Bhakti Basu

The Kdp-ATPase system and its regulation

K+, the dominant intracellular cation, is required for various physiological processes like turgor homeostasis, pH regulation etc. Bacterial cells have evolved many diverse K+ transporters to maintain the desired concentration of internal K+. In E. coli, the KdpATPase (comprising of the KdpFABC complex), encoded by the kdpFABC operon, is an inducible high-affinity K+ transporter that is synthesised under conditions of severe K+ limitation or osmotic upshift. The E. coli kdp expression is transcriptionally regulated by the KdpD and KdpE proteins, which together constitute a typical bacterial two-component signal transduction system. The Kdp system is widely dispersed among the different classes of bacteria including the cyanobacteria. The ordering of the kdpA, kdpB and kdpC is relatively fixed but the kdpD/E genes show different arrangements in distantly related bacteria. Our studies have shown that the cyanobacterium Anabaena sp. strain L-31 possesses two kdp operons, kdp1 and kdp2, of which, the later is expressed under K+ deficiency and desiccation. Among the regulatory genes, the kdpD ORF of Anabaena L-31 is truncated when compared to the kdpD of other bacteria, while a kdpE-like gene is absent. The extremely radio-resistant bacterium, Deinococcus radiodurans strain R1, also shows the presence of a naturally short kdpD ORF similar to Anabaena in its kdp operon. The review elaborates the expression of bacterial kdp operons in response to various environmental stress conditions, with special emphasis on Anabaena. The possible mechanism(s) of regulation of the unique kdp operons from Anabaena and Deinococcus are also discussed.

Gamma Radiation-induced Proteome of Deinococcus radiodurans Primarily Targets DNA Repair and Oxidative Stress Alleviation

The extraordinary radioresistance of Deinococcus radiodurans primarily originates from its efficient DNA repair ability. The kinetics of proteomic changes induced by a 6-kGy dose of gamma irradiation was mapped during the post-irradiation growth arrest phase by two-dimensional protein electrophoresis coupled with mass spectrometry. The results revealed that at least 37 proteins displayed either enhanced or de novo expression in the first 1 h of post-irradiation recovery. All of the radiation-responsive proteins were identified, and they belonged to the major functional categories of DNA repair, oxidative stress alleviation, and protein translation/folding. The dynamics of radiation-responsive protein levels throughout the growth arrest phase demonstrated (i) sequential up-regulation and processing of DNA repair proteins such as single-stranded DNA-binding protein (Ssb), DNA damage response protein A (DdrA), DNA damage response protein B (DdrB), pleiotropic protein promoting DNA repair (PprA), and recombinase A (RecA) substantiating stepwise genome restitution by different DNA repair pathways and (ii) concurrent early up-regulation of proteins involved in both DNA repair and oxidative stress alleviation. Among DNA repair proteins, Ssb was found to be the first and most abundant radiation-induced protein only to be followed by alternate Ssb, DdrB, indicating aggressive protection of single strand DNA fragments as the first line of defense by D. radiodurans, thereby preserving genetic information following radiation stress. The implications of both qualitative or quantitative and sequential or co-induction of radiation-responsive proteins for envisaged DNA repair mechanism in D. radiodurans are discussed.

Methyl viologen responsive proteome dynamics of Anabaena sp. strain PCC7120

A proteomic approach was employed to elucidate the response of an agriculturally important microbe, Anabaena sp. strain PCC7120, to methyl viologen (MV). Exposure to 2 μM MV caused 50% lethality (LD50) within 6 h and modified the cellular levels of several proteins. About 31 proteins increased in abundance and 24 proteins decreased in abundance, while 55 proteins showed only a minor change in abundance. Of these, 103 proteins were identified by MS. Levels of proteins involved in ROS detoxification and chaperoning activities were enhanced but that of crucial proteins involved in light and dark reactions of photosynthesis declined or constitutive. The abundance of proteins involved in carbon and energy biogenesis were altered. The study elaborated the oxidative stress defense mechanism deployed by Anabaena, identified carbon metabolism and energy biogenesis as possible major targets of MV sensitivity, and suggested potential biotechnological interventions for improved stress tolerance in Anabaena 7120.

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.

Repression of the glucose-inducible outer- membrane protein OprB during utilization of aromatic compounds and organic acids in Pseudomonas putida CSV86

Pseudomonas putida CSV86 shows preferential utilization of aromatic compounds over glucose. Protein analysis and [¹⁴C]glucose-binding studies of the outer membrane fraction of cells grown on different carbon sources revealed a 40 kDa protein that was transcriptionally induced by glucose and repressed by aromatics and succinate. Based on 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis, the 40 kDa protein closely resembled the porin B of P. putida KT2440 and carbohydrate-selective porin OprB of various Pseudomonas strains. The purified native protein (i) was estimated to be a homotrimer of 125 kDa with a subunit molecular mass of 40 kDa, (ii) displayed heat modifiability of electrophoretic mobility, (iii) showed channel conductance of 166 pS in 1 M KCl, (iv) permeated various sugars (mono-, di- and tri-saccharides), organic acids, amino acids and aromatic compounds, and (v) harboured a glucose-specific and saturable binding site with a dissociation constant of 1.3 µM. These results identify the glucose-inducible outer-membrane protein of P. putida CSV86 as a carbohydrate-selective protein OprB. Besides modulation of intracellular glucose-metabolizing enzymes and specific glucose-binding periplasmic space protein, the repression of OprB by aromatics and organic acids, even in the presence of glucose, also contributes significantly to the strains ability to utilize aromatics and organic acids over glucose.

Modulation of Glucose Transport Causes Preferential Utilization of Aromatic Compounds in Pseudomonas putida CSV86

Pseudomonas putida CSV86 utilizes aromatic compounds in preference to glucose and coutilizes aromatics and organic acids. Protein analysis of cells grown on different carbon sources, either alone or in combination, revealed that a 43-kDa periplasmic-space protein was induced by glucose and repressed by aromatics and succinate. Two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis identified this protein as closely resembling the sugar ABC transporter of Pseudomonas putida KT2440. A partially purified 43-kDa protein showed glucose binding activity and was specific for glucose. The results demonstrate that the aromatic- and organic acid-mediated repression of a periplasmic-space glucose binding protein and consequent inhibition of glucose transport are responsible for this strains ability to utilize aromatics and organic acids in preference to glucose.

A novel serralysin metalloprotease from Deinococcus radiodurans

A hypothetical protein (DR2310) from the radiation resistant organism Deinococcus radiodurans harbors highly conserved Zn+2-binding (HEXXH) domain and Met-turn (SVMSY), characteristic of the serralysin family of secreted metalloproteases from Gram negative bacteria. Deletion mutagenesis of DR2310 confirmed that the ORF is expressed in Deinococcus radiodurans as a secreted protease of 85 kDa. Biochemical analysis revealed DR2310 to be a Ca+2 and Zn+2-requiring metalloprotease. Unique features such as a long N-terminus, replacement of the highly conserved C-terminal glycine rich Ca+2-binding repeats with a single N-terminal aspartate rich eukaryotic thrombospondin type-3 Ca+2-binding repeat and absence of C-terminal secretion signals make it a novel member of serralysin family. This is the first report of a functional serralysin family metalloprotease from a Gram positive organism.

Depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite toxicity in Deinococcus radiodurans

Oxidative stress resistant Deinococcus radiodurans surprisingly exhibited moderate sensitivity to tellurite induced oxidative stress (LD50 = 40 μM tellurite, 40 min exposure). The organism reduced 70% of 40 μM potassium tellurite within 5 h. Tellurite exposure significantly modulated cellular redox status. The level of ROS and protein carbonyl contents increased while the cellular reduction potential substantially decreased following tellurite exposure. Cellular thiols levels initially increased (within 30 min) of tellurite exposure but decreased at later time points. At proteome level, tellurite resistance proteins (TerB and TerD), tellurite reducing enzymes (pyruvate dehydrogense subunits E1 and E3), ROS detoxification enzymes (superoxide dismutase and thioredoxin reductase), and protein folding chaperones (DnaK, EF‐Ts, and PPIase) displayed increased abundance in tellurite‐stressed cells. However, remarkably decreased levels of key metabolic enzymes (aconitase, transketolase, 3‐hydroxy acyl‐CoA dehydrogenase, acyl‐CoA dehydrogenase, electron transfer flavoprotein alpha, and beta) involved in carbon and energy metabolism were observed upon tellurite stress. The results demonstrate that depletion of reduction potential in intensive tellurite reduction with impaired energy metabolism lead to tellurite toxicity in D. radiodurans.

Proteomic analysis reveals contrasting stress response to uranium in two nitrogen-fixing Anabaena strains, differentially tolerant to uranium

Two strains of the nitrogen-fixing cyanobacterium Anabaena, native to Indian paddy fields, displayed differential sensitivity to exposure to uranyl carbonate at neutral pH. Anabaena sp. strain PCC 7120 and Anabaena sp. strain L-31 displayed 50% reduction in survival (LD50 dose), following 3h exposure to 75μM and 200μM uranyl carbonate, respectively. Uranium responsive proteome alterations were visualized by 2D gel electrophoresis, followed by protein identification by MALDI-ToF mass spectrometry. The two strains displayed significant differences in levels of proteins associated with photosynthesis, carbon metabolism, and oxidative stress alleviation, commensurate with their uranium tolerance. Higher uranium tolerance of Anabaena sp. strain L-31 could be attributed to sustained photosynthesis and carbon metabolism and superior oxidative stress defense, as compared to the uranium sensitive Anabaena sp. strain PCC 7120. SIGNIFICANCE Uranium responsive proteome modulations in two nitrogen-fixing strains of Anabaena, native to Indian paddy fields, revealed that rapid adaptation to better oxidative stress management, and maintenance of metabolic and energy homeostasis underlies superior uranium tolerance of Anabaena sp. strain L-31 compared to Anabaena sp. strain PCC 7120.

In situ real-time evaluation of radiation-responsive promoters in the extremely radioresistant microbe Deinococcus radiodurans.

ABSTRACTA third generation promoter probe shuttle vector pKG was constructed, using the green fluorescent protein as a reporter, for in situ evaluation of Deinococcal promoter activity in Escherichia coli or Deinococcus radiodurans. The construct yielded zero background fluorescence in both the organisms, in the absence of promoter sequences. Fifteen Deinococcal promoters, either harbouring Radiation and Desiccation Response Motif (RDRM) or not, were cloned in vector pKG. Only the RDRM-promoter constructs displayed (i) gamma radiation inducible GFP expression in D. radiodurans, following gamma irradiation, (ii) DdrO-mediated repression of GFP expression in heterologous E. coli, or (iii) abolition in GFP induction following gamma irradiation, in pprI mutant of D. radiodurans. Utility of pKG vector for real-time in situ assessment of Deinococcal promoter function was, thus, successfully demonstrated.