Development of efficient modules for recombinant protein expression and periplasmic localisation in Pseudomonas bharatica CSV86T.

Escherichia coli has been widely employed as a host for heterologous protein expression. However, due to certain limitations, alternative hosts like Pseudomonas, Lactococcus and Bacillus are being explored. Pseudomonas bharatica CSV86T, a novel soil isolate, preferentially degrades wide range of aromatics over simple carbon sources like glucose and glycerol. Strain also possesses advantageous eco-physiological traits, making it an ideal host for engineering xenobiotic degradation pathways, which necessitates the development of heterologous expression systems. Based on the efficient growth, short lag-phase and rapid metabolism of naphthalene, Pnah and Psal promoters (regulated by NahR) were selected for expression. Pnah was found to be strong and leaky as compared to Psal, using 1-naphthol 2-hydroxylase (1NH, ∼66 kDa) as reporter gene in strain CSV86T. The Carbaryl hydrolase (CH, ∼72 kDa) from Pseudomonas sp. C5pp was expressed under Pnah in strain CSV86T and could successfully be translocated to the periplasm due to the presence of the Tmd + Sp sequence. The recombinant CH was purified from the periplasmic fraction and the kinetic characteristics were found to be similar to the native protein from strain C5pp. These results potentiate the suitability of P. bharatica CSV86T as a desirable host, while Pnah and the Tmd + Sp can be employed for overexpression and periplasmic localisation, respectively. Such tools find application in heterologous protein expression and metabolic engineering applications.

Taxonomic, metabolic traits and species description of aromatic compound degrading Indian soil bacterium Pseudomonas bharatica CSV86T.

A soil bacterium, strain CSV86T isolated from a petrol station in Bangalore, India displays a unique carbon source utilization hierarchy with preferential utilization of various genotoxic aromatic compounds over glucose. Cells were Gram-negative, motile rods, oxidase- and catalase-positive. Strain CSV86T possess a 6.79 Mb genome with 62.72 G + C mol%. 16S rRNA gene phylogeny relates strain CSV86T to the genus Pseudomonas, with highest similarity to Pseudomonas japonica WLT (99.38%). Multi-locus sequence analyses of gyrB-rpoB-rpoD-recA and 33 ribosomal proteins (rps) displayed overall low similarities to its phylogenetic relatives with poor similarity score (6%). Average nucleotide identity (ANI) and in-silico DNA-DNA hybridization (DDH) showed poor (87.11% and 33.2%, respectively) genomic relatedness of strain CSV86T to its closest relatives, indicating genomic distinctiveness. The major cellular fatty acids were 16:0, 17:0cyclo, summed-feature-3 (16:1ω7c/16:1ω6c) and -8 (18:1ω7c). Further, differential abundance of 12:0, 10:0 3-OH and 12:0 3-OH and phenotypic differences distinguished strain CSV86T from closest relatives, hence designated as Pseudomonas bharatica. The unique aromatic degradation ability, resistance to heavy metals, efficient nitrogen-sulfur assimilation, beneficial eco-physiological traits (production of indole acetic acid, siderophore and fusaric acid efflux) and plasmid-free genome suggest strain CSV86T to be a model organism for bioremediation and ideal host for metabolic engineering.

Eco-physiological portrait of a novel Pseudomonas sp. CSV86: an ideal host/candidate for metabolic engineering and bioremediation.

Pseudomonas sp. CSV86, an Indian soil isolate, degrades wide range of aromatic compounds like naphthalene, benzoate and phenylpropanoids, amongst others. Isolate displays the unique and novel property of preferential utilization of aromatics over glucose and co-metabolizes them with organic acids. Interestingly, as compared to other Pseudomonads, strain CSV86 harbours only high-affinity glucokinase pathway (and absence of low-affinity oxidative route) for glucose metabolism. Such lack of gluconate loop might be responsible for the novel phenotype of preferential utilization of aromatics. The genome analysis and comparative functional mining indicated a large genome (6.79 Mb) with significant enrichment of regulators, transporters as well as presence of various secondary metabolite production clusters, suggesting its eco-physiological and metabolic versatility. Strain harbours various integrative conjugative elements (ICEs) and genomic islands, probably acquired through horizontal gene transfer events, leading to genome mosaicity and plasticity. Naphthalene degradation genes are arranged as regulonic clusters and found to be part of ICECSV86nah . Various eco-physiological properties and absence of major pathogenicity and virulence factors (risk group-1) in CSV86 suggest it to be an ideal candidate for bioremediation. Further, strain can serve as an ideal chassis for metabolic engineering to degrade various xenobiotics preferentially over simple carbon sources for efficient remediation.

A unique global metabolic trait of Pseudomonas bharatica CSV86T: metabolism of aromatics over simple carbon sources and co-metabolism with organic acids.

Hierarchical utilization of substrate by microbes (utilization of simple carbon sources prior to complex ones) poses a major limitation to the efficient remediation of aromatic pollutants. Aromatic compounds, being complex and reduced in nature, appear to be a deferred choice as the carbon source in the presence of a plethora of simple organic compounds in the environment. The soil bacterium Pseudomonas bharatica CSV86T displays a unique carbon source utilization hierarchy. It preferentially utilizes aromatics over glucose and co-metabolizes them with succinate or pyruvate (Basu et al., 2006, Applied and Environmental Microbiology, 72 : 22226-2230). In the present study, the substrate utilization hierarchy for strain CSV86T was tested for additional simple carbon sources such as glycerol, acetate, and tri-carboxylic acid (TCA) cycle intermediates like α-ketoglutarate and fumarate. When grown on a mixture of aromatics (benzoate or naphthalene) plus glycerol, the strain displayed a diauxic growth profile with significantly high activity of aromatic utilization enzymes (catechol 1,2- or 2,3-dioxygenase, respectively) in the first-log phase. This suggests utilization of aromatics in the first-log phase followed by glycerol in the second-log phase. On a mixture of an aromatic plus organic acid (acetate, α-ketoglutarate or fumarate), the strain displayed a monoauxic growth profile, indicating co-metabolism. Interestingly, the presence of glycerol, acetate, α-ketoglutarate or fumarate does not repress metabolism/utilization of the aromatic. Thus, the substrate utilization hierarchy of strain CSV86T is aromatics=organic acids>glucose/glycerol, which is unique as compared to other Pseudomonas species, where degradation of aromatics is repressed by glycerol, glucose, acetate or organic acids, including TCA cycle intermediates. This novel substrate utilization hierarchy appears to be a global metabolic phenomenon in strain CSV86T, thus implying it to be an ideal host for metabolic engineering as well as for its potential application in bioremediation.

Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle.

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane ß-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.

Degradation strategies and associated regulatory mechanisms/features for aromatic compound metabolism in bacteria.

As a result of anthropogenic activity, large number of recalcitrant aromatic compounds have been released into the environment. Consequently, microbial communities have adapted and evolved to utilize these compounds as sole carbon source, under both aerobic and anaerobic conditions. The constitutive expression of enzymes necessary for metabolism imposes a heavy energy load on the microbe which is overcome by arrangement of degradative genes as operons which are induced by specific inducers. The segmentation of pathways into upper, middle and/or lower operons has allowed microbes to funnel multiple compounds into common key aromatic intermediates which are further metabolized through central carbon pathway. Various proteins belonging to diverse families have evolved to regulate the transcription of individual operons participating in aromatic catabolism. These proteins, complemented with global regulatory mechanisms, carry out the regulation of aromatic compound metabolic pathways in a concerted manner. Additionally, characteristics like chemotaxis, preferential utilization, pathway compartmentalization and biosurfactant production confer an advantage to the microbe, thus making bioremediation of the aromatic pollutants more efficient and effective.

Atrazine Bioremediation and Its Influence on Soil Microbial Diversity by Metagenomics Analysis.

Pesticide accumulation in agricultural soils is an environmental concern, often addressed through distinct bioremediation strategies. This study has tried to analyze various soil bioremediation options viz., biostimulation, bioaugmentation, and natural attenuation in terms of efficiency and the response of autochthonous microbial flora by using atrazine as a model contaminant. Soil mesocosms were established with 100 kg of soil simulating the field conditions. The soil previously exposed to the herbicide was used for the bioaugmentation strategy undertaken in this study. We have tried to analyze how the microbial community responds to a foreign compound, both in terms of taxonomic and functional capacities? To answer this, we have analyzed metagenome of the mesocosms at a time point when 90% atrazine was degraded. Bioaugmentation for bioremediation proved to be efficient with a DT90 value of 15.48 ± 0.79 days, in comparison to the natural attenuation where the DT90 value was observed to be 41.20 ± 1.95 days. Metagenomic analysis revealed the abundance of orders Erysipelotrichales, Selemonadales, Clostridiales, and Thermoanaerobacterales exclusively in SBS mesocosm. Besides Pseudomonas, bacterial genera such as Achromobacter, Xanthomonas, Stenotrophomonas, and Cupriavidus have emerged as the dominant members in various bioremediation strategies tested in this study. Inclusive results suggest that inherent microbial flora adjust their community and metabolic machinery upon exposure to the pollutant. The site under pollutant stress showed efficient microbial communities to bio-remediate the newly polluted terrestrial ecologies in relatively less time and by economic means.

Structural modulation of a periplasmic sugar-binding protein probes into its evolutionary ancestry.

Substrate-binding proteins (SBPs) are periplasmic proteins consisting of two α/ß domains joined by a hinge region with specificity towards cognate ligands. Based on three-dimensional fold, sugar-specific SBPs have been classified into cluster B and cluster D-I. The analysis of sequences and structures of sugar-binding pocket of cluster D-I SBPs revealed the presence of extra residues on two loops (L1, L2) and a helix (H1) in few members of this family, that binds specifically to monosaccharides. Presence of conserved histidine in L2 and tryptophan in H1 can be considered as the identity marks for the cluster D-I monosaccharide-binding SBPs. A glucose binding protein (ppGBP) from Pseudomonas putida CSV86 was found to contain a structural fold similar to oligosaccharide-binding cluster D-I SBPs, but functionally binds to only glucose due to constriction of its binding pocket mainly by L2 (375-382). ppGBP with partial deletion of L2 (ppGBPΔL2) was created, crystallized and biochemical characterization was performed. Compared to wild type ppGBP, the ppGBPΔL2 structure showed widening of the glucose-binding pocket with ∼80% lower glucose binding. Our results show that the substrate specificity of SBPs can be altered by modulating the size of the binding pocket. Based on this, we propose a sub classification of cluster D-I SBPs into (i) cluster D-I(a)-monosaccharide-binding SBPs and (ii) cluster D-I(b)-oligosaccharide-binding SBPs. This study also provides the direct structural and functional correlation indicating that divergence of proteins may occur through insertions or deletions of sequences in the already existing SBPs leading to evolution at the functional level.

Carbaryl as a Carbon and Nitrogen Source: an Inducible Methylamine Metabolic Pathway at the Biochemical and Molecular Levels in Pseudomonas sp. Strain C5pp.

Carbaryl is the most widely used carbamate family pesticide, and its persistent nature causes it to pollute both soil and water ecosystems. Microbes maintain the Earth's biogeochemical cycles by metabolizing various compounds present in the matter, including xenobiotics, as a sole source of carbon, nitrogen, and energy. Soil isolate Pseudomonas sp. strain C5pp metabolizes carbaryl efficiently as the carbon source. Periplasmic carbaryl hydrolase catalyzes the conversion of carbaryl to 1-naphthol and methylamine. 1-Naphthol was further used as a carbon source via gentisate, whereas the metabolic fate of methylamine is not known. Here, we demonstrate that strain C5pp showed efficient growth on carbaryl when supplied as a carbon and nitrogen source, suggesting that the methylamine generated was used as the nitrogen source. Genes involved in the methylamine metabolism were annotated and characterized at the biochemical and molecular level. Transcriptional and enzyme activity studies corroborate that the γ-glutamylmethylamide/N-methylglutamate (GMA/NMG) pathway is involved in the metabolism of carbaryl and methylamine as a nitrogen source. Compared to carbaryl, methylamine was found to be an effective inducer for the metabolic and transporter genes. Strain C5pp also harbored genes involved in sarcosine metabolism that were cotranscribed and induced by sarcosine. The presence of inducible pathways for metabolism of carbaryl as a nitrogen and carbon source helps in complete and efficient mineralization of carbaryl by strain C5pp, thereby maintaining the biogeochemical cycles.IMPORTANCE The degradation of xenobiotics plays a significant role in the environment to maintain ecological systems as well as to prevent the imbalance of biogeochemical cycles via carbon-nitrogen cycling. Carbaryl is the most widely used pesticide from the carbamate family. Pseudomonas sp. strain C5pp, capable of utilizing carbaryl as a carbon and nitrogen source for its growth, subsequently helps in complete remediation of carbaryl. Thus, it maintains the ecosystem by balancing the biogeochemical cycles. The metabolic versatility and genetic diversity of strain C5pp for the transformation of contaminants like carbaryl and 1-naphthol into less harmful products make it a suitable candidate from the perspective of bioremediation.

Compartmentalization of the Carbaryl Degradation Pathway: Molecular Characterization of Inducible Periplasmic Carbaryl Hydrolase from Pseudomonas spp.

Pseudomonas sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of mcbA, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of Escherichia coli The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of E. coli expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent Km and Vmax in the range of 98 to 111 µM and 69 to 73 µmol · min-1 · mg-1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of Pseudomonas spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.IMPORTANCE Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of Pseudomonas spp. Predicted transmembrane domain and signal peptide sequences of Pseudomonas were found to be functional in Escherichia coli and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.

High Resolution Structures of Periplasmic Glucose-binding Protein of Pseudomonas putida CSV86 Reveal Structural Basis of Its Substrate Specificity.

Periplasmic substrate-binding proteins (SBPs) bind to the specific ligand with high affinity and mediate their transport into the cytoplasm via the cognate inner membrane ATP-binding cassette proteins. Because of low sequence identities, understanding the structural basis of substrate recognition by SBPs has remained very challenging. There are several structures available for the ligand-bound sugar SBPs, but very few unliganded structures are reported. No structural data are available for sugar SBPs fromPseudomonassp. to date. This study reports the first high resolution crystal structures of periplasmic glucose-binding protein fromPseudomonas putidaCSV86 (ppGBP) in unliganded form (2.5 Å) and complexed with glucose (1.25 Å) and galactose (1.8 Å). Asymmetric domain closure of ppGBP was observed upon substrate binding. The ppGBP was found to have an affinity of ∼ 0.3 µmfor glucose. The structural analysis showed that the sugars are bound to the protein mainly by hydrogen bonds, and the loss of two strong hydrogen bonds between ppGBP and galactose compared with glucose may be responsible for lowering its affinity toward galactose. The higher stability of ppGBP-glucose complex was also indicated by an 8 °C increase in the melting temperature compared with unliganded form and ppGBP-galactose complex. ppGBP binds to monosaccharide, but the structural features revealed it to have an oligosaccharide-binding protein fold, indicating that during evolution the sugar binding pocket may have undergone structural modulation to accommodate monosaccharide only.

Carbon Source-Dependent Inducible Metabolism of Veratryl Alcohol and Ferulic Acid in Pseudomonas putida CSV86.

Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz, veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the ß-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer, ver, and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O-demethylases, viz, VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products.IMPORTANCEPseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O-demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the metabolic pathway and gene pool for lignin degradation in bacteria. The biochemical and genetic characterization of phenylpropanoid metabolism makes it a prospective system for its application in producing valuable products, such as vanillin and vanillic acid, from lignocellulose. This study supports the immense potential of P. putida CSV86 as a suitable candidate for bioremediation and biorefinery.

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.

Insights into metabolism and sodium chloride adaptability of carbaryl degrading halotolerant Pseudomonas sp. strain C7.

Pseudomonas sp. strain C7 isolated from sediment of Thane creek near Mumbai, India, showed the ability to grow on glucose and carbaryl in the presence of 7.5 and 3.5% of NaCl, respectively. It also showed good growth in the absence of NaCl indicating the strain to be halotolerant. Increasing salt concentration impacted the growth on carbaryl; however, the specific activity of various enzymes involved in the metabolism remained unaffected. Among various enzymes, 1-naphthol 2-hydroxylase was found to be sensitive to chloride as compared to carbaryl hydrolase and gentisate 1,2-dioxygenase. The intracellular concentration of Cl- ions remained constant (6-8 mM) for cells grown on carbaryl either in the presence or absence of NaCl. Thus the ability to adapt to the increasing concentration of NaCl is probably by employing chloride efflux pump and/or increase in the concentration of osmolytes as mechanism for halotolerance. The halotolerant nature of the strain will be beneficial to remediate carbaryl from saline agriculture fields, ecosystems and wastewaters.

Metabolic engineering of Pseudomonas bharatica CSV86T to degrade Carbaryl (1-naphthyl-N-methylcarbamate) via the salicylate-catechol route.

Pseudomonas bharatica CSV86T displays the unique property of preferential utilization of aromatic compounds over simple carbon sources like glucose and glycerol and their co-metabolism with organic acids. Well-characterized growth conditions, aromatic compound metabolic pathways and their regulation, genome sequence, and advantageous eco-physiological traits (indole acetic acid production, alginate production, fusaric acid resistance, organic sulfur utilization, and siderophore production) make it an ideal host for metabolic engineering. Strain CSV86T was engineered for Carbaryl (1-naphthyl-N-methylcarbamate) degradation via salicylate-catechol route by expression of a Carbaryl hydrolase (CH) and a 1-naphthol 2-hydroxylase (1NH). Additionally, the engineered strain exhibited faster growth on Carbaryl upon expression of the McbT protein (encoded by the mcbT gene, a part of Carbaryl degradation upper operon of Pseudomonas sp. C5pp). Bioinformatic analyses predict McbT to be an outer membrane protein, and Carbaryl-dependent expression suggests its probable role in Carbaryl uptake. Enzyme activity and protein analyses suggested periplasmic localization of CH (carrying transmembrane domain plus signal peptide sequence at the N-terminus) and 1NH, enabling compartmentalization of the pathway. Enzyme activity, whole-cell oxygen uptake, spent media analyses, and qPCR results suggest that the engineered strain preferentially utilizes Carbaryl over glucose. The plasmid-encoded degradation property was stable for 75-90 generations even in the absence of selection pressure (kanamycin or Carbaryl). These results indicate the utility of P. bharatica CSV86T as a potential host for engineering various aromatic compound degradation pathways.IMPORTANCEThe current study describes engineering of Carbaryl metabolic pathway in Pseudomonas bharatica CSV86T. Carbaryl, a naphthalene-derived carbamate pesticide, is known to act as an endocrine disruptor, mutagen, cytotoxin, and carcinogen. Removal of xenobiotics from the environment using bioremediation faces challenges, such as slow degradation rates, instability of the degradation phenotype, and presence of simple carbon sources in the environment. The engineered CSV86-MEC2 overcomes these disadvantages as Carbaryl was degraded preferentially over glucose. Furthermore, the plasmid-borne degradation phenotype is stable, and presence of glucose and organic acids does not repress Carbaryl metabolism in the strain. The study suggests the role of outer membrane protein McbT in Carbaryl transport. This work highlights the suitability of P. bharatica CSV86T as an ideal host for engineering aromatic pollutant degradation pathways.

Metabolic regulation and chromosomal localization of carbaryl degradation pathway in Pseudomonas sp. strains C4, C5 and C6.

Pseudomonas sp. strains C4, C5 and C6 degrade carbaryl (1-naphthyl N-methylcarbamate) via 1-naphthol, 1,2-dihydroxynaphthalene, salicylate and gentisate. Carbon source-dependent metabolic studies suggest that enzymes responsible for carbaryl degradation are probably organized into 'upper' (carbaryl to salicylate), 'middle' (salicylate to gentisate) and 'lower' (gentisate to TCA cycle) pathway. Carbaryl and 1-naphthol were found to induce all carbaryl pathway enzymes, while salicylate and gentisate induce middle and lower pathway enzymes. The strains were found to harbor plasmid(s), and carbaryl degradation property was found to be stable. Genes encoding enzymes of the degradative pathway such as 1-naphthol 2-hydroxylase, salicylaldehyde dehydrogenase, salicylate 5-hydroxylase and gentisate 1,2-dioxygenase were amplified from chromosomal DNA of these strains. The gene-specific PCR products were sequenced from strain C6, and phylogenetic tree was constructed. Southern hybridization and PCR analysis using gel eluted DNA as template supported the presence of pathway genes onto the chromosome and not on the plasmid(s).

Life Within a Contaminated Niche: Comparative Genomic Analyses of an Integrative Conjugative Element ICEnahCSV86 and Two Genomic Islands From Pseudomonas bharatica CSV86T Suggest Probable Role in Colonization and Adaptation.

Comparative genomic and functional analyses revealed the presence of three genomic islands (GIs, >50 Kb size): ICEnahCSV86, Pseudomonas bharatica genomic island-1 (PBGI-1), and PBGI-2 in the preferentially aromatic-degrading soil bacterium, Pseudomonas bharatica CSV86T. Site-specific genomic integration at or near specific transfer RNAs (tRNAs), near-syntenic structural modules, and phylogenetic relatedness indicated their evolutionary lineage to the type-4 secretion system (T4SS) ICEclc family, thus predicting these elements to be integrative conjugative elements (ICEs). These GIs were found to be present as a single copy in the genome and the encoded phenotypic traits were found to be stable, even in the absence of selection pressure. ICEnahCSV86 harbors naphthalene catabolic (nah-sal) cluster, while PBGI-1 harbors Co-Zn-Cd (czc) efflux genes as cargo modules, whereas PBGI-2 was attributed to as a mixed-function element. The ICEnahCSV86 has been reported to be conjugatively transferred (frequency of 7 × 10-8/donor cell) to Stenotrophomonas maltophilia CSV89. Genome-wide comparative analyses of aromatic-degrading bacteria revealed nah-sal clusters from several Pseudomonas spp. as part of probable ICEs, syntenic to conjugatively transferable ICEnahCSV86 of strain CSV86T, suggesting it to be a prototypical element for naphthalene degradation. It was observed that the plasmids harboring nah-sal clusters were phylogenetically incongruent with predicted ICEs, suggesting genetic divergence of naphthalene metabolic clusters in the Pseudomonas population. Gene synteny, divergence estimates, and codon-based Z-test indicated that ICEnahCSV86 is probably derived from PBGI-2, while multiple recombination events masked the ancestral lineage of PBGI-1. Diversifying selection pressure (dN-dS = 2.27-4.31) imposed by aromatics and heavy metals implied the modular exchange-fusion of various cargo clusters through events like recombination, rearrangement, domain reshuffling, and active site optimization, thus allowing the strain to evolve, adapt, and maximize the metabolic efficiency in a contaminated niche. The promoters (Pnah and Psal) of naphthalene cargo modules (nah, sal) on ICEnahCSV86 were proved to be efficient for heterologous protein expression in Escherichia coli. GI-based genomic plasticity expands the metabolic spectrum and versatility of CSV86T, rendering efficient adaptation to the contaminated niche. Such isolate(s) are of utmost importance for their application in bioremediation and are the probable ideal host(s) for metabolic engineering.

Functional genome mining and taxono-genomics reveal eco-physiological traits and species distinctiveness of aromatic-degrading Pseudomonas bharatica sp. nov.

Assistive eco-physiological traits are necessary for microbes to adapt and colonize at polluted niches, enabling efficient clean-up. To demarcate species distinctiveness and eco-physiological traits of aromatic compounds metabolizing Pseudomonas sp. CSV86T (earlier identified as Pseudomonas putida), an Indian isolate from a petrol station soil, comparative genome mining, taxono-genomic, and physiological analyses were performed. A 6.79 Mbp genome (62.72 G + C mol%) of CSV86T encodes 6798 CDS and 238 unique genes. Naphthalene metabolism and Co-Zn-Cd resistance gene clusters were part of distinct genomic islands. Abundance of transporters (aromatics, organic acids, amino acids, and metals) and mobile elements (integrases, transposases, conjugative proteins) differentiated CSV86T from its closest relatives. Enhanced siderophore production for Fe-uptake during aromatic metabolism, indole acetic acid production, and fusaric acid resistance wasvalidated by genomic attributes. Full-length 16S-rRNA phylogeny revealed Pseudomonas japonica WLT as a closest relative of CSV86T . However, lower genomic indices (<97% gyrB-rpoB-rpoD homology, <90% ANI, <50% DNA-DNA relatedness) and taxonomic differences (assimilation of organic acids, amino acids, fatty acids composition) substantially differentiated CSV86T from its closest relatives, indicating it to be a novel species as Pseudomonas bharatica. Preferential metabolism of aromatics with advantageous eco-physiological traits renders CSV86T an ideal candidate for bioremediation and host for metabolic engineering.

Glucose-6-Phosphate Dehydrogenase, ZwfA, a Dual Cofactor-Specific Isozyme Is Predominantly Involved in the Glucose Metabolism of Pseudomonas bharatica CSV86T.

Glucose-6-phosphate dehydrogenase (Zwf) is an important enzyme in glucose metabolism via the Entner-Doudoroff pathway and the first enzyme in the oxidative pentose-phosphate pathway. It generates NAD(P)H during the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, thus aiding in anabolic processes, energy yield, and oxidative stress responses. Pseudomonas bharatica CSV86T preferentially utilized aromatic compounds over glucose and exhibited a significantly lower growth rate on glucose (0.24 h-1) with a prolonged lag phase (~10 h). In strain CSV86T, glucose was metabolized via the intracellular phosphorylative route only because it lacked an oxidative (gluconate and 2-ketogluconate) route. The genome harbored three genes zwfA, zwfB, and zwfC encoding three Zwf isozymes. The present study aimed to understand gene arrangement, gene expression profiling, and molecular and kinetic properties of the purified enzymes to unveil their physiological significance in the strain CSV86T. The zwfA was found to be a part of the zwfA-pgl-eda operon, which was proximal to other glucose transport and metabolic clusters. The zwfB was found to be arranged as a gnd-zwfB operon, while zwfC was present independently. Among the three, zwfA was transcribed maximally, and the purified ZwfA displayed the highest catalytic efficiency, cooperativity with respect to G6P, and dual cofactor specificity. Isozymes ZwfB and ZwfC were NADP+-preferring and NADP+-specific, respectively. Among other functionally characterized Zwfs, ZwfA from strain CSV86T displayed poor catalytic efficiency and the further absence of oxidative routes of glucose metabolism reflected its lower growth rate on glucose compared to P. putida KT2440 and could be probable reasons for the unique carbon source utilization hierarchy. IMPORTANCE Pseudomonas bharatica CSV86T metabolizes glucose exclusively via the intracellular phosphorylative Entner-Doudoroff pathway leading the entire glucose flux through Zwf as the strain lacks oxidative routes. This may lead to limiting the concentration of downstream metabolic intermediates. The strain CSV86T possesses three isoforms of glucose-6-phosphate dehydrogenase, ZwfA, ZwfB, and ZwfC. The expression profile and kinetic properties of purified enzymes will help to understand glucose metabolism. Isozyme ZwfA dominated in terms of expression and displayed cooperativity with dual cofactor specificity. ZwfB preferred NADP+, and ZwfC was NADP+ specific, which may aid in redox cofactor balance. Such beneficial metabolic flexibility facilitated the regulation of metabolic pathways giving survival/fitness advantages in dynamic environments. Additionally, multiple genes allowed the distribution of function among these isoforms where the primary function was allocated to one of the isoforms.

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.