readily transformed with
Haemophilus DNA; uptake requires the hUS, consisting of a
conserved 9-bp core within an extended 29-bp consensus sequence. The
H. influenzae strain Rd KW20 (hereafter referred to as Rd)
genome, which has been sequenced (17), contains 1,465 hUSs. While most of these occur as single copies, 17% occur as pairs
of inverted repeats in a stem-loop configuration. Stem-loop hUS pairs
tend to occur at the ends of transcription units and may function as
transcription terminators (44). At least one horizontally
transferred island has been found inserted within a stem-loop hUS pair,
suggesting that paired hUSs may play an additional role as potential
targets for the insertion of virulence-associated genes, possibly
mediated by phage integrases (10, 30).
N. meningitidis is a gram-negative bacterium
phylogenetically distant from H. influenzae; N. meningitidis
is classified in the 
subdivision and H. influenzae is
classified in the 
subdivision of the proteobacteria. In sub-Saharan
Africa, N. meningitidis serogroup A causes epidemic
meningitis, whereas in the developed world serogroups B and C cause
endemic invasive disease (29, 47). Natural transformation
is the primary means of genetic exchange among neisseriae; it is so
frequent that lineage boundaries are blurred, requiring populations to
be depicted as networks rather than as distinct clades
(45). Transformation employs a neisseria-specific 10-bp
uptake sequence (nUS) distinct from the hUS (14).
To identify NTHi pathogenicity genes, we initiated whole genome
comparisons between Int1 and Rd, a nonpathogenic laboratory strain.
Here we report the identification of lav, a candidate pathogenicity gene whose inferred translation product is homologous to
virulence-associated autotransporters (AT) and whose DNA sequence is
similar to NMB1527 (also called orf2 and nmrep3)
and NMA1725 of N. meningitidis (24, 36, 37,
48). We present evidence that lav arose by fusion of
segments from different organisms and has recently been transferred
from Haemophilus to Neisseria.
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MATERIALS AND METHODS |
Bacteria.
H. influenzae was grown as previously
described (30, 31); strains are described in Table
1.
Differential cloning of Int1 sequences.
An M13mp18 library
was prepared from a partial Sau3A digest of Int1 DNA size
fractionated to yield fragments between 0.75 and 1.5 kb. Bacterial
clones containing recombinant plasmids were arrayed on nylon filters
(1,200 colonies each). Replicate filters were probed at high stringency
with digoxigenin-labeled Rd and Int1 genomic DNAs, using M13 clones
from Pseudomonas aeruginosa, a G+C-rich organism as
background controls. Clones hybridizing with Int1 but not with Rd were
picked, and inserts were initially sequenced in one direction with a
universal primer.
DNA isolation and PCR.
H. influenzae DNA
isolation and long PCR were done as previously described
(31). The holB-tmk region was
amplified with forward primer 5'-CTTTAATCAGCACAGCATGATGCC,
corresponding to H. influenzae Rd nucleotides 477248 to 477272, and reverse primer 5'-TGTTCAAGCTGATATTGAAAGTGC,
corresponding to H. influenzae nucleotides 477396 to
477373 (17). DNA from BPF isolates R2140 and R2141 was
amplified using the same reverse primer but with forward primer 5'-CACATTTTCAAACTGGCTTGAC.
DNA blotting.
The genomic blots, hybridization, procedures
and stringent wash conditions used have been previously described
(31). The lav probe was a gel-purified 1.4-kb
PCR fragment made using Int1 lav internal primers
5'-GCTTTTGGCTGTTGATTACG and 5'-CCGCCCATTAAGCCAACGG, that was then digoxigenin labeled.
DNA sequencing.
PCR fragments were gel purified and
sequenced directly or following subcloning into a phagemid vector; both
strands were sequenced as previously described (31).
Sequences were analyzed using BLASTX, and BLASTN (1, 2)
and the GCG and Omiga packages.
Nucleotide sequence accession number.
The sequences reported
in this paper have been deposited in GenBank and assigned accession no.
AF385403 (lav) and AF385404 (las).
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RESULTS |
Identification of lav.
We constructed a bank of
clones from the pathogenic NTH: strain Int1 and screened them by
hybridization against whole genomic DNA from strain Int1 and, as a
comparison, from nonpathogenic strain Rd. This differential screening
was intended to identify genes specifically present in Int1 which
potentially encode pathogenicity-related proteins. Of an initial 1,200 clones prepared from Int1 DNA, ~10% were novel relative to Rd.
Sequence 122 bore the 3' end of an open reading frame (ORF) homologous
to a family of AT, which includes VirG of Shigella flexneri
(27), AIDA-I of Escherichia coli
(4), VapA/nmrep2 of N. meningitidis
(37), and PerT of Bordetella pertussis
(15). These virulence factors are outer membrane proteins involved in adhesion, invasion, intercellular spread, or immune evasion
(21, 28). Each consists of a carboxyl-terminal -barrel domain, which forms a pore in the outer membrane; a linker peptide; a
passenger effector domain, which is secreted through the pore; and an
N-terminal signal sequence. The partial Int1 ORF was ~90% identical
in DNA sequence to orf2/nmrep3/NMB1525 of N. meningitidis strain MC58 (serogroup B), previously identified by
its homology to other AT (24, 37).
Downstream from the presumed AT gene, sequence 122 contained a junction
with 93% DNA homology to a gene in strain Rd, the 3' end of H. influenzae gene HI 0456 (tmk), encoding thymidylate kinase. In Rd, the gene immediately downstream from tmk is
H. influenzae gene HI 0455 (holB), which encodes
the 
subunit of DNA polymerase III. The initiator codon of
holB overlaps the terminator codon of tmk; a
paired hUS in (+/
) inverted repeat configuration extends across the
region of overlap (Fig. 1A).
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FIG. 1.
(A) Arrangement of lav homologs in H. influenzae and N. meningitidis MC58 genomes. Neisserial
genes are depicted as filled arrows (indicating orientation); H. influenzae-derived genes are depicted as open arrows. Hairpin
symbols represent paired hUSs in stem-loop configuration. Relative to
that in H. influenzae-Rd, the Int1 lav island is
inserted within a stem-loop hUS pair, resulting in partial duplication
of the hUS pair flanking the site of insertion within an Int1 ancestor.
Fragments of H. influenzae holB and tmk in
Neisseria are indicated in parentheses. (B)
Repeat-containing regions of lav homologs in Int1 (hi
lav), N. meningitidis MC58 (nm) (24,
37), N. gonorrhoeae, (ng) and biotype aegyptius (hae)
reference strain R1967. (C) Inferred amino acid sequences of regions of
Lav homologs, aligned with MEGALIGN (DNAStar). aa 1 to 40 are conserved
sequences at the N terminus, including the predicted signal sequence
cleavage site (arrow). aa 121 to 156 represent a less conserved region
within the presumptive passenger domain, including a sequence bracketed
by conserved cysteine residues (asterisks).
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Rd-specific primers to the 5' end of holB and the 3' end of
tmk were used to amplify Int1 genomic DNA by PCR. The
predicted 125-bp fragment was obtained with Rd and a Hib (Eagan)
template, but a 2.4-kb fragment was obtained with Int1, including 2.3 kb of novel sequence (Fig. 1A). The novel DNA is located within the (+/) hUS pair at the holB-tmk junction, 18 bp of which are
now found duplicated at either end of the island. The insert contains a
single, 2,078-bp potential ORF termed lav (for like a VirG). The putative initiating ATG is in a different reading frame from the
downstream coding region; 10 bp downstream are 19 copies of the
tetranucleotide GCAA. A gain of one GCAA repeat creates a 2,082-bp ORF,
potentially encoding a 693-amino-acid (aa) Lav polypeptide (pI = 9.3) with a 54-aa signal sequence. Lav is homologous at its carboxyl
terminus to VirG and other AT.
Horizontal transfer of lav.
The complete DNA
sequence of H. influenzae lav was 89% similar to
orf2/nmrep3 of N. meningitidis MC58. The
meningococcal gene is located in a region containing rfaF
(encoding a heptosyl transferase involved in lipo-oligosaccharide
biosynthesis) between a gene (orf1) whose inferred product
is a small protein B homolog and a gene (dld) encoding a
D-lactate dehydrogenase (4, 18, 48). N. meningitidis serotype B orf2 is part of a 2.45-kb
island of similarity to H. influenzae (Fig. 1A). Four GCAA
repeats follow the initiator codon of orf2/nmrep3, placing
its translated product out of reading frame; loss of a GCAA would
permit translation of a 678-aa protein. A homologous gene (NMA1725)
95% identical to N. meningitidis serotype B. orf2, but with three GCAA repeats, is found at the same site
in the genome of N. meningitidis serogroup A strain Z2491
(36).
The unfinished Neisseria gonorrhoeae genome
(http://www.genome.ou.edu) was searched for homology with
N. meningitidis orf2. A gonococcal sequence 91% identical
to N. meningitidis orf2 and 89% identical to H. influenzae lav was found on contig 140, at the same chromosomal
site as in N. meningitidis. Endpoints of the island of
homology with H. influenzae are identicalexactly 57 bp
downstream from orf1 and 35 bp downstream from
dldin the N. meningitidis and N. gonorrhoeae genomes. N. gonorrhoeae orf2 is preceded by
two GCAA repeats after the initiating codon (Fig. 1B), is also out of
reading frame, and contains an in-frame stop codon and a frameshift
mutation. Inferred proteins encoded by lav homologs contain
highly conserved regions interspersed with divergent regions (Fig. 1C).
The N-terminal region surrounding the signal sequence is highly
conserved, as is the C-terminal end of the -domain; the passenger
domain contains two conserved cysteine residues which can potentially
form a short loop of variable sequence (Fig. 1C).
Three considerations indicate that transfer from Haemophilus
to Neisseria has occurred. (i) The neisserial island of
similarity to H. influenzae includes a sequence upstream of
orf2 which matches hUS consensus at all 29 bp and a paired
(+/) uptake sequence downstream of orf2 which matches
consensus at all but 1 bp. These hUSs align with those in Int1. (ii)
The N. gonorrhoeae island includes 116 bp from H. influenzae holB and 225 bp from H. influenzae tmk in
the intergenic spaces flanking orf2. The N. meningitidis island also includes 116 bp from H. influenzae
holB, but it has only 77 bp from H. influenzae tmk. The
77-bp tmk sequence aligns with 225 bp of the Rd sequence but
with a 148-bp internal deletion. The presence in the meningococcal
genome of nonfunctional bits of adjacent H. influenzae genes
is highly suggestive of the direction of transfer. (iii) The G+C
content of N. meningitidis serotype B orf2 is
40.0%, and that of N. gonorrhoeae is 39.7%. These values are much closer to the H. influenzae genomic average of
38.2% than to the neisserial average of 51.8%.
Recent transfer of lav from Haemophilus to
Neisseria.
Genetic similarity suggests that the
Haemophilus lav gene and the Neisseria orf2 gene
diverged relatively recently, not long before orf2 genes
diverged in Neisseria. As a more reliable indicator of
genetic distance, we compared noncoding, intergenic DNA sequence, which
was expected to be subject to fewer selective constraints than coding
sequence. The 256-bp sequence immediately upstream of lav
can be aligned for all four bacterial genomes (Fig. 2a). It includes 19 codons of holB, but the remaining 206 bp in
H. influenzae (and all 256 bp in Neisseria) are
noncoding. The H. influenzae sequence is 94.5% identical to
that of N. gonorrhoeae and 93.8% identical to that of
N. meningitidis, whereas sequences from the two neisserial
species are 96.9% identical. The distance between the sequences for
H. influenzae and Neisseria is approximately twice that between the sequences for neisserial species (Fig. 2b).
Similar relative distances (but higher absolute divergences) were found
for the lav region including the -barrel plus the linker
(Fig. 2b). In comparison, the DNA sequence of an evolutionarily conserved regulatory gene, rpoD (encoding the major sigma
factor), is only 47% identical between H. influenzae and
Neisseria, yet rpoD similarity between the two
Neisseria sequences is 97% (Fig. 2b).
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FIG. 2.
(a) Alignment (MEGALIGN) of DNA sequence 256 bp upstream
of lav homologs. Strain designations are as described for
Fig. 1, except that NM-B is MC58 and NM-A is Z2491. The initiating
codon (complementary strand) of holB is indicated (left
arrow), and the sequence includes 19 codons at the N terminus-encoding
portion of of holB. The start of lav is also
indicated (right arrow). An 18-bp sequence from an hUS is underlined;
this sequence has been duplicated from a paired hUS at the
tmk end of the Int1 lav island. (b) Phylograms of
aligned sequences from the 256-bp upstream sequence (5' to
lav), from the -barrel region (last 385 codons, including
the predicted linker region), and from rpoD (17, 36,
48). Phylogenies were derived with the ClustalV program of
MEGALIGN, using a combination of the unweighted pair group method using
arithmetic averages algorithm and the neighbor-joining method
(39, 46). (c) H. influenzae lav is anomalously
similar to its N. meningitidis homologs relative to pairwise
comparisons between housekeeping genes shared by H. influenzae and N. meningitidis. Linear regression of
inferred amino acid sequence identity on DNA identity of 12 housekeeping genes (open squares) in N. meningitidis
serotype B and H. influenzae Rd is shown. Filled circles
(arrows) indicate Int1 lav compared to homologs in N. meningitidis serotype B and N. meningitidis serotype A,
in ascending order of DNA similarity. Genes compared, in ascending
order of DNA similarity, were tpiA, lig (lig-1 in
N. meningitidis serotype B) dnaG, mutS, gyrA, rpoD,
cysK, aroG, rpoB, recA, sucD, and ilvD
(ilvD-1 in N. meningitidis serotype B). The
sequences were compared using ClustalV (MEGALIGN).
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The atypically high similarity of lav homologs, relative to
that of other genes shared by Haemophilus and
Neisseria, is evident in Fig. 2c, which shows a comparison
of 12 pairs of evolutionarily conserved housekeeping genes shared by
these species to provide a baseline. The mean DNA similarity between
Haemophilus and Neisseria was 47.3% (standard
deviation, 8.7%; range, 35.0 to 60.2%). The values for the
lav homologs lie above the range of DNA similarity represented for these highly conserved genes. With respect to DNA
similarity, the Lav amino acid sequence is more divergent than that of
or typical housekeeping genes, presumably because lav is
exposed to a different type of selection (Fig. 2c).
Chimeric origin of lav as evidence for domain
shuffling.
A scan of G+C content across the lsi-dld
region revealed additional detail (Fig. 3). The island
of similarity between H. influenzae and neisserial
chromosomes coincides with a dip in G+C content to a level approaching
the H. influenzae genomic average; the profile is similar in
detail for all three species, again indicating a recent common origin.
G+C content is not uniform across lav but rather rises
sharply at 850 ± 50 bp after the GCAA repeat region. Thus, Int1
lav is composed of two distinct segments, a 5' segment of
283 codons averaging 35.1% G+C and a 3' segment of 376 codons
averaging 40.8% G+C.
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FIG. 3.
G+C content of regions containing lav
homologs, scanned with a sliding window of 100 bp, in 25-bp increments.
hi, H. influenzae Int1; nm, N. meningitidis
serotype A Z2491; ng, N. gonorrhoeae. The interval on the
abscissa corresponds to bp 113 to 5147 of N. gonorrhoeae
contig 140 aligned with N. meningitidis serotype A interval
1660274 to 1654800 (which has a deletion within tmk) by
creating a 148-bp gap in the N. meningitidis serotype A
sequence starting at bp 4033 and the entire Int1 sequence between
tmk and holB.
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The two segments defined by G+C content approximately coincide with
predicted boundaries of the two major structural and functional domains
of an AT. The closest homolog to Lav with functionally characterized
domains is the 1,286-aa E. coli AIDA-I preprotein (31). Amino acid sequence homology begins near the
processing site at AIDA-I Leu840 and includes the linker
region and the 14-strand -barrel, which begins at AIDA-I
Ala1002. On the basis of alignment with AIDA-I, the
-domain of Int1 lav is predicted to extend from codon 385 to 659 codon and the linker is predicted to extend from codon ~290 to
codon 384. The low-G+C segment of lav (first 283 codons)
thus corresponds roughly to the predicted passenger domain, and the
high-G+C segment (last 376 codons) corresponds to the -barrel domain
plus linker.
The two segments of lav have evolved at markedly different
rates. The role of selection in fixing mutations can be assessed by
comparing silent-substitution rates (Ks = synonymous changes per 100 synonymous sites) to nonsilent-substitution
rates (Ka = nonsynonymous changes per 100 nonsynonymous sites) (32); the Ks/Ka ratio is usually >10 for
conserved housekeeping genes. Silent-substitution rates in the C
terminus-encoding segment are consistent with recent common origin of
these genes (Table 2). The Ks
values derived from a comparison of Int1 and Neisseria are
only 1.2- to 1.5-fold higher than those derived from a comparison of
N. meningitidis and N. gonorrhoeae. However,
nonsynonymous-substitution rates differ substantially between the
segments encoding the N and C termini within each lineage. The
Ks/Ka ratio is 
4 for the segment encoding the C terminus, but it is 
1 for the segment encoding the N
terminus-segment (Table 2). These differences suggest that the two
major segments of the gene are subject to different evolutionary constraints.
Distribution of lav.
Various H. influenzae strains and clinical isolates, including NTHi and
representatives from each capsular serotype, were examined by PCR and
genomic Southern analyses for the presence of an insert at the
holB-tmk junction. The junction was amplified in 26 strains,
including 8 from encapsulated lineages (Rd, Hib [Eagan], and
reference type strains of serotypes a to f), 9 respiratory NTHi
isolates, and 9 NTHi isolates from patients with invasive disease
(Table 1). A 2.4-kb fragment containing a lav-related sequence was found at the holB-tmk site in six strains,
representing just two clades of NTHi. Southern analysis confirmed the
absence of a lav sequence in the other strains or at other
chromosomal sites. Strains having lav-related islands
included Int1 and two respiratory isolates previously identified as
clonally related to Int1 (T. Mutangadura and M. Golomb, unpublished
data), 1128 (isolated from a patient with otitis media)
(13) and AAr160 (isolated from a tracheal aspirate)
(12). Partial sequencing revealed lav genes at
identical sites in both isolates. The sequence of the AAr160
lav island was identical to that of Int1 for 600 bp,
starting at position 1 in Fig. 2a, with the exception of the number of
GCAA repeats and a G
T polymorphism at Int1 position 382, which
conservatively changes an L to an F within the lav-encoded signal sequence. A 2.4-kb island was found at the same chromosomal site
in R1967, the reference biotype aegyptius strain, and in R2140 and
R2141, clonally related biotype aegyptius strains isolated from
patients with BPF (Table 3).
Domain shuffling and the emergence of a novel biotype
aegyptius AT gene.
The holB-tmk island of R1967
(biotype aegyptius) had endpoints identical to those in Int1. The
island contained a single potential ORF of 2,102 bp, at a position
aligned with Int1 lav. Downstream from the predicted
initiating ATG were 25 GCAA repeats, placing the coding sequence out of
reading frame. Gain of a single repeat would allow translation of a
702-aa polypeptide with a predicted 61-aa signal polypeptide. The
C-terminal -domain of this polypeptide is strongly homologous to
Lav. We refer to the putative biotype aegyptius gene at this site as
las. Partial sequencing of the R2140 and R2141 islands (BPF)
revealed las homologs (Table 3).
Like lav, biotype aegyptius las is a mosaic of
low- and high-G+C segments (Fig. 4a). Although the
region immediately upstream from biotype aegyptius las and
the las -domain are highly homologous (~90%) to
corresponding regions in lav (Fig. 2), the N
terminus-encoding portion of the gene, following the GCAA repeats, has
no significant homology to lav or to any sequence in the
databanks at either the DNA or amino acid sequence level (Fig. 4b).
Dispersed motifs within the inferred N-terminal region of Las suggest a
distant common ancestry with H. influenzae Lav; thus, both
proteins share the sequence SLWEPR(W/F)NS at corresponding sites (aa
222 to 230 in Int1 and aa. 226 to 234 in biotype aegyptius). The
junction with the high-homology segment (Fig. 4a, a) is near the G+C
transition point, which coincides with that in lav (~850
bp or 283 codons). High amino acid sequence similarity starts at the
position corresponding to lav codon 277 (biotype aegyptius
codon 280). Evidently the lav and las genes
diverged from an ancestral H. influenzae sequence by
recombinational fusion of a novel passenger domain to the H. influenzae -domain near the predicted passenger-linker
boundary.
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FIG. 4.
(a) G+C bias of biotype aegyptius las codons,
computed using CodonPreference with a 25-codon window. The abscissa
shows nucleotide position in las following the GCAA repeat
region. The horizontal line indicates average H. influenzae
codon usage. (b) Homology between H. influenzae lav and
H. influenzae las, using a 10-bp window, as a function of
nucleotide position.
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DISCUSSION |
Lateral acquisition of lav by NTHi.
The restricted
distribution of lav could be explained either by transfer
from another species or by inheritance of the gene from an H. influenzae ancestor followed by loss within multiple lineages.
Genomic comparisons favor lateral transfer. In Rd, holB and
tmk overlap by 4 bp, with the terminating TGA codon of
tmk overlapping the initiating ATG codon of holB.
A diverse set of bacterial species have similar arrangements (Fig.
5A). Discussion In Pasteurella, sister genus
to Haemophilus (38), the genes are contiguous
but not overlapping
(http://www.cbc.umn.edu/ResearchProjects/AGAC/Pm/pmhome.html). The tmk and holB genes are also contiguous
in Buchnera, a gram-negative endocellular parasite of aphids
that has a subset of the genes found in E. coli.
Buchnera is believed to have diverged before the common
ancestor of H. influenzae and E. coli appeared
(41). The contiguity of holB and tmk
in other gram-negative bacteria and even in Mycoplasma
(phylogenetically closer to gram-positive bacteria) suggests that it is
the ancestral arrangement for eubacteria. Thus, lav was most
likely inserted into the chromosome of an Int1 ancestor.
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FIG. 5.
(A) Arrangement of tmk and holB
in various genomes. Nucleotides at the junction of the two genes are
shown. In H. influenzae Rd and E. coli, the
reading frame of holB is offset 2 relative to that of
tmk; in all others shown, the relative reading frame of
holB is offset 1. In P. multocida
(http://www.cbc.umn.edu/ResearchProjects/AGAC/Pm/pmhome.html), the two
genes are separated by 4 bp. GenBank accession numbers and sources are
as follows: Rd, reference 17 E. coli, reference
5; P. multocida, contig 186, nucleotides 22421 to 23201; Yersinia pestis, accession no. AF065312
(11); Mycoplasma pneumoniae, accession no.
AE000016 (22); and Mycoplasma fermentans,
accession no. AF100324 (8). (B) Proposed scenario for
origin of lav homologs in various organisms. The original
form of the gene in H. influenzae is arbitrarily shown as
Int1 lav but could have been either a lav or a
las ancestor. nm, N. meningitidis; ng, N. gonorrhoeae; hi, H. influenzae; Hae, biotype
aegyptius.
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The present distribution of lav requires at least three
interspecies transfers (Fig. 5B). The extrageneric origin of
lav is supported by its limited distribution within
Haemophilus, by G+C content analysis, and by analysis of
junctions with adjoining genes. Although the lav segment
does not differ in G+C content from the H. influenzae
average, it is actually composed of two segments, one atypically low
and one atypically high in G+C content. This suggests that the gene
originated in another, unidentified, organism by fusion between
segments from different species. The chimeric gene was then laterally
acquired by H. influenzae-and inserted within a stem-loop
hUS pair originally present in single copy in a chromosome resembling
that of Rd. The stem-loop pair was partially duplicated during this
integration, resulting in direct repeats flanking the lav
island in an ancestor of Int1. Insertion of virulence-associated genes
within hUS pairs has previously been noted for the tna
cluster (30); the targeting of regions of high potential
secondary structure suggests that paired hUSs, like tRNA genes, may
serve as targets for phage integrases in Haemophilus.
Subsequently, the lav island with adjoining bits of
holB and tmk was incorporated by
Neisseria, most likely by transformation. Perhaps the
fragment recombined within a region of weak, fortuitous homology,
though none has been identified. (The two hUS pairs, located internally
within the transferred fragment, presumably played no role in
neisserial uptake of Haemophilus DNA. Their presence in
Neisseria merely reflects the recent origin of this fragment
in Haemophilus.) The strongest evidence indicating polarity of transfer is the presence of pseudogene fragments of the adjoining Haemophilus holB and tmk genes. (Functional,
endogenous holB and tmk genes are located at
different chromosomal sites in Neisseria.) Notably, the
usual criteria for direction of transfer (G+C content, within-species
distribution, and presence of signature sequences like the hUS) are
less reliable and may be deceptive at times. For instance, the G+C
content of the Lav-encoding part of the H. influenzae island
is species typical not because of its origin within H. influenzae but because it is an artifact of averaging across two
dissimilar segments. In general, detailed sequence analysis is needed
to infer direction of lateral transfer.
The appearance of lav in both N. meningitidis and
N. gonorrhoeae could be explained either by preexistence in
a common ancestor or by acquisition by one lineage followed by
homologous transformation into the other (Fig. 5B). A third
possibility, independent acquisition by N. meningitidis and
N. gonorrhoeae from two different H. influenzae lineages, is much less likely because the junction of neisserial sequence with the H. influenzae island is identical, to the
base pair, in N. gonorrhoeae and N. meningitidis.
The presence of lav (orf2) in all N. meningitidis strains surveyed to date (37) is
consistent with its preexistence in an N. meningitidis
ancestor, though it could also be explained by transmission across
N. meningitidis lineages and strong selective advantage.
DNA sequence similarity attests that the meningococcus and the
gonococcus have diverged relatively recently, although no time frame
estimates have been published. Most genes are shared by both pathogens
and are on average 
98% identical (47, 49). If emergence
of N. gonorrhoeae as a sexually transmitted pathogen required relatively high human population densities, divergence may
have occurred as recently as the Neolithic period, 10,000 years ago.
Supposing that lav preexisted in the common ancestor of
N. gonorrhoeae and N. meningitidis, it must have
emerged shortly before species divergence, since it is only slightly
less similar between H. influenzae and Neisseria
than between neisserial species. Once in Neisseria, lav
evolved rapidly, especially within its passenger domain. Nonsilent- and
silent-mutation rates are approximately equal within the passenger
domains, suggesting either selective neutrality or (more likely)
diversifying selection acting on variable regions interspersed within a
more conserved framework, as suggested by the data presented in Fig.
1C.
In Haemophilus, phase variation by repeat slippage is
associated with surface molecules exposed to immune surveillance
(23, 51), and slippage rates increase with the number of
repeats (20). The 19 GCAA repeats of Int1 lav
and the 25 GCAA repeats of biotype aegyptius las should be
relatively unstable in vivo, consistent with an actively expressed
gene. We have detected phase variants ranging from 18 to 22 repeats in
laboratory populations (data not shown). Although the Int1
lav variant described here is out of reading frame, in-frame
variants would arise readily in natural populations and are represented
by two clinical isolates (Table 3). Compared to Haemophilus
orf2 genes, the N. meningitidis orf2 genes have fewer
GCAA repeats; among 41 N. meningitidis isolates possessing
orf2, the number of repeats ranged from 1 to 12 (37).
A mosaic origin for lav was inferred from a G+C content
transition at the boundary of its presumed passenger domain with the linker and -barrel domains. Similarly, the junction of nonhomology between lav and las coincides with the G+C
transition and inferred domain boundaries of both genes. On the basis
of quite different evidence (discordance between phylogenies based on
individual domains), Loveless and Saier have proposed that AT proteins
evolve by domain shuffling (28). A functionally novel AT
can arise by linking a new passenger activity to a generic -barrel
pore. Our analysis provides independent evidence for the combinatorial origin and subsequent reshuffling of at least one AT protein.
Within the small sample of H. influenzae strains examined,
lav was restricted to a few NTHi strains with unusual
virulence potential. Although Int1 lacks the type b polysaccharide
capsule which protects against lysis by human serum, it is more serum resistant than most NTHi strains (B. Williams and A. L. Smith, unpublished data). Int1, AAr160, and 1128 constitute a clade by various
criteria, including the results of pulsed-field gel electrophoresis after restriction with rare-cutting enzymes (Mutangadura and Golomb, unpublished). All three strains have the phage HP2, otherwise present
in only a small minority of NTHi strains (Williams and Smith,
unpublished). The biotype aegyptius is associated with unusual
virulence because it includes BPF isolates.
As biotype aegyptius strains and Int1 belong to different phylogenetic
subgroups, it is unlikely that they inherited lav from a
common ancestor. Rather, it is likely that the first H. influenzae clade to acquire the gene passed it to one or more
other clades by transformation and homologous recombination within
flanking DNA. Once a laterally transferred fragment has been acquired
by a population of naturally transformable bacteria, it can readily be
assimilated into the species by co-opting linked homologous sequence
and uptake signals. Interstrain and interspecies transfer implies a
shared selective advantage in certain host environments.
This is the second report of gene transfer from Haemophilus
to Neisseria (25) and the first of a gene
belonging to a family widely associated with virulence. Gene flow from
Neisseria to Haemophilus has not yet been
documented and may be rarer than gene flow in the opposite direction.
Natural transformation is more frequent and promiscuous in
Neisseria than in Haemophilus, and the
requirement for the nUS can be bypassed experimentally (6). In contrast, the requirement of
Haemophilus transformation for the hUS is stringent
(42). Many genes in the N. meningitidis genome
appear to be of external origin, as judged by anomalous G+C content; in
contrast, the H. influenzae Rd genome contains fewer islands
of unusual G+C content (16, 17, 36). One way to discover a
sequence of external origin is to search a genome for heterologous
uptake sequences. When Kroll et al. (25) searched the Rd
genome with the 10-bp nUS, its only occurrence was within an ORF
believed to have been transferred from Haemophilus to
Neisseria. When the small Int1-specific library was searched
with the nUS, a single hit was found. It consisted of two oppositely
oriented nUS sequences in the /+ configuration, separated by 71 bp,
within a slightly longer region of dyad symmetry:
TTTCAGACGGCATN67ATGCCGTCTGAAA (inverted repeat nUSs are underlined and N signifies any base). The probability that two nUSs will occur by chance within 100 bp of
each other is <10
4. Inverse PCR identified a >2.5-kb
island without significant homology to Rd (data not shown). The two
nUSs are within a 1,431-bp ORF that is 65% identical in deduced amino
acid sequence to the Vibrio cholerae ORF VC1769 product, a
presumed methyltransferase (HsdM) subunit of a type I restriction
endonuclease system (20). G+C content of this Int1 ORF is
46.1%, a value midway between H. influenzae and neisserial
averages. The Vibrio ORF is 43% G+C and part of an island
of anomalously low G+C content. Despite possession of a neisserial
signature, this ORF did not match sequence entries for N. meningitidis serotype A, N. meningitidis serotype B, or
N. gonorrhoeae. We conclude that the Int1 nUS-bearing
ORF, although laterally derived, is unlikely to be from
Neisseria.
Humans are the exclusive natural host of H. influenzae and
the meningococcus. Virulence genes are likely to be recent
specializations for competitive adaptation to a human host, and their
most obvious sources are other human pathogens and commensals.
Repeat-regulated genes such as lav are well equipped for
horizontal mobility, since their regulatory system is self-contained.
In contrast to selfish operons, which must be transferred as a unit
(26), contingency genes are controlled without the need
for accessory proteins or for extensive compatibility between
regulatory elements from different species. The emergence of invasive
NTHi strains, such as those causing BPF and meningitis in
immunocompetent children, suggests that contingency gene transfer
between dangerous respiratory pathogens may be an ongoing process.
We are grateful to Robert Munson, Janet Gilsdorf, and
Loek van Alphen for provision of bacterial strains, to Michael Calcutt for helpful discussions, and to Stephen Lory for advice and help in
constructing the M13 library. We thank Arnie Kas, University of
Washington, for an unpublished G+C scan program. Incompletely sequenced
microbial genomes were accessed on the World Wide Web from the
Gonococcal Sequencing Project at Oklahoma University and the
Pasteurella multocida Sequencing Project at the University of Minnesota.
This work was supported by NIH grant 5RO1 HG01475 to Maynard
Olson, University of Washington, by NIH grant AI 44002 to A.L.S., and
by University of Missouri Research Board grants to M.G. and A.L.S.
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Abstract
Introduction
