Engineering non-conserved salt bridges in GH11 xylanase from Bacillus pumilus SSP34 for improved thermal stability: an in-silico evaluation.

GH11 xylanases are commercially important enzymes for degradation of xylan fibers. We have identified the presence of nine non-conserved and five conserved salt bridges in GH11 xylanase from Bacillus pumilus SSP34. We have designed two sets of mutants viz., (1) substitution mutants in which non-conserved charged amino acid residues have been replaced with appropriate hydrophobic residues based on side chain occupancy and hydrophobicity and (2) deletion mutants where non-conserved charged residues have been deleted. The stability of the mutants has been evaluated in-silico by analyzing the contributions of non-covalent interactions like hydrophobic interaction clusters and salt bridges. The stability of the resultant mutants was evaluated using parameters such as radius of gyration, solvent accessible surface area, root mean square deviation, root mean square fluctuations and protein unfolding measurements using molecular dynamic simulations. The deletion of certain charged residues resulted in mutants having lowered radius of gyration and decreased surface areas. However, RMSD and RMSF measurements indicated lowered stability in comparison to substitution mutants. Of the substitution mutants, the SBM 3 was the most stable mutant as indicated by Rg, SASA, RMSF and simulated protein unfolding measurements. The major contributing factors for improved stability could be strengthening of hydrophobic interactions in the GH11 xylanase from B. pumilus. These in-silico stability measurements of salt bridge mutants may lead to better design of GH11 xylanases for commercial applications.Communicated by Ramaswamy H. Sarma.

Design of mutants of GH11 xylanase from Bacillus pumilus for enhanced stability by amino acid substitutions in the N-terminal region: an in silico analysis.

GH11 xylanases are versatile small-molecular-weight single-polypeptide chain monofunctional enzymes. This family of glycoside hydrolases has important applications in food, feed and chemical industries. We designed mutants for improved thermal stability with substitutions in the first six residues of the N-terminal region and evaluated the stability in silico. The first six residues RTITNN of native xylanase have been mutated accordingly to introduce ß structure, increase hydrophobic clusters and enhance conformational rigidity in the molecule. To design stable mutants, the approach consisted of constructing root mean square fluctuation (RMSF) plots of both mesophilic and thermophilic xylanases to check the localized backbone displacement maxima, identify the hydrophobic interaction cluster in and around the peaks of interest, construct mutants by substituting appropriate residues based on beta propensity, hydrophobicity, side chain occupancy and conformational rigidity. This resulted in the decreased number of possible substitutions from 19 to 6 residues. Introduction of conformational rigidity by substitution of asparagine residues at 5th and 6th residue position with proline and valine enhanced the stability. Deletion of N-terminal region increased the stability probably by reducing entropic factors. The structure and stability of GH11 xylanase and resultant mutants were analyzed by root mean square deviation, RMSF, radius of gyration and solvent accessible surface area analysis. The stability of the mutants followed the order N-del > Y1P5 >Y1V5 > ATRLM. The contribution of N-terminal end to overall stability of the molecule is significant because of the proximity of the C-terminal end to the N-terminal end which reinforces long-range interactions. Communicated by Ramaswamy H. Sarma.

Evaluation of an in-house LipL32 polymerase chain reaction for diagnosis of leptospirosis and its correlation with various serological diagnostic techniques.

BACKGROUND: Leptospirosis is a zoonotic disease of ubiquitous distribution. During rainy seasons, in spring and summer and also during harvest times, the risk of leptospirosis increases as there are chances of frequent contact with infected rat population which is common in Karnataka as farming is a main source of income to the people here. There is a paucity of data regarding the prevalent serovars from Karnataka. This study was undertaken as an attempt to compare a battery of tools such as immunochromatographic test (ICT), microscopic agglutination test (MAT), immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) and in-house polymerase chain reaction (PCR) to detect leptospirosis. SETTINGS AND DESIGN: This study using consecutive sampling technique was conducted in a tertiary care centre, Mysore, Karnataka. SUBJECTS AND METHODS: Samples from 783 suspected cases of leptospirosis in and around Mysore between April 2013 and April 2016 were processed. Samples from 783 patients suspected of leptospirosis were subjected to ICT, IgM ELISA, MAT and in-house PCR. STATISTICAL ANALYSIS USED: The statistical analysis was carried out using SPSS software version. RESULTS: Among 783 samples tested, only 14 (1.7%) were positive by ICT, 341 (44%) were positive by IgM ELISA, 368 (47%) were positive by MAT and 393 (50.2%) were positive by in-house PCR. CONCLUSIONS: Mysore can be considered endemic for leptospirosis. The in-house PCR based on LipL32 gene proved to be useful in the early diagnosis of leptospirosis.

The pearl millet mitogen-activated protein kinase PgMPK4 is involved in responses to downy mildew infection and in jasmonic- and salicylic acid-mediated defense.

Plant mitogen-activated protein kinases (MPKs) transduce signals required for the induction of immunity triggered by host recognition of pathogen-associated molecular patterns. We isolated a full-length cDNA of a group B MPK (PgMPK4) from pearl millet. Autophosphorylation assay of recombinant PgMPK4 produced in Escherichia coli confirmed it as a kinase. Differential accumulation of PgMPK4 mRNA and kinase activity was observed between pearl millet cultivars 852B and IP18292 in response to inoculation with the downy mildew oomycete pathogen Sclerospora graminicola. This increased accumulation of PgMPK4 mRNA, kinase activity as well as nuclear-localization of PgMPK protein(s) was only detected in the S. graminicola resistant cultivar IP18292 with a ~tenfold peak at 9 h post inoculation. In the susceptible cultivar 852B, PgMPK4 mRNA and immuno-detectable nuclear PgMPK could be induced by application of the chemical elicitor ß-amino butyric acid, the non-pathogenic bacteria Pseudomonas fluorescens, or by the phytohormones jasmonic acid (JA) or salicylic acid (SA). Furthermore, kinase inhibitor treatments indicated that PgMPK4 is involved in the JA- and SA-mediated expression of three defense genes, lipoxygenase, catalase 3 and polygalacturonase-inhibitor protein. These findings indicate that PgMPK/s contribute to pearl millet defense against the downy mildew pathogen by activating the expression of defense proteins.

A Network Map of FGF-1/FGFR Signaling System.

Fibroblast growth factor-1 (FGF-1) is a well characterized growth factor among the 22 members of the FGF superfamily in humans. It binds to all the four known FGF receptors and regulates a plethora of functions including cell growth, proliferation, migration, differentiation, and survival in different cell types. FGF-1 is involved in the regulation of diverse physiological processes such as development, angiogenesis, wound healing, adipogenesis, and neurogenesis. Deregulation of FGF-1 signaling is not only implicated in tumorigenesis but also is associated with tumor invasion and metastasis. Given the biomedical significance of FGFs and the fact that individual FGFs have different roles in diverse physiological processes, the analysis of signaling pathways induced by the binding of specific FGFs to their cognate receptors demands more focused efforts. Currently, there are no resources in the public domain that facilitate the analysis of signaling pathways induced by individual FGFs in the FGF/FGFR signaling system. Towards this, we have developed a resource of signaling reactions triggered by FGF-1/FGFR system in various cell types/tissues. The pathway data and the reaction map are made available for download in different community standard data exchange formats through NetPath and NetSlim signaling pathway resources.

Polygalacturonase-inhibitor proteins in pearl millet: possible involvement in resistance against downy mildew.

Polygalacturonase-inhibitor protein (PGIP) is a defense protein found in plant cell walls. It prevents the degradation of pectin by modulating the endo-polygalacturonase activity. The present study has used heterologous anti-bean PGIP probes to investigate the role of PGIP in pearl millet [Pennisetum glaucum (L) R. Br.] resistance against downy mildew caused by oomycete pathogen Sclerospora graminicola (Sacc.) Schroet. Northern blot analysis using bean pgip2 DNA fragment as probe showed an early and marked induction of transcripts (∼1.2 kb) upon pathogen-inoculation in pearl millet cultivar resistant to downy mildew, with the maximum level observed at 24 and 48 h post-inoculation (h.p.i.). Western blot analysis of pearl millet total cell wall proteins using antibodies against bean PGIP showed the presence of a major band of ∼43 kDa, and several minor ones. The protein accumulation was higher in resistant seedlings than in susceptible seedlings with a differential expression observed only in the case of incompatible interaction. Immunocytochemical localization in epidermal peelings of coleoptiles and tissue-printing showed a similar trend in the PGIP accumulation. PGIP was found to localize in the epidermal as well as in the vascular regions of tissues. Higher accumulation was observed in the stomatal guard cells of resistant cultivar inoculated with the pathogen. PGIP activity of pearl millet total protein extracts when assayed against Aspergillus niger PG displayed differential PG inhibitory activities between the resistant and suceptible cultivars with resistant sample showing the highest inhibition of 16%, post-pathogen treatment. Thus, PGIP appeared to be an important player in pearl millet-S. graminicola interaction leading to host resistance.

Isolation and characterisation of a protein elicitor from Sclerospora graminicola and elicitor-mediated induction of defence responses in cultured cells of Pennisetum glaucum.

Sclerospora graminicola (Sacc.) Schroet., an oomycete pathogen of Pennisetum glaucum (L.) R.Br. infects the meristematic tissues of young seedlings. The motile zoospores from the sporangia encyst, germinate and penetrate the plant tissue. Resistance to the invading pathogen is governed by the specific recognition of conserved pathogen-associated proteins or elicitors. In the present study, a zoospore protein was isolated and purified to homogeneity by a combination of size exclusion and high-performance liquid chromatography (HPLC). The crude fractionated protein was able to elicit an array of defence responses in resistant and susceptible cells of pearl millet. Treatment of cultured cells of pearl millet with partially purified elicitor protein resulted in a rapid loss of cell viability in the resistant cells and the percentage of cell death was higher in the resistant than in the susceptible cells. Cultures of resistant cells showed a sharp increase in the extra cellular pH compared with susceptible cells when treated with the crude elicitor. Increased oxidative burst was also recorded in the cells treated with the crude elicitor. The purified elicitor showed unique properties. The purified protein was acidic with a pI of 5.6 as revealed by isoelectric focusing (IEF) and matrix-assisted laser desorption ionisation (MALDI) analysis showed that the elicitor had a molecular mass of 7040 daltons. The primary structure determined by N-terminal Edman degradation and searches with BLAST did not reveal similarities to any known plant pathogenic or oomycete elicitor. Higher activities of the important defence-related enzymes phenylalanine ammonia lyase (PAL) and peroxidase in the resistant cell cultures than in the susceptible cell cultures treated with the purified elicitor were clearly evident. Studies of gene expression by northern blotting with heterologus peroxidase, PAL and oxalate oxidase probes showed that the mRNA transcripts were strongly up-regulated in resistant cell cultures within 30 min of elicitor treatment. The purified elicitor also demonstrated a very strong concentration-dependent sterol binding. The purified elicitor protein belongs to a class of low molecular weight oomycete elicitors with sterol carrier properties. The identified low molecular weight protein elicitor displays unique properties that can be exploited for synthesis of novel molecules for eco-friendly crop protection.