Detection, distribution and characterization of novel superoxide dismutases from Yersinia enterocolitica Biovar 1A.

BACKGROUND: Superoxide dismutases (SODs) cause dismutation of superoxide radicals to hydrogen peroxide and oxygen. Besides protecting the cells against oxidative damage by endogenously generated oxygen radicals, SODs play an important role in intraphagocytic survival of pathogenic bacteria. The complete genome sequences of Yersinia enterocolitica strains show presence of three different sod genes. However, not much is known about the types of SODs present in Y. enterocolitica, their characteristics and role in virulence and intraphagocytic survival of this organism. METHODOLOGY/PRINCIPAL FINDINGS: This study reports detection and distribution of the three superoxide dismutase (sodA, sodB and sodC) genes in 59 strains of Y. enterocolitica and related species. The majority (94%) of the strains carried all three genes and constitutive expression of sodA and sodB was detected in 88% of the strains. Expression of sodC was not observed in any of the strains. The sodA, sodB and sodC genes of Y. enterocolitica were cloned in pET28a (+) vector. Recombinant SodA (82 kDa) and SodB (21 kDa) were expressed as homotetramer and monomer respectively, and showed activity over a broad range of pH (3.0-8.0) and temperature (4-70°C). SodA and SodB showed optimal activity at 4°C under acidic pH of 6.0 and 4.0 respectively. The secondary structures of recombinant SodA and SodB were studied using circular dichroism. Production of YeSodC was not observed even after cloning and expression in E. coli BL21(DE3) cells. A SodA(-) SodB(-) Escherichia coli strain which was unable to grow in medium supplemented with paraquat showed normal growth after complementation with Y. enterocolitica SodA or SodB. CONCLUSIONS/SIGNIFICANCE: This is the first report on the distribution and characterization of superoxide dismutases from Y. enterocolitica. The low pH optima of both SodA and SodB encoded by Y. enterocolitica seem to implicate their role in acidic environments such as the intraphagocytic vesicles

Maternal vitamin D levels in hypertensive disorders of pregnancy

Background: Current study was conducted with the objective to evaluate maternal vitamin D levels in patients with hypertensive disorders of pregnancy.Methods: The present prospective observational study was conducted among 200 pregnant women attending gynaecological OPD of Subharti medical college, Meerut, over a period of two years and satisfying the inclusion criteria were enrolled. On admission, patient demographic profile, complete history was recorded, and comprehensive clinical examination was done. In all the patients, blood samples for routine examination along with LFT, RFT, random blood sugar, serum electrolytes, serum uric acid and serum vitamin D were drawn, and serum levels of these biochemical parameters were determined according to standard laboratory procedures. Subjects were classified into three categories according to serum vitamin D level i.e. >20 ng/ml (mild), 10-20 ng/ml (moderate deficiency), <10 ng/ml (severe deficiency).Results: Insignificant difference was found between case (hypertensive) and control (normotensive) group when compared in relation to age, education, parity and socioeconomic status. The mean vitamin D level of women with HDP was 5.7+2.99 ng/ml as compared to 17.34+5.79 ng/ml in normotensive women with statistically significant difference as p<0.001. No correlation was found between severity of vitamin D deficiency and maternal complications (p=0.318).Conclusions: The results of the present study concluded that women with hypertension had significantly lower vitamin D level as compared to normotensive women

Molecular weight, activity and pI analysis of recombinant SODs:

<p>(<b>a</b>) SDS–PAGE of recombinant <i>Ye</i>SodA and <i>Ye</i>SodB expressed in pET 28a (+) (samples were resolved on 15% polyacrylamide gel and stained with Coomassie Brilliant Blue R-250). The purified SodA and SodB showed a single band each of 23 KDa and 21 kDa respectively. M1 and M2: Protein marker; Lane 1: SodA; Lane 2 SodB. (<b>b</b>) Molecular weight determination of <i>Ye</i>SodA (82 kDa) and <i>Ye</i>SodB (21 kDa) by Sephacryl S-200 molecular sieve chromatography. The molecular weight of marker proteins (SigmaAldrich) were as follows: β-Amylase (200 kDa), Alcohol dehydrogenase (150 kDa), BSA (66 kDa), Carbonic anhydrase (29 kDa) and Cytochrome C (12.4 kDa). (<b>c</b>) Zymogram analysis showing achromatic bands of <i>Ye</i>SodA and <i>Ye</i>SodB against a dark background. Lane 1: <i>Ye</i>SodA; Lane 2: <i>Ye</i>SodB. (<b>d</b>) Isoelectric point (pI) of purified recombinant <i>Ye</i>SodA and <i>Ye</i>SodB stained with coomassie brilliant blue. M: pI marker; Lane 1: <i>Ye</i>SodA; Lane 2: <i>Ye</i>SodB.</p

Zymogram analysis of superoxide dismutases:

<p>(<b>a</b>) Representative zymogram showing two acromatic bands of crude bacterial lysate from <i>Yersinia</i> spp. (<b>b</b>) Effect of different inhibitors on SOD isoforms of <i>Y. enterocolitica</i>. Lane A: Cell lysate without inhibitor; B: with 5 mM H₂O₂; C: with 5 mM NaN₃; D: with DDTC; E: with chloroform:ethanol.</p

Effect of physical parameters on recombinant SOD activity:

<p>(<b>a</b>) Optimum temperature of <i>Ye</i>SodA and <i>Ye</i>SodB was 4°C (<b>b</b>) while optimum pH was 4.0 and 6.0 respectively. The results are expressed as percent change in the activity of the respective enzyme with the value at optimum temperature and pH taken as 100%.</p

Proposed three dimensional structure:

<p>Predicted 3D structure of (<b>a</b>) SodA and (<b>b</b>) SodB showing metal binding ligands: His27, His82, Asp169 and His 173 in SodA and, His27, His74, Asp157 and His161 in <i>Ye</i>SodB.</p

Sequence homology:

<p>Multiple sequence alignment (MSA) of (<b>a</b>) <i>Ye</i>SodA with <i>E. coli</i> (PDB id: 1VEW), <i>Deinococcus radiodurans</i> (PDB id: 2CDY), <i>B. anthracis</i> (PDB id: 1XUQ) and <i>B. subtilis</i> (PDB id: 2RCV); (<b>b</b>) <i>Ye</i>SodB with <i>E. coli</i> (PDB id: 2NYB<i>), Aliivibrio salmonicida</i> (PDB id: 2W7W), <i>Pseudomonas ovalis</i> (PDB id: 1DT0) and <i>Francisella tularensis</i> (PDB id: 3H1S) drawn using ESPript 2.2. Symbols α and β indicate alpha helices and beta sheets, respectively; η represents turns and TT denotes sharp turns in the structure.</p

Bacterial strains and plasmids used for cloning and expression in this study.

a<p>Prof. Joan S. Valentine, University of California Los Angeles.</p

Secondary structure analysis using circular dichroism:

<p>Far-UV spectra of (<b>a</b>) <i>Ye</i>SodA and (<b>b</b>) <i>Ye</i>SodB at pH 7 and 28°C. Far-UV spectra of (<b>c</b>) <i>Ye</i>SodA and (<b>d</b>) <i>Ye</i>SodB at different pH.</p

Details of primers and the PCR conditions.

<p>Nucleotide bases in bold represent restriction sites. NA: the primer was designed using the sequence of <i>sodC</i> (accession no. JX204785) from strain IP27366 as the template.</p