Pran K. Chakrabartty

Production and Characterization of Nisin-Like Peptide Produced by a Strain of Lactococcus lactis Isolated from Fermented Milk

An isolate of Lactococcus lactis from fermented milk was found to produce a bacteriocin peptide. The isolate could grow in a medium with an initial pH of 11.0, in which it produced the bacteriocin extracellularly at the highest level. The level of the bacteriocin in the medium increased in parallel to the bacterial growth and reached its peak during the late exponential phase; thereafter it plateaued. The bacteriocin had a broad antibacterial spectrum similar to that of nisin and inhibited several related species of lactic acid bacteria and other Gram-positive bacteria. The inhibitory activity of the bacteriocin was found to be stable over a wide range of pH and temperature. The molecular weight of the peptide was judged to be 2.5 kDa by SDS-polyacrylamide gel electrophoresis.

Iron acquisition during growth in an iron-deficient medium by Rhizobium sp. isolated from Cicer arietinum

A catechol-type siderophore was produced extracellularly by Rhizobium sp. strain BICC 651 isolated from Cicer arietinum during growth in iron-deficient medium. Production of the siderophore was fully repressed in the presence of 50 μM Fe3+. The siderophore was purified and characterized as containing 2,3-dihydroxybenzoic acid (DHBA) as the core compound and threonine as its conjugate. The siderophore was able to reverse growth inhibition of the strain induced by ethylenediamine-di(o-hydroxyphenyl-acetic acid) (EDDA). A high-affinity iron-transport system capable of transporting 59Fe-siderophore complex was also induced in Rhizobium BICC 651 grown under iron deficiency. Two protein bands of molecular masses 76 and 82 kDa were also inductively synthesized in the outer membrane of the cells. A partially purified ferrireductase enzyme of Rhizobium BICC 651 catalysed reductive release of iron from the ferric chelate of DHBA. The enzyme had a K m of 0.3 mM for ferri-DHBA, was constitutive in nature, and was present in the cytosolic fraction during growth under both iron-deficient and iron-sufficient conditions. The enzyme occurred as two isoenzymes with R F values of 0.48 and 0.51, respectively, in a nondenaturing polyacrylamide gel.

Effect of Aluminum on the Production of Siderophore by Rhizobium sp. (Cicer arietinum)

Abstract.Rhizobium sp. strain BICC 651 in the presence of 100 μM Al3+ produced a threefold higher level of siderophore than in the control culture under iron limitation during the stationary phase. Al3+ in increasing concentrations resulted in decreased growth, and the effect was alleviated by the addition of iron. Siderophore production decreased gradually in Al3+-treated culture as well as in the control with the addition of increasing concentrations of Fe3+, and at 50 μM Fe3+ the level of siderophore was practically undetectable. The siderophore binds Fe3+ and also Al3+. The outer membrane protein profiles of the bacteria grown in the presence or absence of Al3+ were indistinguishable.

Succinate-mediated catabolite repression of enzymes of glucose metabolism in root-nodule bacteria

Enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways were detected in strains ofRhizobium andBradyrhizobium cultured on glucose. The enzymes, except glyceraldehyde-3-phosphate dehydrogenase, were present only in trace amounts in succinategrown cells. The enzymes of the pentose phosphate pathway, being absent inBradyrhizobium, were detected only in glucose-grown cells ofRhizobium. The presence of the glucose-catabolic enzymes in cells only during growth on glucose suggests that they are inducible in nature. Succinate repressed the glucose catabolic enzymes, and the repression appeared to be similar to catabolite repression. Exogenous addition of cAMP caused no change in the activity of these enzymes, demonstrating that the repression was unlikely to be mediated via cAMP.

Cytochromes in free-living rhizobia

Difference spectra of the crude cell-free extracts of 22 strains of fast-growing and slow-growing rhizobia, including members of the cowpea group, reveal the presence of cytochromes c, b, aa3, o, and a soluble CO-reactive hemoprotein P-428 in all of them. No strict correlation between the growth properties of the strains and their cytochrome contents are observed. All the pigments are present in varying quantities at all stages of growth ofRhizobium meliloti SU 216 andBradyrhizobium species (Lupinus) RL3001. A progressive increase in the level of the pigments is observed with the progress of bacterial growth until an optimum concentration is reached, whereupon the level tends to decrease. However, the ratio of cytochrome c to cytochrome b increases linearly throughout the growth period. Cytochromes b, c552, aa3, and o are particle bound, whereas cytochrome c550 and P-428 are soluble.

Metabolism of glucose and gluconate in fast- and slow-growing rhizobia

Abstract Enzymatic evidence was sought for the operation of pathways involved in glucose and gluconate catabolisms in fast- and slow-growing Rhizobium species including members of the cowpea group. Enzymes of the Entner-Doudoroff pathway, pentose phosphate pathway and tricarboxylic acid cycle were detected in fast-growing rhizobia but the pentose phosphate pathway was absent in slow-growers, regardless of the carbon source used. When analysed for enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways in glucose-grown cells, the pathways were found to operate simultaneously in rhizobia.

Genome size variation of rhizobia

Slow-growing species ofRhizobium have a genome size almost double that of the fast-growing species. This provides additional support in favor of their being placed in separate genera.

Cloning, overexpression, and characterization of a novel alkali-thermostable xylanase from Geobacillus sp. WBI

An endo‐β‐1,4‐xylanase gene xynA of a thermophilic Geobacillus sp. WBI from “hot” compost was isolated by PCR amplification. The gene encoding 407 residues were overexpressed in E. coli and purified by Ni‐NTA chromatography. The purified enzyme (47 kDa) had a broad pH optimum of 6.0 to 9.0, and was active between 50 and 90 °C. The enzyme retained 100% of its activity when incubated at 65 °C for 1 h under alkaline condition (pH 10.0) and retained 75% activity at pH 11.0. The Km and Vmax of the enzyme were 0.9 mg ml−1 and 0.8 µmol ml−1 min−1, respectively. In molecular dynamics simulation at 338 K (65 °C), the enzyme was found to be stable. At an elevated temperature (450 K) specific α‐helix and β‐turns of the proteins were most denatured. The denaturation was less in WBI compared with its highest homolog G. stearothermophilus T‐6 xylanase with difference of six residues. The results predict that these regions are responsible for the improved thermostability observed over related enzymes. The present work encourages further experimental demonstration to understand how these regions contribute thermostability to WBI xylanase. The study noted that WBI produces a xylanase with unique characteristics, specifically alkali‐thermostability.

Enzymes of carbohydrate metabolism in root‐nodule bacteria during growth on acetate

The key enzymes of the Embden‐Meyerhof‐Parnas (EMP), Entner‐Doudoroff (ED) and pentose phosphate (PP) pathways as well as those of the tricarboxylic acid (TCA) cycle and gluconeogenesis were assayed in the cells of Rhizobium and Bradyrhizobium grown on acetate. Except for glyceraldehyde‐3‐phosphate dehydrogenase, the activities of all the enzymes of the EMP, ED and PP pathways were found to be low in acetate grown cells in the present study as compared to those of glucose grown cells reported by us earlier (Mandal and Chakrabartty 1993) indicating a probable repression of the enzymes. The enzymes of the tricarboxylic acid cycle were detected in high levels in the acetate grown cells of the root‐nodule bacteria. The operation of the glyoxylate pathway for acetate metabolism in the root‐nodule bacteria were reported previously by us (Mandal and Chakrabartty 1992). The evidences taken together point to the simultaneous operation of both the TCA cycle and the glyoxylate cycle for acetate metabolism in these bacteria.

Characterization of an rpoN mutant of Mesorhizobium ciceri

Aims:  To study the genetic basis of C4‐dicarboxylate transport (Dct) in relation to symbiotic nitrogen fixation in Mesorhizobium ciceri.