Characterization of a novel Salmonella Typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate

Article abstract of DOI:10.1093/glycob/cwq174

Salmonella contain genes annotated as chitinases; however, their chitinolytic activities have never been verified. We now demonstrate such an activity for a chitinase assigned to glycoside hydrolase family 18 encoded by the SL0018 (chiA) gene in Salmonella enterica Typhimurium SL1344. A C-terminal truncated form of chiA lacking a putative chitin-binding domain was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3) with an N-terminal (His)6 tag. The purified enzyme hydrolyzes 4-nitrophenyl N,N′-diacetyl - d-chitobioside, 4-nitrophenyl -d-N,N′,N″-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl - d-glucosaminide, peptidoglycan or 4-nitrophenyl -d-cellobioside. Enzyme activity was also characterized by directly monitoring product formation using ¹H-nuclear magnetic resonance which showed that chitin is a substrate with the release of N,N′-diacetylchitobiose. Hydrolysis occurs with the retention of configuration and the enzyme acts on only the -anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Gal1 → 4GlcNAc-O-(CH₂)₈CONH(CH ₂)₂NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.

Full text of DOI:10.1093/glycob/cwq174

Glycobiology vol. 21 no. 4 pp. 426–436, 2011
doi:10.1093/glycob/cwq174
Advance Access publication on November 9, 2010
Characterization of a novel Salmonella Typhimurium
chitinase which hydrolyzes chitin, chitooligosaccharides
and an N-acetyllactosamine conjugate
Tanja Larsen2,3, Bent O Petersen3, Birgit G Storgaard2,3
Jens Ø Duus3, Monica M Palcic3, and Jørgen
J Leisner1,2,3
2Department of Veterinary Disease Biology, Faculty of Life Sciences,
University of Copenhagen, Grønnegårdsvej 15, 1870 Frederiksberg C.,
Denmark and 3Carlsberg Laboratory, Gamle Carlsberg Vej 10, 2500 Valby,
Denmark
,
biopolymer in nature and the most abundant carbohydrate in
the aquatic biosphere. Chitin is found in the cell walls of
fungi, the cuticles of arthropods and the peritrophic mem-
branes of annelids and some arthropods (Gooday 1990;
Cauchie 2002). Many bacteria produce enzymes that hydro-
lyze chitin and the majority of these belong to glycoside
hydrolase family 18 (GH-18; www.cazy.org).
Bacterial chitinases play important roles in chitin cycling
especially in the marine environment. Vibrio cholerae and
other Vibrio spp. chitinases are involved in nutrient acquisition
and promote increased environmental survival (Keyhani and
Roseman 1999; Meibom et al. 2004). Bacterial chitinases can,
however, also contribute to human and animal infections as
shown for Legionella pneumophila where secreted chitinase
promotes bacterial infection in the lung of mice (DebRoy et al.
2006). Listeria monocytogenes chitinase expression is upregu-
lated in the mouse intestine after infection (Toledo-Arana et al.
2009) and is under the control of a central virulence regulator
(Larsen et al. 2010). For the Salmonella SL1344 strain, a micro
array study has revealed that the expression of the GH-18
SL0018 gene is strongly upregulated during the infection of
murine mouse macrophage cells (Eriksson et al. 2003).
However, chitinase activity has not yet been demonstrated for
Salmonella (Clarke and Tracey 1956; O’Brien and Colwell
1987; Norhana et al. 2009). Using bioinformatics, we now
show that several Salmonella strains contain a gene for a chiti-
nase that is a member of GH-18 though the gene product lacks
a signal peptide. To gain a better understanding of the possible
biological roles of this chitinase, we cloned the corresponding
SL1344 gene SL0018 (chiA) to verify the chitinolytic activity
of the gene product and to investigate its reaction specificity
with a panel of substrates.
Received on March 19, 2010; revised on October 22, 2010; accepted on
October 25, 2010
Salmonella contain genes annotated as chitinases; however,
their chitinolytic activities have never been verified. We
now demonstrate such an activity for a chitinase assigned
to glycoside hydrolase family 18 encoded by the SL0018
(chiA) gene in Salmonella enterica Typhimurium SL1344.
A C-terminal truncated form of chiA lacking a putative
chitin-binding domain was amplified by PCR, cloned and
expressed in Escherichia coli BL21 (DE3) with an N-term-
inal (His)6 tag. The purified enzyme hydrolyzes 4-nitrophe-
nyl N,N′-diacetyl-β-D-chitobioside, 4-nitrophenyl β-D-N,N′,
N″-triacetylchitotriose and carboxymethyl chitin Remazol
Brilliant Violet but does not act on 4-nitrophenyl N-acetyl-
β-D-glucosaminide, peptidoglycan or 4-nitrophenyl β-D-cel-
lobioside. Enzyme activity was also characterized by
directly monitoring product formation using 1H-nuclear
magnetic resonance which showed that chitin is a substrate
with the release of N,N′-diacetylchitobiose. Hydrolysis
occurs with the retention of configuration and the enzyme
acts on only the β-anomers of chitooligosaccharide
substrates. The enzyme also released N-acetyllactosamine
disaccharide from Galβ1 → 4GlcNAcβ-O-(CH2)8CONH
(CH2)2NHCO-tetramethylrhodamine, a model substrate
for LacNAc terminating glycoproteins and glycolipids.
Keywords: Enterobacteriaceae / family 18 hydrolase /
kinetics / N-acetyllactosamine / NMR
Results
Examination of bacterial chitinase activity
No chitinolytic activity was observed with the Salmonella
Typhimurium SL1344 strain incubated on a chitin agar
medium under aerobic or anaerobic conditions at 30°C for up
to 12 days.
Introduction
Chitin, an insoluble linear polymer of β1–4-linked
N-acetylglucosamine (GlcNAc), is the second most abundant
Phylogenetic analyses of chiA
1To whom correspondence should be addressed: Tel: +45-35332768; Fax:
+45-35332757; e-mail: jjl@life.ku.dk
We identified a putative chitinase gene, SL0018 (chiA)
(http://xbase.bham.ac.uk/colibase/genome.pl?id=1846),
in
© The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
426

Characterization of a Salmonella chitinase
Fig. 1. Alignment of the amino acid sequence of Salmonella Typhimurium SL1344 ChiA GLH18 domain (in bold) with representative sequences for similar
domains in other species and strains.
Salmonella Typhimurium SL1344 encoding a chitinase
assigned to GH-18. This gene is widely distributed among
genome-sequenced Salmonella strains (Figure 1).
culture could be obtained after chromatography on HisTrap
columns (Figure 2).
The gene product of chiA include a 412 amino acid GH-18
domain (amino acids 53–465) and a 50 amino acid C-terminal
chitin-binding module (CtBM; amino acids 646–696) but no
signature sequence for sec-dependent protein export. The
sequence of the putative catalytic region embedded in the
GH-18 domain could not readily be arranged within any of
the glycoside hydrolase 18 groups as outlined by Svitil and
Kirchman (1998). The GH-18 domain of ChiA is, however,
related to GH-18 domains in gene products annotated as chiti-
nases in other members of the Enterobacteriaceae family,
including Citrobacter rodentium, Cronobacter sakazakii,
Cronobacter turicensis, Photorhabdus luminescens, Sodalis
glossinidius, Xenorhabdus bovienii, Xenorhabdus nemato-
phila and Yersinia bercovieri as well as the other bacterial
groups including Burkholderia xenovorans and Rickettsia felis
(Figure 1). As was seen for chiA, the related genes in all these
bacteria also lack a signal peptide sequence.
Activity, kinetic parameters, pH and temperature effects
The activity spectrum of ChiAΔCtBM was investigated by
the use of the chitin pseudo-substrates, 4-nitrophenyl
N-acetyl-β-D-glucosaminide (pNP-GlcNAc), 4-nitrophenyl
N,N′-diacetyl-β-D-chitobioside (pNP-(GlcNAc)2) and 4-
nitrophenyl β-D-N,N′,N″-triacetylchitotriose (pNP-(GlcNAc)3).
The recombinant Salmonella enzyme demonstrated chitinase
activity toward the substrate pNP-(GlcNAc)2. Product amounts
per 1 nM enzyme were 0.09 and 0.14 μM for 0.21 and 0.82
mM substrates, respectively, after incubation at 30°C for 30
min. The enzyme also showed activity toward pNP-(GlcNAc)3
with product amounts of 0.4 and 0.15 μM for 0.21 and 0.82
mM substrates, respectively. Thus, substrate inhibition was
seen for higher concentrations of pNP-(GlcNAc)3. No activity
was measured toward pNP-GlcNAc, the cellulose pseudo-
substrate 4-nitrophenyl β-D-cellobioside or peptidoglycan,
whereas there was low activity toward carboxymethyl chitin.
In the latter case, the specific activity of ChiA was 0.000053
(ΔOD550 min−1, per nmol enzyme). The Salmonella chitinase
has a pH optimum of pH 6.1 and relatively high activity from
pH 4 to more than pH 7.25 (Figure 3). The temperature
optimum was at 37°C and the enzyme remained active
down to 4°C (Figure 4). The kcat for pNP-(GlcNAc)2 was
0.7 0.07/s with a Km of 0.73 0.23 mM at pH 5 where the
activity is nearly as high as observed at the optimal pH 6.1
value (Figure 3).
Cloning and expression of chiAΔCtBM
A truncated form of ChiA was constructed without the puta-
tive CtBM (amino acids 646–696) and three additional
C-terminal amino acids (amino acids 697–699). The 1971 bp
DNA fragment encoding the truncated ChiA chitinase and a
(His)6 tag at the N-terminus was cloned into a pET-46 Ek/LIC
expression vector and expressed in Escherichia coli BL21
(DE3) cells. The predicted mass of the tagged protein
ChiAΔCtBM is 71,365 Da. Expression levels of the recombi-
nant protein were very good and 80–100 mg enzyme/L of cell
The Salmonella chitinase showed activity toward Galβ1 →
4GlcNAcβ-O-(CH2)8CONH(CH2)2NHCO-tetramethylrho
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T Larsen et al.
Fig. 4. Temperature profile for activity of ChiAΔCtBM toward 0.41 mM
pNP-(GlcNAc)2.
Fig. 2. SDS–PAGE of Salmonella chitinase at different stages of purification.
Lanes 1 and 5 standards, lane 2 crude extract, lane 3 HisTrap column eluted
with 0.5 M imidazole and lane 4 HisTrap with a gradient of imidazole from
5 to 250 mM.
Fig. 5. Thin layer chromatography of reactions with fluorescent acceptors.
LacNAc-TMR (lanes 1 and 3) or GlcNAc-TMR (lanes 2 and 4) were
incubated with 5 μL of Salmonella ChiA chitinase (1.7 mg/mL) or T. viride
chitinase (1.7 mg/mL) for 30 min. LacNAc-TMR and GlcNAc-TMR were
substrates for the Salmonella and T. viride chitinases, respectively, releasing
the aglycone HO-(CH2)8CONH(CH2)2NHCO-tetramethylrhodamine
completely from GlcNAc-TMR in the case of T. viride enzyme.
concentrations of LacNAc-TMR; kcat was 0.05 0.005/s with
a Km of 10 1.4 mM at pH 6.
Fig. 3. pH profile at 30°C for activity of ChiAΔCtBM toward 0.41 mM
pNP-(GlcNAc)2.
Nuclear magnetic resonance studies with chitin,
pNP-(GlcNAc)2, chitooligosaccharides (GlcNAc)3
and LacNAc-TMR
damine (LacNAc-TMR) when examined by thin layer chrom-
atography releasing the linking arm HO-(CH2)8CONH
(CH2)2NHCO-tetramethylrhodamine (Figure 5). This activity
Chitin was shown to be a substrate for the Salmonella chiti-
nase with the slow release of N,N′-diacetylchitobiose
1
was
inhibited
by
the
addition
of
1.8 mM
((GlcNAc)2) as monitored by H-nuclear magnetic resonance
hexa-N-acetylchitohexaose where only a trace of linking arm
was observed suggesting that the activity is due to the chiti-
nase rather than a contaminating enzyme. Trichoderma viride
chitinase did not act on LacNAc-TMR but was very active on
GlcNAc-β-O-(CH2)8CONH(CH2)2NHCO-tetramethylrhoda-
mine (GlcNAc-TMR) with the complete release of the
linking arm. The LacNAc isomer, Galβ1 → 3GlcNAcβ-
(NMR) (Figure 6). The same slow release was seen for col-
loidal chitin (data not shown). The stereochemical course of
hydrolysis of pNP-(GlcNAc)2 was studied at 15°C to reduce
the rate of anomerization of the released saccharides
(Figure 7). At 800 MHz, all the N-acetyl groups of
pNP-(GlcNAc)2, (GlcNAc)2 and GlcNAc have distinct chemi-
cal shifts (Figure 7B). After 15 min, new N-acetyl signals for
the β-anomer of (GlcNAc)2 are observed confirming the reac-
tion occurs with the retention of configuration and after 20
min half of the substrate had been depleted. This is followed
by the slow mutarotation of the released (GlcNAc)2 giving
O-(CH2)8CONH(CH2)2NHCO-tetramethylrhodamine
(Type
I-TMR), was not a substrate for either enzyme (data not
shown). Kinetic constants were determined by capillary elec-
trophoresis (CE) analysis of reactions at different
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Characterization of a Salmonella chitinase
Fig. 6. Hydrolysis of chitin by Salmonella ChiA chitinase. NMR spectra at 25°C showing the conversion of crab shell chitin (2 mg) to (GlcNAc)2 over time; the
top spectrum is that of a (GlcNAc)2 reference, whereas the other spectra were acquired after addition of 0.1 mg of enzyme.
the α-anomer after 1 h. After 15 min, traces of β-GlcNAc
and pNP-GlcNAc are also produced. These can arise from
the hydrolysis of (GlcNAc)2 and/or the terminal GlcNAc of
The hydrolysis of LacNAc-TMR also occurs with the release
of LacNAc with the retention of configuration followed by the
slower mutarotation of β-LacNAc to α-LacNAc (Figure 9).
This reaction occurs with 50% conversion to product in 280
min. LacNAc-(CH2)8CO2CH3 was also hydrolyzed releasing
disaccharide though at a much slower rate than LacNAc-TMR
taking 24 h for 50% conversion (results not shown).
pNP-(GlcNAc)2. pNP-GlcNAc is not
a
substrate for
Salmonella chitinase; thus, this signal remains constant after
3 h when all of the pNP-(GlcNAc)2 has been depleted. At 15
min, a small amount of transglycosylation products have been
produced which are hydrolyzed. The amount of GlcNAc at
3h20min is greater than that of pNP-GlcNAc demonstrating
that (GlcNAc)2 has also been hydrolyzed to GlcNAc. In all
cases, reactions occur with the retention of configuration since
β-anomers are the only products of enzyme-catalyzed steps.
(GlcNAc)3 was also studied at 15°C since the rate of anom-
erization in spectra acquired at 25°C was too rapid to deter-
mine anomeric preference. Figure 8B shows the rapid
decrease in the N-acetyl signals for the β-trisaccharide with
50% depletion in 40 min and with its complete depletion at
150 min. The rate of decrease in the α-trisaccharide anomer is
subject to a lag followed by a slow constant decrease due to
its anomerization to β (GlcNAc)3, which is the preferred sub-
strate for the enzyme (Figure 8B). For up to 50 min, there is
formation of equal amounts of β-(GlcNAc)2 and β-GlcNAc
along with a lag in the formation of α-(GlcNAc)2 and
α-GlcNAc, which are only formed by anomerization as
expected for hydrolysis with the retention of configuration.
However, at the end of the reaction, there is a somewhat
larger amount of both α- and β-GlcNAc than α- and
β-(GlcNAc)2, which is attributed to the slow hydrolysis of
(GlcNAc)2. All enzymatic and nonenzymatic steps for
(GlcNAc)3 hydrolysis are shown in Figure 8A.
Discussion
In this study, we were unable to show chitinolytic activity for
the Salmonella Typhimurium SL1344 strain using a standard
assay. Indeed, while Salmonella Typhimurium LT2 has been
reported to be able to hydrolyse (GlcNAc)2 and to use this
substrate for growth (Keyhani and Roseman 1997), genuine
chitinolytic activity by this species has not been demonstrated
previously (Clarke and Tracey 1956; O’Brien and Colwell
1987; Norhana et al. 2009). Salmonella encode, however,
both GH-18 and GH-19 enzymes annotated as chitinases. A
chitinolytic phenotype has been readily observed by the use
of similar assays for chitinolytic bacteria such as Vibrio spp.
for which this activity is important for survival in the environ-
ment (Keyhani and Roseman 1999; Meibom et al. 2004). We
speculated whether the Salmonella Typhimurium SL1344
ChiA enzyme is not expressed under conditions related to the
environmental survival and more particularly whether the bio-
logical role of the ChiA chitinase might instead be associated
with the infection of insects and/or vertebrates. Indeed,
Salmonella is associated with chitin-containing insects, such
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T Larsen et al.
Fig. 7. (A) An overview of the products formed during the hydrolysis of pNP-(GlcNAc)2 by Salmonella ChiA chitinase. (B) Partial 1H-NMR spectra of the
N-acetyl groups of substrate (1 mg) and products during hydrolysis by 0.14 mg of enzyme monitored at 15°C.
as cockroaches, chironomus midges and flies, and these hosts
may carry the bacteria as possible dispersal vectors (Kopanic
et al. 1994; Olsen and Hammack 2000; Moore et al. 2003;
Holt et al. 2007). In addition, Salmonella Typhimurium is a
well-known infectious pathogen of many vertebrate species
including humans (Jay 2000).
This hypothesis prompted us to clone and overexpress the
enzyme in a heterologous host with a view to purify it and
test the catalytic activity toward substrates representing
models of chitin as well as GlcNAc-containing glycans found
on or in eukaryotic cells. We employed a C-terminal truncated
version of the protein omitting the chitin-binding domain in
order to facilitate a kinetic comparison with an L. monocyto-
genes ChiA GH-18 enzyme with no chitin-binding domain
that was simultaneously cloned, expressed and purified in our
laboratory (Leisner et al. 2009). Some of the chitinases related
to ChiA produced by other bacteria do also not contain such a
domain (Figure 1).
430

Characterization of a Salmonella chitinase
Fig. 8. (A) An overview of the enzymatic products of the hydrolysis of (GlcNAc)3 by Salmonella ChiA chitinase. At the start of the reaction, there is a 60:40
ratio of α- to β-(GlcNAc)3; however, only the β-anomer is a substrate for the enzyme. (B) Plots of the changes in N-acetyl signals of α-(GlcNAc)3 (a-t) and
β-(GlcNAc)3 (b-t) during the hydrolysis of 1 mg of substrate by 0.14 mg of Salmonella chitinase monitored at 15°C. Up to 40 min, the only products are
β-(GlcNAc)2 (b-d) and β-GlcNAc (b-m). α-(GlcNAc)2 (a-d) and α-GlcNAc (a-m) are slowly formed upon anomerization of their corresponding β-anomers.
The recombinant Salmonella enzyme showed activity
toward both pNP-(GlcNAc)2 and pNP-(GlcNAc)3 in agree-
ment with the hydrolysis reaction starting two and three units
from the nonreducing end of the chitin pseudo-substrate as
shown for an Aeromonas caviae chitinase (Wang et al. 2003)
but without a markedly reduced hydrolysis at the three unit
position. The activity toward pNP-(GlcNAc)3 was lowest at
the highest concentration, which may indicate some kind of
substrate inhibition of the enzyme. Such a result has been
reported by Den Tandt and Scharpe (1997), when measuring
the activity of a human plasma specific chitinase toward
methylumbelliferyl-tetra-N-acetyl-β-D-chitotetraoside, a chitin
pseudo-substrate. For this reason, we only measured kinetic
values for pNP-(GlcNAc)2 and found that the observed Km
values were similar to other chitinases, whereas the kcat value
was at the lower end (www.brenda-enzymes.info/, Department
of Bioinformatics and Biochemistry, Technical University of
Braunschweig, Germany; Leisner et al. 2009). The effect of
the carbohydrate-binding domain on the activity of the
ChiAΔCtBM enzyme remains to be studied. This type of
domain appears to support the activity of other chitinases
toward α-chitin and colloidal chitin (Watanabe et al. 1994;
Svitil and Kirchman 1998; Wang et al. 2003) but it was not
entirely necessary for activity toward chitin in our study as
verified by monitoring the slow release of (GlcNAc)2 by
1H-NMR (Figure 6) and the hydrolysis of carboxymethyl
chitin Remazol Brilliant Violet (CM-Chitin-RBV) solution. In
the latter case, the specific activity of ChiA was, however,
markedly less than observed for Serratia marcescens A, B
and C1 chitinases (Δ0.0035–0.066 OD550 min−1, per pmol
enzyme, Synstad et al. 2008), although it should be noted that
incubation temperature (30 vs. 37°C) and substrate
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T Larsen et al.
Fig. 9. (A) Hydrolysis of LacNAc-TMR catalyzed by Salmonella ChiA chitinase with the proton signals being monitored in the NMR experiment highlighted
in bold. (B) Partial 1H-NMR spectra showing the anomeric signals for the starting substrate (0.3 mg) and the terminal galactose of LacNAc that is released
in the reaction. Reaction was at 15°C with 0.7 mg of enzyme.
432

Characterization of a Salmonella chitinase
concentration (2 vs. 0.6 mg/mL) differed. Both S. marcescens
chitinases B and C possess chitin-binding domains contrary
to the Salmonella ChiAΔCtBM enzyme investigated here. We
intend to conduct further studies on a nontruncated version of
the Salmonella ChiA enzyme in order to further evaluate its
chitinolytic activity.
We explored further the catalytic activity of the Salmonella
enzyme toward pNP-(GlcNAc)2 by means of NMR. As
expected, the enzyme exhibited a retaining mechanism
(Figures 7 and 8) in accord with the classification of this
enzyme as a GH-18 member. The activity toward (GlcNAc)3
was somewhat lower than that toward pNP-(GlcNAc)2
since the complete hydrolysis of the β-anomer of
(GlcNAc)3 required 150 min rather than just over 1 h for
pNP-(GlcNAc)2. Penta-N-acetylchitopentaose ((GlcNAc)5)
was completely hydrolyzed to (GlcNAc)2 and (GlcNAc)3 and
a trace of GlcNAc in <7 min under identical conditions
(Supplementary data, Figure S1).
The enzyme activity of Salmonella chitinase toward
pNP-(GlcNAc)2 had a broad pH optimum and a temperature
range (Figures 3 and 4). The enzyme showed a relatively high
pH optimum, a value in accord with gut pH values commonly
observed for many but not all insects (Dow 1992; Johnson
and Felton 1996; Clark 1999).
The enzyme showed no activity toward the related sub-
strates, 4-nitrophenyl β-D-cellobioside, a cellulose pseudo-
substrate and peptidoglycan. The latter result is supported by
the observation that for the Salmonella Typhimurium LT2
strain, a gene (STM0018) identical to the chiA gene of the
SL1344 strain is not essential for building of the cell wall.
Thus, a mutant with this gene deleted exhibited relatively
similar, although not entirely identical, growth with the wild-
type strain during incubation at 30 or 37°C in Brain
Heart Infusion Broth or Luria Broth (B.G. Storgaard and
J.J. Leisner, unpublished results). Previously, however, bifunc-
tional chitinases with chitinase and lysozyme activity have
been reported (Wang and Chang 1997).
The ability of the Salmonella enzyme to release LacNAc
from LacNAc-TMR is an unusual observation. The activity
exhibited a low kcat but a high Km value compared with the
activity toward pNP-(GlcNAc)2. As expected the
enzyme showed no activity toward GlcNAc-TMR, a substrate
analog to pNP-GlcNAc. This type of activity has only been
described for a chitinase from Amycolatopsis orientalis, an
Actinobacteria where the disaccharide was released from
pNP-LacNAc (Murata et al. 2005). Chitinases from T. viride
(Figure 5) and L. monocytogenes (ChiA; data not shown) were
inactive on this substrate, and none of the enzymes acted on
Type I-TMR that has a Galβ1 → 3GlcNAc linkage rather than
Galβ1 → 4GlcNAc of LacNAc. LacNAc-TMR is a model sub-
strate for LacNAc terminating glycoproteins and glycolipids
on vertebrate cells. Further studies are needed to elucidate
whether this activity indeed fulfils a biological role but it raises
the interesting possibility that the Salmonella enzyme is target-
ing LacNAc-containing glycans in the host organism. The
observation that the ChiA gene expression is upregulated
during the infection of murine macrophage cell lines (Eriksson
et al. 2003) strongly suggest the feasibility of this possibility.
Indeed, LacNAc-containing glycans are common in the human
glycome (Cummings 2009). It has previously been demon-
strated that LacNAc glycosides can be important for bacterial
pathogenesis exemplified by E. coli pathogenic strains that
bind to the human intestinal epithelium by LacNAc-specific
lectin activity (Humphries et al. 2009).
The potential role of the Salmonella GH-18 chitinase for
the infection of insects is supported by the observation that it
has an amino acid sequence similar to chitinases encoded by
a range of related bacterial species (Figure 1) known to have
interactions, symbiotic or pathogenic, with invertebrates, e.g.
P. luminescens that live in nematodes (Ciche et al. 2008) and
S. glossinidius that live as a secondary endosymbiont in the
mid-gut of tsetse flies. Sodalis glossinidius chitinase activity
is necessary for invasion of the bacterium into the insect host
(Dale and Welburn 2001).
The GH-18 enzyme encoded by these organisms, including
Salmonella, lacks a signal peptide essential for export by the
general secretory pathway (Thanassi and Hultgren 2000;
Figure 1) but they are still, however, predicted as secretory
proteins when examined by the use of the CBS-DTU secre-
tome 2.0 software (www.cbs.dtu.dk/services/secretomeP;
results not shown). This finding suggest that these enzymes
are not secreted into the environment and therefore do not
play a role for survival outside a suitable host. It is possible
that they instead may be effector proteins secreted by the type
III secretory pathway that is employed during the infection of
insects as well as mammals (Dale et al. 2001; Cornelis 2006)
but further research is needed to confirm this.
This study demonstrates for the first time that a Salmonella
GH-18 chitinase indeed possesses activity toward chitin as
well as toward LacNAc-TMR. This observation raises intri-
guing questions regarding the potential biological roles for the
enzyme that deserves further research. Thus, it will be of
importance to define under which conditions and locations in
relation to host cells the enzyme is expressed during the infec-
tion of insect and vertebrate host organisms. It also remains to
be studied which biological substrates might be a target for
the LacNAc-TMR hydrolytic activity. Finally, the potential
synergism between the GH-18 chitinase included in this study
and other Salmonella chitinases that belong to the GH-19
(www.cazy.org) remains to be clarified. Our laboratories are
currently conducting research to resolve these issues.
Materials and methods
Bacterial strains, standard growth medium and examination
of bacterial chitinase activities
The Salmonella Typhimurium SL1344 strain was obtained
from Dr John Elmerdahl Olsen, Department of Veterinary
Disease Biology, Faculty of Life Sciences, University of
Copenhagen, whereas the E. coli BL21 strain was purchased
from Novagen (Germany). Both bacteria were propagated in
Luria Broth (Merck, Germany) at 37°C.
Investigation of the chitinolytic phenotype of Salmonella
Typhimurium 1344 was done as described by Leisner et al.
(2008) using a chitin agar medium that contained per liter
10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl,
15.0 g of agar and 2.5 g of chitin from crab shells (Sigma
C9752, Germany). Cultures were incubated under aerobic and
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T Larsen et al.
anaerobic conditions at 30°C and scored for hydrolytic ability
(clearing zones) for up to 12 days.
For protein purification, 1 L of cells were grown at 30°C
to A600 = 0.4 before induction with 2 mM IPTG. After
induction, growth was continued for 21 h at 30°C. The cells
were harvested by centrifugation and resuspended in 50 mL
of loading buffer, 20 mM MOPS, pH 7.5, containing 0.5 M
NaCl and 5 mM imidazole. The cells were disrupted using
a Constant Systems cell disruptor at 4°C with the pressure
of 1.36 Kbar. The lysate was centrifuged at 4°C for 1.5 h at
48,000 × g and the filtered supernatant applied to a 5 mL
HisTrap column (GE Healthcare, UK) with a flow rate of
ꢀ5 mL/min. The column was washed with 80 mL of
loading buffer before being eluted with 20 mL of 100 mM
MOPS, pH 7.8, 0.5 M NaCl and 0.5 M imidazole. Fractions
Identification and phylogenetic analyses of genes encoding
putative chitinases in Salmonella
We used the following online resources to identify genes
encoding putative chitinases in published genome sequences
of Salmonella: www.cazy.org and http://xbase.bham.ac.uk.
The translated amino acid sequence of the chitinase chiA
gene (http://xbase.bham.ac.uk/colibase/genome.pl?id=1846)
from
the
genome-sequenced
Salmonella
enterica
Typhimurium SL1344 was used as probe to search for similar
sequences using the microbial resource Blast at National
Center for Biotechnology Information (ncbi; www.ncbi.nlm.
nih.gov/). A selection of sequences most similar to the chiA
gene was included for further phylogenetic analyses in
addition to sequences representative of other bacterial groups.
Protein domain predictions including the determination of the
glycosyl hydrolase family 18 (GLH18) domains were
obtained from http://smart.embl-heidelberg.de/. Signal peptide
predictions were performed using SignalP 3.0 from Center for
Biological Sequence Analysis, BioCentrum-DTU, Technical
University of Denmark (www.cbs.dtu.dk/services/SignalP/).
GLH18 domain sequences selected from BLAST searches
were aligned to the chiA sequence. Phylogenetic trees were
constructed by the neighbor-joining method using the Clustal
X version 1.81 (Thompson et al. 1997) and the MEGA
version 4 (Tamura et al. 2007) software packages with default
settings.
containing enzyme were combined, concentrated in
a
Vivaspin (30,000 Da cutoff), exchanged into loading buffer
and re-chromatographed on a 5 mL HisTrap column with a
gradient of imidazole from 5 to 250 mM imidazole. The
fractions containing enzyme were dialysed against 50 mM
sodium phosphate buffer, pH 6.0, at 4°C. The protein con-
centration in the sample was determined by the Bradford
method using a commercial kit (Bio-Rad, CA) using bovine
γ-globulin as a protein reference standard.
Enzyme assays
The activity of ChiAΔCtBM were determined using
pNP-GlcNAc, pNP-(GlcNAc)2 and pNP-(GlcNAc)3 as sub-
strates. In a standard assay, 5 µL of enzyme (19.9 mg/mL)
diluted 100- or 10,000-fold in 50 mM sodium phosphate
buffer, pH 6.0, and 45 µL of either 0.21 or 0.81 mM substrate
dissolved in Sigma buffer (A4855, pH 5.0) and incubated at
30°C for 30 min. The reaction was quenched by adding 100
µL of sodium carbonate. Absorbance was measured in a plate
reader at 405 nm and corrected for absorption in a control
sample with added sodium phosphate buffer, pH 6.0, instead
of enzyme. Linearity was ensured by monitoring product for-
mation at different time intervals and absorption values were
converted into concentrations by the use of a p-nitrophenol
(Sigma) standard curve. Kinetic parameters were determined
for pNP-(GlcNAc)2 by measuring initial rates of reaction at
eight different substrate concentrations ranging from 0.1 to 4
mM. Km and Vmax were calculated using the software
GraphPad Prism 4.0 (GraphPad Software) and kcat estimated
by dividing Vmax by enzyme concentration.
The effect of temperature was determined using 0.41 mM
pNP-(GlcNAc)2 as a substrate incubated with 10,000-fold
diluted enzyme (0.00199 mg/mL) for 30 min at 4, 10, 15, 22,
30, 37 and 40°C. For pH dependency measurements, the
same substrate concentration and incubation temperature (30°
C) in either phosphate buffer (pH 3, 4, 5, 6.1 and 7.2) or
glycine buffer (pH 7.25 and 9.4) were used.
Chitinase activity was also measured using the soluble
polymeric substrate CM-Chitin-RBV (LOEWE Biochemica,
Munich, Germany) as described previously (Synstad et al.
2008) with the following modifications. The reaction mixture
consisted of 100 µL of 2 mg/mL CM-Chitin-RBV, 90 µL of
5 mM sodium phosphate buffer, pH 6.0, and 10 µL of
1000-fold diluted enzyme (0.0199 mg/mL) incubated at 30°C
for 30 min. The reaction was terminated by adding 50 µL of
1 M hydrochloric acid.
Cloning, recombinant expression and purification
of ChiAΔCtBM
Salmonella enterica Typhimurium SL1344 DNA was purified
using a commercial kit (FastDNA) according to the manufac-
turer (MP Biomedicals, LLC, OH). Primers designed to
amplify the chiA gene omitting the C-terminal chitin-binding
domain (amino acids 646–696) were 5′-GAC GAC GAC AAG
ATC GCT ACA AGC AAA CTG ATT CAA G-3′ (forward
primer) and 5′-GA GGA GAA GCC CGG TTA GTA AGG
TGA CGT TTT GTC ATC TG-3′ (reverse primer). PCR was
done using the Roche (Germany) expand high fidelity PCR
system including the following cycle steps: initial denaturing
(95°C, 30 s), 25 cycles of denaturing (95°C, 30 s), annealing
(60°C, 1 min), extension (68°C, 5 min) and final extension
(68°C, 10 min). The PCR product was purified using the
Qiagen (Germany) MinElute PCR purification kit and the pre-
dicted sizes subsequently verified by the use of a 1.5% agarose
gel. The amplified sequence was cloned into the N-terminal
(His)6 tag pET-46 Ek/LIC vector (Novagen) and expressed in
E. coli BL21 (DE3) as described by the manufacturer. The
presence of correct insert DNA in clones was verified by
sequencing (Agowa, Germany) using T7 promoter and termin-
ator sequences as primers. The induction of the IPTG inducible
T7 promoter was verified by growing cells overnight in Luria
Broth at 30°C with 2 mM IPTG and 50 µL/mL of carbenicillin
and subsequent lysis using Novagen Popculture reagent as
described by the manufacturer. The presence of ChiAΔCtBM
was verified by SDS–PAGE as well as by enzymatic activity as
described below under Enzyme assays.
434

Characterization of a Salmonella chitinase
Activity toward 4-nitrophenyl β-D-cellobioside (Sigma)
was measured as above for the chromogenic chitin
pseudo-substrates. Lysozyme activity was measured by using
Micrococcus lysodeikticus peptidoglycan as a substrate. The
procedure was according to the manufacturer’s instructions
(Sigma) with an enzyme dilution of 100-fold (0.199 mg/mL)
for assays with these two substrates.
The spectra at 800 MHz were obtained on the Bruker
Avance 800 spectrometer of the Danish Instrument Center for
NMR Spectroscopy of Biological Molecules.
Conflict of interest
None declared.
Fluorescent substrates, either LacNAc-TMR (1 µL of 0.2
mg/200 µL H2O), GlcNAc-TMR (0.5 µL of 0.4 mg/mL) or
Type I-TMR (0.5 µL of a 0.4 mg/200 µL H2O) were incu-
bated with 5 µL of enzyme (1.7 mg/mL) at ambient tempera-
ture. Reaction progress was monitored by removing 0.5 µL
aliquots for thin-layer chromatography on silica gel plates
developed with CHCl3/MeOH/H2O (65/35/5).
Abbreviations
CE, capillary electrophoresis; CM-Chitin-RBV, carboxymethyl
chitin Remazol Brilliant Violet; CtBM, C-terminal
chitin-binding module; GH-18, glycoside hydrolase family 18;
GlcNAc, N-acetylglucosamine; GH-18, glycosyl hydrolase
family
18;
LacNAc-TMR,
Galβ1 → 4GlcNAcβ-O-
Kinetic constants for LacNAc-TMR were determined by
monitoring product formation with 10 different concentrations
of substrate ranging from 0.080 to 10.5 mM at 30°C by CE.
The reaction volume was 10 µL in 50 mM sodium phosphate
buffer, pH 6.0. Aliquots (1 µL) were removed at 120 min,
quenched with 50 µL of CE running buffer (50 mM borate, pH
9.3, and 150 mM sodium dodecyl sulfate). Analyses were per-
formed on an automated PrinCE 560 CE system from PrinCE
Technologies B.V. (Emmen, The Netherlands). Separations
were carried out in an uncoated fused-silica capillary of 75 µm
i.d. with CE running buffer. TMR-labeled compounds were
detected and quantitated using an Argos 250B fluorescence
detector (Flux Instruments, Switzerland) equipped with
an excitation filter of 546.1/10 nm and an emission filter of
570 nm. All experiments were carried out at a normal polarity,
i.e. inlet anodic. Data were processed by PrinCE 7.0 software.
(CH2)8CONH(CH2)2NHCO-tetramethylrhodamine; GlcNAc-
TMR, GlcNAc-β-O-(CH2)8CONH(CH2)2NHCO-tetramethylr-
hodamine; (GlcNAc)2, N,N′-diacetylchitobiose; (GlcNAc)3, N,
N′,N″-triacetylchitotriose; (GlcNAc)5, penta-N-acetylchitopen-
taose; pNP-GlcNAc, 4-nitrophenyl N-acetyl-β-D-glucosami-
nide; pNP-(GlcNAc)2, 4-nitrophenyl N,N′-diacetyl-β-D-
chitobioside; pNP-(GlcNAc)3, 4-nitrophenyl β-D-N,N′,N″-
triacetylchitotriose; Type I-TMR, Galβ1 → 3GlcNAcβ-O-
(CH2)8CONH(CH2)2NHCO-tetramethylrhodamine.
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