Transcriptional Modulation of Transport- and Metabolism-Associated Gene Clusters Leading to Utilization of Benzoate in Preference to Glucose in Pseudomonas putida CSV86.

The effective elimination of xenobiotic pollutants from the environment can be achieved by efficient degradation by microorganisms even in the presence of sugars or organic acids. Soil isolate Pseudomonas putida CSV86 displays a unique ability to utilize aromatic compounds prior to glucose. The draft genome and transcription analyses revealed that glucose uptake and benzoate transport and metabolism genes are clustered at the glc and ben loci, respectively, as two distinct operons. When grown on glucose plus benzoate, CSV86 displayed significantly higher expression of the ben locus in the first log phase and of the glc locus in the second log phase. Kinetics of substrate uptake and metabolism matched the transcription profiles. The inability of succinate to suppress benzoate transport and metabolism resulted in coutilization of succinate and benzoate. When challenged with succinate or benzoate, glucose-grown cells showed rapid reduction in glc locus transcription, glucose transport, and metabolic activity, with succinate being more effective at the functional level. Benzoate and succinate failed to interact with or inhibit the activities of glucose transport components or metabolic enzymes. The data suggest that succinate and benzoate suppress glucose transport and metabolism at the transcription level, enabling P. putida CSV86 to preferentially metabolize benzoate. This strain thus has the potential to be an ideal host to engineer diverse metabolic pathways for efficient bioremediation.IMPORTANCEPseudomonas strains play an important role in carbon cycling in the environment and display a hierarchy in carbon utilization: organic acids first, followed by glucose, and aromatic substrates last. This limits their exploitation for bioremediation. This study demonstrates the substrate-dependent modulation of ben and glc operons in Pseudomonas putida CSV86, wherein benzoate suppresses glucose transport and metabolism at the transcription level, leading to preferential utilization of benzoate over glucose. Interestingly, succinate and benzoate are cometabolized. These properties are unique to this strain compared to other pseudomonads and open up avenues to unravel novel regulatory processes. Strain CSV86 can serve as an ideal host to engineer and facilitate efficient removal of recalcitrant pollutants even in the presence of simpler carbon sources.

Putative DNA modification methylase DR_C0020 of Deinococcus radiodurans is an atypical SAM dependent C-5 cytosine DNA methylase.

BACKGROUND: Control of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans. METHODS: C-5 methyl cytosines (m5C) were detected by immuno-blotting with m5C-specific antibody. Site of cytosine methylation by DR_C0020 encoded protein was investigated by bisulfite sequencing. The DR_C0020 knockout mutant (Δdcm), constructed by site directed mutagenesis, was assessed for effect on growth, radiation resistance and proteome. Proteins were identified by mass spectrometry. RESULTS: Methylated cytosines were detected in the D. radiodurans genome. The DR_C0020 encoded protein (Dcm, NCBI accession: WP_034351354.1), whose amino acid sequence resembles m4C methylases, was shown to be the lone SAM-dependent C-5 cytosine methyltransferase. Purified Dcm protein was found to methylate CpN sequence with a preference for methylation of two consecutive cytosines. The Δdcm strain completely lost m5C modification from its genome, had no effect on growth but became radiation sensitive. The Δdcm cells exhibited minor alterations in the abundance of several proteins involved primarily in protein homeostasis, oxidative stress defense, metabolism, etc. CONCLUSION: DR_C0020 encoded SAM-dependent methyltransferase Dcm is solely responsible for C-5cytosine methylation at CpN sites in the genome of D. radiodurans and regulates protein homeostasis under normal growth conditions. The protein is an unusual case of an amino methyltransferase that has evolved to producing m5C. GENERAL SIGNIFICANCE: Although, dispensable under optimal growth conditions, the presence of m5C may be important for recognition of parent strand and, thus, could contribute to the extraordinary DNA repair in D. radiodurans.

A novel glutaredoxin domain-containing peroxiredoxin 'All1541' protects the N2-fixing cyanobacterium Anabaena PCC 7120 from oxidative stress.

Prxs (peroxiredoxins) are ubiquitous thiol-based peroxidases that detoxify toxic peroxides. The Anabaena PCC 7120 genome harbours seven genes/ORFs (open reading frames) which have homology with Prxs. One of these (all1541) was identified to encode a novel Grx (glutaredoxin) domain-containing Prx by bioinformatic analysis. A recombinant N-terminal histidine-tagged All1541 protein was overexpressed in Escherichia coli and purified. Analysis with the protein alkylating agent AMS (4-acetamido-4'-maleimidyl-stilbene-2,2'-disulfonate) showed All1541 to form an intra-molecular disulfide bond. The All1541 protein used glutathione (GSH) more efficiently than Trx (thioredoxin) to detoxify H(2)O(2). Deletion of the Grx domain from All1541 resulted in loss of GSH-dependent peroxidase activity. Employing site-directed mutagenesis, the cysteine residues at positions 50 and 75 were identified as peroxidatic and resolving cysteine residues respectively, whereas both the cysteine residues within the Grx domain (positions 181 and 184) were shown to be essential for GSH-dependent peroxidase activity. On the basis of these data, a reaction mechanism has been proposed for All1541. In vitro All1541 protein protected plasmid DNA from oxidative damage. In Anabaena PCC 7120, all1541 was transcriptionally activated under oxidative stress. Recombinant Anabaena PCC 7120 strain overexpressing All1541 protein showed superior oxidative stress tolerance to H(2)O(2) as compared with the wild-type strain. The results suggest that the glutathione-dependent peroxidase All1541 plays an important role in protecting Anabaena from oxidative stress.

Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC7120.

The heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 displayed two superoxide dismutase (SOD) activities, namely FeSOD and MnSOD. Prolonged exposure of Anabaena PCC7120 cells to methyl viologen mediated oxidative stress resulted in loss of both SOD activities and induced cell lysis. The two SOD proteins were individually overexpressed constitutively in Anabaena PCC7120, by genetic manipulation. Under nitrogen-fixing conditions, overexpression of MnSOD (sodA) enhanced oxidative stress tolerance, while FeSOD (sodB) overexpression was detrimental. Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress. The results demonstrate a nitrogen status-dependent protective role of individual superoxide dismutases in Anabaena PCC7120 during oxidative stress.

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 strain's ability to utilize aromatics and organic acids over glucose.

GroEL of the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31 exhibits GroES and ATP-independent refolding activity.

The nitrogen-fixing cyanobacterium, Anabaena L-31 has two Hsp60 proteins, 59 kDa GroEL coded by the second gene of groESL operon and 61 kDa Cpn60 coded by cpn60 gene. Anabaena GroEL formed stable higher oligomer (>12-mer) in the presence of K(+) and prevented thermal aggregation of malate dehydrogenase (MDH). Using three protein substrates (MDH, All1541 and green fluorescent protein), it was found that the refolding activity of Anabaena GroEL was lower than that of Escherichia coli GroEL, but independent of both GroES and ATP. This correlated with in vivo data. GroEL exhibited ATPase activity which was enhanced in the presence of GroES and absence of a denatured protein, contrary to that observed for bacterial GroEL. However, a significant role for ATP could not be ascertained during in vitro folding assays. The monomeric Cpn60 exhibited much lower refolding activity than GroEL, unaffected by GroES and ATP. In vitro studies revealed inhibition of the refolding activity of Anabaena GroEL by Cpn60, which could be due to their different oligomeric status. The role of GroES and ATP may have been added during the course of evolution from the ancient cyanobacteria to modern day bacteria enhancing the refolding ability and ensuring wider scope of substrates for GroEL.

Functional roles of N-terminal and C-terminal domains in the overall activity of a novel single-stranded DNA binding protein of Deinococcus radiodurans.

Single-stranded DNA binding protein (Ssb) of Deinococcus radiodurans comprises N- and C-terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: SsbN (N-terminal without connector), SsbNC (N-terminal with connector) and SsbC (C-terminal), each harboring one OB fold. Both SsbN and SsbNC displayed weak single-stranded DNA (ssDNA) binding activity, compared to the full-length Ssb (SsbFL). The level of ssDNA binding activity displayed by SsbC was intermediate between SsbFL and SsbN. SsbC and SsbFL predominantly existed as homo-dimers while SsbNC/SsbN formed different oligomeric forms. In vitro, SsbNC or SsbN formed a binary complex with SsbC that displayed enhanced ssDNA binding activity. Unlike SsbFL, Ssb variants were able to differentially modulate topoisomerase-I activity, but failed to stimulate Deinococcal RecA-promoted DNA strand exchange. The results suggest that the C-terminal OB fold is primarily responsible for ssDNA binding. The N-terminal OB fold binds weakly to ssDNA but is involved in multimerization.

DNA adenine hypomethylation leads to metabolic rewiring in Deinococcus radiodurans.

The protein encoded by DR_0643 gene from Deinococcus radiodurans was shown to be an active N-6 adenine-specific DNA methyltransferase (Dam). Deletion of corresponding protein reduced adenine methylation in the genome by 60% and resulted in slow-growth phenotype. Proteomic changes induced by DNA adenine hypomethylation were mapped by two-dimensional protein electrophoresis coupled with mass spectrometry. As compared to wild type D. radiodurans cells, at least 54 proteins were differentially expressed in Δdam mutant. Among these, 39 metabolic enzymes were differentially expressed in Δdam mutant. The most prominent change was DNA adenine hypomethylation induced de-repression of pyruvate dehydrogenase complex, E1 component (aceE) gene resulting in 10 fold increase in the abundance of corresponding protein. The observed differential expression profile of metabolic enzymes included increased abundance of enzymes involved in fatty acid and amino acid degradation to replenish acetyl Co-A and TCA cycle intermediates and diversion of phosphoenolpyruvate and pyruvate into amino acid biosynthesis, a metabolic rewiring attempt by Δdam mutant to restore energy generation via glycolysis-TCA cycle axis. This is the first report of DNA adenine hypomethylation mediated rewiring of metabolic pathways in prokaryotes.

Pyrroloquinoline-quinone: a reactive oxygen species scavenger in bacteria.

Transgenic Escherichia coli expressing pyrroloquinoline-quinone (PQQ) synthase gene from Deinococcus radiodurans showed superior survival during Rose Bengal induced oxidative stress. Such cells showed significantly low levels of protein carbonylation as compared to non-transgenic control. In vitro, PQQ reacted with reactive oxygen species with rate constants comparable to other well known antioxidants, producing non-reactive molecular products. PQQ also protected plasmid DNA and proteins from the oxidative damage caused by gamma-irradiation in solution. The data suggest that radioprotective/oxidative stress protective ability of PQQ in bacteria may be consequent to scavenging of reactive oxygen species per se and induction of other free radical scavenging mechanism.

N-terminal processing of membrane-targeted MnSOD and formation of multiple active superoxide dismutase dimers in the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC7120.

Anabaena sp. strain PCC7120 expresses a 30 kDa manganese-dependent superoxide dismutase (MnSOD) comprising a hydrophobic region (signal peptide + linker peptide) attached to a catalytic unit. Bioinformatics predicted cleavage of the signal peptide at (25)CQPQ by signal peptidase and of the linker peptide by an Arg-C-like protease at the Arg52/Arg59 residue. The three predicted forms of MnSOD were immunodetected in Anabaena, with the 30 kDa MnSOD found exclusively in the membrane and the shorter 27 and 24 kDa forms found both in the membrane and soluble fractions. The corresponding sodA gene was truncated for (a) the first eight residues, or, (b) the signal peptide, or (c) the entire hydrophobic region, or (d) the Arg52/Arg59 residues were modified to serine. Overexpression of these MnSOD variants in recombinant Anabaena strains revealed that (a) the 30 kDa membrane-targeted MnSOD was cleaved by membrane-localized signal peptidase either during or after its transport through the membrane to release the 27 kDa form, either in the cytosol or in the periplasmic/thylakoid lumen, (b) the 27 kDa form was further cleaved to the 24 kDa form by Arg-C-like protease, both in the cytosol and in the periplasmic/thylakoid lumen, (c) deletion of signal peptide localized the MnSOD forms in the cytosol, and (d) alteration of the signal/linker peptide cleavage sites interfered with MnSOD localization and processing. Homo/heterodimerization of the 24 and 27 kDa forms of MnSOD and the cytosolic iron-dependent SOD results in multiple SOD activities, from a single MnSOD gene (sodA), in different cellular compartments of Anabaena.

Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli.

The involvement of signal transduction in the repair of radiation-induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline-quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline-quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline-quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10-fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline-quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild-type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild-type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation-induced DNA strand break repair and homologous recombination in E. coli.

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 strain's ability to utilize aromatics and organic acids in preference to glucose.

Preferential utilization of aromatic compounds over glucose by Pseudomonas putida CSV86.

Pseudomonas putida CSV86, a naphthalene-degrading organism, exhibited diauxic growth on aromatic compounds plus glucose, with utilization of aromatics in the first log phase and of glucose in the second log phase. Glucose supplementation did not suppress the activity of degrading enzymes, which were induced upon addition of aromatic compounds. The induction was inhibited by chloramphenicol, suggesting that de novo protein synthesis was essential. Cells showed cometabolism of aromatic compounds and organic acids; however, organic acids suppressed glucose utilization.

An exonuclease I-sensitive DNA repair pathway in Deinococcus radiodurans: a major determinant of radiation resistance.

Deinococcus radiodurans R1 recovering from acute dose of gamma radiation shows a biphasic mechanism of DNA double-strand break repair. The possible involvement of microsequence homology-dependent, or non-homologous end joining type mechanisms during initial period followed by RecA-dependent homologous recombination pathways has been suggested for the reconstruction of complete genomes in this microbe. We have exploited the known roles of exonuclease I in DNA recombination to elucidate the nature of recombination involved in DNA double-strand break repair during post-irradiation recovery of D. radiodurans. Transgenic Deinococcus cells expressing exonuclease I functions of Escherichia coli showed significant reduction in gamma radiation radioresistance, while the resistance to far-UV and hydrogen peroxide remained unaffected. The overexpression of E. coli exonuclease I in Deinococcus inhibited DNA double-strand break repair. Such cells exhibited normal post-irradiation expression kinetics of RecA, PprA and single-stranded DNA-binding proteins but lacked the divalent cation manganese [(Mn(II)]-dependent protection from gamma radiation. The results strongly suggest that 3' (rho) 5' single-stranded DNA ends constitute an important component in recombination pathway involved in DNA double-strand break repair and that absence of sbcB from deinococcal genome may significantly aid its extreme radioresistance phenotype.

Differential expression of the two kdp operons in the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31.

In several types of bacteria, the Kdp ATPase (comprising of the KdpABC complex) is an inducible, high-affinity potassium transporter that scavenges K+ from the environment. The cyanobacterium Anabaena sp. strain L-31 showed the presence of not one but two distinct kdp operons in its genome. The kdp1 consisted of kdpA1B1G1C1D genes, whereas the kdp2 contained the kdpA2B2G2C2 genes. Among the regulatory genes, the kdpD open reading frame of Anabaena sp. strain L-31 was truncated compared to the kdpD of other bacteria, whereas a kdpE-like gene was absent in the vicinity of the two kdp operons. In response to K+ limitation (<0.05 mM external K+), only kdp2 (and not kdp1) expression could be detected as a 5.3-kb transcript on Northern blots, indicating that kdpA2B2G2C2 genes constitute a polycystronic operon. Unlike E. coli, addition of osmolytes like NaCl, or a change in pH of the medium did not enhance the kdp expression in Anabaena sp. strain L-31. Interestingly, the Anabaena sp. strain L-31 kdp2 operon was strongly induced in response to desiccation stress. The addition of K+ to K+ -starved cultures resulted in repression and degradation of kdp2 transcripts. Our results clearly show that kdp2 is the major kdp operon expressed in Anabaena sp. strain L-31 and may play an important role in adaptation to K+ limitation and desiccation stress.

Pleiotropic effects of potassium deficiency in a heterocystous, nitrogen-fixing cyanobacterium, Anabaena torulosa.

Omission of potassium from the growth medium caused multiple metabolic impairments and resulted in cessation of growth of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anabaena torulosa, during both diazotrophic and nitrogen-supplemented growth. Prominent defects observed during potassium deprivation were: (i) the loss of photosynthetic pigments, (ii) impairment of photosynthetic functions, (iii) reduced synthesis of dinitrogenase reductase (Fe-protein), (iv) inhibition of nitrogenase activity, and (v) specific qualitative modifications of protein synthesis leading to the repression of twelve polypeptides and synthesis and accumulation of nine novel polypeptides. The observed metabolic defects were reversible, and growth arrested under prolonged potassium deficiency was fully restored upon re-addition of potassium. Such pleiotropic effects of potassium deficiency demonstrate that apart from its well-known requirement for pH and turgor homeostasis, K+ plays other vital specific roles in cyanobacterial growth and metabolism.

Pyrroloquinoline-quinone synthesized in Escherichia coli by pyrroloquinoline-quinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization.

Deinococcus radiodurans, an extremely radioresistant bacterium, synthesizes coenzyme pyrroloquinoline-quinone (PQQ) but exhibits a negative phenotype for mineral phosphate solubilization. Gene for the putative PQQ synthesizing protein was PCR amplified and cloned from Deinococcus, sequenced, and expressed in Escherichia coli, under an inducible E. coli promoter. The transgenic E. coli expressed PQQ synthase protein of 42kDa and complemented the mineral phosphate solubilization phenotype of E. coli, suggesting the synthesis of an active protein. The cells expressing high levels of this protein showed increased protection against photodynamically produced reactive oxygen species. The effect could be attributed to the upregulation of antioxidant enzymes such as catalase and superoxide dismutase by PQQ in transgenic E. coli through an unknown mechanism. The study elucidates a hitherto unknown possible function of PQQ in bacteria.