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(Received for publication, July 17, 1995; and in revised form, September 8, 1995) From the
The HtrB protein was first identified in Escherichia coli as a protein required for cell viability at high temperature, but
its expression was not regulated by temperature. We isolated an htrB homologue from nontypable Haemophilus influenzae strain (NTHi) 2019, which was able to functionally complement the E. coli htrB mutation. The promoter for the NTHi 2019 htrB gene overlaps the promoter for the rfaE gene, and the two
genes are divergently transcribed. The deduced amino acid sequence of
NTHi 2019 HtrB had 56% homology to E. coli HtrB. In vitro transcription-translation analysis confirmed production of a
protein with an apparent molecular mass of 32-33 kDa. Primer
extension analysis revealed that htrB was transcribed from a
Lipopolysaccharide (LPS) ( The htrB gene was first
identified in Escherichia coli as encoding a protein essential
for cell viability at a temperature above 33 °C(2) . Unlike
other heat shock proteins, however, its expression is not regulated by
temperature(3) . Bacteria with a mutation in the htrB gene, when exposed to nonpermissive temperatures in rich media,
cease to divide and lose viability within 2 h(2) . They also
show morphological changes similar to those of cells with double
mutations in two of the cell wall synthesis genes, pbpA and pbpB, suggesting a role of the HtrB protein in cell wall
synthesis(3, 4) . The E. coli htrB mutation
does not affect the mobility of LPS on SDS gels, but the silver-stained
LPS has a dramatically reduced intensity and a conversion from a black
to a brown coloration(1) . One study of htrB suppressor genes has shown that htrB mutant bacteria
accumulate a high level of phospholipid at 42 °C and that
spontaneous mutations in the accBC operon, which is involved
in fatty acid biosynthesis, cause a decrease in phospholipid
biosynthesis, restoring the balance between the two(5) . This
study suggested that htrB may be part of a link between growth
rate and the regulation of phospholipid biosynthesis. Haemophilus influenzae is a causative agent of many
childhood diseases, including meningitis and respiratory tract
infections. Nontypable strains of H. influenzae (NTHi), which
are commonly present in the nasopharynx of 50-80% of healthy
carriers, are recently recognized to be important human pathogens, and
evidence has shown that their lipo-oligosaccharide (LOS) is important
in pathogenesis. Haemophilus LOS differs from enterobacterial
LPS in that it does not contain repeating O-antigen units and
is more similar to those from Neisseria and Bordetella(6) . We have been studying genes involved
in LOS biosynthesis in Haemophilus. In the process of
sequencing the upstream region of the NTHi rfaE gene, which is
responsible for ADP-heptose synthesis, we identified the htrB homologue(7) . These two genes have overlapping promoter
regions and are transcribed into diverse orientations. In an effort to
understand the function of HtrB in Haemophilus, we constructed
and characterized NTHi 2019 htrB mutants. Structural analysis
of the LOS from an htrB mutant revealed a modification of the
ratio of hexose to phosphoethanolamine in the LOS core structure and
the loss of one or both myristic acid substitutions of the lipid A.
Figure 1:
Restriction map of the region of the
NTHi 2019 htrB gene. The arrows indicate open reading
frames. Two triangles marked on the map show mini-Tn3
insertion sites in isogenic htrB mutants (open triangle for NTHi B28 and solid triangle for NTHi B29). B, BamHI; H, HindIII; P, PstI; RV, EcoRV; S, ScaI.
Primer extension analysis was carried out using the Promega Primer
Extension kit following the manufacturer's suggestions. 20 µg
of RNA were used for each reaction, and the reaction products were
precipitated in ethanol, dissolved in loading dye, and loaded on a 6%
sequencing gel. The gel was dried and exposed to an x-ray film with an
intensifying screen at -70 °C. The dideoxy-sequencing ladder
obtained with the same primer was used as a marker to confirm the
position of the primer-extended products.
Both lipid A
samples were then directly analyzed by liquid secondary ion mass
spectrometry (LSIMS) in the negative ion mode. Lipid samples were
redissolved in CH
To construct a plasmid
containing the htrB ORF, we carried out PCR using two
oligonucleotides, one upstream of the promoter region
(5`-aagcatcacatcgcctaatacaa-3`) and the other the universal forward
primer, with pHIE2 as a template(7) . The resulting 1.3-kb PCR
fragment was cloned into a vector, pCRII, yielding pHIH. When the E. coli htrB mutant strains, MLK48 and MLK217, were
transformed with pHIH, they were able to grow at 37 °C as well as
the wild type parent MLK2, indicating that the function of NTHi 2019
HtrB is analogous to that of E. coli HtrB. Disruption of the htrB ORF in pB28 and pB29 by mini-Tn3 abolished the
complementing ability of these plasmids (data not shown). In pHIE0 (Fig. 1), we also found a partial ORF located downstream of the htrB gene. A homology search of data banks revealed that the
deduced amino acid sequence of this partial ORF was highly homologous
to E. coli topoisomerase IV subunit B (73% identity and 86%
similarity over 100 amino acids) by BESTFIT software analysis. A rho-independent transcription termination sequence was found
between these two ORFs, suggesting that htrB is transcribed as
a monocistronic message.
Figure 2:
In vitro transcription-translation analysis of the NTHi 2019 htrB gene. Lane 1, protein molecular mass standards; lane
2, pCRII; lane 3, pHIH; lane 4, pHIE0; lane
5, pHIE7; lane 6, pGEM-7Zf(+). The open circle indicates the protein product corresponding to rfaE; the solid circle indicates that corresponding to htrB;
the open square indicates that corresponding to the ampicillin
resistance gene; the solid square indicates that corresponding
to the kanamycin resistance gene. The sizes of the molecular mass
standards are shown on the left.
Figure 3:
Primer extension analysis of the NTHi 2019 htrB (A) and rfaE (B) genes. The
position of the transcription start site is indicated with an asterisk on the sequence, and the position for the extended
product is indicated with an arrow. The DNA sequencing ladders
shown (GATC) were obtained using the same primer as that used in the
primer extension reaction. Lanes 1, no RNA; lanes 2,
NTHi 2019 grown at 30 °C; lanes 3, NTHi 2019 grown at 37
°C; lanes 4, NTHi B29 at grown 30 °C; lanes
5, NTHi B29 grown at 37 °C.
The E. coli htrB protein is required for cell
viability at high temperatures, but it has been reported that htrB gene expression is not under heat shock regulation(3) . To
determine if the same is true in Haemophilus, we measured the htrB transcript level in RNA from NTHi 2019 grown at 30 and 37
°C by primer extension analysis. The results indicated that the
transcript level of htrB was not affected by temperature (Fig. 3A, lanes 2 and 3). Because the E. coli htrB mutation phenotype is very diverse and complex (2, 3, 5, 21, 22) and our
previous data suggested that it may play a role in regulation of gene
expression(7) , we measured htrB and rfaE expression levels in RNA from NTHi 2109 and B29 grown at 30 and 37
°C. Temperature did not affect htrB expression in either
strain. The level of expression of htrB, however, increased in
B29 as compared with NTHi 2019 at both temperatures by 4-6-fold
when quantitated using an Ambis 4000 gel scanner (Ambis Inc, San Diego,
CA). Similar results were observed for rfaE in that rfaE expression increased in htrB
Previous reports
have suggested that htrB is involved in LPS synthesis and/or
cell wall formation, subsequently changing membrane permeability to
various compounds(21) . Therefore, we tested NTHi htrB
Figure 4:
Sensitivity of NTHi 2019 and isogenic htrB mutant to temperature and kanamycin. Overnight cultures
grown in sBHI broth at 30 °C were diluted to 10 Klett units and
incubated in the absence or presence of kanamycin (5 µg/ml) at 30 (A) or 37 °C (B). Open square, NTHi
2019; solid square, NTHi 2019 with kanamycin; open
circle, NTHi B29; solid circle, NTHi B29 with
kanamycin.
The sensitivity pattern of B28 and B29 to
deoxycholate was similar to kanamycin. At 30 and 37 °C, NTHi 2019
grew well in the presence of 1000 µg/ml of deoxycholate and still
showed some growth in the presence of 2500 µg/ml of deoxycholate
(data not shown). At 30 °C, B28 and B29 cells grew as well as the
wild type cells in the presence of 250 µg/ml of deoxycholate but
began to show sensitivity at 500 µg/ml and failed to grow at 1000
µg/ml. At 37 °C, B28 and B29 began to show growth inhibition at
50 µg/ml of deoxycholate and almost complete inhibition at 250
µg/ml of deoxycholate. To confirm that the htrB gene
alone can complement the phenotypes of B28 and B29, we constructed a
plasmid expressing htrB. The BamHI-PstI fragment carrying the htrB gene from pHIH was cloned into pGHH. Because strain B29 carried
the chloramphenicol acetyltransferase gene in mini-Tn3, we cloned a
kanamycin cassette into the PstI site of pGHH, resulting in
pKHH. pKHH was transformed into B29 by electroporation and selected on
kanamycin (15 µg/ml) at 30 °C to increase the probability to
rescue transformants. pKHH carries the p15A origin, which is known to
replicate in Haemophilus(23) , but we were unable to
detect any plasmid DNA in mini-plasmid DNA preparations from B29 cells
transformed with pKHH. To detect any presence of plasmid DNA, we also
transformed these mini DNA preparations into E. coli DH5
Figure 5:
SDS-polyacrylamide gel analysis of LOS
from NTHi 2019 and isogenic htrB mutants. The arrow indicates the direction of sample migration. Lanes 1 and 4, NTHi 2019; lane 2, NTHi B29; lane 3, NTHi
B28.
We also performed immunoblotting
analysis of LOS using the anti H. influenzae LOS mAb 6E4 (15) to determine if a change in LOS epitope structure occurred
in the NTHi htrB mutants. Our studies demonstrated binding of
this mAb to H. influenzae strain 2019 LOS at 30 and 37 °C.
LOS from B29 and B28 did not bind to mAb 6E4 in the first few passages
of the mutants. As the temperature sensitivity began to be repressed,
we could show that mAb 6E4 reacted weakly with both B29 and B28 at 37
°C, whereas the antibody failed to react with the mutants at 30
°C. Reconstitution of B29 with pKHH reverted the mAb 6E4 phenotype
of the corrected mutant to the wild type pattern.
Figure 6:
Electrospray-mass spectrum of the O-deacylated LOS from wild type NTHi 2019 (A) and the
isogenic mutant B29 (B). Inset histograms show
proposed composition differences among LOS glycoforms relative to the
number of hexose(1, 2, 3, 4, 5, 6) and
phosphoethanolamine (1, 2) units. In both cases a core
Hep
Figure 7:
Partial negative ion LSIMS spectra of the
lipid A of the wild type 2019 showing deprotonated molecular ions for
the di- and monophosphoryl hexaacyl lipid A at m/z 1823 and 1743 and various fragment ions (A), and
deprotonated molecular ions formed from di- and monophosphoryl
pentaacyl and tetraacyl lipid As from the mutant B29 at m/z 1613, 1533, 1403, and 1323 (B). H.
influenzae lipid A structures shown in the spectrum are those
taken from Helander et al.(26) . The minor ion not
discussed in the text can be rationalized as follows: m/z 1725 and 1727 as loss of HPO
In contrast, the
LSIMS spectrum shown in Fig. 7B for the lipid A
preparation obtained from the mutant B29 strain lacks molecular ions
corresponding to the wild type hexaacyl lipid A species. This spectrum
contains two high mass ions at m/z 1613 and 1533 (M We have identified a homologue of the E. coli htrB gene in NTHi 2019, which is transcribed in the opposite direction
and immediately downstream of the rfaE gene. Studies of the htrB gene in E. coli have shown that this gene is
associated with exquisite heat sensitivity but is not heat-inducible (3) . Studies using E. coli htrB suppressor genes
indicated that the phospholipid content in the bacterial outer membrane
is elevated and that the protein content, including porin proteins, is
reduced at 42 °C in htrB mutant cells(5) . This
suggested that HtrB may be involved in phospholipid biosynthesis.
Modifications in the lipid A structure of the htrB mutants
might be a factor in the temperature sensitivity. Clementz and
co-workers have shown that E. coli htrB and msbB encode KDO-dependent acyltransferases(27) . Mass
spectrometric analysis of the lipid A from the NTHi htrB mutant B29 indicated that modification of lipid A had occurred.
The lipid A of the parent strain NTHi 2019 is hexaacyl. The analysis of
the lipid A from B29 shows two species, a tetraacyl and a pentaacyl
species, indicating loss of one or both of the myristic acid
substitutions. It is interesting to speculate that in the low passage htrB mutants, the predominance of the tetraacyl lipid A
species may account for the temperature susceptibility. As the
suppressors, msbB(21) and msbA(22) , of the htrB mutation are induced,
they partially restore the myristic acid substitution and correct the
temperature sensitivity. The NTHi HtrB homologue has
several other characteristics of the E. coli protein. It
encodes a basic protein with an isoelectric point of 10.31 and
molecular mass of 36,066 Da, which is slightly smaller than the E.
coli HtrB protein. NTHi htrB mutants were
temperature-sensitive upon initial isolation but, unlike E. coli mutants, upon passage at 30 °C developed a temperature profile
similar to that of the wild type strain. This suggests that factor(s)
similar to the E. coli msbA, msbB, and accBC genes
may be operative that are suppressing the htrB mutation(5, 21, 22) . In our study,
NTHi htrB mutants showed hypersensitivity to kanamycin and
deoxycholate compared with the wild type strain. In contrast to our
results, E. coli htrB mutants exhibit higher resistance to
deoxycholate than does the wild type(21) . A difference in
phenotype between Haemophilus and E. coli mutants and
the corresponding wild type strains was also found in
SDS-polyacrylamide gel patterns of LPS/LOS. LPS from both species were
changed in color on silver-stained gels, indicating that their
structures were modified. The LOS from the NTHi mutant, however,
migrated faster than that from its parent strain, whereas LPS from the E. coli mutant strain did not show any change in migration on
SDS gels(1) . ESI-MS analysis of LOS from the NTHi htrB mutant confirmed modification of the LOS and revealed a 50%
reduction in the LOS species containing two phosphoethanolamines as
well as a shift to higher molecular weight species not seen with LOS
from the NTHi 2019 parent strain. These findings suggest that the
degree of phosphorylation of heptose may be affecting chain progression
from specific heptose moieties. In addition, elongation of these chains
may be related to the degree of phosphorylation. A reduction in
phosphate on heptose moieties has also been observed in LPS from rfaP mutant strains. rfaP mutants were initially
isolated from S. typhimurium and characterized with respect to
their sensitivity to hydrophobic antibiotics and
detergents(26) . The most striking feature of these mutants is
the lack of a phosphate group linked to heptose I of the LPS core
structure. RfaP is believed to have an LPS kinase-like
function(28) . There is no evidence that HtrB acts as an LPS
kinase, but it may indirectly regulate phosphorylation of LOS. rfaP mutants are also moderately heat-sensitive, but the unique
temperature profile of htrB mutant strains cannot be explained
solely by dephosphorylation of LOS. At present, little is known about
the enzymology or regulation of phosphate or phosphoethanolamine
incorporation into LOS in H. influenzae. No one has identified
an rfaP homologue in H. influenzae. We are currently
pursuing studies in these areas. Several studies indicate that the
assembly and organization of the bacterial outer membrane require
specific interactions between proteins and LPS and that the core
structure is the region that is responsible for these
interactions(29) . It was also suggested that sensitivity to
hydrophobic agents is not directly associated with truncations in the
carbohydrate structure but with loss of phosphate groups on heptose
moieties, indicating that the presence of phosphate residues may be
even more important than that of the addition of saccharide groups.
Nikaido and Vaara (29) also suggested that the bridging of
negatively charged phosphate groups appears to be important in LPS-LPS
interactions, which serve as a very effective barrier against
hydrophobic molecules. The hypersensitivity of NTHi htrB mutants to kanamycin and deoxycholate could be explained in the
context of dephosphorylation of LOS. This idea is also supported by the
observation of Ray et al. (30) that LPS from Pseudomonas syringae, isolated in Antarctica, was more
phosphorylated when grown at higher temperatures than when grown at
lower temperatures and was more sensitive to cationic antibiotics such
as kanamycin when grown at low temperatures. The greater sensitivity of
NTHi htrB mutants to kanamycin at the higher temperature may
be due to the synergistic effect of these two factors. The
susceptibility of the htrB mutant to deoxycholate and
kanamycin did not change with the restoration of the temperature
stability. This indicates that the mechanisms controlling these
phenotypes are different. The temperature sensitivity is related to
modification in the lipid A and the changes in cell membrane
permeability to modifications in phosphoethanolamine content of the
LOS. This hypothesis is also supported by our observation that the
NTHi htrB mutants are more heat-sensitive than the NTHi rfaD mutant lacking heptose (and, thus, lacking a phosphate
group) ( The increased
expression of htrB and rfaE in the htrB As described, htrB mutant strains
exhibit very diverse and complex phenotypes. It is not likely that the
HtrB protein directly exerts all of these separate functions, but these
may be the results of indirect effects regulated by HtrB. Our results
strongly suggest that H. influenzae HtrB is a multifunctional
protein with either acyltransferase activity or the ability to regulate
this activity. In addition, it appears to play an important role in
controlling cell responses to environmental changes including
temperature.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s)
U17642[GenBank].
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 27151-27159
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-dependent consensus promoter and its expression was
not affected by temperature. The expression of htrB and rfaE was 2.5-4 times higher in the NTHi htrB mutant B29 than in the parental strain. In order to study the
function of the HtrB protein in Haemophilus, we generated two
isogenic htrB mutants by shuttle mutagenesis using a mini-Tn3.
The htrB mutants initially showed temperature sensitivity, but
they lost the sensitivity after a few passages at 30 °C and were
able to grow at 37 °C. They also showed hypersensitivity to
deoxycholate and kanamycin, which persisted on passage.
SDS-polyacrylamide gel electrophoresis analysis revealed that the
lipo-oligosaccharide (LOS) isolated from these mutants migrated faster
than the wild type LOS and its color changed from black to brown as has
been described for E. coli htrB mutants. Immunoblotting
analysis also showed that the LOS from the htrB mutants lost
reactivity to a monoclonal antibody, 6E4, which binds to the wild type
NTHi 2019 LOS. Electrospray ionization-mass spectrometry analysis of
the O-deacylated LOS oligosaccharide indicated a modification
of the core structure characterized in part by a net loss in
phosphoethanolamine. Mass spectrometric analysis of the lipid A of the htrB mutant indicated a loss of one or both myristic acid
substitutions. These data suggest that HtrB is a multifunctional
protein and may play a controlling role in regulating cell responses to
various environmental changes.
)is a component of the
outer membrane of Gram-negative bacteria. It consists of lipid A linked
by 2-keto-3-deoxyoctulosonic acid (KDO) to a heterogeneous sugar
polymer and repeating O-antigen units. LPS plays an important
role in pathogenicity and virulence. It also serves as a building block
for the outer membrane and permeation barrier to hydrophobic
compounds(1) . Salmonella typhimurium LPS deep core
mutants show increased sensitivity to various hydrophobic reagents and
to elevated temperatures.
Strains, Plasmids, and Culture Conditions
The
bacterial strains and plasmids used in this study are described in Table 1and Fig. 1. E. coli strains were grown in
LB medium containing appropriate antibiotics at 30 or 37 °C. NTHi
strains were cultured on brain-heart infusion medium agar plates
supplemented with 2% Fildes reagent (Difco Laboratories) (sBHI) or in
sBHI broth at 30 or 37 °C with agitation. For E. coli strains, antibiotics were added when necessary at a final
concentration of 50 µg/ml for ampicillin, 30 µg/ml for
kanamycin, and 40 µg/ml for chloramphenicol. For NTHi strains, 1.5
µg/ml of chloramphenicol or 15 µg/ml of kanamycin was added
when needed.
DNA Manipulations
Restriction and DNA-modifying
enzymes were purchased from New England Biolabs and Promega. Standard
DNA recombinant procedures were performed as described(8) . For
cloning of DNA fragments made by PCR into a vector DNA, the TA cloning
system from Invitrogen was used. Transformation of E. coli strains with plasmid DNA was routinely done by the
CaCl
method (9) or by electroporation using the
Life Technologies Cell Porator. NTHi strains were transformed by the M
IV method (10) or by electroporation(11) .Transposon Mutagenesis
Mutagenesis of the htrB gene was carried out by shuttle mutagenesis using a mini-Tn3 as
described by Seifert et al.(12) . The locations of
transposon insertion sites were determined by DNA sequencing of
plasmids and genomic Southern hybridization.Genomic Southern Hybridization
NTHi 2019 genomic
DNA was digested with restriction enzymes, resolved on a 1% agarose
gel, transferred to a Hybond-N membrane (Amersham Corp.) by capillary
blotting overnight, and cross-linked to the membrane using the
Stratalinker (Stratagene). After prehybridization in 6 
SSC, 1%
SDS, 0.5% dry milk, the membrane was hybridized with a
digoxigenin-dUTP-labeled DNA probe at 65 °C and washed. The
hybridized probe was detected using the DIG Luminescent Detection Kit
(Boehringer Mannheim), and the membrane was exposed to a Kodak X-Omat
AR film at room temperature.Primer Extension Analysis
NTHi strains grown
overnight in sBHI broth at 30 °C were diluted 10-fold with fresh
medium, grown to OD 0.2, and then shifted to the
desired temperature and allowed to grow for 1 h. Cells were then
harvested, and RNA was extracted as described by Verwoerd et
al.(13) , followed by DNase I treatment and ethanol
precipitation. The purified RNA was quantitated spectrophotometrically,
and the quality of RNA was confirmed on a formaldehyde-agarose gel
stained with ethidium bromide. Oligonucleotides used as primers were
synthesized using the 391 DNA Synthesizer (Applied Biosystems).
In Vitro Transcription-Translation
Analysis
Plasmids were purified by CsCl gradient (8) and
used for in vitro transcription-translation (Promega). The
translation products were labeled using translation grade
[S]methionine (Amersham Corp.). The reaction
mixtures were separated on a 11% SDS-polyacrylamide gel(14) ,
which was dried and exposed to an x-ray film.
C-labeled
Rainbow standards (Amersham Corp.) were used as protein molecular
weight standards.Analysis of Cell Growth and Sensitivity to
Antibiotics
To determine temperature sensitivity, NTHi strains
were grown overnight in 5 ml of sBHI broth at 30 °C with agitation,
and the cultures were diluted to 45 klett units. 1-ml aliquots of the
diluted cultures were inoculated into 50 ml of fresh media and allowed
to grow at the desired temperature while being monitored for growth.
For kanamycin sensitivity, overnight cultures grown in sBHI broth at 30
°C were diluted to 10 Klett units with fresh sBHI and allowed to
grow at 30 or 37 °C with or without kanamycin (5 µg/ml). To
measure sensitivity to deoxycholate, the overnight cultures grown in
sBHI broth at 30 °C were serially diluted, and 5 µl of each was
spotted on a series of sBHI plates containing different concentrations
of deoxycholate. The plates were incubated at 30 or 37 °C overnight
before reading the results.Immunoblot Analysis
Immunoblot analysis of
colonies lifted after overnight growth was performed with monoclonal
antibody 6E4. mAb 6E4 recognizes a nonphase varying KDO-like epitope on
the LOS of all H. influenzae strains we have
studied(15) . Colonies grown on sBHI plates were transferred
onto a nitrocellulose filter (Micron Separations Inc.) and air-dried
completely. The filter was blocked by soaking in 3% gelatin solution
and probed with mAb 6E4 (15) overnight at room temperature. The
filter was then incubated with protein A conjugated with horseradish
peroxidase (Zymed Laboratories). A chromogenic substrate,
4-chloro-1-naphthol (Bio-Rad), was used as the developing reagent.LPS Gel Analysis
LPS was prepared by proteinase K
digestion as described(16) , separated on a 14% polyacrylamide
gel containing SDS(14) , and visualized by silver-staining as
described previously (17) . Large scale preparations of LOS
were obtained using the procedure of Darveau and Hancock(18) .Electrospray Ionization-Mass Spectrometry of O-Deacylated
LOS
O-Deacylated LOS was prepared from approximately
2-3 mg of LOS from NTHi 2019 and the isogenic htrB mutant B29 by mild hydrazine treatment (37 °C, 20 min) as
described previously(19) . To determine molecular masses, O-deacylated LOS from both strains were subjected to mass
analysis by electrospray ionization-mass spectrometry
(ESI-MS)(20) . Briefly, O-deacylated LOS was first
solubilized in water at a concentration of 10 µg/µl; a
1-µl aliquot of this solution was then taken and diluted with 4
µl of the ESI-MS running solvent prior to injection. Mass analysis
was performed using a VG/Fison Platform single quadrupole mass
spectrometer operation in the negative ion mode with a running solvent
of H
O/acetonitrile (1:1, v/v) containing 1% acetic acid at
a flow rate of 10 µl/min. Mass calibration was accomplished with an
external mass reference of CsNO
, which provided cluster
masses from m/z 256.9,
[Cs(NO)] to m/z 2011.1,
[Cs
(NO
)]
.
Preliminary compositions were assigned to the LOS by computer search of
potential monosaccharide compositions using the program Gretta Carbos
developed by W. Hines.
Fractionation and Mass Spectrometric Characterization of
Lipid A
Approximately 32 mg of LOS from NTHi strain 2019 and 6.6
mg of LOS from the isogenic mutant B29 were hydrolyzed in 1% acetic
acid for 2 h at 100 °C at a concentration of 2 mg/ml. The
hydrolysates were centrifuged at 5,000 g for 20 min at
4 °C, and the supernatants were removed. The pellets were washed
twice with 0.5 ml of HO followed by centrifugation at 5,000
g for 20 min at 4 °C. The water-insoluble crude
lipid A fractions were suspended in chloroform/methanol/HO
(2:1:1, v/v/v) and centrifuged at 5,000 g for 15 min,
and the lower organic layer plus the middle emulsion layer containing
the lipid A were removed and evaporated to dryness.Cl/CHOH (3:1,
v/v), and 1 µl of nitrobenzylalcohol/triethanolamine (1:1, v/v) was
applied as the liquid matrix. Samples were then analyzed on a Kratos MS
50S mass spectrometer retrofitted with a cesium ion source operating at
a resolution of 2000 (m/
m, 10% valley). A
primary ion beam of 10 keV was used to ionize the samples, and
secondary ions were accelerated at 10 keV. Scans were acquired at 300
s/decade and recorded on a Gould electrostatic recorder.
Ultramark 1206 was used for manual calibration to an
accuracy better than ± 0.2 Da.
Cloning and Identification of the NTHi 2019 htrB
Gene
We isolated the rfaE gene from NTHi 2019 by
complementing an rfaE mutant of S. typhimurium LT2(7) . Sequence analysis of the upstream region of the rfaE gene revealed an open reading frame (ORF) highly
homologous to the E. coli htrB gene. The deduced amino acid
sequences of these two genes shared homology (56% identity and 73%
similarity). The NTHi 2019 HtrB homologue also showed homology (27%
identity and 54% similarity) with the E. coli MsbB protein,
which is a multicopy suppressor of the htrB mutation(21) . This is similar to the homology between the E. coli HtrB and MsbB proteins.Identification of the htrB Gene Product
To confirm
that the htrB ORF synthesizes a protein with the molecular
mass estimated from the nucleotide sequence, we carried out in
vitro transcription-translation analysis (Fig. 2). pCRII,
the vector used to construct pHIH, produced two bands corresponding to
the 31.5-kDa 
-lactamase precursor from its ampicillin resistance
gene and the 29.6-kDa aminoglycoside phosphotransferase from its
kanamycin resistance gene (Fig. 2, lane 2). pHIH
produced one more band, presumably from the htrB gene, above
the -lactamase precursor (Fig. 2, lane 3). The
estimated molecular mass of HtrB was 36 kDa, but the peptide
synthesized from pHIH migrated with an apparent molecular mass of
32-33 kDa (Fig. 2, lane 3). Based on
complementation data (see below), we believe that this is the authentic
HtrB. pHIE0 produced three proteins in addition to -lactamase from
the vector pSH-lox-1 (Fig. 2, lane 4), one
corresponding to HtrB and another corresponding to RfaE as seen with
pHIE7 (Fig. 2, lane 5).
Construction of NTHi 2019 htrB Mutants
In order to
investigate the function of HtrB in Haemophilus, we
constructed isogenic htrB mutants by shuttle mutagenesis. The
2.4-kb BglII fragment from pHIE0 was cloned into pHSS6,
resulting in pHSBglII, which was used for mini-Tn3
mutagenesis(12) . Two plasmids, pB28 and pB29, each with a
mini-Tn3 transposon inserted into the htrB ORF at a different
location, were obtained (Fig. 1). Sequence analysis and
restriction mapping of these plasmids indicated that in both plasmids,
mini-Tn3 was inserted with the chloramphenicol acetyltransferase gene
in the same orientation as htrB. Both plasmids were used to
transform NTHi 2019, and cells were selected on chloramphenicol (1.5
µg/ml) plates. Because the E. coli htrB mutant is
temperature-sensitive above 33 °C, we incubated cells at 30 °C.
Mutant strains from each plasmid were designated NTHi B28 and B29,
respectively. The locations of the mini-Tn3 insertions in the NTHi 2019
chromosome were confirmed by genomic Southern hybridization using the
2.4-kb BglII fragment as a probe. BglII digestion
yielded a 2.4-kb fragment with NTHi 2019 DNA but produced 4.0-kb
fragments with NTHi B28 and B29 DNAs, indicating that they contained
the 1.6-kb transposon (data not shown). The 2.4-kb band remained the
same in NTHi 2019 DNA upon BglII and EcoRI double
digestion, but the 4.0-kb BglII bands of NTHi B28 and B29 were
cut into two fragments by EcoRI, which is present in mini-Tn3. mRNA Expression of htrB and rfaE in htrB Mutant
Strains
Primer extension analysis was carried out to determine
the promoter region of the htrB gene (Fig. 3A), and a single transcription start site was
found using a primer (5`-ccaatatggcgcaaaataggatagggaagac-3`)
complementary to the 5` region of the htrB ORF. The first
nucleotide transcribed was a C residue located at 21 base pairs
upstream of the putative translation start site ATG. The region
upstream of the transcription start site contained a sequence (TAAACT)
similar to the consensus -10 region of the bacterial
-dependent promoters at an appropriate distance (6
base pairs). An element (TTACCA) resembling the consensus sequence of
the -35 region (TTGACA) was found. There was a 16-base pair
spacer between this region and the -10 region. This -35
element partially overlapped with the -10 region of the rfaE gene.
cells and was
not dependent on temperature (Fig. 3B). The increase in rfaE expression in htrB
cells was
2-2.5-fold higher than that found in htrB
cells.
Characterization of the NTHi htrB Mutants
NTHi B28
and B29 strains were initially selected at 30 °C and were not able
to grow at 37 °C. After they were passed a few times at 30 °C,
however, they began to lose this temperature sensitivity and showed
growth at 37 °C. The growth rates of B28 and B29 were comparable
with that of 2019 at 30 °C, but slower growth was observed at 37
°C. The temperature sensitivity of these htrB strains was greater at 38.5 °C, so
that their growth reached a maximum at 6 h and then started to
decrease, suggesting cell lysis (data not shown).
strains for sensitivity to kanamycin
and deoxycholate. Overnight cultures grown at 30 °C were diluted
and allowed to grow in the absence or presence of kanamycin (5
µg/ml) at 30 or 37 °C. At 30 °C, no difference was observed
in the growth rate between the wild type and htrB
strains B28 and B29 in the absence of
kanamycin, but the growth of htrB
cells was
significantly inhibited by addition of kanamycin, whereas the wild type
cell growth was not affected (Fig. 4A). The effect of
kanamycin on the htrB mutant strains was greater at 37 °C
because there was essentially no growth in the presence of kanamycin (Fig. 4B).
with selection for kanamycin resistance but did not get any
transformants. A genomic Southern blot of the pKHH-transformed B29
cells, however, indicated that the kanamycin cassette had been
integrated into the chromosome (data not shown). B29 transformed with
pKHH behaved similarly to NTHi 2019 in sensitivity to deoxycholate at
both temperatures.LOS Analysis of NTHi 2019 htrB Mutants
It has been
described that LPS of the E. coli htrB mutant silver-stained
weakly on SDS-polyacrylamide gels but its migration pattern was not
affected(1) . The LOS from B28 and B29 migrated faster than
that from NTHi 2019 on silver-stained SDS-polyacrylamide gels (Fig. 5), and the color of the band had changed from black to
brown. Reconstitution of mutant strain B29 with pKHH carrying the htrB gene restored the mobility and the color of LOS to those
of the wild type (data not shown).
Electrospray Ionization-Mass Spectrometry Analysis of
NTHi htrB Mutant LOS
ESI-MS analysis of the O-deacylated LOS from the NTHi B29 strain provided a molecular
mass profile of the LOS components that was similar but not identical
to the wild type NTHi 2019 strain (see Fig. 6A).
Despite their similarities, two differences can be readily discerned:
one, a decrease in the level of LOS species containing two
phosphoethanolamines and two, a shift to higher molecular mass LOS
species containing more hexoses. This trend can be best seen by noting
the most abundant species present in the B29 strain relative to the
NTHi 2019 wild type. In the NTHi 2019 strain, this species is seen as
the triply charged peak at m/z 799,
(M-3H), with a mass of 2,400 Da, corresponding to
the previously reported LOS species containing a lactose disaccharide
attached to a Hep
KDO(P)-Lipid A core with two PEA
groups(24) . In contrast, the ESI-MS spectrum of the B29 mutant O-deacylated LOS shows a triply charged base peak at m/z 853 (M
= 2562),
corresponding to a trihexose substituted core LOS with a much more
prominent species at lower mass containing only one PEA group, i.e. (M-3H) at m/z 812 with a M
of 2439. Indeed, if one examines the relative
proportion of LOS species containing one or two PEA groups between the
wild type and mutant strains (Fig. 6, A and B, insets), it becomes clear that the PEA content has decreased
significantly in the B29 strain. Further chemical analysis will be
required to completely delineate the structural difference between
these two strains, such as linkage positions of the singly and doubly
phosphorylated core LOS and the nature of the LOS containing three or
more hexoses. Nevertheless, the ESI-MS analysis clearly points to the
major trends inherent in the htrB mutant LOS structures.
KDO-lipid A is present.
Mass Spectrometric Analysis of Lipid A
LSIMS
analysis of the wild type 2019 strain produced a spectrum containing
two abundant deprotonated molecular ions, (M-H) at m/z 1823 and 1743, with several lower mass ions (see Fig. 7A). The two higher mass ions are consistent with
a hexaacyl lipid A structure containing either one (hexaacyl
monophosphoryl lipid A, M
= 1744) or two
phosphates (hexaacyl diphosphoryl lipid A, M = 1824). Moreover, this spectrum is essentially identical
to that previously reported for the major lipid A structure isolated
from the LOS of Haemophilus ducreyi strain 35000 (25) and is also consistent with the hexaacyl lipid A structure
determined by NMR from a deep rough mutant H. influenzae strain I-69 Rd/B
(26) (see structure inset to Fig. 7A). The lower mass fragment ions can be explained
by the loss of myristic acid (-210 and/or 228 Da, m/z 1613/1595 and 1533/1515) and myristoylmyristic
acid (-436 and/or 454 Da, m/z 1387/1369 and
1307/1289) as the ketene and/or protonated acids. These ions most
likely arise through LSIMS-induced fragmentation of the higher mass
mono- and diphosphorylated molecular ion species.
and
HPO, respectively, from (M-H) at m/z 1823; m/z 1517 as loss
of
-hydroxymyristic acid as the ketene from (M-H) at m/z 1823. All ions are listed as their
nominal masses to the next lowest integer (e.g. m/z 1823.7 is listed as 1823 for the (M-H)
ion
corresponding to the wild type diphosphoryl hexaacyl lipid A
species).
of 1614 and 1534) that correspond to the
molecular ions for a di- and monophosphoryl pentaacyl lipid A species
missing one myristic acid moiety. Because there are two myristic acid
moieties (and four -hydroxymyristic acids) on the parent wild type
lipid A, it is not clear if these pentaacyl lipid A species originate
from a single specific -myristic acid deletion at one of two
possible attachment sites or as a mixture formed by deletion at both
sites. At lower masses, two additional molecular ions species are
observed at m/z 1403 and 1323 that correspond to a
mono- and diphosphoryl tetraacyl lipid A species lacking both myristic
acid moieties. One should note that it is often difficult to
distinguish between ions that are formed by LSIMS-generated
fragmentation of higher mass parent ions losing fatty acyl groups as
their corresponding ketenes from their isobaric molecular ion
counterparts present in the original lipid A mixture lacking these same
fatty acids. However, the presence of ions that are formed only through
fragmentation processes can be used to help differentiate between these
two possibilities. For example, the loss of myristic acid as the
neutral free acid from the intact lipid A species (i.e. -228 Da as HOOC(CH)CH
)
is characteristic of gas phase LSIMS fragmentation in the wild type
lipid A spectrum. Ions of this type can be seen as ion pairs at m/z 1595/1515 and m/z 1385/1305 in Fig. 7A. These ions are largely absent in the LSIMS
spectrum of the mutant B29 lipid A, supporting our interpretation that
the tetraacyl lipid A ions at m/z 1403 and 1323 are
formed primarily from molecular lipid A species (see Fig. 7B) and not as gas phase ketene losses of myristic
acid (i.e. -210 Da as
O=C=CH-(CH)CH
) from
the higher mass pentaacyl lipid A molecular ions.
)and that this heat sensitivity is lost upon passage
suggests that the temperature profile of htrB mutants may not
be directly associated with dephosphorylation of LOS. strains cannot be explained at this
time. Motif analysis of HtrB failed to indicate the presence of DNA
binding domains. It is interesting, however, that the htrB gene shares the promoter region with the rfaE gene coding
for ADP-heptose synthase involved in LOS biosynthesis and both of the
gene expressions are elevated in htrB
strains, suggesting that these two genes are transcriptionally related.
It is also tempting to speculate that HtrB may down-regulate these
genes (and others) and, thus, play an important role in the regulation
of LOS biosynthesis.
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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