Cadmium biosorption by yeast, Candida tropicalis CBL-1, isolated from industrial wastewater

The present study is aimed at assessing the ability of metal-resistant yeast, Candida tropicalis CBL-1, to uptake metal from liquid medium. The minimum inhibitory concentration of Cd(II) against Candida tropicalis CBL-1 was 2,800 mg/L. The yeast could also tolerate Zn(II) (3,100 mg/L), Hg(II) (2,400 mg/L), Ni(II) (2,200 mg/L), Cr(VI) (2,000 mg/L), Pb(II) (1,100 mg/L), and Cu(II) (2,200 mg/L). The yeast isolate showed typical growth curves but lag and log phases extended in the presence of cadmium. The yeast isolate showed optimum growth at 30oC and pH 7. The metal processing ability of the isolate was determined in a medium containing 100 mg/L of Cd(II). Candida tropicalis CBL-1, could reduce Cd(II) 59%, 64% and 70% from the medium after 48, 96 and 144 h, respectively. C. tropicalis CBL-1 was also able to remove Cd(II) 46% and 60% from the wastewater after 6 and 12 days, respectively. Cd produced an increase in glutathione and non-protein thiols level by 37% (17.50±0.8-24.0±1.2) and 18% (3.30±0.7- 3.90±0.8) at 100 mg/L concentration, respectively. Metal tolerance and accumulation together with changes in the GSH status and non-protein thiols under Cd exposure were studied in C. tropicalis.     

Rhizobacterial potential to alter auxin content and growth of <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.)

Potential of non-symbiotic plant growth promoting rhizobacteria (PGPR) to influence the endogenous indole-3-acetic acid (IAA) content and growth of Vigna radiata (L.) was evaluated. The bacterial strains used belonged to Pseudomonas, Escherichia, Micrococcus and Staphylococcus genera. All strains were able to produce IAA (1.16–8.22 μg ml⁻¹) in the presence of 1,000 μg ml⁻¹ of l-tryptophan as revealed by gas chromatography and mass spectrometric (GC–MS) analysis. However, strains exhibited variable results for other growth promoting traits such as phosphate solubilization and siderophore or hydrogen cyanide production. Bacterial IAA production showed significant positive correlation with endogenous IAA content of roots (r = 0.969; P = 0.01) and leaves (r = 0.905; P = 0.01) under axenic conditions. Bacterization of V. radiata seeds significantly enhanced shoot length (up to 48.10%) and shoot fresh biomass (up to 43.80%) under fully axenic conditions. Bacterial strains applied under wire-house conditions also improved shoot length, number of pods, and grain weight up to 58, 65, and 17.15% respectively, over control. Hence, free living (non-symbiotic) PGPR have the ability to influence endogenous IAA content and growth of leguminous plants.     

Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway

Riboflavin (vitamin B₂) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction.Riboflavin is the substrate for biosynthesis of the essential flavocoenzymes FMN and FAD, which occur in all kingdoms of life and have roles in diverse redox reactions as well as in other processes such as DNA repair, light sensing, and bioluminescence (Fischer and Bacher, 2005). Plants and many microorganisms can synthesize riboflavin, but humans and other animals cannot, so they must obtain it from the diet (Powers, 2003). Plant foods are important sources of riboflavin for humans, and the riboflavin pathway is a target for engineering biofortified crops (Fitzpatrick et al., 2012).Riboflavin biosynthesis proceeds via the same pathway in bacteria and plants (Fischer and Bacher, 2005; Roje, 2007). This pathway starts from GTP, which is converted by GTP cyclohydrolase II (named RibA in Escherichia coli) to the pyrimidine derivative 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-P. Deamination of the pyrimidine ring then yields 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-P, and subsequent reduction of the ribosyl moiety gives 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-P. After dephosphorylation, this product is condensed with 3,4-dihydroxy-2-butanone 4-P to give 6,7-dimethyl-8-ribityllumazine, whose dismutation yields riboflavin. Figure 1 shows the first four steps of this pathway.Open in a separate windowFigure 1.The first four steps of the riboflavin biosynthesis pathway in bacteria and plants. The enzymes involved are GTP cyclohydrolase II (RibA), pyrimidine deaminase (Deam), pyrimidine reductase (Red), and a specific phosphatase (Pase). Enzymes for which the plant genes are not known are colored red. Intermediates are as follows: 1, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-P; 2, 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-P; 3, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-P; 4, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.In E. coli, the deamination and reduction steps are catalyzed by a single bifunctional enzyme, RibD, which has N-terminal deaminase and C-terminal reductase domains that retain their respective activities when expressed separately (Richter et al., 1997; Magalhães et al., 2008). The situation in plants seems superficially similar but is in fact more complex (Gerdes et al., 2012). The bidomain bacterial RibD protein has two types of homologs in plants (Fischer et al., 2004; Chatwell et al., 2006; Chen et al., 2006), here called PyrD and PyrR, both with apparent deaminase and reductase domains (Fig. 2A). Only PyrD, represented by At4g20960, has been studied biochemically; it was found to have pyrimidine deaminase but not reductase activity (Fischer et al., 2004). The function of PyrR, represented by At3g47390, is unknown, although it has been inferred to have reductase activity (Chatwell et al., 2006; Chen et al., 2006; Ouyang et al., 2010) and perhaps to lack deaminase activity (Ouyang et al., 2010). Another mystery surrounding PyrR proteins is the presence of an extra C-terminal domain of unknown function (COG3236 in the Clusters of Orthologous Groups database; Fig. 2A); this domain occurs as a stand-alone protein in many bacteria. One possibility is that it catalyzes the dephosphorylation that follows the pyrimidine reduction step in the pathway (Fig. 1). The phosphatase involved is most likely substrate specific, but it has not been identified in plants or any other organism (Roje, 2007; Gerdes et al., 2012), and genes for enzymes in the same pathway, especially for successive steps, are quite commonly fused (Suhre, 2007). A mutation (phs1) that deleted the COG3236 domain from Arabidopsis (Arabidopsis thaliana) PyrR resulted in a photosensitive phenotype that could be rescued by supplied FAD (Ouyang et al., 2010).Open in a separate windowFigure 2.Structure and phylogeny of plant PyrD and PyrR proteins. A, Domain architectures. The examples shown are Arabidopsis At4g20960 and At3g47390; the predicted plastid targeting peptide (TP) varies in length between species. B, Phylogenetic tree of PyrD and PyrR proteins. Sequences were aligned with ClustalW; the tree was built by the neighbor-joining method with MEGA5. Bootstrap values (%) for 1,000 replicates are next to the nodes. Only the tree topology is shown. Note that the PyrD proteins of green algae (underlined) lack a reductase domain. C, Alignments showing the conservation of the zinc-binding residues (arrowheads) in the deaminase domain of PyrD but not PyrR proteins and the conservation of the predicted substrate-binding residues (asterisks) in the reductase domain of PyrR but not PyrD proteins. The deaminase sequences correspond to residues 45 to 85 of B. subtilis RibD (synonym RibG); the reductase sequences correspond to residues 150 to 210 and (separated by dots) 288 to 292 of B. subtilis RibD. Identical zinc- or substrate-binding residues are black, and conservative replacements are gray. Dashes indicate gaps that maximize the alignment.The plant riboflavin synthesis pathway is considered to be plastidial (Roje, 2007), but this location is based almost solely on bioinformatics and high-throughput proteome analyses (Gerdes et al., 2012). In only one case is there more definitive experimental support: in vitro chloroplast import data for the pathway’s penultimate enzyme, 6,7-dimethyl-8-ribityllumazine synthase (Jordan et al., 1999). Similarly, clear genetic support for the function of most plant riboflavin synthesis enzymes is lacking (Gerdes et al., 2012), the exception being an Arabidopsis RibA homolog (Hedtke and Grimm, 2009).The work reported here established, using maize (Zea mays) and Arabidopsis, that PyrR is indeed the missing pyrimidine reductase, that it lacks deaminase activity, and that its COG3236 domain is not essential for pyrimidine reductase activity and most likely lacks phosphatase activity. We also demonstrated the import of PyrR and PyrD into chloroplasts in vitro and confirmed that the gene for PyrR is essential.     

Aeromonas punctata PNS-1: a promising candidate to change the root morphogenesis of Arabidopsis thaliana in MS and sand system

A model system of sand, comprising Arabidopsis plants inoculated with Aeromonas punctata PNS-1 strain, was used to evaluate the bacterial effect in modulation of plant root structure at second-order lateral root level. In MS media, the root morphogenesis was changed only at first-order lateral root level when inoculated with PNS-1 strain. Inoculation with PNS-1 strain was subjected to significant (P < 0.01) increase in primary root length and lateral root density in both MS and sand system. However, this strain modulated the root structure in the sand environment in a complex manner that may be helpful for incitation of the plant–microbe interaction close to natural environment. In order to determine whether this change in root morphology was due to bacterial auxin, Arabidopsis transgenic line (DR5:GUS) was used to reveal the change in homeostasis of endogenous auxin. In PNS-1 inoculated transgenic seedlings of Arabidopsis plant (DR5:GUS), endogenous auxin in primary root apices and lateral roots was enhanced. For confirmation, PNS-1 was evaluated for auxin production in vitro, showed an increase in auxin production after supplementation of l-tryptophan. The presence of ACC deaminase activity in PNS-1 showed its possible involvement in primary root elongation. In the present study Aeromonas punctata PNS-1 is the potential candidate for triggering the change in root morphogenesis of Arabidopsis thaliana with the involvement of auxin and ACC deaminase production.     

Spodoptera frugiperda FKBP‐46 is a consensus p53 motif binding protein

p53 protein, the central molecule of the apoptosis pathway, is mutated in 50% of the human cancers. Of late, p53 homologues have been identified from different invertebrates including Drosophila melanogaster, Caenorhabditis elegans, Squid, and Clams. We report the identification of a p53‐like protein in Spodoptera frugiperda (Sf9) insect cells, which is activated during oxidative stress, caused by exposure to UV‐B or H₂O₂, and binds to p53 consensus DNA binding motifs as well as other p53 cognate motifs. Sf9 p53 motif‐binding protein is similar to murine and Drosophila p53 in terms of molecular size, which is around 50–60 kDa, as evident from UV cross‐linking, and displays DNA binding characteristics similar to both insect and vertebrate p53 as seen from electrophoretic mobility shift assays. The N‐terminal sequencing of the purified Sf9 p53 motif‐binding protein reveals extensive homology to the pro‐apoptotic FK‐506 binding protein (FKBP‐46), earlier identified in Sf9 cells as a factor which interacts with murine casein kinase. FKBP, an evolutionarily conserved protein of mammalian origin functions as a pro‐apoptotic factor. Identification of FKBP‐46 as a novel p53 motif‐binding protein in insect cells adds a new facet to our understanding of the mechanisms of apoptosis under oxidative stress in the absence of a typical p53 homologue. J. Cell. Biochem. 114: 899–907, 2013. © 2012 Wiley Periodicals, Inc.     

The translation initiation factor, PeIF5B, from Pisum sativum displays chaperone activity

We earlier documented the structural and functional characterization of PeIF5B factor from Pisum sativum that shows strong homology to the universal translation initiation factor eIF5B (Rasheedi et al., 2007, 2010 [12] and [13]). We now show that PeIF5B is an unusually thermo-stable protein resisting temperatures up to 95 °C. PeIF5B prevents thermal aggregation of heat labile proteins, such as citrate synthase (CS) and NdeI, under heat stress or chemical denaturation conditions and promotes their functional folding. It also prevents the aggregation of DTT induced insulin reduction. GTP appears to stimulate PeIF5B-mediated chaperone activity. In-vivo, PeIF5B over expression significantly enhances, the viability of Escherichia coli cells after heat stress (50 °C). These observations lead us to conclude that PeIF5B, in addition to its role in protein translation, has chaperone like activity and could be likely involved in protein folding and protection from stress.     

A New Coarse-Grained Model for E. coli Cytoplasm: Accurate Calculation of the Diffusion Coefficient of Proteins and Observation of Anomalous Diffusion

A new coarse-grained model of the E. coli cytoplasm is developed by describing the proteins of the cytoplasm as flexible units consisting of one or more spheres that follow Brownian dynamics (BD), with hydrodynamic interactions (HI) accounted for by a mean-field approach. Extensive BD simulations were performed to calculate the diffusion coefficients of three different proteins in the cellular environment. The results are in close agreement with experimental or previously simulated values, where available. Control simulations without HI showed that use of HI is essential to obtain accurate diffusion coefficients. Anomalous diffusion inside the crowded cellular medium was investigated with Fractional Brownian motion analysis, and found to be present in this model. By running a series of control simulations in which various forces were removed systematically, it was found that repulsive interactions (volume exclusion) are the main cause for anomalous diffusion, with a secondary contribution from HI.     

Molecular structure of serum transferrin at 3.3-A resolution

Serum transferrin is a metal-binding glycoprotein, molecular weight ca. 80,000, whose primary function is the transport of iron in the plasma of vertebrates. The X-ray crystallographic structure of diferric rabbit serum transferrin has been determined to a resolution of 3.3 A. The molecule has a beta alpha structure of similar topology to human lactoferrin and is composed of two homologous lobes that each bind a single ferric ion. Each lobe is further divided into two dissimilar domains, and the iron-binding site is located within the interdomain cleft. The iron is bound by two tyrosines, a histidine, and an aspartic acid residue. The location of the 19 disulfide bridges is described, and their possible structural roles are discussed in relation to the transferrin family of proteins. Mapping of the intron/exon splice junctions onto the molecule provides some topological evidence in support of the putative secondary role for transferrin in stimulating cell proliferation.     

Electron donation between copper containing nitrite reductases and cupredoxins: the nature of protein-protein interaction in complex formation.

In denitrifying organisms with copper containing dissimilatory nitrite reductases, electron donation from a reduced cupredoxin is an essential step in the reduction of nitrite to nitric oxide. Copper nitrite reductases are categorised into two subgroups based on their colour, green and blue, which are found in organisms where the cupredoxins are pseudoazurins and azurins, respectively. In view of this and some in vitro electron donation experiments, it has been suggested that copper nitrite reductases have specific electron donors and that electron transfer takes place in a specific complex of the two proteins. We report results from the first comprehensive electron donation experiments using three copper nitrite reductases, one green and two blue, and five cupredoxins, one pseudoazurin and four azurins. Our data show that pseudoazurin can readily donate electrons to both blue and green copper nitrite reductases. In contrast, all of the azurins react very sluggishly as electron donors to the green nitrite reductase. These results are discussed in terms of surface compatibility of the component proteins, complex formation, overall charges, charge distribution, hydrophobic patches and redox potentials. A docking model for the complexes is proposed.     

Polyphasic Taxonomic Analysis Establishes Mycobacterium indicus pranii as a Distinct Species

Background

Mycobacterium indicus pranii (MIP), popularly known as Mw, is a cultivable, non-pathogenic organism, which, based on its growth and metabolic properties, is classified in Runyon Group IV along with M. fortuitum, M. smegmatis and M. vaccae. The novelty of this bacterium was accredited to its immunological ability to undergo antigen driven blast transformation of leukocytes and delayed hypersensitivity skin test in leprosy patients, a disease endemic in the Indian sub-continent. Consequently, MIP has been extensively evaluated for its biochemical and immunological properties leading to its usage as an immunomodulator in leprosy and tuberculosis patients. However, owing to advances in sequencing and culture techniques, the citing of new strains with almost 100% similarity in the sequences of marker genes like 16S rRNA, has compromised the identity of MIP as a novel species. Hence, to define its precise taxonomic position, we have carried out polyphasic taxonomic studies on MIP that integrate its phenotypic, chemotaxonomic and molecular phylogenetic attributes.

Methodology/Principal Findings

The comparative analysis of 16S rRNA sequence of MIP by using BLAST algorithm at NCBI (nr database) revealed a similarity of ≥99% with M. intracellulare, M. arosiense, M. chimaera, M. seoulense, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis and M. bohemicum. Further analysis with other widely used markers like rpoB and hsp65 could resolve the phylogenetic relationship between MIP and other closely related mycobacteria apart from M. intracellulare and M. chimaera, which shares ≥99% similarity with corresponding MIP orthologues. Molecular phylogenetic analysis, based on the concatenation of candidate orthologues of 16S rRNA, hsp65 and rpoB, also substantiated its distinctiveness from all the related organisms used in the analysis excluding M. intracellulare and M. chimaera with which it exhibited a close proximity. This necessitated further analysis of MIP with more sensitive and segregating parameters to ascertain its precise taxonomic position as a new species. The analysis of MIP and its comparison with other mycobacterial reference strains based on cellular and biochemical features, growth characteristics and chemotaxonomic studies like FAME profiling confirmed that MIP is uniquely endowed with diverse metabolic attributes that effectively distinguishes it from all the closely related mycobacteria including M. intracellulare and M. chimaera.

Conclusion

The results presented in this study coupled with the non-pathogenic nature and different biochemical and immunomodulatory properties of MIP affirm it as a distinct species belonging to M. avium complex (MAC). It is further proposed to use an earlier suggested name Mycobacterium indicus pranii for this newly established mycobacterial species. This study also exemplifies the growing need for a uniform, consensus based broader polyphasic frame work for the purpose of taxonomy and speciation, particularly in the genus Mycobacterium.