(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
-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.
Lipopolysaccharide (LPS) ()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.
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 CHCl
/CH
OH (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.
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 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.
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 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).
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
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.
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
HepKDO-lipid A is present.
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 and
H
PO
, 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).
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 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.
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) ()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.
The increased
expression of htrB and rfaE in the htrB 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.
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].