G. Archana

Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate.

Keratinases play an important role in biotechnological applications such as improvement of feather meal, enzymatic dehairing and production of amino acids or peptides from high molecular weight substrates. Bacillus subtilis P13, isolated from Vajreshwari hot spring (45–50°C) near Mumbai, India, produces a neutral serine protease and has an optimum temperature of 65°C. This enzyme preparation was keratinolytic in nature and could disintegrate whole chicken feathers, except for the remnants of shafts. The enzyme preparation also exhibited depilation of goat hides with the recovery of intact animal hair. The enzyme preparation could release peptides from ground feathers and bring about their weight reduction; however, similar action on hair was relatively weak. A single major PMSF-sensitive protease band could be detected upon zymogram analysis, indicating that a single enzyme may be responsible for feather degradation and hide depilation. The importance of these findings in the biotechnological application for feather and leather industries is discussed.

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 μM 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 μM, 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.

Metabolic channeling of glucose towards gluconate in phosphate-solubilizing Pseudomonas aeruginosa P4 under phosphorus deficiency

Most phosphate-solubilizing bacteria (PSB), including the Pseudomonas species, release P from sparingly soluble mineral phosphates by producing high levels of gluconic acid from extracellular glucose, in a reaction catalyzed by periplasmic glucose dehydrogenase, which is an integral component of glucose catabolism of pseudomonads. To investigate the differences in the glucose metabolism of gluconic acid-producing PSB pseudomonads and low gluconic acid-producing/non-PSB strains, several parameters pertaining to growth and glucose utilization under P-sufficient and P-deficient conditions were monitored for the PSB isolate Pseudomonas aeruginosa P4 (producing approximately 46 mM gluconic acid releasing 437 microM P) and non-PSB P. fluorescens 13525. Our results show interesting differences in the channeling of glucose towards gluconate and other catabolic end-products like pyruvate and acetate with respect to P status for both strains. However, PSB strain P. aeruginosa P4, apart from exhibiting better growth under both low and high Pi conditions, differed from P. fluorescens 13525 in its ability to accumulate gluconate under P-solubilizing conditions. These alterations in growth, glucose utilization and acid secretion are correlated with glucose dehydrogenase, glucose-6-phosphate dehydrogenase and pyruvate carboxylase activities. The ability to shift glucose towards a direct oxidative pathway under P deficiency is speculated to underlie the differential gluconic acid-mediated P-solubilizing ability observed amongst pseudomonads.

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.

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.

Role of Siderophores in Crop Improvement

Iron is critical for life and poses the living system with a number of challenges. Its low solubility in the environment is of major concern which tends to create problems during its acquisition and transport. Its rich redox chemistry that underlies many of iron’s essential biological functions has resulted in highly evolved mechanisms of iron transport in living system. Iron, although available in plenty in rhizospheric soil, is seldom in its bioavailable form at neutral and alkaline pH, hence there always prevails an “iron stressed” condition. Siderophore is the Greek phrase for “iron bearer” and is applied to molecules that can bind metal at very high affinity. One of the major mechanisms evolved for iron acquisition to combat the iron-stressed condition is synthesis of high-affinity siderophore-mediated iron transport systems. Two main components – siderophore production, and synthesis of membrane receptor molecules which mediate internalization of iron bound to siderophores – form part of this high-affinity transport system. Better exploration and exploitation of soil resources require increasing the efficiency of nutrient uptake by plants/microorganisms and decreasing nutrient augmentation in the soil from outside. Achieving this condition requires understanding of intimate processes and factors that govern nutrient availability to plants and microorganisms. The present chapter addresses this issue with special emphasis on siderophore-mediated iron acquisition system, the significance of iron–siderophore affinity in niche colonization by specific group of microorganisms, significance of possessing diverse ferri-siderophore uptake system and its impact in competitive survival of microorganisms in the rhizosphere, and how all these factors contribute to the plant growth.

Response of ammonia-oxidizing archaea and bacteria to long-term industrial effluent-polluted soils, Gujarat, Western India

Soil nitrifiers have been showing an important role in assessing environmental pollution as sensitive biomarkers. In this study, the abundance and diversity of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were investigated in long-term industrial waste effluent (IWE) polluted soils. Three different IWE polluted soils characterized as uncontaminated (R1), moderately contaminated (R2), and highly contaminated (R3) were collected in triplicate along Mahi River basin, Gujarat, Western India. Quantitative numbers of ammonia monooxygenase α-subunit (amoA) genes as well as 16S rRNA genes indicated apparent deleterious effect of IWE on abundance of soil AOA, AOB, bacteria, and archaeal populations. Relatively, AOB was more abundant than AOA in the highly contaminated soil R3, while predominance of AOA was noticed in uncontaminated (R1) and moderately contaminated (R2) soils. Soil potential nitrification rate (PNR) significantly (P < 0.05) decreased in polluted soils R2 and R3. Reduced diversity accompanied by apparent community shifts of both AOB and AOA populations was detected in R2 and R3 soils. AOB were dominated with Nitrosospira-like sequences, whereas AOA were dominated by Thaumarchaeal “group 1.1b (Nitrososphaera clusters).” We suggest that the significant reduction in abundance and diversity AOA and AOB could serve as relevant bioindicators for soil quality monitoring of polluted sites. These results could be further useful for better understanding of AOB and AOA communities in polluted soils.

Functional expression of Escherichia coli fhuA gene in Rhizobium spp. of Cajanus cajan provides growth advantage in presence of Fe3+: ferrichrome as iron source.

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the EscherichiacolifhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 ΔfhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.

Plasmid load adversely affects growth and gluconic acid secretion ability of mineral phosphate-solubilizing rhizospheric bacterium Enterobacter asburiae PSI3 under P limited conditions

Effect of the metabolic load caused by the presence of plasmids on mineral phosphate-solubilizing (MPS) Enterobacter asburiae PSI3, was monitored with four plasmid cloning vectors and one native plasmid, varying in size, nature of the replicon, copy number and antibiotic resistance genes. Except for one plasmid, the presence of all other plasmids in E. asburiae PSI3 resulted in the loss of the MPS phenotype as reflected by the failure to bring about a drop in pH and release soluble P when grown in media containing rock phosphate (RP) as the sole P source. When 100 μM soluble P was supplemented along with RP, the adverse effects of plasmids on MPS phenotype and on growth parameters was reduced for some plasmid bearing derivatives, as monitored in terms of specific growth rates, glucose consumed, gluconic acids yields and P released. When 10 mM of soluble P as the only P source, was added to the medium all transformants showed growth and pH drop comparable with native strain. It may be concluded that different plasmids impose, to varying extents, a metabolic load in the phosphate-solubilizing bacterium E. asburiae PSI3 and results in diminishing its growth and P-solubilizing ability in P deficient conditions.

Broad-host-range plasmid-mediated metabolic perturbations in Pseudomonas fluorescens 13525

Genetic engineering of fluorescent pseudomonads for various industrially, agriculturally and environmentally important bioprocesses often involves the use of suitable plasmids. Plasmid-mediated alterations in host physiology and metabolism are poorly understood for this group of organisms. Thus, we investigated the metabolic perturbations in Pseudomonas fluorescens 13525 due to the independent and combined presence of broad-host-range plasmids, pBBR1MCS-2 (copy number 30) and pUCPM18 derived pAB4 and pAB8 (copy number 14-16). Presence of pAB4 and pAB8 not only significantly increased the growth rate and glucose utilization of P. fluorescens 13525, but also increased glucose dehydrogenase activity and gluconic acid production indicating enhanced direct oxidative pathway for glucose catabolism. Additionally, increased secretion of pyruvic, acetic, and citric acids caused faster media acidification in presence of pAB4 and pAB8. Simultaneous presence of pAB4/pAB8 in Pf (pAB48) and pAB4/pBBR1MCS-2 in Pf (pAB4BBR1MCS-2) reduced their respective copy numbers to nearly half. Pf (pAB48) demonstrated further increase in direct oxidation pathway without altering growth and glucose depletion rates, as compared with single transformants. Conversely, pBBR1MCS-2 plasmid did not greatly alter P. fluorescens 13525 metabolism when present independently but masked the effects imposed by pAB4 when present in its combination. In conclusion, P. fluorescens 13525 redesigns its metabolism in response to the presence of plasmids irrespective of their nature, by enhancing anaplerosis with a simultaneous reduction in catabolism as indicated by increased pyruvate carboxylase and decreased citrate synthase activities, respectively. Such information will be helpful for vector designing during genetic engineering of fluorescent pseudomonads.