(Received for publication, August 25, 1995; and in revised form, December 1, 1995)
From the
The lpcA locus has been identified in Escherichia
coli K12 novobiocin-supersensitive mutants that produce a short
lipopolysaccharide (LPS) core which lacks glyceromannoheptose and
terminal hexoses. We have characterized lpcA as a single gene
mapping around 5.3 min (246 kilobases) on the E. coli K12
chromosome and encoding a 22.6-kDa cytosolic protein. Recombinant
plasmids containing only lpcA restored a complete core LPS in
the E. coli strain 711. We show that this strain has an
IS5-mediated chromosomal deletion of 35 kilobases that
eliminates lpcA. The LpcA protein showed discrete similarities
with a family of aldose/ketose isomerases and other proteins of unknown
function. The isomerization of sedoheptulose 7-phosphate, into a
phosphosugar presumed to be D-glycero-D-mannoheptose
7-phosphate, was detected in enzyme reactions with cell extracts of E. coli lpcA
and of lpcA mutants
containing the recombinant lpcA gene. We concluded that LpcA
is the phosphoheptose isomerase used in the first step of
glyceromannoheptose synthesis. We also demonstrated that lpcA is conserved among enteric bacteria, all of which contain
glyceromannoheptose in the inner core LPS, indicating that LpcA is an
essential component in a conserved biosynthetic pathway of inner core
LPS.
LPS, ()an integral component of the outer membrane of
Gram-negative bacteria, consists of lipid A attached to a core
oligosaccharide, and in some microorganisms, contains an O-specific surface polysaccharide which is subsequently
attached to the terminal residues of the core(1, 2) .
The core oligosaccharide has an inner domain made of
3-deoxy-D-manno-octulosonic acid and L-glycero-D-mannoheptose, and an outer domain
composed of hexoses and N-acetylglucosamine. The structure of
the inner core is relatively highly conserved among enteric (3) and non-enteric bacteria(4) . Most of the genes
involved in the biosynthesis and assembly of the core oligosaccharide
are located within the rfa cluster, at about 81 min on the
chromosome in Escherichia coli K12 and 79 min in Salmonella enterica LT2(1) . However, the genes
involved in the early steps of the synthesis of L-glycero-D-mannoheptose are not located in the rfa region and they have not been characterized as yet.
LPS plays an important role in maintaining the structural integrity of the outer membrane by interacting with other components of the outer membrane and providing a physical barrier against the entry of deleterious compounds and some bacteriophages(3) . E. coli LPS mutants with defects in the inner core display a dramatic reduction in porin proteins (5) and are unable to grow in media containing detergents, bile salts, or hydrophobic antibiotics, all of which normally have a reduced permeability across the outer membrane and are toxic only in high concentrations(6) . Since these mutants lack an attachment site for the rest of the core oligosaccharide, they are resistant to LPS core-specific bacteriophages (6) and survive poorly within the host environment(7) .
Early work by Tamaki et al.(6) resulted in the isolation of mutations conferring supersensitivity to novobiocin which mapped to two different regions on the E. coli K12 chromosome: between ara and lac (1-10 min) next to the proAB genes, and between 55 and 60 min; they were designated as lpcA (LPS-core synthesis) and lpcB, respectively(6) . Similar mutations were also identified by Havekes et al.(8) as F plasmid conjugation-deficient mutants. The LPS of both lpcA and lpcB mutants lacks heptose(6) , suggesting these loci are involved in synthesis of the inner core domain, but since their original discovery, their precise function has not been established.
This study reports the molecular analysis of the lpcA locus, and the biochemical characterization of its gene product. We conclude that lpcA encodes a phosphoheptose isomerase used in the first step of the biosynthesis of the inner core LPS precursor, ADP-L-glycero-D-mannoheptose. We also demonstrate that lpcA is widely conserved among enteric bacteria, suggesting that its function is part of a conserved pathway for LPS biosynthesis.
The 14-kb EcoRI DNA fragment of pJB1 was labeled with DIG-11-dUTP using
a DIG DNA Labeling and Detection kit from Boehringer Mannheim.
Chromosomal DNAs from E. coli strains 711 and
705
were cleaved with EcoRI and electrophoresed in a 0.6% agarose
gel for 18-20 h at 15 mA. Southern blots were hybridized at 42
°C for 18-20 h and bands developed using a colorimetric
detection system as recommended by the manufacturer (Boehringer
Mannheim).
Direct evidence
of an altered LPS structure was obtained by a comparative analysis of
the LPS profiles of strains 711 and
705 (Fig. 1).
Strain
705 produces a core oligosaccharide identical to that of
the prototypic E. coli K12 strain Y10 (Fig. 1, lanes 1 and 4) whereas strain
711 produces a
much shorter core (Fig. 1, lane 2). LPS of E. coli strains D21e7, CS2051, and D31m4, containing different mutations
in rfa genes, were examined and compared with the
711
core. The LPS core of strains D21e7 and CS2051 was shorter than the
wild-type core but still longer than the
711 core (Fig. 1, lanes 5 and 6), whereas the LPS core of D31m4
migrated the same distance in the gel as the LPS core of
711 (Fig. 1, lanes 7 and 2). Since strain D31m4
produces a heptoseless LPS made of only
3-deoxy-D-manno-octulosonic acid and lipid A (32) we
conclude that
711 also lacks heptose in its core LPS.
Figure 1:
Silver-stained 16.4% (w/v) T (total
acrylamide), 1.9% (w/v) C (bisacrylamide) Tricine SDS-polyacrylamide
gel showing the LPS profiles of E. coli K12 strains. 1, 705; 2,
711; 3,
711(pJB2); 4, Y10; 5, D21e7 (rfa-1); 6, CS2051 (has a deletion eliminating rfaG, rfaP, rfaM, rfaN, and rfaB); 7, D31m4 (rfa-229, rfa-230).
Figure 2: Physical map of the lpcA region. Vector sequences are not shown. pE4021 is a cosmid clone containing chromosomal DNA from E. coli W3110, including the region from 4.9 to 5.8 min. RNHQ, PEPD, GPTA, PHOE, and PROAB indicate the location of sequenced genes. pJB1 contains a 14-kb EcoRI fragment cloned from pE4021. pJB2 and pJB8 contain a 3-kb BamHI fragment cloned from pJB1 into different vectors. pJB2-9 to pJB2-34 indicate the various deletions of pJB2 spanning the lpcA region. pJB15 indicates the DNA insert used for construction of DIG-labeled riboprobes. ORF1 and ORF2 are two open reading frames found on opposite strands of the DNA. The direction of transcription of lpcA is indicated by the arrow beneath ORF2. The complementation of the novobiocin supersensitivity phenotype by the deletion clones is indicated: R, successful complementation; S, unsuccessful complementation. Restriction enzymes indicated are: A, AvaII; B, BamHI; Bs, BstEII; E, EcoRI; Ev, EcoRV; Hc, HincII; P, PvuI.
Figure 3: Nucleotide sequence and deduced amino acid sequence of lpcA. The underlined sequence AGGA denotes the possible ribosomal binding site (rbs). The putative -10 consensus sequence is indicated by double underlining. The deletion end points of pJB2-10 and pJB2-25 (Fig. 2) are indicated by arrows followed by the numbers 10 and 25 in parentheses; the direction of the arrows indicates the sequence contained within pJB2-10 and pJB2-25, respectively. A putative transcription termination signal is indicated by arrows beneath the sequence downstream of the termination codon TAA. Lines above the sequences GCCG and CGGC indicate the complementary sequences forming the stem of the hairpin loop structure. Boxed sequences denote the location of the repetitive extragenic palindromic sequence.
The % G + C content of lpcA was 51%, similar to the reported values for % G + C content of the E. coli genome, and the codon usage was typical for E. coli genes. The sequence AGGA found 8 bp upstream of the AUG codon may correspond to the ribosomal binding site (Fig. 3). The sequence TATAAT located 146 bp upstream of the AUG codon has similarities with a -10 consensus sequence (Fig. 3). A repetitive extragenic palindromic sequence was noted 54 bp downstream of the termination codon of lpcA (Fig. 3). Inverted repeats with a potential for a hairpin secondary structure consistent with a transcriptional termination signal were also noticed between the repetitive extragenic palindromic sequence and the termination codon (Fig. 3).
DNA and deduced amino acid sequences of lpcA were compared to sequences from EMBL/GenBank. No homologies with known genes involved in LPS synthesis were noted. A region of the LpcA polypeptide spanning 45 amino acids showed significant similarities with a family of isomerases described as glutamine:fructose-6-phosphate amidotransferases, the lincomycin biosynthesis gene product LmbN from Streptomyces lincolnensis, and two other proteins, KpsF (E. coli) and Orfb (Clostridium perfringens) with no assigned functions (Table 1). The potential significance of these similarities is discussed below.
The expression of the lpcA gene product in vivo and in vitro identified a 22.6-kDa polypeptide as the LpcA protein (data not shown), which is in agreement with the predicted molecular mass of 20.6 kDa. A hydropathy profile of the LpcA protein (34) deduced from the nucleotide sequence of lpcA, did not reveal any significant regions of hydrophobicity compatible with membrane domains suggesting that LpcA is a cytosolic protein.
Figure 5:
Reversed-phase high performance liquid
chromatography analyses of carbohydrates synthesized by E. coli strains 711 and
711(pJB2) cell extracts following
incubation with 1.0 µmol of sedoheptulose 7-phosphate. Panel
A,
711 incubated 60 min. Panel B,
711(pJB2)
incubated 2 min. Panel C,
711(pJB2) incubated 60 min
without sedoheptulose 7-phosphate. Panel D,
711(pJB2)
boiled extract incubated 60 min. Large arrow indicates the
retention peak of the phosphorylated product. Small arrow indicates the retention peak of sedoheptulose
7-phosphate.
Figure 4:
A
schematic representation of the proposed events leading to the
chromosomal deletion of the lpcA locus in E. coli strain 711. Panel A, chromosomal map of E. coli K12 strain
705. RNHQ, LPCA, PROAB, IS30A, IS5A,
IS1B, and IS30 indicate the location of sequenced genes. Panel B, Southern blot showing chromosomal DNA profiles of E. coli strains
711 and
705 probed with a 14-kb EcoRI DIG-11-dUTP-labeled DNA probe. M,
HindIII molecular weight markers; 1,
711 DNA
digested with EcoRI; 2,
705 DNA digested with EcoRI; 3, pJB2 digested with BamHI; 4, pJB1 digested with EcoRI. Panel C,
restriction maps of pJB1 and pJB16 showing identical nucleotide
sequence (hatched box) and the IS5 element (open
box). Panel D, transposition of the IS5A insertion element from approximately 5.9 min to 5.2 min followed
by replication of the element, and chromosomal map of E. coli strain
711 showing the resulting deletion of the lpcA locus. Restriction endonucleases indicated are: E, EcoRI.
Glutamine:fructose-6-phosphate amidotransferase belongs to both
ketose/aldose isomerase and amidotransferase
groups(36, 37) . The amidotransferase reaction
requires residues located in the NH-terminal sequence of
these enzymes, notably an NH
-terminal Cys which is the
active residue for the glutamine amide transfer function(38) .
This region is conserved among the various
glutamine:fructose-6-phosphate amidotransferases investigated to
date(38) ; the absence of this region in LpcA suggests that
this protein is not an amidotransferase.
To test whether lpcA catalyzes an isomerization reaction, cell extracts of E. coli strain 711, E. coli strain
711(pJB2), and E. coli strain
711 (pJB18, pREP4) were used in an assay
containing sedoheptulose 7-phosphate. The reaction products were
examined by high performance liquid chromatography (HPLC) after
derivatization with aminobenzoic ethyl ester (ABEE) to facilitate their
detection with UV light. Chromatograms from reactions with cell
extracts of
711(pJB2) and
711(pJB18,pREP4) revealed the
appearance of a new peak with a retention time of 8.2 min (Fig. 5B and data not shown) after 2 min of incubation
with the enzyme. After 60 min incubation, the peak corresponding to
sedoheptulose 7-phosphate decreased considerably, 39 and 93% of initial
amount for
711(pJB2) and
711(pJB18,pREP4), respectively
(data not shown), suggesting that the substrate was consumed, whereas
the level of sedoheptulose 7-phosphate remained constant for 60 min
when incubated with boiled extract (Fig. 5D). Upon
incubation of
711 cell extract with sedoheptulose 7-phosphate (Fig. 5A), a peak with 8.2 min retention as found with
711(pJB2) and
711(pJB18,pREP4) was not apparent, although
other peaks with retention times ranging from 8.5 to 10.5 min were
observed. These extra peaks could be due to the conversion of
sedoheptulose 7-phosphate into fructose 6-phosphate and erythrose
4-phosphate by a transaldolase activity (39) in the extracts,
or into D-ribose 5-phosphate and D-xylulose
5-phosphate by a transketolase activity(39) , or the
conversions of triose phosphates and glucose 6-phosphate which are
present as contaminants in the sedoheptulose 7-phosphate preparation.
From this experiment we concluded that the 8.2-min peak corresponded to the reaction product, presumably D-glycero-D-mannoheptose 7-phosphate, however, this could not be verified directly since this phosphosugar is not commercially available. To prove that the product was a phosphorylated form of D-glycero-D-mannoheptose, reaction components were dephosphorylated by treatment with alkaline phosphatase prior to HPLC analysis. A peak with a retention time of 10.5 min corresponding to the retention time of authentic glyceromannoheptose was detected (Fig. 6). A peak with a retention time of 8.2 min corresponding to the reaction product in the absence of alkaline phosphatase was not detected in this experiment (Fig. 6, arrow). Therefore, we concluded that the product of the reaction in the presence of sedoheptulose 7-phosphate is a phosphorylated form of glyceromannoheptose. The two peaks in the glyceromannoheptose standard (Fig. 6) probably indicate the two anomeric forms of the sugar.
Figure 6:
Effect of alkaline phosphatase treatment
in the reaction products analyzed by reversed-phase high performance
liquid chromatography. Upper panel, HPLC profile of
711(pJB2) extract incubated with 1.0 µmol of sedoheptulose
7-phosphate (SED-7-P) and treated with alkaline phosphatase (4
units) prior to derivatization with ABEE. Arrow indicates the
location of the reaction peak of the reaction product in the absence of
alkaline phosphatase treatment. Lower panel, HPLC profile of
authentic glyceromannoheptose derivatized with ABEE. ABEE,
p-aminobenzoic ethyl ester; AP, alkaline phosphatase; GMH, glyceromannoheptose.
The HPLC analysis, however, did not allow us to determine if the
product seen in the HPLC in the absence of alkaline phosphatase
treatment is D-glycero-D-mannoheptose 7-phosphate or D-glycero-D-mannoheptose 1-phosphate. The latter is
the product of the second reaction in the biosynthetic pathway of
ADP-L-glycero-D-mannoheptose which is catalyzed by a
phosphomutase (Fig. 7). However, since the phosphomutase
reaction takes place after the formation of D-glycero-D-mannoheptose 7-phosphate and a peak
corresponding to a phosphorylated D-glycero-D-mannoheptose is not present in the
711 extract with sedoheptulose 7-phosphate (Fig. 5A) we conclude that it must correspond to D-glycero-D-mannoheptose 7-phosphate and the LpcA
protein must be the phosphoheptose isomerase.
Figure 7:
A schematic diagram of the biosynthetic
pathway of the nucleotide precursor
ADP-L-glycero-D-mannoheptose, following Eidels and
Osborn(28) , Coleman(5) , and
Sirisena et al.(47)
.
Sequencing and mapping of the cloned lpcA locus revealed a single gene, lpcA, that is located at 5.3 min (246 kb) on the chromosomal map of E. coli. This gene is physically unlinked to the majority of genes used in core biosynthesis that are found within the rfa cluster at 81 min on the chromosomal map. LpcA was expressed in vitro and in vivo as a protein of 22.6 kDa molecular mass. Results of preliminary fractionation experiments and the absence of characteristic features of membrane proteins suggested that LpcA is a soluble protein present in the cytoplasmic fraction.
The chromosomal deletion of the lpcA locus in E. coli strain 711 appeared to be the
result of an IS5-mediated DNA rearrangement. Since an IS5 element is not present in the 14-kb EcoRI fragment of the
parent strain
705 and
711 lacks proAB, we propose
a transposition of an IS5 insertion element originally located
in the vicinity of proAB within a region particularly rich in
IS sequences(40) . Recombination of the original IS5 insertion element and the newly replicated IS5 element
may have resulted in the removal of the looped out DNA (Fig. 4D). IS5-mediated rearrangements have
also been implicated in chromosomal DNA inversions in other E. coli K12 derivatives(41) . It is also interesting that some
mutations in other LPS genes have resulted from IS5 movements
within the bacterial chromosome(42, 43) .
In this
study, we provide genetic and biochemical evidence that lpcA is necessary for heptose biosynthesis of inner core
lipopolysaccharide in E. coli. Eidels and
Osborn(28, 44) , proposed a biosynthetic scheme for L-glycero-D-mannoheptose that uses the conversion of
sedoheptulose 7-phosphate into
ADP-L-glycero-D-mannoheptose. The four reactions
needed for the synthesis of
ADP-L-glycero-D-mannoheptose shown in Fig. 7,
include: 1) conversion of sedoheptulose 7-phosphate to D-glycero-D-mannoheptose 7-phosphate by a
phosphoheptose isomerase, 2) conversion of D-glycero-D-mannoheptose 7-phosphate to D-glycero-D-mannoheptose 1-phosphate by a
phosphoheptose mutase, 3) conversion of D-glycero-D-mannoheptose 1-phosphate with ATP to
ADP-D-glycero-D-mannoheptose and PP by an
ADP-heptose synthase, and 4) racemization by an epimerase of
ADP-D-glycero-D-mannoheptose to the L-isomer. The completed
ADP-L-glycero-D-mannoheptose is then used for the
transfer of its sugar moiety onto the inner core LPS by a specific
transferase. The only genes of this pathway fully characterized to date
are rfaD and rfaC, encoding the epimerase (45) and the transferase(46, 47) ,
respectively. Work by Sirisena et al.(47) in Salmonella typhimurium suggests that rfaE encodes the
ADP-heptose synthase since addition of ADP-glyceromannoheptose to cell
extracts of rfaE mutants restores the synthesis of a complete
core LPS, but a similar gene in E. coli has not been
identified.
Our data demonstrate that lpcA restores the
expression of a complete core LPS by the heptoseless mutant, E.
coli strain 711, and encodes the phosphoheptose isomerase
used in the biosynthesis of
ADP-L-glycero-D-mannoheptose. Biochemical evidence
for a phosphoheptose isomerase includes: 1) appearance of a new product
upon incubation with sedoheptulose 7-phosphate with a concomitant
reduction of substrate concentration, 2) the new product is a
phosphorylated sugar, 3) upon dephosphorylation, the product has the
same HPLC retention time as authentic glyceromannoheptose, 4) the new
product does not appear in reactions with cell extracts of the lpcA mutant. In addition, a region of the LpcA protein has amino acid
sequence homology with a family of aldo/keto isomerases.
Sedoheptulose 7-phosphate has been isolated from plants such as Sedum spectabile(48) , and from animal tissues such as rat liver (49) and chicken muscle(50) , as an intermediate in the nonoxidative portion of the pentose phosphate pathway(39) . To date there are no reports of an isomerase in plants or animals that uses sedoheptulose 7-phosphate as a substrate. Thus, its conversion into D-glycero-D-mannoheptose 7-phosphate could be interpreted as a specialized branch of the pentose phosphate pathway for LPS synthesis. This branch is likely to be present in many if not all Gram-negative bacteria.
The degree of
similarity of LpcA with the lincomycin biosynthetic gene product LmbN
of S. lincolnensis, suggests that lmbN may
encode an isomerase needed for the synthesis of this antibiotic.
Chemical synthesis of 8-carbon sugar derivatives as potential
intermediates leading to the production of methyl
6-amino-6,8-dideoxy-1-thi-D-erythro--D-galacto-octopyranoside,
the carbohydrate moiety of lincomycin, appears to require an
isomerization step(51) .
The fact that glyceromannoheptose is a very common component of the inner core LPS of many enteric (52) and non-enteric bacteria (4) and the conservation of lpcA among enteric bacteria suggests that this gene has an essential function in a conserved pathway for ADP-L-glycero-D-mannoheptose synthesis. Although the lpcA homolog was not identified in Pseudomonas, we believe that this function does exist in this genus but lack of hybridization with the probe reflects the high hybridization stringency used in this experiment.
In general, genes necessary for synthesis of the conserved lipid A and inner core components are found scattered in the E. coli K12 chromosome: lipid A synthesis genes (lpxA, lpxB, and lpxD) are found at 4 min, 3-deoxy-D-manno-octulosonic acid pathway genes (kdsA and kdsB) are found at 27 and 85 min, respectively. From the heptose pathway, only rfaD and rfaC are located at one end of the rfa cluster at 81 min (4) next to the other genes for synthesis of the structurally more variable outer core components whereas the lpcA gene is located outside of the rfa cluster. The G + C content of lpcA is similar to the G + C content of the lipid A and 3-deoxy-D-manno-octulosonic acid pathway genes, rfaC and rfaD genes, and is close to the average G + C content for E. coli and other enteric bacteria. This conservation of G + C supports the suggestion that biosynthesis genes required for lipid A and inner core may have been part of a common enterobacterial genome, and that outer core biosynthesis genes, which have a lower G + C content, evolved later(4) .
This study reports the first molecular characterization of a novel phosphoheptose isomerase in prokaryotes that uses sedoheptulose 7-phosphate as a substrate, and it is needed for the first reaction committed to the biosynthesis and assembly of inner core lipopolysaccharide in enteric bacteria. Our findings have relevance to the area of infection since bacterial strains with defects in core LPS are more susceptible to the killing effect of serum complement and phagocytosis(7) . The molecular details of the enzyme-substrate activity, currently being assessed in our laboratory, will provide further information about the use of sedoheptulose 7-phosphate as an intermediary component of the pentose phosphate pathway, and will lead to the design of enzyme inhibitors which may serve as novel antimicrobial agents.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U32590[GenBank].