Vitreoscilla hemoglobin promotes biofilm expansion and mitigates sporulation in Bacillus subtilis DK1042.

Biofilm formation is considered as a stress combating strategy adopted by bacteria in response to variety of cellular and environmental signals. Impaired respiration due to low oxygen concentrations is one such signal that triggers wrinkling and robust biofilm formation in Bacillus subtilis. Vitreoscilla hemoglobin (VHb) improves microaerobic growth and bioproduct synthesis in a variety of bacteria by supplying oxygen to the respiratory chain. Present study was carried out to determine the effect of VHb on multicellularity of B. subtilis. Thus, B. subtilis DK1042 (WT) was genetically modified to express vgb and gfp genes under the control of P43 promoter at amyE locus by double cross over events. Biofilm formation by the integrant NRM1113 and WT was monitored on Lysogeny broth (LB) and LB containing glycerol and manganese (LBGM) medium. The WT produced more wrinkled colonies than NRM1113 on LB and LBGM medium. Concomitantly, biofilm-associated sporulation and production of pulcherriminic acid was decreased in NRM1113 as compared to WT on LB as well as LBGM. Expression studies of genes encoding structural components of biofilms revealed ~ 70% down-regulation of bslA gene in NRM1113 on both LB and LBGM which is correlated with reduced wrinkling in NRM1113. Moreover, NRM1113 showed increased colony expansion compared to WT in LB, LBGM and high osmolarity conditions. VHb expression alters various processes in different host cells, our study represents that VHb modulates biofilm formation, sporulation and pulcherriminic acid formation in B. subtilis DK1042.

Colonization by multi-potential Pseudomonas aeruginosa P4 stimulates peanut (Arachis hypogaea L.) growth, defence physiology and root system functioning to benefit the root-rhizobacterial interface.

The beneficial associations between Arachis hypogaea L. (peanut) and fluorescent Pseudomonas species have been poorly explored despite their predominance in the peanut rhizosphere. The present study explores the mutually beneficial interactions between peanut roots and P. aeruginosa P4 (P4) in terms of their impact on plant growth, defence physiology and the root-rhizobacterial interface. The efficient phosphate solubilizer P4 exhibited biocontrol abilities, including the production of siderophores, pyocyanin, indole-3-acetic acid and hydrogen cyanide. The bacterization of peanut seeds with multi-potential P4 significantly enhanced in vitro seed germination and seedling vigour. Under sand-based gnotobiotic (10 days post-inoculation) and sterile soil-based cultivation systems (30 days post-inoculation), sustained P4 colonization enhanced the peanut root length and dry plant biomass. The subsequent increase in catalase, polyphenol oxidase and phenylalanine ammonia lyase activities with increased phenolic contents in the peanut roots and shoots suggested the systemic priming of defences. Consequently, the altered root exudate composition caused enhanced chemo-attraction towards P4 itself and the symbiotic N2-fixing Bradyrhizobium strain. Co-inoculating peanuts with P4 and Bradyrhizobium confirmed the improved total bacterial colonization (∼2 fold) of the root tip, with the successful co-localization of both, as substantiated by scanning electron microscopy. Collectively, the peanut-P4 association could potentially model the beneficial Pseudomonas-driven multi-trophic rhizosphere benefits, emphasizing the plausible role of non-rhizobium PGPR in promoting N2 fixation.

Involvement of topoisomerase mutations and qnr and aac(6')Ib-cr genes in conferring quinolone resistance to clinical isolates of Vibrio and Shigella spp. from Kolkata, India (1998-2009).

OBJECTIVES: Quinolone antibiotics have been widely used to treat diarrhoeal diseases caused by bacterial agents such as those belonging to the genera Vibrio and Shigella. As these pathogens are accumulating quinolone resistance, treating infections caused by them has become complicated. METHODS: In this study, Vibrio and Shigella spp. isolates obtained from diarrhoeal patients from Kolkata, India, over a period of 12 years (1998-2009) were analysed for quinolone resistance. A total of 27 Vibrio spp. (9 Vibrio cholerae, 11 Vibrio fluvialis and 7 Vibrio parahaemolyticus) and 10 Shigella spp. isolates (7 Shigella flexneri, 2 Shigella dysenteriae and 1 Shigella sonnei) showing reduced susceptibility to quinolones were studied to unravel the genetic factors responsible for quinolone resistance. RESULTS: Antimicrobial susceptibility testing showed a wide spectrum and varying degree of resistance to different generations of quinolones. Genotypic characterisation revealed the involvement of GyrA(S83I) and ParC(S85L) mutations in V. cholerae and V. fluvialis, whereas Shigella spp. isolates showed the mutations S83L and/or D87N/Y in GyrA and S80I or E84K in ParC. Analysis of plasmid-mediated quinolone resistance genes showed that qnrVC5 was detected in three V. fluvialis isolates, aac(6')-Ib-cr in one V. fluvialis isolate and qnrS1 in a S. flexneri isolate. CONCLUSIONS: These results emphasise that quinolone resistance is widespread and therefore quinolones should be used prudently. To the best of our knowledge, this is the first study where resistance to various generations of quinolones in Vibrio and Shigella spp. has been examined in terms of detailed genotype-phenotype correlation.

Genetically engineered Escherichia coli Nissle 1917 synbiotic counters fructose-induced metabolic syndrome and iron deficiency.

Consumption of fructose leads to metabolic syndrome, but it is also known to increase iron absorption. Present study investigates the effect of genetically modified Escherichia coli Nissle 1917 (EcN) synbiotic along with fructose on non-heme iron absorption. Charles foster rats weighing 150-200 g were fed with iron-deficient diet for 2 months. Probiotic treatment of EcN (pqq) and EcN (pqq-glf-mtlK) was given once per week, 109 cells after 2 months with fructose in drinking water. Iron levels, blood, and liver parameters for oxidative stress, hyperglycemia, and dyslipidemia were estimated. Transferrin-bound iron levels in the blood decreased significantly after 10 weeks of giving iron-deficient diet. Probiotic treatment of EcN (pqq-glf-mtlK) and fructose together led to the restoration of normal transferrin-bound iron levels and blood and hepatic antioxidant levels as compared to iron-deficient control group. The probiotic also led to the restoration of body weight along with levels of serum and hepatic lipid, blood glucose, and antioxidant in the blood and liver as compared to iron-deficient control group. Restoration of liver injury marker enzymes was also seen. Administration of EcN-producing PQQ and mannitol dehydrogenase enzyme together with fructose led to increase in the transferrin-bound iron levels in the blood and amelioration of consequences of metabolic syndrome caused due to fructose consumption.

Ensifer meliloti overexpressing Escherichia coli phytase gene (appA) improves phosphorus (P) acquisition in maize plants.

The Escherichia coli phytase gene appA encoding enzyme AppA was cloned in a broad host range plasmid pBBR1MCS2 (lac promoter), termed pVA1, and transformed into the Ensifer meliloti 1020. Transformation of pVA1 in Ensifer meliloti {E. m (pVA1)} increased its phosphatase and phytase activity by ∼9- and ∼50-fold, respectively, compared to the transformants containing empty plasmid as control {E. m (pBBR1MCS2)}. The western blot experiments using rabbit anti-AppA antibody showed that AppA is translocated into the periplasm of the host after its expression. Ensifer meliloti harboring AppA protein {E. m (pVA1)} and {E. m (pBBR1MCS2)} could acidify the unbuffered phytate minimal media (pH 8.0) containing Ca-phytate or Na-phytate as sole organic P (Po) source to below pH 5.0 and released P. However, both {E. m (pVA1)} and {E. m (pBBR1MCS2)} neither dropped pH of the medium nor released P when the medium was buffered at pH 8.0 using Tris-Cl, indicating that acidification of medium was important for the enzymatic hydrolysis of phytate. Further experiments proved that maize plants inoculated with {E. m. (pVA1)} showed increase in growth under sterile semi solid agar (SSA) medium containing Na-phytate as sole P source. The present study could be helpful in generating better transgenic bioinoculants harboring phosphate mineralization properties that ultimately promote plant growth.

Amelioration of cadmium- and mercury-induced liver and kidney damage in rats by genetically engineered probiotic Escherichia coli Nissle 1917 producing pyrroloquinoline quinone with oral supplementation of citric acid.

OBJECTIVE: Antioxidants, chelating agents, and probiotics are used to manage the toxic effects of cadmium (Cd) and mercury (Hg). The aim of this study was to investigate the combined effects of antioxidants, chelating agents, and probiotics against heavy metal toxicity. METHOD: Genetically modified probiotic Escherichia coli Nissle 1917 (EcN-20) producing a potent water soluble antioxidant pyrroloquinoline quinone (PQQ) was supplemented with oral citric acid and compared with another genetically modified probiotic EcN-21 producing PQQ and citric acid against oxidative stress induced by Cd and Hg. Rats were independently given 100 ppm Cd and 80 ppm Hg in drinking water for 4 wk. RESULTS: EcN-20 was found to be more effective than EcN-2 (EcN strain with genomic integration of vgb and gfp genes) with orally given PQQ against oxidative stress induced by Cd and Hg. EcN-20 supplemented with oral citric acid was more effective against Cd and Hg toxicity compared with EcN-2+citric acid (oral), EcN-2+PQQ (oral), EcN-2+PQQ (oral)+citric acid (oral), EcN-20, and EcN-21. However, protection shown by EcN-21 was similar to EcN-20. CONCLUSION: The combination therapy involving probiotic EcN-20 producing PQQ with citric acid given orally was found to be a moderately effective strategy against toxicity induced by Cd and Hg, whereas the protective effect of EcN-21 was the same as EcN-20.

Characterization of arsenite tolerant Halomonas sp. Alang-4, originated from heavy metal polluted shore of Gulf of Cambay.

Arsenite [As(III)]-oxidizing bacteria were isolated from heavy metal contaminated shore of Gulf of Cambay at Alang, India. The most efficient bacterial strain Alang-4 could tolerate up to 15 mM arsenite [As(III)] and 200 mM of arsenate [As(V)]. Its 16S rRNA gene sequence was 99% identical to the 16S rRNA genes of genus Halomonas (Accession no. HQ659187). Arsenite oxidase enzyme localized on membrane helped in conversion of As(III) to As(V). Arsenite transporter genes (arsB, acr3(1) and acr3(2)) assisted in extrusion of arsenite from Halomonas sp. Alang-4. Generation of ROS in response to arsenite stress was alleviated by higher activities of catalase, ascorbate peroxidase, superoxide dismutase and glutathione S-transferase enzymes. Down-regulation in the specific activities of nearly all dehydrogenases of carbon assimilatory pathway viz., glucose-6-phosphate, pyruvate, α-ketoglutarate, isocitrate and malate dehydrogenases, was observed in presence of As(III), whereas, the specific activities of phosphoenol pyruvate carboxylase, pyruvate carboxylase and isocitrate lyase enzymes were found to increase two times in As(III) treated cells. The results suggest that in addition to efficient ars operon, alternative pathways of carbon utilization exist in the marine bacterium Halomonas sp. Alang-4 to overcome the toxic effects of arsenite on its dehydrogenase enzymes.

Functional microbial diversity dynamics in common effluent treatment plants of South Gujarat and hydrocarbon degradation.

Common effluent treatment plants (CETPs) of South Gujarat region, India, process wastewater generated by more than 2500 industries because of the nonfeasibility of processing at the individual industrial unit. This study assessed functional microbial diversity in wastewater samples of CETPs over a geological belt using Ecoplate®, isolation of the most abundant bacteria, and screening for hydrocarbon degradation. The high evenness (EPielou) values (0.9) in almost all samples indicated a highly even community structure. Principal component analysis of carbon source utilization showed a cluster of all inlet samples except E1 and another cluster of all outlet samples; aeration tank community samples were dispersed. In spite of the high richness found in microbial communities, 60 morphologically similar organisms were observed and isolated; 46 out of them were subjected to amplified ribosomal DNA restriction analysis with MboI, HaeIII, and TaqI enzyme, followed by UPGMA clustering. In screening the most abundant bacteria from each cluster, one of the cultures showed a high potential for hydrocarbon degradation and was identified as Pseudomonas citronellolis by 16S rDNA sequencing. Because of its highly adapted inherent nature, this bacterium may help augment the conventional procedure in wastewater treatment and efficiently decrease the organic load.

Protection against 1,2-di-methylhydrazine-induced systemic oxidative stress and altered brain neurotransmitter status by probiotic Escherichia coli CFR 16 secreting pyrroloquinoline quinone.

Exposure to environmental pollutant 1,2-dimethylhydrazine (DMH) is attributed to systemic oxidative stress and is known to cause neurotropic effect by altering brain neurotransmitter status. Probiotics are opted as natural therapeutic against oxidative stress and also have the ability to modulate gut-brain axis. Pyrroloquinoline quinone (PQQ) is water-soluble, heat-stable antioxidant molecule. Aim of the present study was to evaluate the antioxidant efficacy of PQQ-producing probiotic E. coli CFR 16 on DMH-induced systemic oxidative damage and altered neurotransmitter status in rat brain. Adult virgin Charles Forster rats (200-250 g) were given DMH dose (25 mg/kg body weight, s.c.) for 8 weeks. Blood lipid peroxidation levels exhibited a marked increase while antioxidant enzyme activities of superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase and glutathione peroxidase were found to be reduced in DMH-treated rats. Likewise, brain serotonin and norepinephrine levels displayed a significant decrease, whereas epinephrine levels demonstrated a marked increase in brain of these rats. PQQ-producing E. coli CFR 16 supplementation reduced systemic oxidative stress and also restored brain neurotransmitter status. However, E. coli CFR 16 did not show any effect on these parameters. In contrast, E. coli CFR 16:: vgb-gfp and E. coli CFR 16:: vgb-gfp vector exhibited some degree of protection again oxidative stress but they were not able to modulate neurotransmitter levels. In conclusion, continuous and sustained release of PQQ by probiotic E. coli in rat intestine ameliorates systemic oxidative stress and restored brain neurotransmitter levels.

Artificial citrate operon and Vitreoscilla hemoglobin gene enhanced mineral phosphate solubilizing ability of Enterobacter hormaechei DHRSS.

Mineral phosphate solubilization by bacteria is mediated through secretion of organic acids, among which citrate is one of the most effective. To overproduce citrate in bacterial systems, an artificial citrate operon comprising of genes encoding NADH-insensitive citrate synthase of E. coli and Salmonella typhimurium sodium-dependent citrate transporter was constructed. In order to improve its mineral phosphate solubilizing (MPS) ability, the citrate operon was incorporated into E. hormaechei DHRSS. The artificial citrate operon transformant secreted 7.2 mM citric acid whereas in the native strain, it was undetectable. The transformant released 0.82 mM phosphate in flask studies in buffered medium containing rock phosphate as sole P source. In fermenter studies, similar phenotype was observed under aerobic conditions. However, under microaerobic conditions, no citrate was detected and P release was not observed. Therefore, an artificial citrate gene cluster containing Vitreoscilla hemoglobin (vgb) gene under its native promoter, along with artificial citrate operon under constitutive tac promoter, was constructed and transformed into E. hormaechei DHRSS. This transformant secreted 9 mM citric acid under microaerobic conditions and released 1.0 mM P. Thus, incorporation of citrate operon along with vgb gene improves MPS ability of E. hormaechei DHRSS under buffered, microaerobic conditions mimicking rhizospheric environment.

Heterologous expression of pyrroloquinoline quinone (pqq) gene cluster confers mineral phosphate solubilization ability to Herbaspirillum seropedicae Z67.

Gluconic acid secretion mediated by the direct oxidation of glucose by pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) is responsible for mineral phosphate solubilization in Gram-negative bacteria. Herbaspirillum seropedicae Z67 (ATCC 35892) genome encodes GDH apoprotein but lacks genes for the biosynthesis of its cofactor PQQ. In this study, pqqE of Erwinia herbicola (in plasmid pJNK1) and pqq gene clusters of Pseudomonas fluorescens B16 (pOK53) and Acinetobacter calcoaceticus (pSS2) were over-expressed in H. seropedicae Z67. Transformants Hs (pSS2) and Hs (pOK53) secreted micromolar levels of PQQ and attained high GDH activity leading to secretion of 33.46 mM gluconic acid when grown on 50 mM glucose while Hs (pJNK1) was ineffective. Hs (pJNK1) failed to solubilize rock phosphate, while Hs (pSS2) and Hs (pOK53) liberated 125.47 µM and 168.07 µM P, respectively, in minimal medium containing 50 mM glucose under aerobic conditions. Moreover, under N-free minimal medium, Hs (pSS2) and Hs (pOK53) not only released significant P but also showed enhanced growth, biofilm formation, and exopolysaccharide (EPS) secretion. However, indole acetic acid (IAA) production was suppressed. Thus, the addition of the pqq gene cluster, but not pqqE alone, is sufficient for engineering phosphate solubilization in H. seropedicae Z67 without compromising growth under nitrogen-fixing conditions.

Remodulation of central carbon metabolic pathway in response to arsenite exposure in Rhodococcus sp. strain NAU-1.

Arsenite-tolerant bacteria were isolated from an organic farm of Navsari Agricultural University (NAU), Gujarat, India (Latitude: 20°55'39.04″N; Longitude: 72°54'6.34″E). One of the isolates, NAU-1 (aerobic, Gram-positive, non-motile, coccobacilli), was hyper-tolerant to arsenite (As(III), 23 mM) and arsenate (As(V), 180 mM). 16S rRNA gene of NAU-1 was 99% similar to the 16S rRNA genes of Rhodococcus (Accession No. HQ659188). Assays confirmed the presence of membrane bound arsenite oxidase and cytoplasmic arsenate reductase in NAU-1. Genes for arsenite transporters (arsB and ACR3(1)) and arsenite oxidase gene (aoxB) were confirmed by PCR. Arsenite oxidation and arsenite efflux genes help the bacteria to tolerate arsenite. Specific activities of antioxidant enzymes (catalase, ascorbate peroxidase, superoxide dismutase and glutathione S-transferase) increased in dose-dependent manner with arsenite, whereas glutathione reductase activity decreased with increase in As(III) concentration. Metabolic studies revealed that Rhodococcus NAU-1 produces excess of gluconic and succinic acids, and also activities of glucose dehydrogenase, phosphoenol pyruvate carboxylase and isocitrate lyase were increased, to cope with the inhibited activities of glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase enzymes respectively, in the presence of As(III). Enzyme assays revealed the increase in direct oxidative and glyoxylate pathway in Rhodococcus NAU-1 in the presence of As(III).

Repression of mineral phosphate solubilizing phenotype in the presence of weak organic acids in plant growth promoting fluorescent pseudomonads.

Two phosphate solubilizing bacteria (PSB), M3 and SP1, were obtained from the rhizosphere of mungbean and sweet potato, respectively and identified as strains of Pseudomonas aeruginosa. Their rock phosphate (RP) solubilizing abilities were found to be due to secretion high amount of gluconic acid. In the presence of malate and succinate, individually and as mixture, the P solubilizing ability of both the strains was considerably reduced. This was correlated with a nearly 80% decrease in the activity of the glucose dehydrogenase (GDH) but not gluconate dehydrogenase (GAD) in both the isolates. Thus, GDH enzyme, catalyzing the periplasmic production of gluconic acid, is under reverse catabolite repression control by organic acids in P. aeruginosa M3 and SP1. This is of relevance in rhizospheric conditions and is a new explanation for the lack of field efficacy of such PSB.

Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene.

Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant approximately 2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2- and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3- and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525. Significantly low extracellular citrate levels as compared to the intracellular levels in Pf(pAB7) suggested a probable limitation of efficient citrate transport.

Variation in the nature of organic acid secretion and mineral phosphate solubilization by Citrobacter sp. DHRSS in the presence of different sugars.

A novel phosphate solubilizing bacterium (PSB) was isolated from the rhizosphere of sugarcane and is capable of utilizing sucrose and rock phosphate as the sole carbon and phosphate source, respectively. This PSB exhibited mineral phosphate solubilizing (MPS) phenotype on sugars such as sucrose and fructose, which are not substrates for enzyme glucose dehydrogenase (GDH), along with GDH substrates, viz., glucose, xylose, and maltose, as carbon sources. PCR amplification of the rRNA gene and sequence analysis identified this bacterium as Citrobacter sp. DHRSS. On sucrose and fructose Citrobacter sp. DHRSS liberated 170 and 100 microM free phosphate from rock phosphate and secreted 49 mM (2.94 g/L) and 35 mM (2.1 g/L) acetic acid, respectively. Growth of Citrobacter sp. DHRSS on sucrose is mediated by an intracellular inducible neutral invertase. Interestingly, in the presence of GDH substrates like glucose and maltose, Citrobacter sp. DHRSS produced approximately 20 mM (4.36 g/L) gluconic acid and phosphate released was 520 and 570 microM, respectively. Citrobacter sp. DHRSS GDH activity was found when grown on GDH and non-GDH substrates, indicating that it is constitutive and could act on a wide range of aldose sugars. This study demonstrates the role of different organic acids in mineral phosphate solubilization by rhizobacteria depending on the nature of the available carbon source.

Substrate specificity of glucose dehydrogenase (GDH) of Enterobacter asburiae PSI3 and rock phosphate solubilization with GDH substrates as C sources.

Enterobacter asburiae PSI3 is a rhizospheric isolate that solubilizes mineral phosphates by the action of a phosphate starvation-inducible GDH (EC 1.1.5.2). We report here that GDH activity of this isolate shows broad substrate range, being able to act on mono and disaccharides. Enterobacter asburiae PSI3 was proficient at bringing about a drop in pH and solubilization of RP with the use of 75 mmol/L of each of the GDH substrate sugars tested as the sole C source. It liberated amounts of P ranging from 450 micromol/L (on arabinose) to 890 micromol/L (on glucose). When grown on a mixture of 7 GDH substrates at concentrations of 15 mmol/L each, the bacterium solubilized RP equivalent to 46% of the value when 75 mmol glucose/L was the C source. HPLC analysis of the culture supernatant under these conditions showed that the acidification of the media is primarily due to the production of organic acids. The significance of these results on the efficacy of E. asburiae PSI3 at solubilizing phosphates under rhizospheric conditions is discussed.