Centro de Engenharia Biológica e
Química, Instituto Superior Técnico, 1049-001 Lisbon,
Portugal
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Bacterial strains of the new genus
Sphingomonas (47) are relatively ubiquitous in
soil, water, and sediments, have broad catabolic capabilities (12,
17, 33, 35), and produce at least eight extracellular acid
heteropolysaccharides that have similar but not identical
structures (9, 31). These polysaccharides, the
sphingans (after the genus), exhibit properties which make them
candidates for food and industrial applications, such as thermoreversible gel formation and solution viscosity (9,
34). The industrial strain Sphingomonas paucimobilis
ATCC 31461 (formerly Pseudomonas elodea) synthesizes in high
yields from different carbon sources and from cheese whey a new gelling
agent, gellan gum (15, 23, 36). The commercial utility of
gellan (9, 34) has been a stimulus for the study of its biosynthesis.
The cloning and functional analysis of genes essential for gellan
synthesis are indispensable in attempting the genetic and environmental
manipulation of its biosynthetic pathway in order to develop new
polysaccharides with distinct structural and physical properties. Among
the gellan biosynthetic enzymes (29), phosphoglucomutase (PGM; EC 5.4.2.2) plays a pivotal role, being an ideal target for
metabolic engineering. Indeed, PGM catalyzes the interconversion of
D-glucose-6-phosphate (G6P) and
D-glucose-1-phosphate (G1P), representing a branch point in
carbohydrate metabolism. G6P enters catabolic processes to yield energy
and reducing power, whereas G1P is the precursor of sugar nucleotides
that are used by the cells in the synthesis of various polysaccharides.
In gellan gum biosynthesis, G1P is required for the synthesis of three
sugar nucleotides, UDP-D-glucose,
dTDP-L-rhamnose, and UDP-D-glucuronic acid,
that are activated precursors for the synthesis of the repeating tetrasaccharide unit in gellan gum (29).
The objective of the present work was to identify the PGM gene from
S. paucimobilis ATCC 31461 (pgmG gene) to be used
as a target in the metabolic engineering of the gellan pathway.
DNA manipulations.
Plasmid DNA was purified from
Escherichia coli cultures by the alkaline lysis procedure
(4) or with a QIAprep spin plasmid kit (Qiagen, Santa
Clarita, Calif.). DNA restriction, agarose gel electrophoresis, and
cloning procedures were carried out by established protocols
(43). Nonradioactive probes were used in Southern and colony
hybridizations. The Gene Images system (Amersham Pharmacia Biotech,
Carnaxide, Portugal) was used for labeling and detecting nucleic acids
with fluorescein as the nonradioactive label. Plasmid DNA was
introduced into E. coli strains either by a standard
transformation procedure or by electrotransformation (43).
Cloning and sequence analysis of the S. paucimobilis
pgmG gene.
The pgmG gene was cloned based on the
PCR amplification of a DNA fragment of 670 bp from S. paucimobilis ATCC 31461 genomic DNA using degenerate primers from
conserved regions of the phosphohexosemutase protein sequences in
databases: the synthetic oligonucleotides PGM1 (sense)
(5'-ACCGSCAGCCABAAYCCG-3') and PGM2 (antisense)
(5'-BSCGCTCATYTCGCC-3'), purchased from Pharmacia (Uppsala,
Sweden) (Table 1). Each PCR mixture
contained, in a final volume of 50 µl, 200 ng of S. paucimobilis ATCC 31461 DNA, 10 mM each deoxynucleoside
triphosphate (Promega Corporation, Madison, Wis.), 300 pmol of each
primer, 2.5 U of Taq DNA polymerase (Boehringer GmbH,
Mannheim, Germany), 5 µl of 10× Taq DNA polymerase
buffer, and 2.5 mM MgCl2. PCR amplification was performed
using a PTC-100 thermocycler (MJResearch Inc., Watertown, Mass.) under
the following conditions: 30 cycles consisting of 60 s at 95°C,
30 s at 55.5°C, and 60 s at 72°C. The PCR products yielded were analyzed on a 1% (wt/vol) low-melting-temperature agarose
gel (FMC Bioproducts, Rockland, Maine). The amplification product was
recovered from the gel, purified by using a WizardPCR Preps DNA
purification kit (Promega), and finally cloned into vector plasmid
pCR2.1 (Invitrogen, San Diego, Calif.), yielding plasmid pPV1. The PCR
product was sequenced, and analysis of the deduced amino acid sequence
confirmed that it contained an incomplete open reading frame and that
the deduced amino acid sequence was homologous to PGM and
phosphomannomutase (PMM) protein sequences in data banks.
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TABLE 1.
Degenerate oligonucleotide primers used in this work to
amplify a specific pgm probe from S. paucimobilis ATCC 31461 genomic DNA
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The PGM1 and PGM2 primer sites are located in two conserved regions in
the PGM or the PGM and PMM proteins of the gram-negative species listed
in Table 1, corresponding to the active-site region and the substrate
specificity region, respectively (10, 40). The 670-bp
labeled pgmG probe was used to screen, by colony
hybridization, the 1,200 clones of the genomic library of S. paucimobilis ATCC 31461 constructed to clone the gellan gum
gene cluster(s). To prepare this gene bank, high-molecular-weight
DNA was extracted from S. paucimobilis ATCC 31461 by the method of Goldberg and Ohman (19), partially digested
with Sau3AI, and size fractionated by continuous sucrose
gradient (10 to 40% [wt/vol]) ultracentrifugation (43).
Fractions containing DNA fragments of approximately 30 to 40 kb were
selected and ligated to the BamHI-digested and
dephosphorylated Hypercos.1 vector plasmid (Stratagene, La Jolla,
Calif.). The ligation product was packaged in vitro into a lambda phage
particle packing kit (Boehringer) and used to infect E. coli
NM554 (38). Eight clones of the gene bank thus obtained
hybridized with the probe mentioned above; restriction analysis of the
inserts and cross-hybridization analysis revealed the presence of
overlapping insertions. One of the clones (pF6), with an 
35-kb
insert, was retained for further characterization.
Subcloning of pF6 into pKK233-3 (8) led to the
identification of pPV233, a subclone with a 2,900-bp PstI
fragment insert. Southern hybridization of S. paucimobilis genomic DNA digested with PstI revealed
that the above-mentioned pgmG internal region hybridized
under high-stringency conditions with a single 2,900-bp genomic
fragment encompassing the complete pgmG gene sequence (data
not shown).
Nucleotide sequence accession number.
A nucleotide sequence of
1,563 bp within the pPV233 insert was determined on both strands at the
DNA Core Facility, University of Missouri, Columbia. This
sequence has been deposited in the GenBank database under
accession no. AF167367.
Sequence data were analyzed with DNASIS 3.0 software (Hitachi Software
Engineering, Hitachi America, Ltd., Brisbane, Calif.), revealing the
presence of a unique open reading frame, designated pgmG and
1,388 nucleotides long, starting at a putative ATG start codon. The
average G+C content of pgmG (66.8%) is characteristic of
Sphingomonas genes, and a high frequency of G or C in the
third codon was also observed (48). Preceding the start
codon (6 nucleotides upstream), a putative ribosome-binding site
(5'-GGGAGG-3') was found; possible promoter sequences,
TATGCTG for the 
35 region and TATTAA for the
10 region, were observed in the 5'-flanking region. The protein
deduced from the pgmG sequence is composed of 462 residues,
with a calculated molecular mass of 50,059 Da and a predicted pI of
4.85. Analysis of the amino acid composition revealed that there are
67% nonpolar residues and 33% polar residues; among the charged
residues, 42% are basic.
The deduced amino acid sequence encoded by pgmG was
compared with data in the GenBank database using the BLAST
network service (2) at the National Center for Biotechnology
Information, Bethesda, Md. Alignments to determine protein
similarities and for construction of a phylogenetic tree were
performed with the CLUSTAL method (20) (Genetics Computer
Group, University of Wisconsin) through a computer link to the European
Molecular Biology Network (EMBnet) [Portuguese node]
at the Gulbenkian Institute of Science, Oeiras, Portugal. The following
high levels of identity and similarity with other PGM and/or PMM
proteins from a variety of organisms were found: 59% identity and 71%
similarity with PmmA of Prochlorothrix hollandica
(13), 53% identity and 63% similarity with ExoC of Azospirillum brasilense (EMBL accession
no. 695163), 38% identity and 52% similarity with AlgC of
Pseudomonas aeruginosa (51), 38% identity and
53% similarity with the PGM of Neisseria gonorrhoeae (50), and 37% identity and 52% similarity with ExoC of
Rickettsia prowazekii (3). Lower levels of
homology were noted with phosphoglucosamine mutases, including
Staphylococcus aureus GlmM (25% identity and 39%
similarity) (21), E. coli GlmM (25% identity and
39% similarity) (11), and Pseudomonas syringae
GlmM (23% identity and 39% similarity) (41).
With the CLUSTAL V alignment program (20), the predicted
amino acid sequence of S. paucimobilis PgmG was compared
with the primary structures of PGM and/or PMM enzymes from prokaryotic and eukaryotic organisms (Fig. 1A). The
primary structures of all the proteins compared display a high degree
of similarity in length, and the three highly conserved domains were
confirmed (box I, box II, and box III) (Fig. 1A). According to the
observations first described for rabbit muscle PGM (10, 40),
box I (T-X-S-H-N-P; Fig. 1A) was assumed to correspond to the
active-site region, in which the serine residue (S 104 for S. paucimobilis PgmG) is phosphorylated during the catalytic action
of hexosephosphate mutases (10, 22, 32, 39); box II
(D-X-D-X-D-R; Fig. 1A) contains a metal-ion-binding loop; and box III
(G-E-X-S; Fig. 1A) may be responsible for interaction with the
substrate (10, 39).
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FIG. 1.
(A) Multiple amino acid alignment of the three
characteristic consensus sequences present in 14 proteins with PGM
and/or PMM activities from different organisms. Boxes I to III indicate
the functional domains exhibiting highest similarity among the
polypeptides. The amino acid sequences in boxes I, II, and III are
known to be critical for PGM activity, based on the study of rabbit
muscle PGM (10, 40). The box I motif contains the catalytic
site of the enzyme; the box II motif contains a metal-ion-binding loop;
and the box III motif interacts with the substrate. Asterisks below the
sequences indicate residues conserved in all the enzymes. Numbers of
intervening amino acids and accession numbers are given in parentheses.
(B) Phylogenetic tree based on the multiple-sequence analysis of the
fully sequenced PGM and PMM proteins. The CLUSTAL program
(20) was used for the sequence alignment and the
phylogenetic tree construction.
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A phylogenetic tree for the PGM and/or PMM family of 14 proteins was
constructed, including S. paucimobilis PgmG described here
for the first time (Fig. 1B). S. paucimobilis PgmG was
included in the subclass of phosphohexomutases with other bifunctional PMM and/or PGM proteins, such as AlgC from P. aeruginosa
(51), XanA from Xanthomonas campestris
(24), the N. gonorrhoeae PGM (44, 50),
PmmA from P. hollandica (13), the RFK7 and ManB enzymes from E. coli (showing PMM activity) (28,
45), or the putative PMMs from A. brasilense
(ExoC) (EMBL accession no. 695163) and R. prowazekii
(ExoC) (3). The PGM enzymes of E. coli and Acetobacter xylinum (CelB) (7, 25, 27) were very
similar to the PGM enzymes of Saccharomyces cerevisiae
(Pgm1) (5) and rabbit muscle (PgmU) (39) and are
considered to be highly specific for phosphoglucose. Although these two
subclasses share active-site and metal-ion-binding domains (box I and
II) in box III [GE(M or E)S] that may control substrate specificity
(10), the subclass of specific PGMs exhibits the E residue,
while in the subclass of bifunctional enzymes, including PgmG, this
residue is substituted by M (Fig. 1A).
Overexpression of pgmG in E. coli and
purification of the PgmG protein.
To overexpress the S. paucimobilis pgmG gene in E. coli, plasmid pLC1 was
constructed by inserting this gene behind the bacteriophage T7 RNA
polymerase promoter in the pET29a translation vector (Novagen Inc.,
Madison, Wis.) after specific amplification by PCR. Synthetic oligonucleotide primers used for amplification were PGM3 (sense) (5'-GGGGATTCATGACGCACCGTTTCGAT-3') and PGM4
(antisense) (5'-GGAAGCTTTCAATGCGCCGCCTGCTC-3'), designed
also to introduce BamHI and HindIII sites
(underlined) at the 5' ends of the PGM3 and PGM4 primers, respectively.
Pwo DNA polymerase (Boehringer) was used for amplification
of the pgmG gene. PgmG was overproduced as an N-terminal
fusion with the S-tag epitope of the S-protein portion of ribonuclease
A in E. coli BL21(DE3) transformed with pLC1. E. coli
strain BL21(DE3) carries the gene encoding T7 RNA polymerase under
the control of the inducible lacUV5 promoter
(46). E. coli transformants were cultivated in 25 ml of Lennox broth containing 0.5% (wt/vol) glucose and 150 mg of
ampicillin per liter and grown at 37°C until the culture optical
density at 600 nm reached 0.6. pgmG transcription was then
induced by adding isopropyl-
-D-thiogalactopyranoside (IPTG) (0.1 mM), followed by an additional period of 3 h of
growth. Cells were recovered by centrifugation, resuspended in 2.5 ml of cold 20 mM Tris-HCl (pH 7.5) with 0.15 M NaCl and 0.1% (wt/vol) Triton X-100 (Sigma Chemical Co., St. Louis, Mo.), disrupted by sonication (VibraCell; Sonics Material Inc., Danbury, Conn.)
(29), and centrifuged at 18,000 × g for 45 min at 4°C. Upon induction with IPTG, PGM and PMM activities in
E. coli crude extracts were significantly increased due to
recombinant PgmG (Table 2).
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TABLE 2.
PGM and PMM activities in crude extracts from cells of
E. coli BL21(DE3) harboring the cloning vector pET29a or the
recombinant plasmid pLC1, with the S. paucimobilis pgmG
gene, induced (3 h) or not induced with IPTG
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The specific activities of PGM and PMM were assayed with cell crude
extracts and with the sample resulting from PgmG purification under the
conditions described by Martins and Sá-Correia (29) and Sá-Correia et al. (42), as modified by
Leitão et al. (26). The increase in the optical
density at 340 nm due to the reduction of NADP at 30°C in coupled
reaction systems was recorded by using a double-beam spectrophotometer
(model U-2000; Hitachi Ltd., Tokyo, Japan). Enzyme activities were
calculated from the initial linear rates of cofactor reduction after
subtraction of endogenous activity (measured in enzyme assays lacking
the substrate). Control assays lacking only the extracts were also
carried out. All enzymes used in coupled reactions, phosphorylated
sugars, sugar nucleotides, NADP, dithiothreitol (DTT),
phenylmethylsulfonyl fluoride (PMSF), and bovine serum albumin were
from Sigma. Under the assay conditions used, one unit of enzyme
activity was defined as the amount of enzyme needed to reduce 1 µmol
of NADP per min. Protein concentration was determined by the method
described by Bradford (6) with bovine serum albumin fraction
V as the standard. PGM and PMM specific activities are the means of the
values of at least three enzyme assays and three protein determinations
in samples resulting from a growth and purification experiment
representative of the two complete independent experiments carried out.
Under standard conditions, the reaction mixture for the PGM assay
contained 15 mM Tris-Cl buffer (pH 7.6), 10 mM MgCl2, 5 mM
DTT, 0.1 mM glucose 1,6-diphosphate, 1 mM G1P, 1 U of
G6P dehydrogenase (zwf), and 1 mM NADP. The
reaction was started by the addition of 100 µl of the enzyme solution
to a final volume of 1 ml. PMM activity was assayed using an identical reaction mixture supplemented with 1 U each of phosphoglucose isomerase and phosphomannose isomerase and with 1 mM
mannose-1-phosphate (M1P) instead of G1P. The reaction was started by
the addition of 100 µl of the enzyme solution to a final volume
of 1 ml.
Heterologous PgmG was purified to homogeneity (Table
3) from cell crude extracts prepared from
IPTG-induced cells by using the S-tag-thrombin purification system
(Novagen). The purified enzyme fraction was eluted with a solution
containing 20 mM Tris-HCl (pH 8.4) with 0.15 M NaCl and 2.5 mM
CaCl2, dialyzed overnight at 4°C against 300 ml of 100 mM
Tris-HCl (pH 7.6) with 2 mM DTT and 0.5 mM PMSF, and immediately used
for enzyme assays. The proteins in cell crude extracts obtained by
sonication or in the sample obtained after purification were separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using
12.5% (wt/vol) polyacrylamide gels and samples loaded at a
concentration of 30 or 2 µg, respectively (Fig.
2). The S. paucimobilis
enzyme, capable of using both G1P and M1P, was purified over 12-fold
with a 31% yield (Table 3). Under denaturing conditions, purified PgmG
migrated as a single polypeptide with the expected molecular mass of 50 kDa (Fig. 2).
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FIG. 2.
Coomassie blue-stained proteins separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis during the
purification of S. paucimobilis PgmG overproduced in cells
of E. coli BL21(DE3) harboring plasmid pLC1 and induced with
IPTG. Lanes: A, molecular markers in kilodaltons; B, crude extracts
from cells of E. coli BL21(DE3) harboring plasmid pLC1 and
harvested after 3 h of IPTG (0.1 mM) induction; C, fraction
resulting from S-tag purification of PgmG.
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Heterologous complementations.
The functionality of the
S. paucimobilis pgmG gene-encoded protein as PGM
and PMM enzymes was confirmed by heterologous complementation of
both an E. coli mutant defective in the pgm gene
(1; kindly provided by the E. coli
Genetic Stock Center [CGSC], Yale University, New Haven, Conn.) and a
P. aeruginosa mutant defective in alginate synthesis due to
an algC gene mutation (51; kindly
provided by A. M. Chakrabarty, University of Illinois, Chicago).
The recombinant plasmid pPV233 slightly complemented the E. coli PGM1 defect (CGSC 5527) (1). This finding was
judged by the ability of PGM1 transformed with pPV233 to grow more
efficiently on MacConkey agar base plates (Difco Laboratories)
supplemented with 0.5% (wt/vol) galactose, yielding darker purple-red
colonies than E. coli PGM1 transformed with the
cloning vector pKK233-3 after incubation at 37°C for 2 days
(results not shown). The slight change in size and color of the
colonies demonstrated the recovery of the ability of the pgm-complemented mutant to produce acid from growth in galactose.
For the complementation experiments with the nonmucoid P. aeruginosa 8858 mutant with a defect in the algC gene
(53), the recombinant plasmid pLC100 was constructed by
subcloning PCR-amplified pgmG with Pwo DNA
polymerase, using primers PGM5 (sense)
(5'-GGGAATTCCACGTAACATTTTGCCGG-3') and PGM4
(antisense) (indicated above), designed also to introduce an
EcoRI site (underlined), into the broad-host-range
vector pMMB66(EH) (18). This recombinant plasmid was
mobilized into P. aeruginosa 8858 by triparental filter
mating using E. coli HB101/pRK2013 as the helper
strain (14, 16, 43). Individual transconjugants, isolated by
spreading the mating mixture onto Pseudomonas isolation agar
supplemented with carbenicillin, were examined visually for alginate
production on this same medium after 2 days of incubation at 30°C.
The introduction of plasmid pLC100 led to the recovery of alginate
biosynthetic ability, as did the introduction of plasmid pNZ49 with the
P. aeruginosa algC gene into the same cloning vector (51); the nonmucoid phenotype of the host algC
mutant was maintained after the introduction of the cloning vector
(Fig. 3).
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FIG. 3.
(A) Complementation of the nonmucoid
algC mutant strain P. aeruginosa 8858 by plasmid pLC100, containing the S. paucimobilis pgmG gene
in the cloning vector pMMB66(EH). (B and C) Positive (B) and negative
(C) controls were also prepared by the mobilization of pNZ49 or the
cloning vector pMMB66(EH) into P. aeruginosa 8858.
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Kinetic properties of PgmG.
The kinetic constants
Km and Vmax,
corresponding to the PGM or PMM activities of purified PgmG for G1P
(range, 15 to 1,000 µM) or M1P (range, 50 to 3,000 µM),
respectively, were calculated based directly on the Michaelis-Menten
equation; the rectangular hyperbolic function was solved using
iterative procedures (computer program: Solver from Microsoft Excel).
kcat values (minute
1) were
calculated on the basis of a molecular mass of 50 kDa for PgmG. Thus,
kcat values were obtained by dividing
Vmax (micromoles minute1
milligram1) by 0.02 to convert protein concentration to
molarity. The purified PgmG enzyme converted G1P into G6P and M1P into
M6P, as indicated by the results shown in Tables 3 and 4 and in Fig.
4. Although S. paucimobilis
PgmG is bifunctional, under the assay conditions used here the enzyme
exhibits a marked preference for G1P (Fig. 4 and Table
4). Indeed, the catalytic efficiency,
based on the kcat/Km
ratio, was about 50-fold higher for G1P than it was for M1P (Table 4).
The estimated apparent Km values for G1P and M1P were high, 330 and 1,270 µM, respectively.
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FIG. 4.
Saturation curves of purified PgmG with the indicated
concentrations of G1P or M1P, conforming to Michaelis-Menten kinetics.
The apparent Michaelis constant (Km) and
Vmax (Table 4) were calculated based directly on
the Michaelis-Menten equation.
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pgmG gene and the gellan pathway.
The S. paucimobilis pgmG gene identified in the present work is
presumably involved in the formation of G1P, required for the synthesis
of the three sugar precursors for the synthesis of the repeating
tetrasaccharide unit in gellan. However, this gene is not located in
the cluster of genes involved in gellan synthesis, recently identified
in our laboratory by PCR amplification (P. A. Videira, A. M. Fialho, and I. Sá-Correia, unpublished data), based on the gene
cluster involved in the synthesis of the exopolysaccharide (EPS)
sphingan S-88 by Sphingomonas S-88 (48). The
organization of the gellan gene cluster was found to be
essentially identical to that reported for the S-88 gene cluster and
includes genes essential for the assembly and secretion of EPS and a
four-gene operon needed for the synthesis of
dTDP-L-rhamnose. Interestingly, the algC gene of
P. aeruginosa, encoding the bifunctional PGM and PMM protein
of the alginate biosynthetic pathway, also does not map in the alginate
cluster, where algA and algD, the other genes
involved in GDP-mannuronic acid formation, are located (30).
However, in X. campestris, the gene
xanA, encoding the bifunctional PGM and PMM protein of the
xanthan gum biosynthetic pathway, is located in the xanthan cluster of
biosynthetic genes (24).
The bifunctional protein PgmG exhibits apparent
Km values for both substrates (Table 4) higher
than those reported for the majority of other PGM or PGM and PMM
enzymes characterized before. These values are 8 µM (G1P) for PgmU
of rabbit muscle (39), 17 µM (M1P) and 22 µM (G1P)
for AlgC of P. aeruginosa (49), and 20 µM (G1P)
for the PGM of maize leaves (37). However, these Km values are below the value calculated
for the specific PGM of A. xylinum (2,600 µM) [G1P]
(25). Interestingly, this protein was included in the
subclass of enzymes specific for G1P that is distinct from the subclass
of phosphohexosemutases formed by S. paucimobilis PgmG and
other bifunctional enzymes, such as AlgC from P. aeruginosa (51), XanA from X. campestris
(24), N. gonorrhoeae PGM (44, 50), and
PmmA from P. hollandica (13) (Fig. 1B). Although
the back reaction has not been studied for A. xylinum PGM,
the high Km (G1P) value calculated was
considered consistent with the involvement of this enzyme in the
production of extracellular cellulose, since a high
Km (G1P) favors metabolic flux toward polymer
synthesis rather than catabolic pathways (25).
Although the catalytic efficiency
(kcat/Km) of S. paucimobilis PgmG was about 50-fold higher for G1P than it was for
M1P, the pgmG gene cloned into pMMB66(EH) led to the
recovery of alginate biosynthetic ability when introduced into a
P. aeruginosa mutant with a defective
algC gene. This observation clearly indicates that PgmG
can indeed efficiently convert M6P into M1P in the initial steps of
alginate biosynthesis and, together with the other results reported,
suggests that PgmG may convert G6P into G1P in the gellan pathway. The
next step in this work will be the characterization of gellan gum
biosynthesis after pgmG disruption in S. paucimobilis ATCC 31461, whether the gene is nonessential and the
respective deletion mutant can be obtained, or in recombinant strains
in which the expression of this gene is increased.
This work was supported by JNICT/FCT, FEDER, and the PRAXIS XXI
Program (grant PRAXIS/2/2.1/BIO/1125/95 and Ph.D. and M.Sc. scholarships to P.A.V. and L.L.C., respectively).
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