Indian J Med Res 131, April 2010, pp 565-570
Detection, amplification & sequence homology of sodC in clinical
isolates of Salmonella sp.
M.K. Sanjay, S.M. Srideshikan*, M.S. Usha, A. PhilipRaj, S.M. Gaddad & C.T. Shivannavar
Department of Microbiology, Gulbarga University, Gulbarga & *Department of Biochemistry, Indian Institute
of Science, Bangalore, India
Received December 17, 2008
Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is
an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative
burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown
to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug
resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified,
cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were
compared with the standard strain salmonella Typhimurium 14028.
Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonella enterica serovar Paratyphi (S.
Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened
for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC)
was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were
compared using CLUSTAL X.
Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic
DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons
respectively. Single mutational difference at position 489 was observed between the sodC of S. Typhi and
S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino
acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that
of standard strain S. Typhimurium 14028.
Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as
phylogenetic marker.
Key words Cloning - Cu,Zn-SOD - Gram-negative bacteria - Salmonella - sequence homology - sodC
and are located in the cytosol and catalyse dismutation
of O2− generated during aerobic metabolism and a third
SOD is cofactored by copper and zinc (Cu,Zn-SOD)
and has been identified in the periplasm of a wide
range of Gram-negative bacteria viz., Caulobacter
Superoxide dismutases (SODs) are virtually
ubiquitous in aerobic bacteria, catalyzing the
conversion of oxygen radical (O2−) into hydrogen
peroxide and oxygen1. Three types of SODs are found
in bacteria, two are cofactored by manganese or iron
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INDIAN J MED RES, April 2010
crescentus2, Photobacterium leiognathi3,, Brucella
abortus4, Haemophilus species5, Legionella species6,
Actinobacillus and Pasteurella species7, Escherichia
coli8,9, Salmonella Typhimurium, S. choleraesuis
and S. dublin10,11, and in some of the Gram-positive
bacteria like Mycobacterium tuberculosis12. The
role of Cu,Zn-SOD as an important protective agent
against the extracellular superoxide radicals has
been demonstrated by using null mutants of different
pathogens which showed attenuated virulence and
increased sensitivity13-17. An insight into the nucleotide
and amino acid sequence of sodC and their homology
among the pathogenic bacteria may clarify the role of
these enzymes in virulence and may also reveal the
phylogenetic relatedness among these pathogens. In
the present investigation we detected the presence of
sodC in pathogenic S. Typhi and S. Paratyphi isolates
and nucleotide and amino acid sequence homology of
sodC was compared with a standard S. Typhimurium
14028 strain.
Material & Methods
Collection of samples: Blood samples (n=150) from
patients suffering from enteric fever were collected
(August 2005 to September 2006) from different
hospitals and diagnostic centres in Gulbarga city,
Karnataka, India. Salmonella sp. were isolated using
clot culture method by enriching the blood clots in bile
salt broth containing streptokinase and then culturing
on Wilson and Blair bismuth sulphate medium18. The
isolated bacteria were identified based on the cultural,
morphological, biochemical and serological tests19,20.
Salmonella enterica serovar Typhimurium 14028
(ATCC) was used as the standard reference strain.
Screening of Salmonella sp. for Cu,Zn-superoxide
dismutase: Preparation of periplasmic extracts Periplasmic extracts from the bacterial isolates grown
in Luria-Bertani (LB) broth (Hi-Media, India) for
30 h were prepared by the lysozyme-EDTA method
described by Battistoni and Rotilio21, briefly 250 ml of
30 h culture was centrifuged (Sigma 3K-30, USA) at
6000 g for 15 min. The pellet was re-suspended in 5 ml
of the isotonic solution comprising Tris-HCl (30 mM;
pH 8.0), sucrose (20% wt/vol) and EDTA (1 mM). To
this, 2 ml of lysozyme solution (10 mg/ml) was added
and mixed by inversion, incubated for 10 min on ice
and centrifuged at 13,200 g for 10 min. The supernatant
was used for the analysis of SodC.
Visualization of SODs in PAGE - Superoxide
dismutase was detected by non-denaturing
polyacrylamide gel (7.5%) electrophoresis (PAGE)22
with 5 per cent stacking gel pH 6.8, 7.5 per cent
separating gel pH 8.8 and tris-glycine (pH 8.3) buffer
system of Davis23. SOD activity in PAGE gels was
visualized by modified method of Beauchamp and
Fridovich24 as described by Steinman25. In a two-step
staining procedure, the gels were incubated in 2.45
mM Nitroblue tetrazolium chloride (NBT) in dark for
20 min and in a solution containing 28 µM riboflavin
and 28 mM N,N,N’,N’ –tetramethylethylenediamine
(TEMED) in a 36 mM potassium phosphate buffer
(pH 7.8) for 10 min in dark. The Cu,Zn-SOD band was
identified by specific inhibition by 2mM potassium
cyanide (KCN)26,27. The achromatic band indicating the
zone of SOD activity was developed by illumination.
Amplification of sodC from Salmonella sp.
- The genomic DNAs of isolates S. Typhi and S.
Paratyphi and standard strain S. Typhimurium 14028
were isolated28. In vitro amplification of sodC DNA
sequence was carried out by polymerase chain reaction
(PTC-150 Minicycler-MJ Research Inc., USA) using
oligonucleotide primers (Sigma-Aldrich, USA),
designed based on the existing sodC sequence from
database (S. Typhimurium NCBI GenBank Accession
Number AF056931).
Salfor
5`-ATGAAGCGATTAAGTTTAGCGATGG3`(Forward primer)
Salrev5`-TTTAATGACTCCGCAGGCGTAACGC
3`(Reverse primer).
-
The PCR mix (50 µl) contained 50-80 ng of genomic
DNA (template), 200 nM primers (Sigma-Aldrich) and
1.5 U XT-5 polymerase (Bangalore Genie, Bangalore)
in 1x reaction buffer. Samples were processed through
35 cycles of initial denaturation at 94°C for 3 min,
denaturation at 94°C for 30 sec, annealing at 56°C
for 45 sec, elongation at 72°C for 30 sec and final
elongation at 72°C for 15 min. The sodC amplicons
were purified by using the GenElute Gel Extraction Kit
(Sigma-Aldrich, USA).
Cloning of sodC amplicons of Salmonella sp.: Ligation
of sodC amplicons into cloning vector pJET1 - The
purified sodC DNA amplicon was ligated into pJET1
using GeneJET PCR Cloning kit (MBI, Fermentas,
Inc., MD, USA) by blunt end ligation with T4 DNA
ligase.
Transformation of sodC cloned pJET1 into E.
coli DH5α - Competent E. coli DH5α cells29 were
transformed with 10 µl of the ligated mix using
Sanjay et al: sodC in Salmonella sp.
567
TransformAid Bacterial Transformation Kit (MBI,
Fermentas) and the transformants were screened on
LB agar medium containing 100 mg/l ampicillin. The
recombinant clones were analysed for the presence
and orientation of the DNA insert using restriction
digestion analysis using XhoI and XbaI. The plasmid
DNA was isolated from an overnight bacterial culture
(transformants) by Miniprep method using the
GeneElute Plasmid Miniprep kit (Sigma-Aldrich).
Sequencing of the cloned sodC - After confirming
the sodC clones of Salmonella sp. (pJET1-14028,
pJET1-34, pJET1-35) by restriction digestion analysis,
the amplicons were submitted for sequencing (MWG
Biotech Pvt. Ltd., Bangalore, India). Multiple alignments
of nucleotide and protein sequences of S. Typhimurium
14028, S. Typhi-34 and S. Paratyphi-35 were carried out
with CLUSTAL X30. The homology of the sequences
was done using reference sequences from the database
based on homology BLAST search of SodC protein
sequences of bacteria. The sequence alignment was done
using CLUSTAL X and the dendrogram (phylogenetic
tree) was constructed using BioEdit (USA) and MEGA
software programmes (The Biodesign institute, Arizona
State University, Tempe, USA).
Fig. 1. Achromatic bands against the dark background indicate the
presence of SOD enzymes in periplasmic extracts from isolates:
Lane 1. S. Typhi 34; Lane 2. S. Paratyphi 35; Lane 3. S. Typhimurium
14028; Lanes 4,5,6. enzyme extracts of above strains treated with
2mM KCN indicating the inhibition of Cu,Zn-SOD activity.
Results
Visualization of SodC: The presence of SodC was
confirmed by the absence of the specific band on KCN
treated gel (Fig. 1). The SodC was detected in 7 (3-S.
Typhi and 4-S. Paratyphi) of a total of 20 Salmonella
sp. isolated.
PCR amplification of sodC: Amplification of sodC of
S. typhimurium 14028, S. typhi 34 and S. paratyphi 35
resulted in 519, 519 and 515 bp amplicons respectively
on 1 per cent agarose gel (Fig. 2).
Cloning of sodC amplicons of Salmonella sp.: The
PCR sodC amplicons of S. Typhi 34, S. Paratyphi 35
and S. Typhimurium 14028 were successfully cloned
into pJET1 and the recombinants were designated
respectively as pJET1-34, pJET1-35 and pJET1-14028.
The restriction digestion analysis of the plasmid DNA
isolated from the recombinants with XhoI or XbaI and
both resulted in 3.7 Kb band with single digestion and
3.2 and 0.5 Kb bands with double digestion confirming
the insertion of the sodC into the 3.2 K pJET1 vector
(Fig. 3).
Sequencing of the clones: The nucleotide sequences
of sodC of the three Salmonella sp. was subjected
for BLAST analysis with sodC sequences of
Fig. 2. Ethidium bromide stained 1 per cent agarose gel showing
the amplicons of sodC gene by PCR Lane 1. DNA ladder; Lane 2.
sodC amplicon of S. Typhimurium 14028; Lane 3. sodC amplicon
of S. Typhi 34; Lane 4. sodC amplicon of S. Paratyphi 35.
S. Typhimurium retrieved from NCBI GenBank.
The protein translation of the nucleotide sequences
was done in EXPASY. The nucleotide sequences
of all the three Salmonella sp., S. Typhimurium
14028 (standard strain), S. Typhi 34 isolate, S.
Paratyphi 35 isolate are deposited in NCBI GenBank
with accession numbers EU158189, EU181430,
EU181429 respectively.
Sequence homology of sodC from Salmonella sp.: The
sodC sequence of S. Typhi and S. Paratyphi isolates
differed at only one position at 489, while the sodC
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INDIAN J MED RES, April 2010
sequence of S. Typhimurium 14028 differed with the
other two at six positions (57, 213, 297, 318, 374 and
494) (Figs 4 and 5). Hundred per cent amino acid
sequence homology was observed between the two
isolates compared to the standard S. Typhimurium
14028. Three amino acid variations were found at
position 71 asparagine (N) was replaced by lysine (K),
at position 125, serine (S) was replaced by threonine
(T), and at position 165, methionine (M) was replaced
by threonine (T).
Comparison of amino acid sequences of Salmonella
isolates with other sodC containing bacteria: Multiple
alignments of amino acid sequence of SodC from isolated
S. Typhi, S. Paratyphi and standard strain S. Typhimurium
14028 with the SodC sequences of Enterobacteriaceae,
upper respiratory tract pathogens and M. tuberculosis
were carried out. The sequence homology among
these SodC sequences is represented in the form of a
dendrogram (Fig. 6). In this dendrogram isolated S.
Paratyphi 35, S. Typhi 34 and S. Typhi CT18 showed
maximum homology and formed a single cluster along
with standard S. Typhimurium 14028 and S. Paratyphi
A9150. Similarly upper respiratory tract infecting
bacteria along with Vibrio cholerae and S. Typhimurium
LT2 formed another cluster and M. tuberculosis alone
formed a separate cluster. The results indicate that
Enterobacteriaceae members except S. Typhimurium
LT2 and B. abortus showed more homogeneity in their
amino acid sequence of SodCs.
Discussion
Fig. 3. Ethidium bromide stained 1 per cent agarose gel showing
restriction digestion analysis of sodC cloned in pJET1 (pJET114028, pJET1-34 and pJET1-35).
Lane 1. uncut pJET1-14028; Lane 2. XhoI digested pJET1-14028;
Lane 3. XbaI digested pJET1-14028; Lane 4. XhoI and XbaI
digested pJET1-14028; Lane 5. DNA ladder; Lane 6. XhoI and
XbaI digested pJET1-34; Lane 7. XbaI digested pJET1-34; Lane
8. XhoI digested pJET1-34; Lane 9. uncut pJET1-34; Lane 10.
uncut pJET1-35; Lane 11. XhoI digested pJET1-35; Lane 12. XbaI
digested pJET1-35; Lane 13. XhoI and XbaI digested pJET1-35;
Lane 14. DNA ladder.
SodC has been reported from a variety of pathogenic
Gram-negative and Gram-positive bacteria2,3,9,10,12,31.
Importance of SodC in protecting the pathogens against
the superoxide radicals generated by inflammatory and
phagocytic cells during infections has been emphasized32.
In this study SodC was detected in 7 of the 20 Salmonella
isolates. Non-detection of SodC in the remaining isolates
may be due to their low expression and/or the instability
of the enzyme due to proteolysis10.
Nucleotide sequence difference between the sodCs
of S. Typhi 34, S. Paratyhi 35, S. Typhimurium 14028
were observed. Transitional and transversional mutations
S. Typhimurium 14028 ST
S. Paratyphi 35
S. Typhi 34
Consensus
S. Typhimurium 14028 ST
S. Paratyphi 35
S. Typhi 34
Consensus
S. Typhimurium 14028 ST
S. Paratyphi 35
S. Typhi 34
Consensus
S. Typhimurium 14028 ST
S. Paratyphi 35
S. Typhi 34
Consensus
Fig. 4. Multiple alignment of sodC nucleotide sequences of S. Typhimurium 14028, S. Typhi 34 and S. Paratyphi 35.
Sanjay et al: sodC in Salmonella sp.
569
S. Typhimurium 14028 ST
S. Paratyphi 35
S. Typhi 34
Consensus
S. Typhimurium 14028 ST
S. Paratyphi 35
S. Typhi 34
Consensus
Fig. 5. Multiple alignment of amino acid sequences of SodC (translated) of S. Typhimurium 14028, S. Typhi 34 and S. Paratyphi 35.
in SodC have been shown to affect the activity of the
enzyme. Though genotypic difference was observed
between the sodC of S. Typhi and S. Paratyphi; no
phenotypic difference was observed in the amino acid
sequences of the enzymes. However, amino acid sequence
of SodC of S. Typhimurium differed. The genotypic and
phenotypic similarities and difference in SodC may
depict the origin of the pathogens. The dendrogram
results of the amino acid sequence multiple alignment of
SodCs from isolated Salmonella sp. including standard
strain with the SodC sequences of Enterobacteriaceae,
upper respiratory tract pathogens and M. tuberculosis
revealed that SodC of enteric bacteria, upper respiratory
tract pathogens and M. tuberculosis are distinguishable
from each other. Therefore SodC of pathogenic bacteria
could be a novel candidate as phylogenetic marker33.
B. abortus is highly pathogenic which survives
and multiplies in an intracellular environment within
host phagocytic cells. Here the bacteria are exposed
to a lethal flux of oxyradicals and the similarity of
S. Typhimurium sodC to B. abortus sequence may
reflect particular functional similarity. Similar to
other pathogenic microorganisms, Salmonella sp.
is an intracellular pathogen, embedded within host
monocyte-macrophages and the expression of sodC
is of pathogenic significance in this organism. Within
phagocytes, organisms must somehow abrogate the
respiratory burst host defence reaction, either by
inhibiting its initiation or by resisting the bactericidal
action of superoxide and other free radicals produced10.
A periplasmic SOD, appropriately located to dismute
superoxide generated exogenously in this way, can
be considered as an enhancer of bacterial virulence.
Periplasmic Cu,Zn-SOD has been shown to have a
protective role in the case of B. abortus34. Thus analysis
of the sodC and its distribution illustrates the ability of
a genotypic perspective to provide important insights
into the evolution of bacterial virulence.
Acknowledgment
Authors thank Dr C. Jayabaskaran, Department of
Biochemistry, Indian Institute of Science, Bangalore, India, for
providing laboratory facilities. The Co-operation of doctors and
staff of Microbiology Division of Basaveshwara hospital, K.B.N.
hospital, Pooja Diagnostics and Hyderabad-Karnataka Diagnostics
and Medical Research Institute Pvt. Ltd., Gulbarga in providing
clinical samples is acknowledged.
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Fig. 6. Dendrogram showing SodC amino acid sequence homology
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Paratyphi 35 isolates with other sodC containing bacteria.
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Reprint requests:Dr C.T. Shivannavar, Department of Microbiology, Gulbarga University, Gulbarga 585 106, Karnataka, India
e-mail: ctshiv@gmail.com