Full text of DOI:10.1093/glycob/11.7.533

Glycobiology vol. 11 no. 7 pp. 533–539, 2001
Redirection of sialic acid metabolism in genetically engineered Escherichia coli
Michael Ringenberg, Carol Lichtensteiger, and Eric Vimr1
exact understanding of how carbohydrates modulate these
processes is frequently unavailable, the subversion of animal
glycoconjugates by microorganisms for the purposes of
adhesion, invasion, and propagation is a central feature of most
host–pathogen interactions (Karlsson, 1998). Thus, ability to
block these interactions with carbohydrate-based therapeutics
would provide new avenues to the treatment or prevention of
infectious diseases. Blocking carbohydrate-based recognition
events may also be useful for treating a variety of apparently
noninfectious diseases requiring cell–cell or cell–molecule
interactions for their progression. Although the chemo-
enzymatic syntheses of oligosaccharides offers an attractive
route to the production of therapeutic blocking reagents (for
example, see Gilbert et al., 1998), the commercial scarcity of
required glycosyltransferases and expense of precursors
impedes progress in the design or large-scale production of
certain oligosaccharides.
The difficulty in producing some oligosaccharide precursors is
especially evident with members of the family of nonulosonates
known as the sialic acids. These unique N- or O-substituted
derivatives of N-acetylneuraminic acid (Neu5Ac) are ubiquitous
in animals of the deuterostome lineage (starfish to humans) but
do not seem to be synthesized by most plants, protostomia,
protists, Archaea, or eubacteria (Warren, 1994). Despite the
limited distribution of sialates in nature, a few (mostly patho-
genic) bacterial strains synthesize Neu5Ac de novo and
incorporate it into cell surface glycolipids (Vimr et al., 1995).
Of medical importance is Escherichia coli K1, the leading
cause of Gram-negative neonatal meningitis in humans, in
addition to a frequent etiologic agent of childhood and adult
urinary tract infections and septicemia (Silver and Vimr,
1990). The polysialic acid (PSA) capsule of E. coli K1 inhibits
host innate defense mechanisms and provides molecular
mimicry of the PSA chains on the neural cell adhesion
molecule (NCAM). The discovery of PSA as both virulence
factor and regulator of central nervous system development
prompted extensive investigations into sialate biosynthesis.
In the mammalian liver, Neu5Ac is synthesized and
activated for transfer to glycoconjugate acceptors (R-OH) in
five sequential steps involving phosphorylated intermediates
(Warren, 1994), beginning with UDP-N-acetylglucosamine
(UDP-GlcNAc):
University of Illinois at Urbana-Champaign, Department of Pathobiology,
College of Veterinary Medicine, University of Illinois at Urbana-Champaign,
2522 VMBSB, 2001 South Lincoln Avenue, Urbana, IL 61802, USA
Received on November 15, 2000; revised on February 14, 2001; accepted on
March 2, 2001
Most microorganisms do not produce sialic acid (sialate),
and those that do appear to use a biosynthetic mechanism
distinct from mammals. Genetic hybrids of nonpathogenic,
sialate-negative laboratory Escherichia coli K-12 strains
designed for the de novo synthesis of the polysialic acid
capsule from E. coli K1 proved useful in elucidating the
genetics and biochemistry of capsule biosynthesis. In this
article we propose a dynamic model of sialometabolism to
investigate the effects of biosynthetic neu (N-acetyl-
neuraminic acid) and catabolic nan (N-acylneuraminate)
mutations on the flux of intermediates through the sialate
synthetic pathway. Intracellular sialate concentrations
were determined by high pH anion exchange chromatog-
raphy with pulsed amperometric detection. The results
indicated that a strain carrying a null defect in the gene
encoding polysialyltransferase (neuS) accumulated > 50 times
more CMP–sialic acid than the wild type when strains were
grown in a minimal medium supplemented with glucose and
casamino acids. Metabolic accumulation of CMP–sialic acid
depended on a functional sialic acid synthase (neuB), as
shown by the inability of a strain lacking this enzyme to
accumulate a detectable endogenous sialate pool. The neuB
mutant concentrated trace sialate from the medium,
indicating its potential value for quantitative analysis of
free sialic acids in complex biological samples. The function
of the sialate aldolase (encoded by nanA) in limiting inter-
mediate flux through the synthetic pathway was deter-
mined by analyzing free sialate accumulation in neuA
(CMP–sialic acid synthetase) nanA double mutants. The
combined results demonstrate how E. coli avoids a futile
cycle in which biosynthetic sialate induces the system for its
own degradation and indicate the feasibility of generating
sialooligosaccharide precursors through targeted manipu-
lation of sialate metabolism.
Key words: sialic acid/metabolic engineering/Escherichia coli
K1/pathway analysis/carbohydrate flux
Introduction
The oligosaccharide chains on glycoconjugates play important
roles in cell adhesion and signaling events, and, though an
The specificity of sialyltransferases for a given acceptor (step 6)
dictates the pattern of sialoglycoconjugates (Neu5Ac-O-R)
synthesized by various cells. The bifunctional enzyme, UDP-
GlcNAc 2-epimerase/N-acetylmannosamine (ManNAc) kinase
1To whom correspondence should be addressed
© 2001 Oxford University Press
533

M. Ringenberg et al.
(Stasche et al., 1997), catalyzes the first two steps of the
pathway, yielding ManNAc-6-phosphate (step 2). Production of
CMP-Neu5Ac in step 5 is tightly regulated through allostery, with
increasing CMP-Neu5Ac concentrations inhibiting the epimerase
that produces ManNAc in step 1 (Kornfeld et al., 1964). In step
3, ManNAc-6-P is condensed with phosphoenolpyruvate (PEP) to
yield Neu5Ac-9-P, with subsequent dephosphorylation by
specific or nonspecific phosphatase(s) in step 4 yielding free
Neu5Ac. Alternative mechanisms for synthesizing Neu5Ac
may involve the direct epimerization of free GlcNAc to
ManNAc (Maru et al., 1996), Neu5Ac recycling from surface
glycoconjugates through
a
potential salvage pathway
involving turnover by lysosomal sialidase and the direct
enzymatic condensation of ManNAc with pyruvate by Neu5Ac
aldolase (Rodriguez-Aparico et al., 1995).
Irrespective of the exact mechanism(s) used by different
eukarya to synthesize sialic acids, synthesis of the activated
sialyl donor, CMP-Neu5Ac, by E. coli K1 does not appear to
be tightly regulated by allosteric inhibition (Steenbergen and
Vimr, 1990; Vimr and Troy, 1985b). Because E. coli lacks
CMP-Neu5Ac hydrolase (Masson and Holbein, 1983), accu-
mulation of CMP-Neu5Ac in a polymerase-defective genetic
background is a physiological dead-end. From a commercial
viewpoint, exploitation of this metabolic blockade for targeted
overproduction of free Neu5Ac or CMP-Neu5Ac offers alter-
natives to the chemoenzymatic syntheses of these compounds.
A solely fermentative source of CMP-Neu5Ac would obviate
the need for expensive starting materials, such as Neu5Ac, PEP,
nucleoside phosphates, or recycling enzymes currently required
for efficient in vitro syntheses of sialooligosaccharides. Our
current results contribute to the understanding of regulation of
sialic acid metabolism by the microbial metabolic engineering
of intermediate flux through the synthetic pathway.
Fig. 1. Proposed model of sialate synthesis and degradation in Escherichia coli.
Intracellular reactions are shown within the rectangle representing a hybrid
E. coli K1 cell. Broken arrows indicate that synthesis of LPS and peptidoglycan
ultimately depends on the availability of UDP-GlcNAc. See text for description
of enzymes or reactants.
by the bacterial synthase to produce free Neu5Ac. These differ-
ences have important implications for designed redirection of
sialometabolism in bacteria.
Effect of nan or neu genotype on sialate overproduction
To better understand how mutations in nan or neu influence
sialate synthesis, intracellular Neu5Ac was determined by high
pH anion exchange chromatography equipped with pulsed
amperometric detection (HPAEC/PAD) after hydrolysis to
convert CMP-Neu5Ac that accumulated intracellularly to the
free sugar. Figure 2 shows that the wild-type strain, EV36, did
not accumulate a detectable sialate pool, estimating the limits
of sialate detection at approximately 0.1 nmol (Table II).
Because EV36 synthesizes PSA and expresses K1 capsule
(Figure 1), we expected that the intermediary metabolites
Neu5Ac and CMP-Neu5Ac would be rapidly converted to
polysialate end product and hence not detected by our assay
(Figure 2). In contrast, sialate accumulation was dramatically
increased in strains carrying a null mutation in neuS (strain
EV136 in Figure 2).
Compared to the virtually undetectable Neu5Ac concentration
in the wild type (Figure 2), polysialyltransferase-deficient
mutant EV136 produced about 30 times more sialate than
EV36 when the strains were grown in Luria Bertani (LB)
medium (Figure 2 and Table II), results consistent with our
previous observations (Steenbergen and Vimr, 1990; Vimr and
Troy, 1985b). The two peaks eluting prior to Neu5Ac were
independent of medium composition or a given strain’s ability
to synthesize Neu5Ac or PSA (compare Figure 2 with Figure 3);
these peaks were not investigated further. When a mutation in
nanA was simultaneously present in the neuS background, as in
strain EV240, there was little increase in sialate concentration
(Table II). Four independent repetitions of this comparison
Results
Bacterial sialometabolism
Recent advances in understanding sialate and PSA synthesis
and degradation (Vimr et al., 1995; Plumbridge and Vimr,
1999), and the results presented in this communication support
our metabolic network model in Figure 1. Although the poten-
tial reversibility of most of the individual reactions has not
been formally investigated, genetic and biochemical analyses
of Neu5Ac flux in mutant and wild-type strains indicate that
the enzymes of the catabolic pathway (nan gene products) do
not function biosynthetically (neu gene products) under
balanced growth conditions (Vimr and Troy, 1985b). This
conclusion is challenged by the suggestion that sialate aldolase
(encoded by nanA) is the sole biosynthetic source of free
Neu5Ac in E. coli and other Gram-negative bacteria
(Rodriguez-Aparico et al., 1995; Ferrero et al., 1996; Barrallo
et al., 1999).
Table I lists the bacterial sialate synthetic and catabolic gene
products depicted in Figure 1 compared with known or
suspected mammalian homologs. This comparison highlights
two likely mechanistic differences between the bacterial and
mammalian modes of Neu5Ac synthesis: (1) organization of the
bacterial genes for UDP-GlcNAc epimerase and putative ManNAc
kinase into distinct synthetic and catabolic operons, respectively,
and (2) use of free ManNAc rather than its 6-phosphate derivative
534

Targeted overproduction of sialic acids
Table I. Known or proposed functions of neu and nan gene products in Escherichia coli and comparison with their mammalian homologs or functional
equivalents
Protein
NanA
NanT
NanE
NanK
NanR
NeuA
NeuB
NeuC
NeuS
Function in E. coli
Sialate aldolase
Homologa (E value)b
Accession no.
NAf
Reference
Fc
Vimr and Troy, 1985a
Martinez et al., 1995
Plumbridge and Vimr, 1999
Stasche et al., 1997
Sialate permease
F
NA
ManNAc-6-P epimerase
ManNAc kinase
None
Hd (0.08)
None
NA
NP005467
NA
Sialate repressor
Plumbridge and Vimr, 1999
Munster et al., 1998
Lawrence et al., 2000
Stasche et al., 1997
CMP-sialate synthase
Sialate synthase
H (4e–14
H (8e–50
H (2e–20
FSe
)
)
)
CAA06915
AAF75261
NP005467
NA
UDP-GlcNAc epimerase
Polysialyltransferase
Steenbergen and Vimr, 1990
aOnly mammalian homologs are indicated.
bE values indicate the likelihood that a match is not due to chance.
cF, functional counterpart of the indicated E. coli protein; no sequence available and thus gene product may or may not be homologous.
dH, homologous with its E. coli counterpart.
eFS, sequenced gene encoding a protein with the same function as its E. coli counterpart, but not homologous.
fNA, not applicable
Table II. Effects of growth medium and genotype on sialate pool size in
genetically engineered E. coli
Experiment Strain
Relevant
genotype
Medium
Neu5Ac
(nmols/
A600/ml)
1
EV36
Wild type
neuS
LB
NDb
4.5
EV136
EV239
EV240
EV136
EV136
EV136
EV136
EV239
EV136
EV136
EV136
EV240
EV240
LB
nanA neuB neuS
nanA neuS
neuS
LB
0.5
LB
4.4
Ba
7.8
neuS
B+Glc
B+CMP
17.8
8.1
neuS
neuS
B+Glc+CMP 13.0
Fig. 2. Accumulation of intracellular sialate in wild-type or mutant E. coli
strains. The indicated strains were grown in LB prior to analysis by
HPAEC/PAD.
nanA neuB neuS
neuS
B+Glc
ND
8.8
2
B+GlcNAc
B+Fru
neuS
9.7c
8.8
neuS
B+Gro
B+Fru
indicated no difference (P > 0.05) in sialate concentration
between EV136 and EV240. This result suggests that aldolase
(NanA) does not normally compete with CMP-Neu5Ac
synthetase (NeuA) for the common substrate, Neu5Ac. That
most of the sialate detected in extracts of the strains examined
in Table II is in the form of CMP-Neu5Ac is confirmed below.
nanA neuS
nanA neuS
22.4c
10.9
B+Gro
aB, basal medium, composed of M63 salts with 1% (w/v) casamino acids
(CAA) and, where indicated, 0.2% (w/v) sugar or nucleotide supplements.
bND, not detectable above the minimal detection limit of 0.1 nmol.
cThe difference between these strains was not significant (see text).
Effect of medium composition on sialate overproduction
LB is an unbuffered medium composed of peptides, amino
acids, vitamins or other cofactors, and trace amounts of carbo-
hydrates, including sialic acid (Steenbergen et al., 1992; Vimr,
1992). We wished to test whether sialate production could be
optimized by simple alterations to a defined minimal growth
medium. As shown in Table II, basal medium supported the
overproduction of Neu5Ac by EV136 to an amount comparable
with that of this strain on LB. Sialate was not dramatically
increased by supplementing the basal medium with CMP
(Table II). In contrast, supplementing basal medium with 0.2%
glucose (Glc) resulted in a nearly fourfold increase in Neu5Ac
relative to EV136 grown in LB (Table II). The apparent stimu-
latory effect of Glc could have resulted from the presence of
this preferred carbon source in the medium, thus augmenting
UDP-GlcNAc pool size for synthesis of essential lipopoly-
saccharide (LPS) and peptidoglycan cell wall components
535

M. Ringenberg et al.
Mechanism of sialate overproduction
To determine if the overproduction of Neu5Ac in neuS mutants
is dependent on a functionally intact biosynthetic system
(Figure 1), we analyzed the sialate pool in the triple mutant
EV239. This mutant has defects in NanA, NeuS, and sialate
synthase (NeuB), suggesting either that the Neu5Ac peak
detected in this strain (Figure 2) arose from an alternate
synthetic pathway or, less likely, a compound unrelated to
Neu5Ac eluted at the same time as authentic Neu5Ac. The first
possibility was considered more likely for two reasons. First,
EV36 extracts contained no detectable sialate (Figure 2),
suggesting that the peak in EV239 was actually Neu5Ac.
Second, it was possible we were observing a phenotype of the
synthase mutant that was being magnified by the simultaneous
polysialyltransferase and aldolase deficiencies in EV239.
Others (Rodriguez-Aparico et al., 1995) concluded that E. coli
K1 relies on NanA to synthesize Neu5Ac by catalyzing the
aldol condensation of pyruvate with ManNAc, suggesting that
the NeuB-independent peak detected in extracts of EV239 may
have arisen from residual nanA expression. However, when
EV239 was grown in defined medium, Neu5Ac was no longer
detected (Figure 3 and Table II). Because LB contains trace
sialate (Steenbergen et al., 1992), we concluded that the peak
detected in EV239 grown on LB (Figure 2) resulted from
intracellular concentration of exogenous Neu5Ac by the sialic
acid–specific permease, NanT (Figure 1).
Fig. 3. Accumulation of intracellular sialate requires NeuB. The indicated
strains were grown in basal salts medium supplemented with Glc and CAA.
Note the absence of a sialate peak from the neuB strain, EV239, compared to
the peak in the EV136 extract.
(Figure 1). Alternatively, or in addition to the effect of carbon
source on cell wall–precursor synthesis, Glc induces catabolite
repression of the nan operon (Vimr and Troy, 1985a). This
response would be expected to reduce expression of nanA and
nanK potentially shunting metabolite flux toward the synthetic
pathway (Figure 1). In an attempt to distinguish between these
possibilities, EV136 was grown in medium B supplemented
with fructose (Fru), GlcNAc, or glycerol (Gro).
NanA limits the accumulation of free Neu5Ac
Assuming our proposed model of sialate biosynthesis in Figure 1
is correct, it should be possible to overproduce free Neu5Ac by
truncating the biosynthetic pathway at neuA. Although the
absence of NanA appeared to have little effect on increasing
the sialate pools in neuS strains (Table II), a defective neuA
gene should allow increased sialate accumulation until full
induction of nanA is reached. The metabolic consequence of
this phenotype (aldolase induction) is predicted to limit the
endogenous sialate pool size. As shown in Figure 4, NanR
negatively regulates the nan operon by acting as a repressor
that is inactivated on binding free Neu5Ac (Plumbridge and
Vimr, 1999). Therefore, on full induction of the nan operon,
NanA would convert the available sialate pool to ManNAc
and, ultimately, back to Fru-6-P (Figure 1). However, in a
nanA neuA double mutant, active aldolase would not exist and
the sialate pool should be stabilized.
To determine the role of nanA in regulating sialate flux, we
created isogenic nanA neuA and nanA+ neuA strains by trans-
ducing the nanA4 allele from EV52 into the neuA recipient,
EV5. As shown in Figure 5, nanA transductants (numbers 1
and 6) accumulated significantly more sialate (P < 0.0001)
than EV5 or its nanA+ neuA transductant (number 12). The
increased sialate pool sizes of the doubly mutated transduct-
ants over those of EV5 or transductant 12 confirm that NanA
regulates the biosynthetic pathway when free Neu5Ac concen-
trations are permitted to rise. The KD (unknown) for Neu5Ac of
NanR presumably defines the regulatory signal, above which
point full induction of the nan operon is achieved. These
results also confirm that the sialate pools detected in the
different neuS mutants described above are in the form of
CMP-Neu5Ac, consistent with direct chemical and functional
analyses showing that this nucleotide sugar accumulates in
As indicated in Figure 1, sugars such as GlcNAc, ManNAc, or
Fru (not shown) are transported as phosphorylated derivatives by
the phosphotransferase uptake system (PTS). Gro (not shown)
enters cells by a facilitated diffusion process and therefore
would not influence PTS-regulated operons. The modest
increases in sialate pool size of EV136 or EV240, when grown in
basal medium containing GlcNAc or Fru, but not Gro (Table II),
suggest that catabolite repression is not critical for sialate over-
production. Furthermore, there was no difference (P > 0.05)
between sialate pools from cells grown with Fru or Glc (data
not shown), suggesting that the type of PTS sugar was
unimportant to sialate overproduction. We infer from these
results that the effect of medium composition on E. coli sialate
pool size is largely a function of final cell density, which for
basal medium supplemented with Glc or Fru is two to three
times greater than that of LB. Therefore, when the carbon
supply is plentiful, there appears to be adequate UDP-GlcNAc
synthesized to support the essential production of peptido-
glycan and LPS while simultaneously allowing overproduction
of sialate (Table II). We have not systematically investigated
whether sialate pool size is affected by leakage or efflux mech-
anisms; however, previous results suggest such losses are
probably minor (Vimr and Troy, 1985b). We conclude that
fermentation in a defined medium produces approximately
20 nmols of CMP-Neu5Ac per A600 per ml. Because growth
yields of 30 absorbance units are routinely attainable (Hoffman
et al., 1995), gram quantities of CMP-Neu5Ac should be
readily available through simple fermentation of appropriate
bacterial strains in defined or complex media.
536

Targeted overproduction of sialic acids
Fig. 4. Regulation of nanA expression in E. coli. Open arrows indicate the genes comprising the nan operon (nanATEKyhcH), and the upstream gene encoding this
operon’s repressor, NanR. Bent arrows indicate putative transcriptional start sites, with the nanA promoter negatively regulated by NanR (circle with internal
negative sign). Endogenously produced Neu5Ac (curved arrows) is the precursor of PSA and functions as the probable inducer of the nan operon by converting
NanR to an inactive state (rectangle). Broken arrows indicate that more than one step is required to synthesize ManNAc or PSA, as shown in Figure 1.
(undefined) growth medium and the relatively insensitive and
nonspecific thiobarbituric acid assay to quantify intracellular
sialate pools. Using HPAEC/PAD under conditions established
for anionic carbohydrate analysis, the current results both
confirm and extend our previous studies, demonstrating that
approximately 20 nmols of CMP-Neu5Ac are obtained per unit
of A600 per ml by fermentation of the appropriate strain in
defined medium. Further increases in yields of targeted
compounds may be achieved by overproducing CTP
synthetase (encoded by pyrG), if the pool size of this nucleo-
tide is limiting for CMP-Neu5Ac synthesis. Alternatively,
eliminating NanK (as demonstrated for NanA in this study) is
also likely to increase flux of sialate intermediates through the
synthetic pathway. Therefore, creating mutants simultaneously
defective in nanA and nanK is predicted to complete the sepa-
ration of sialic acid degradation from biosynthesis, potentially
Fig. 5. Effect of sialic acid aldolase on Neu5Ac accumulation in neuA mutants.
Cells were grown in basal salts medium supplemented with Glc and CAA prior
to analyzing extracts for free Neu5Ac. Results are the concentrations of
maximizing the yield of CMP-Neu5Ac obtainable from a
nanAK neuS triple mutant.
Current methods for the chemoenzymatic synthesis of
sialooligosaccharides require specific precursors and enzymes
to generate CMP-Neu5Ac (Liu et al., 1992; Gilbert et al.,
1998). Fermentation has obvious advantages of simplicity and
nominal cost in comparison to traditional organic synthetic
methods and potentially obviates the need for CMP-Neu5Ac
regeneration in vitro. More important, by expressing the
appropriate sialyltransferase in tandem with CMP-Neu5Ac over-
production, it should be possible to engineer E. coli that synthesize
virtually any sialylated oligosaccharide by coexpression of the
relevant acceptor. The large number of completely sequenced
microbial genomes and availability of E. coli with modified
sialometabolic pathways should greatly facilitate engineering
sialooligosaccharide synthesis in bacteria.
Neu5Ac normalized per unit of A600 the standard deviation of three
independent measurements of each strain.
NeuS-deficient strains (Vimr and Troy, 1985b; Steenbergen
and Vimr, 1990). When taken together, our results demonstrate
the feasibility of fermentation for targeted biosynthesis of
sialooligosaccharide precursors and suggest additional
approaches for metabolic engineering of sialate pathways.
Discussion
Metabolic engineering of sialate metabolism
The ability to overproduce CMP-Neu5Ac is necessary for
metabolic engineering of sialooligosaccharide biosynthesis in
E. coli. Although we had previously shown that E. coli neuS
strains overproduce CMP-Neu5Ac (Vimr and Troy, 1985b;
Steenbergen and Vimr, 1990), these studies used complex
Mechanism of sialic acid biosynthesis
Our current results demonstrate the obligatory requirement of
NeuB for sialate biosynthesis in E. coli. Others failed to detect
this activity in vitro, suggesting that NanA instead of NeuB
537

M. Ringenberg et al.
was the sole biosynthetic source of sialate in E. coli (Rodriguez-
Aparico et al., 1995; Ferrero et al., 1996). Although earlier
studies contradicted this suggestion (Vimr and Troy, 1985b;
Steenbergen et al., 1992; Vimr, 1992), our current results
(Figure 3) allow unambiguous rejection of the hypothesis that
NanA normally functions biosynthetically in E. coli. This
conclusion, together with direct biochemical support (Vann et
al., 1997), demonstrates the versatility and power of
experimentally coupling the relevant physiology of amino
sugar metabolism to systematic genetic and biochemical
analyses of sialic acid synthesis.
cells were shown to synthesize Neu5Ac when provided with an
exogenous source of ManNAc, but did not produce CMP-
Neu5Ac. By capitalizing on the ability of genetically engi-
neered E. coli to synthesize PSA, we have shown that simply
growing the appropriate nonpathogenic strain in defined
medium attains overproduction of sialoglycoconjugate
precursor synthesis. It should be possible to further engineer
this system for synthesis of more complex sialosides.
Materials and methods
In mammals, the bifunctional UDP-GlcNAc 2-epimerase/
ManNAc kinase is likely the pivotal biosynthetic enzyme for
synthesis of ManNAc-6-P in vivo. It is noteworthy that in
E. coli, the putative ManNAc kinase (NanK) is genetically and
functionally separate from the UDP-GlcNAc epimerase
(NeuC). NanK would thus appear to function exclusively in
sialic acid degradation as a product of the nan operon, whereas
its mammalian counterpart appears to be a purely biosynthetic
enzyme. The low yet potentially significant similarity between
the bacterial and mammalian ManNAc kinases (Table I) may
support an ancient origin of sialate metabolic enzymes.
However, because sialate biosynthesis is a sporadic trait in
microbes, the possibility that biosynthesis of this sugar
evolved late remains tenable. Entertaining the speculation that
microbes may have acquired some of their biosynthetic and
catabolic machinery from eukaryotes (Hoyer et al., 1992), it
will be interesting to compare whether select unicellular
eukaryotes such as Candida albicans, which reportedly
synthesizes sialic acid (Soares et al., 2000), utilizes the
prokaryotic or mammalian synthetic mechanism.
Bacterial strains and growth conditions
The bacterial strains used in this study are described in
Table III. Transductants 1, 6, and 12 were constructed by
infecting strain EV5 with bacteriophage P1vir grown on strain
EV52, with selection for a tetracycline resistance element (zgj-
791::Tn10) linked to the nanA4 mutation (Table III). Drug-
resistant transductants were screened for aldolase phenotype
by assessing toxicity to exogenous sialic acid as described
(Vimr and Troy, 1985a).
Bacteria were routinely propagated in LB broth purchased
from Fisher Chemical Co. Defined media were composed of
M63 salts (Miller, 1972) supplemented with 1% CAA from
Sigma and sugars or nucleotides at 0.2% final concentration.
Cultures were grown aerobically in a rotary water bath at
200 r.p.m.. Bacteria were harvested by centrifugation when
cultures reached early stationary phase.
Sample preparation
In Neisseria meningitidis group B, the ortholog of neuC (siaA)
is reported to be a GlcNAc-6-P to ManNAc-6-P epimerase
(Petersen et al., 2000). Though biosynthesis of Neu5Ac in
meningococci (as well as in E. coli) appears to require unphos-
phorylated ManNAc as the hexosamine sialate precursor
(Figure 1), it has been postulated that N. meningitidis uses
specific or nonspecific phosphatase(s) to produce ManNAc
(Petersen et al., 2000). Despite the homology between neuC
and siaA, neuC and its mammalian homolog use UDP-GlcNAc as
ManNAc precursor, whereas SiaA may recognize GlcNAc-6-P.
However, GlmS produces GlcN-6-P instead of GlcNAc-6-P,
with GlcN-6-P subsequently isomerized by GlmM and
converted to UDP-GlcNAc via the bifunctional GlmU (Figure 1).
This microbial pathway for the production of UDP-GlcNAc
implies that there is little intracellular GlcNAc-6-P in the
absence of an exogenous supply of free GlcNAc. If the
proposed function of SiaA is correct, there must be an alternative
source of GlcNAc-6-P in N. meningitidis. Pending purification
of NanK, it should become straightforward to chemoenzy-
matically synthesize radiolabeled ManNAc-6-P for detection
of the predicted specific or nonspecific phosphatase(s) in
N. meningitidis or other organisms.
After harvesting cells (2.5–5.0 ml) by centrifugation, culture
supernatants were discarded and the tube sides wiped to
remove as much residual medium as possible. Water was
added to give a tenfold concentration of the starting culture,
and the A600 of samples determined spectrophotometrically
after appropriate dilution. With our instrument (Beckman DU
640), an A600 of 1.0 corresponds to 6.25 × 108 cells/ml.
Cultures were frozen at –20°C, thawed, and then sonicated
briefly to disrupt bacteria. After cell disruption, TCA was
added to 10% final concentration and samples chilled on ice
for 1 h. The copious precipitate that formed after incubation
was removed by centrifugation; the supernatant was dried
under vacuum in a Savant SpeedVac.
Table III. Bacterial strains used in this study
Strain
EV5
Relevant genotype
neuA22
Reference or source
Vimr et al., 1989
EV36
EV52
EV136
EV239
EV240
Wild type
Vimr and Troy, 1985a
Vimr and Troy, 1985a
Steenbergen and Vimr, 1990
Steenbergen et al., 1992
nanA4 zgj-791::Tn10
neuS::Tn10
Conclusion
nanA4 neuS::Tn10
We have quantified the overproduction of free Neu5Ac and
CMP-Neu5Ac under defined growth conditions and shown
how sialate aldolase diverts the flux of intermediates away
from the synthetic pathway. Recently, Betenbaugh and his
associates cloned and expressed the mammalian equivalent of
neuB in insect cells (Lawrence et al., 2000). The transformed
nanA4 neuB25 neuS::Tn10 Steenbergen et al., 1992
Transductant 1 nanA4 neuA22
Transductant 6 nanA4 neuA22
Transductant 12 neuA22
Present study
Present study
Present study
538

Targeted overproduction of sialic acids
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Sialic acid analysis
For quantitation of Neu5Ac, lyophilized samples of hydro-
lyzed culture products of known volume were reconstituted in
250 µl of deionized water and subjected to ultrafiltration
(Ultrafree-MC 100,000 NMWL Filter Unit, Millipore).
Neu5Ac analysis was carried out with a Dionex model DX-300
high-performance liquid chromatography system equipped
with pulsed amperometric detection. Isocratic runs were parti-
tioned using a Carbopac PA-1 column in 0.1 M NaOH–0.05 M
sodium acetate at a flow rate of 1.0 ml/min. Elution times and
concentrations were compared with a standard curve generated
from commercial Neu5Ac (Sigma). Relative detector responses
shown in Figure 2 and 3 are in nA. Unless indicated otherwise,
data were normalized to express the concentration of sialate
per unit of A600 per ml of culture.
Statistical analyses
Differences in sialate accumulation between E. coli strains
EV136 and EV240 were analyzed by the t-test. Differences in
sialate accumulation among E. coli EV5 and three isogenic
transductants (Table III) were analyzed by analysis of variance
using contrasts to compare the transductants to EV5. P-values
< 0.05 were considered significant.
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Acknowledgment
We thank Willie Vann (FDA, Bethesda, MD) for critically
reviewing the manuscript before submission. The research
reported here was supported by NIH grant RO1 AI42015 to E.V.
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Abbreviations
CAA, casamino acids; Fru, fructose; Glc, glucose; GlcN,
glucosamine; GlcNAc, N-acetylglucosamine; Gro, glycerol;
HPAEC/PAD, high pH anion exchange chromatography with
pulsed amperometric detection; LB, Luria Bertani medium;
LPS, lipopolysaccharide; ManNAc, N-acetylmannosamine;
NCAM, neural cell adhesion molecule; Neu5Ac, N-acetyl-
neuraminic acid; PEP, phosphoenolpyruvate; PSA, polysialic
acid; PTS, phosphotransferase system; Pyr, pyruvate; TCA,
trichloroacetic acid.
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elongation in Escherichia coli K1. Mol. Microbiol., 4, 603–611.
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the sialyltransferase complexes in Escherichia coli K1 and K92. J. Bacteriol.,
174, 1099–1108.
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Purification and characterization of the Escherichia coli K1 neuB gene
product N-acetylneuraminic acid synthetase. Glycobiology, 7, 697–701.
Vimr, E.R. (1992) Selective synthesis and lasbelling of the polysialic acid
capsule in Escherichia coli K1 strains with mutations in nanA and neuB.
J. Bacteriol., 174, 6191–6197.
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