Infection and Immunity, January 1999, p. 455-459, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification and Functional Characterization of
the Neisseria gonorrhoeae lbpB Gene Product
Gour D.
Biswas,1,*
James E.
Anderson,1
Ching-Ju
Chen,1
Cynthia Nau
Cornelissen,2 and
P.
Frederick
Sparling1,3
Department of
Medicine1 and
Department of Microbiology
and Immunology,3 School of Medicine, University
of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and
Department of Microbiology and Immunology, Virginia
Commonwealth University, Richmond, Virginia
232982
Received 10 July 1998/Returned for modification 18 August
1998/Accepted 20 October 1998
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ABSTRACT |
We cloned lbpB, encoding a predicted 80-kDa
lipoprotein, upstream of lbpA. A nonpolar mutant
(LbpB
LbpA+) had normal lactoferrin (LF)
binding and grew normally with LF as an iron source, whereas
LbpB LbpA and LbpB+
LbpA strains had reduced binding of LF and did not grow
with LF as an iron source. LbpB bound LF directly in an affinity
purification, suggesting that LbpB might play a still-uncharacterized
role in the LF iron utilization.
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TEXT |
Most organisms use inorganic iron
(Fe) as a cofactor in carrying out a variety of metabolic functions
(34). In an oxidized environment, ferric iron is highly
insoluble and relatively unavailable. Mammals solubilize and sequester
extracellular Fe by binding to the glycoproteins transferrin (TF) in
serum (34) and lactoferrin (LF) on mucosal surfaces
(22). Since there is essentially no free Fe in humans,
pathogens have evolved mechanisms to scavenge Fe from host proteins.
Many bacteria synthesize and secrete siderophores, low-molecular-weight
compounds which bind and transport Fe into the cell through specific
siderophore receptors (16). Production of siderophores
facilitates infection in several bacterial pathogens (26).
Neisseria gonorrhoeae and several other gram-negative mucosal pathogens including Neisseria meningitidis do not
produce siderophores (10, 23) but produce specific receptors
for each of the glycoproteins TF and LF, as well as hemoglobin (5,
7, 10, 15, 21).
A receptor for LF, LbpA, was identified and the corresponding
lbpA gene was cloned and characterized in N. gonorrhoeae (3) and N. meningitidis
(29). Recently, a second LF receptor protein, LbpB, was
identified in N. meningitidis (6, 20, 28). Here we report the cloning of lbpB from N. gonorrhoeae
and characterization of the role of LbpB in gonococcal LF binding and
Fe acquisition.
Cloning of lbpB.
We previously cloned the entire
lbpA gene on four overlapping clones (3).
pUNCH127 (Fig. 1) included 1,246 bp of
DNA upstream of the 5' end of the lbpA open reading frame
(ORF). To test if the region upstream of lbpA was also
involved in LF utilization, we inactivated it by inserting
(31) an 
cassette (32) into an AvaI
site in pUNCH127, creating pUNCH130. Sequence analysis of pUNCH130
confirmed that the fragment was inserted at the AvaI
site. pUNCH130 DNA was introduced into wild-type gonococcal strain FA19
by transformation (4), resulting in a mutant strain, FA6839.
The presence of the fragment in the AvaI site in FA6839 was confirmed by Southern analysis with the use of an
lbpB-specific oligonucleotide probe (positions 2451 to 2470 in the lbpB sequence in GenBank) or the 2-kb fragment
(32). The mutant FA6839 had a phenotype similar to that of
lbpA mutant FA6775 (3), including loss of ability
to (i) bind LF in a solid-phase assay, (ii) take up Fe from LF, (iii)
utilize LF as an Fe source, and (iv) express LbpA. These results
suggest that insertion of upstream of lbpA had a polar
effect on expression of LbpA.
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FIG. 1.
Physical map of the N. gonorrhoeae chromosome
showing genetic organization of the lbpBA operon region. The
top line represents the restriction map of the cloned FA19 chromosome
fragment. The organization of lbpBA genes including the
direction of transcription as indicated by arrows is shown in the next
line. Plasmid pUNCH192SB is a 1.1-kb Sau3AI fragment.
Plasmids pUNCH142 and pUNCH127 contain 2.3-kb RsaI and
2.5-kb SspI fragments, respectively. The solid inverted
triangle denotes the position of the aphA3 insertion in
strain FA6965(pUNCH142), and the open inverted triangle denotes the
position of the insertion in strain FA6839(pUNCH127). L58 and L62
refer to the oligonucleotides used as probes in isolating the clones
pUNCH142 and pUNCH192SB, respectively.
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DNA upstream of lbpA was cloned in two chromosome walking
steps involving the use of oligonucleotide primers L58 and L62 (Fig. 1). Southern hybridization analysis showed that L58 hybridized to a
2.3-kb RsaI fragment (data not shown). We cloned the 2.3-kb RsaI fragment into the SmaI site of pUNCH615
(32), creating pUNCH142. Hybridization analysis with probe
L62 identified a 1.1-kb Sau3A fragment of FA19 DNA. We
ligated the 1.1-kb Sau3A fragment into the BamHI
site of pMCL210 (27) harboring lacZ, allowing rapid color screening for inserts. Screening by colony hybridization produced two clones, pUNCH192 and pUNCH193, that differed in size. The
clone pUNCH192 in Escherichia coli produced colonies on
X-Gal (5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside)
and IPTG (isopropyl--D-thiogalactopyranoside) plates
that had three phenotypes: slightly blue, white, and blue. Plasmid DNA
from a colony belonging to each class was sequenced. Plasmid DNA from a
slightly blue colony designated pUNCH192SB (Fig. 1) contained an
1,138-bp Sau3AI insert with a complete 5' end of an apparent
2,189-bp-long ORF (Fig. 1). In contrast, the corresponding plasmid
isolated from a white colony was 328 bp shorter than pUNCH192SB. The
missing DNA included 21 bp at the 5' end of the 2,189-bp ORF. Plasmid
DNA from the blue colony contained primarily vector DNA. The clone
pUNCH193 produced stable white colonies on similar media and was stable
on subculture. Interestingly, pUNCH193 lacked 20 bp of DNA that
included 5'-GTC GAA TCA ACG CCG ACC GC-3' at positions 31 to 51 from
the ATG codon start site of the 2,189-bp ORF in pUNCH192SB. To verify
that the DNA in pUNCH192SB represented wild-type FA19, we PCR
amplified and directly sequenced FA19 DNA flanking the deleted 20-bp
region. A comparison of the sequences of FA19 DNA in the amplified
product and the corresponding DNA in pUNCH192SB showed complete
identity, confirming that the clone pUNCH192SB contained wild-type FA19
DNA. Thus, the set of three overlapping clones, pUNCH127, pUNCH142, and
pUNCH192SB, represents 2,493 bp of contiguous FA19 DNA upstream of
lbpA.
Nucleotide sequence analysis.
An examination of the sequences
in clones pUNCH127, pUNCH142, and pUNCH192SB revealed a 2,189-bp
ORF. The last 4 bp (ATGA) of the 2,189-bp ORF partially overlapped with
the ATG initiation codon of the lbpA ORF. We designated the
2,189-bp ORF located upstream of lbpA as lbpB.
Further upstream, we found 170 bp that were 56% identical to the 3'
end of an E. coli era gene that encodes a GTP binding
protein (1). Immediately downstream of the era homolog there was a potential hairpin loop structure at positions 180 to 212. This structure has a 
G (25°C) value of 
20.8
kcal (33) and probably corresponds to a transcription
terminator. In addition, a 10-bp inverted repeat at positions 184 to
208 and another 10-bp sequence at positions 1902 to 1911 matched
gonococcal uptake sequence (12, 14).
Translation of the lbpB coding region predicted a protein of
728 amino acids with a molecular mass of 80 kDa and a pI value of 4.53. Analysis of the deduced N-terminal amino acid sequence of LbpB revealed
an 18-amino-acid signal sequence with the lipoprotein modification
consensus sequence LSAC at positions 16 to 19 (17). These
data suggested that LbpB was a lipoprotein. Based on fluorographic analysis and palmitic acid labeling, Lewis et al. concluded that LbpB
in N. meningitidis is a lipoprotein (20). LbpB
was 31% identical to TbpB of the same gonococcal strain and 81%
identical to the recently reported meningococcal LbpB (6,
28). Comparison of the predicted protein sequence of FA19 LbpB
with that of meningococcal LbpB (6, 28) showed very similar
features that differentiate each from the neisserial TbpB family. Both
gonococcal and meningococcal LbpB proteins contained two stretches of
negatively charged residues that were not present in TbpB family
members: region one, residues 462 to 527, and region two, residues 689 to 708 (Fig. 2). These regions also
exhibit considerable variation between meningococcal and gonococcal
LbpB proteins. There was a deletion in region one in meningococcal LbpB
relative to the gonococcal LbpB, and in region two, there was a
deletion in gonococcal LbpB. Overall, these two charged domains were
only about 50% identical, compared to 81% for the whole proteins.
Each of the LbpB proteins contained an identical 21-amino-acid C
terminus that was absent in all neisserial TbpB family members.
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FIG. 2.
Lineup of negatively charged residues in region one and
region two of the gonococcal (NG) and meningococcal (NM) LbpB proteins.
The numbers indicate the position in the unprocessed protein of the
first amino acid listed. Amino acids identical between the two proteins
are denoted by vertical lines; colons indicate highly conservative
substitutions, and dots indicate less conservative substitutions.
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lbpB gene product.
To study the function of
lbpB, we disrupted lbpB by inserting a nonpolar
aphA3 cassette (24) into an HpaI site
in pUNCH142, creating pUNCH195 (Fig. 1; Table
1) and exchanged this mutant allele into
the gonococcal strain FA19 as described above, creating FA6965.
Sequence analysis confirmed that the cassette was inserted in the
correct orientation and translational frame into pUNCH195. Southern
hybridization with the lbpB-specific probe L66 (positions 1156 to 1176) or the 0.9-kb aphA3 fragment (24)
confirmed the expected insertion in lbpB in FA6965. The
lbpB gene product was identified with the use of polyclonal,
affinity-purified antiserum raised against a synthetic 16-mer peptide,
RTRDNGINLSGNGSTN, from the predicted C terminus of LbpB. We used
isogenic strains FA6815, FA6985, and FA6986 (Table 1) lacking TbpB and
TbpA in order to avoid potential problems due to production of proteins
similar in size and structure to LbpB and LbpA. A Western blot probed with LbpB antibodies reacted with a protein of about 95 kDa in the
parent strain FA6815 (Fig. 3A, lane 1). A
protein of the same size also was produced by FA6985
(lbpA::mTn3 Cm) (Fig. 3A, lane 3) but
not FA6986 (lbpB::aphA3) (Fig. 3A, lane
4). The latter strain did produce the 103-kDa LbpA, as expected (Fig.
3B, lane 4). FA6839 (lbpB::) produced neither
LbpA nor LbpB (Fig. 3, lanes 2). These data showed that the 95-kDa
protein was the lbpB gene product and also provided
experimental evidence that lbpB and lbpA were
part of a single transcriptional unit.
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FIG. 3.
Expression of LbpB and LbpA in N. gonorrhoeae. Shown are Western blots containing total membranes
prepared from Fe-starved cells probed with affinity-purified LbpB
polyclonal antibody (A) and monoclonal LbpA antibody (B)
(3). Lanes: 1, parent strain FA6815 (LbpB+
LbpA+); 2, FA6839 (LbpB LbpA);
3, FA6985 (LbpB+ LbpA); 4, FA6986
(LbpB LbpA+).
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We noticed that the nonpolar mutant FA6986
(lbpB::aphA3) expressed reduced amounts
of LbpA (Fig. 3B, lane 4) compared to the parent strain FA6815
(lbpB+ lbpA+) (Fig. 3B, lane 1). We
confirmed this by comparing the intensities of the LbpA protein bands
in linear range of detection in Western blots. We estimated that the
reduction was approximately fivefold from the level for the parent
strain (data not shown). We concluded that the aphA3
insertion in lbpB in FA6986 caused reduced production of the
downstream lbpA gene product. The reason for reduced
expression of LbpA in the lbpB::aphA3
mutant is not understood. Insertion of the aphA3 cassette in
lbpB clearly did allow transcriptional read-through into
lbpA but may have attenuated transcription downstream of the insertion.
LF binding.
To determine whether LbpB participated in LF
binding, we performed dot blot assays of LF binding to whole cells.
Bound LF was detected with horseradish peroxidase-conjugated anti-human LF antibody (Accurate Chemical & Scientific Corp., Westbury, N.Y.) (3). The results demonstrated that the LbpB
LbpA+ mutant FA6986 bound approximately as much LF as the
LbpB+ LbpA+ parent strain FA6815 (data not
shown). In contrast, no binding was detected for cells lacking LbpA,
regardless of whether LbpB was present. Therefore, the solid-phase
binding assay failed to detect LF binding by gonococci expressing LbpB
alone. LbpB-specific binding was not detected under slightly different
conditions (6, 28) for the solid-phase assay (data not shown).
To better quantitate LF binding, we measured the amount of radiolabeled
LF bound by living cells in an equilibrium-phase binding assay
developed by Cornelissen and Sparling (9). The specific activity of iodinated LF was 2.9 × 105 cpm/µg of
LF. Approximately 1 × 107 to 5 × 107 CFU of gonococcal cells grown in chelexed defined
medium with no added Fe (CDM-0) to induce Fe stress (5) were
mixed with 2 to 100 nM 125I-LF in the presence of 1%
bovine serum albumin and 5 µM unlabeled LF in individual wells of a
Multi-Screen microtiter dish (0.45-µm-pore-size filter; MAHV N45;
Millipore, Cambridge, Mass.). After being allowed to bind for 20 min at
room temperature, unbound LF was removed by filtration, followed by
five washes with CDM-0. Filters were dried, punched out, and counted.
Specific binding was the difference between total binding without cold
LF and binding that occurred in the presence of at least a
50-fold-excess concentration of cold LF. All four strains studied
exhibited specific, saturable LF binding (Fig.
4). The wild-type strain bound
significantly more LF than did each of the other strains. An apparent
trend toward an intermediate level of LF binding in FA6965
(LbpB LbpA+) was noted, but the differences
were not statistically significant by the t test in
conjunction with the Bonferroni procedure (19).
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FIG. 4.
Isotherms for binding of LF to lbp mutants.
The curves represent the amount of specifically bound LF as a function
of LF concentration. Each point represents the mean of four individual
experiments. Symbols:  , FA19 (LbpB+ LbpA+);
 , FA6965 (LbpB LbpA+);  , FA6775
(LbpB+ LbpA);  , FA6839 (LbpB
LbpA).
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The data obtained in the liquid-phase LF-binding experiments were
analyzed with the computer program Receptor Fit Saturation Two-Site
(Lundon Software, Inc., Cleveland Heights, Ohio). This program
generates Kd and copy number estimates based on
the best fit of observed data to progressively more complex models.
Table 2 contains the best estimates of
Kd and copy numbers generated by this program.
Considerable variability was observed for LF binding to all strains;
thus, these estimates are best considered comparisons of LF binding to
isogenic strains rather than absolute indicators of intrinsic receptor
affinity or copy number. Isogenic strains expressing LbpA exhibited
complex binding phenomena consistent with the presence of at least two
populations of binding sites, with Kds ranging
from 5 to 500 nM. In contrast, the isogenic mutants that did not
express LbpA bound LF to a more homogeneous population of receptors,
with a single apparent Kd of approximately 45 nM. This was true of both LbpB+ and LbpB
strains, suggesting that LbpB played no detectable role in LF binding
under the conditions employed in this assay. Somewhat surprisingly, the
mutant that did not express either of the two identified LF-binding
proteins (LbpB and LbpA) retained the ability to bind LF. This
observation suggested that there were uncharacterized LF-binding
components on the gonococcal cell surface.
LF affinity isolation of LbpB.
To determine whether LbpB could
bind LF in the absence of LbpA, total membranes prepared from
Fe-starved FA6815 (LbpB+ LbpA+) and its
isogenic derivatives, FA6985 (LbpB+ LbpA) and
FA6986 (LbpB LbpA+), were subjected to
LF-agarose affinity purification (7, 11). Total membrane
proteins were dissolved in 2% Zwittergent 3, 14 (Calbiochem) and
rocked with a 50% slurry of LF-agarose overnight at 4°C. The unbound
material was removed by centrifugation, and the LF-binding protein was
eluted in a Laemmli sample buffer for analytical sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and Western blotting.
Results (Fig. 5) showed an excellent
recovery of LbpB in the absence of LbpA, indicating that LbpB was
capable of binding LF under these conditions. LbpA also could be
isolated by this procedure from total membranes in the absence of LbpB. The amount of LbpA in membrane preparations from the two strains was
roughly the same (lanes A and C), but less LbpA was purified from the
LbpB LbpA+ strain than from wild type (lanes
D and F). This suggested that LbpA alone did not bind to LF as avidly
as it did in the presence of LbpB.
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FIG. 5.
Western blot of LF-agarose affinity-purified LbpB and
LbpA from total membranes of Fe-starved FA6815 (LbpB+
LbpA+) (lanes A and D), FA6985 (LbpB+
LbpA) (lanes B and E), and FA6986 (LbpB
LbpA+) (lanes C and F). The blot was probed with antibodies
to LbpA and then LbpB. The anti-LbpB antibody used here was raised
against an LbpB His-tagged fusion protein. The 66-kDa band present in
lanes A and B was recognized by antiserum elicited against full-length
recombinant LbpB but not against an LbpB-specific peptide (Fig. 3 and
data not shown). This protein band probably represents a breakdown
product of holo-LbpB. Lanes A to C, total membranes (TM); lanes D to F,
LF-agarose affinity-purified proteins. MW, molecular weight in
thousands.
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LF utilization.
To assess the contribution of LbpB in
utilization of Fe from LF, we compared growth in CDM supplemented with
LF of strains that carried either intact lbpB or inactivated
lbpB. On agar plates or in CDM broth culture (5),
the nonpolar lbpB mutant FA6965 (LbpB
LbpA+) failed to show a growth defect, growing to a level
equal to that of wild-type FA19 (data not shown).
55Fe-LF uptake.
To detect smaller effects of LbpB
in LF utilization, we measured the amount of 55Fe-LF taken
up by the use of established procedures (2, 5). Results
indicated that the nonpolar lbpB::aphA3
mutant FA6965 (LbpB LbpA+) acquired
55Fe from LF at about 60% of wild-type levels
(P < 0.03) (data not shown). In contrast,
LbpA mutants, FA6775 and FA6839, failed to take up a
significant amount of 55Fe from LF (P < 0.01). We could not determine whether reduced Fe uptake from LF in
FA6965 was due to loss of LbpB or to decreased expression of LbpA.
Conclusions.
We cloned an N. gonorrhoeae lbpB gene,
providing genetic evidence for the existence of a second LF receptor
protein in the gonococcus, similar to that described recently for
meningococci (6, 20, 28). Despite difficulties encountered
in isolating the 5' end of the gonococcal lbpB gene, we
succeeded in isolating a clone (pUNCH192SB) with an intact 5' end of
lbpB. Attempts to clone the 5' end of the lbpB
gene in N. meningitidis were unsuccessful (6, 20,
28).
A two-gene lbpB lbpA transcriptional unit was implicated by
the observations that the 3' end of lbpB overlapped the 5'
end of lbpA, and creation of a polar mutation in
lbpB resulted in simultaneous loss of expression of the
lbpB and lbpA genes. Recent experiments by Lewis
et al. (20) showed that the lbpB and
lbpA genes in meningococci are organized in a single
transcriptional unit.
Both the gonococcal and meningococcal LbpB proteins contain two
strongly acidic domains, notably absent in TbpB proteins, which are
rich in aspartic acid and glutamic acid. This suggests that these
regions could be involved in binding of LF, which is highly cationic
(34). These regions are quite dissimilar in primary sequence
in the known examples in gonococci and meningococci, suggesting that
these also could be antigenic domains recognized by the host immune
response. Future experiments hopefully will answer whether these
domains are involved in binding LF and are immunogenic.
The role of LbpB in mediating Fe acquisition from LF is unclear. The
LbpB mutant (LbpB LbpA+)
retained the ability to bind LF, and this was reflected in its capacity
to utilize LF as an Fe source. We demonstrated that LbpB, in the
absence of LbpA, could be isolated by LF-agarose affinity, which is the
best evidence that LbpB is an LF-binding protein.
We found that the LbpB LbpA+ mutant used LF
as an Fe source approximately as well as did the parent, as evidenced
by similar growth patterns. The growth patterns of similar
meningococcal LbpB LbpA+ mutants varied, but
all mutants grew to some extent (28). Differences in growth
exhibited by strains in these reports might be explained on the basis
of varying levels of LbpA expression in the LbpB
LbpA+ strain. For example, negligible growth noted by Lewis
et al. might have resulted from a more than 10-fold reduction in LbpA expression (20). Near-normal growth exhibited by the mutant used by Pettersson et al. was correlated with LbpA expression similar
to that of LbpB (28). The general conclusion is that LbpA is
essential for LF-Fe utilization, whereas LbpB is not essential.
The question of how important LF is to pathogenesis remains. Many
pathogens and nonpathogens can survive on the mucosal surface without
being able to bind LF or to use LF as a source of Fe. For example, all
Haemophilus influenzae strains are LF
(18, 30), and about one-half of gonococci are
LF (3, 25). Indeed, gonococcal strain FA1090
is LF due to a large lbpBA deletion
(references 13 and 20 and data not shown), yet it causes urethritis readily in male human volunteers (8). Future work is required to clarify the role of LF in
bacterial pathogenesis and the roles of LbpB, in particular in
utilization of LF.
Nucleotide sequence accession number.
The GenBank accession
number for the 2,189-bp ORF is AF072890.
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ACKNOWLEDGMENTS |
This work was supported by Public Health Service grants AI31496 and
AI26837 from the National Institute of Allergy and Infectious Diseases.
We are grateful to Chris Elkins for his assistance with preparation of
antipeptide sera and affinity isolation procedures. We also thank Chris
Thomas for his expert computer assistance. We acknowledge the
generosity of P. K. Sen for advice on statistical analysis and
Rishikesh Chakravorty for analyzing the LF binding data. We are
thankful to Bill Shafer for providing plasmid pUC18K and the UNC-CH
Automated DNA Sequencing Facility for DNA sequencing.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine, University of North Carolina at Chapel Hill, CB# 7030, Chapel Hill, NC 27599-7005. Phone: (919) 966-3661. Fax: (919) 966-6714. E-mail: gdbis{at}med.unc.edu.
Editor:
P. E. Orndorff
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REFERENCES |
| 1.
|
Ahnn, J.,
P. E. March,
H. E. Takiff, and M. Inouye.
1986.
A GTP binding protein of Escherichia coli has homology to yeast RAS proteins.
Proc. Natl. Acad. Sci. USA
83:8849-8853[Abstract/Free Full Text].
|
| 2.
|
Anderson, J. E.,
P. F. Sparling, and C. N. Cornelissen.
1994.
Gonococcal transferrin-binding protein 2 facilitates but is not essential for transferrin utilization.
J. Bacteriol.
176:3162-3170[Abstract/Free Full Text].
|
| 3.
|
Biswas, G. D., and P. F. Sparling.
1995.
Characterization of lbpA, the structural gene for a lactoferrin receptor in Neisseria gonorrhoeae.
Infect. Immun.
63:2958-2967[Abstract].
|
| 4.
|
Biswas, G. D.,
K. L. Burnstein, and P. F. Sparling.
1986.
Linearization of donor DNA during plasmid transformation in Neisseria gonorrhoeae.
J. Bacteriol.
168:756-761[Abstract/Free Full Text].
|
| 5.
|
Blanton, K. J.,
G. D. Biswas,
J. Tsai,
J. Adams,
D. W. Dyer,
S. M. Davis,
G. G. Koch,
P. K. Sen, and P. F. Sparling.
1990.
Genetic evidence that Neisseria gonorrohoeae produces specific receptors for transferrin and lacteroferrin.
J. Bacteriol.
172:5225-5235[Abstract/Free Full Text].
|
| 6.
|
Bonnah, R. A., and A. B. Schryvers.
1998.
Preparation and characterization of Neisseria meningitidis mutants deficient in production of the human lactoferrin binding proteins LbpA and LbpB.
J. Bacteriol.
180:3080-3090[Abstract/Free Full Text].
|
| 7.
|
Chen, C.-J.,
P. F. Sparling,
L. A. Lewis,
D. W. Dyer, and C. Elkins.
1996.
Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae.
Infect. Immun.
64:5008-5014[Abstract].
|
| 8.
|
Cornelissen, C. N.,
M. Kelley,
M. M. Hobbs,
J. E. Anderson, and P. F. Sparling.
1998.
The transferrin receptor expressed by gonococcal strain FA1090 is required for the experimental infection of human volunteers.
Mol. Microbiol.
27:611-616[Medline].
|
| 9.
|
Cornelissen, C. N., and P. F. Sparling.
1996.
Binding and surface-exposure characteristics of the gonococcal transferrin receptor are dependent on both transferrin-binding proteins.
J. Bacteriol.
178:1437-1444[Abstract/Free Full Text].
|
| 10.
|
Cornelissen, C. N., and P. F. Sparling.
1994.
Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens.
Mol. Microbiol.
14:843-850[Medline].
|
| 11.
| Elkins, C. Unpublished observations.
|
| 12.
|
Elkins, C.,
C. E. Thomas,
H. S. Seifert, and P. F. Sparling.
1991.
Species-specific uptake of DNA by gonococci is mediated by a 10-base-pair sequence.
J. Bacteriol.
173:3911-3913[Abstract/Free Full Text].
|
| 13.
| Gonococcal Genome Sequence Database.
http://dna1.chem.uoknor.edu/.
|
| 14.
|
Goodman, S. D., and J. J. Scocca.
1988.
Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae.
Proc. Natl. Acad. Sci. USA
85:6982-6986[Abstract/Free Full Text].
|
| 15.
|
Gray-Owen, S., and A. B. Schryvers.
1996.
Bacterial transferrin and lactoferrin receptors.
Trends Microbiol.
5:185-191.
|
| 16.
|
Guerinot, M. L.
1994.
Microbial iron transport.
Annu. Rev. Microbiol.
48:743-772[Medline].
|
| 17.
|
Hayashi, S., and H. C. Wu.
1990.
Lipoproteins in bacteria.
J. Bioenerg. Biomembr.
22:451-471[Medline].
|
| 18.
|
Herrington, D. A., and P. F. Sparling.
1985.
Haemophilus influenzae can use human transferrin as a sole source for required iron.
Infect. Immun.
48:248-251[Abstract/Free Full Text].
|
| 19.
|
Hochberg, Y., and A. Tamhane.
1987.
Multiple comparison procedures, p. 30.
and 363. John Wiley & Sons, Inc., New York, N.Y.
|
| 20.
|
Lewis, L. A.,
K. H. Rhode,
B. Behrens,
E. Gray,
S. I. Toth,
B. A. Roe, and D. Dyer.
1998.
Identification and molecular analysis of lbpBA, which encodes the two-component meningococcal lactoferrin receptor.
Infect. Immun.
66:3017-3023[Abstract/Free Full Text].
|
| 21.
|
Lewis, L. A.,
E. Gray,
Y.-P. Wang,
B. A. Roe, and D. Dyer.
1997.
Molecular characterization of hpuAB, the haemoglobin-heptaglobin-utilization operon of Neisseria meningitidis.
Mol. Microbiol.
23:737-749[Medline].
|
| 22.
|
Masson, P. L.,
J. F. Heremans, and C. H. Dive.
1966.
An iron-binding protein common to many external secretions.
Clin. Chim. Acta
14:735-739.
|
| 23.
|
McKenna, W. R.,
P. A. Mickelsen,
P. F. Sparling, and D. W. Dyer.
1988.
Iron uptake from lactoferrin and transferrin by Neisseria gonorrhoeae.
Infect. Immun.
56:785-791[Abstract/Free Full Text].
|
| 24.
|
Menard, R.,
P. J. Sansonetti, and C. Parsot.
1993.
Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells.
J. Bacteriol.
175:5899-5906[Abstract/Free Full Text].
|
| 25.
|
Mickelsen, P. A.,
E. Blackman, and P. F. Sparling.
1982.
Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species to obtain iron from lactoferrin.
Infect. Immun.
35:915-920[Abstract/Free Full Text].
|
| 26.
|
Mietzner, T. A., and S. A. Morse.
1994.
The role of iron binding proteins in the survival of pathogenic bacteria.
Annu. Rev. Nutr.
14:471-493[Medline].
|
| 27.
|
Nakano, Y.,
Y. Yoshida,
Y. Yamashita, and T. Koga.
1995.
Construction of a series of pACYC-derived plasmid vectors.
Gene
162:157-158[Medline].
|
| 28.
|
Pettersson, A.,
T. Prinz,
A. Umar,
J. Biezen, and J. Tommassen.
1998.
Molecular characterization of lbpB, the second lactoferrin-binding protein of Neisseria meningitidis.
Mol. Microbiol.
27:599-610[Medline].
|
| 29.
|
Pettersson, A.,
A. Maas, and J. Tommassen.
1994.
Identification of the iroA gene product of Neisseria meningitidis as a lactoferrin receptor.
J. Bacteriol.
176:1764-1766[Abstract/Free Full Text].
|
| 30.
|
Pidcock, K. A.,
J. A. Wooten,
B. A. Daley, and T. L. Stull.
1988.
Iron acquisition by Haemophilus influenzae.
Infect. Immun.
56:721-725[Abstract/Free Full Text].
|
| 31.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 32.
|
Thomas, C. E., and P. F. Sparling.
1994.
Identification and cloning of a fur homologue from Neisseria meningitidis.
Mol. Microbiol.
11:725-737[Medline].
|
| 33.
|
Tinoco, I., Jr.,
P. N. Borer,
B. Dengler,
M. D. Levine,
O. C. Uhlenbeck,
D. M. Crothers, and J. Gralla.
1973.
Improved estimation of secondary structure in ribonucleic acids.
Nature (London) New Biol.
246:40-41.
|
| 34.
|
Welch, S.
1992.
The chemistry and biology of iron, p. 41-59.
In
S. Welch (ed.), Transferrin: the iron carrier. CRC Press, Inc., Boca Raton, Fla.
|
Infection and Immunity, January 1999, p. 455-459, Vol. 67, No. 1
0019-9567/99/$04.00+0
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