Mutant Gossypium universal stress protein-2 (GUSP-2) gene confers resistance to various abiotic stresses in E. coli BL-21 and CIM-496-Gossypium hirsutum

Gossypium arboreum is considered a rich source of stress-responsive genes and the EST database revealed that most of its genes are uncharacterized. The full-length Gossypium universal stress protein-2 (GUSP-2) gene (510 bp) was cloned in E. coli and Gossypium hirsutum, characterized and point mutated at three positions, 352–354, Lysine to proline (M1-usp-2) & 214–216, aspartic acid to serine (M2-usp-2) & 145–147, Lysine to Threonine (M3-usp-2) to study its role in abiotic stress tolerance. It was found that heterologous expression of one mutant (M1-usp-2) provided enhanced tolerance against salt and osmotic stresses, recombinant cells have higher growth up to 10-5dilution in spot assay as compared to cells expressing W-usp-2 (wild type GUSP-2), M2-usp-2 and M3-usp-2 genes. M1-usp-2 gene transcript profiling exhibited significant expression (8.7 fold) in CIM-496-Gossypium hirsutum transgenic plants and enhance drought tolerance. However, little tolerance against heat and cold stresses in bacterial cells was observed. The results from our study concluded that the activity of GUSP-2 was enhanced in M1-usp-2 but wipe out in M2-usp-2 and M3-usp-2 response remained almost parallel to W-usp-2. Further, it was predicted through in silico analysis that M1-usp-2, W-usp-2 and M3-usp-2 may be directly involved in stress tolerance or function as a signaling molecule to activate the stress adaptive mechanism. However, further investigation will be required to ascertain its role in the adaptive mechanism of stress tolerance.

Bioinformatics Investigations of Universal Stress Proteins from Mercury-Methylating Desulfovibrionaceae

The presence of methylmercury in aquatic environments and marine food sources is of global concern. The chemical reaction for the addition of a methyl group to inorganic mercury occurs in diverse bacterial taxonomic groups including the Gram-negative, sulfate-reducing Desulfovibrionaceae family that inhabit extreme aquatic environments. The availability of whole-genome sequence datasets for members of the Desulfovibrionaceae presents opportunities to understand the microbial mechanisms that contribute to methylmercury production in extreme aquatic environments. We have applied bioinformatics resources and developed visual analytics resources to categorize a collection of 719 putative universal stress protein (USP) sequences predicted from 93 genomes of Desulfovibrionaceae. We have focused our bioinformatics investigations on protein sequence analytics by developing interactive visualizations to categorize Desulfovibrionaceae universal stress proteins by protein domain composition and functionally important amino acids. We identified 651 Desulfovibrionaceae universal stress protein sequences, of which 488 sequences had only one USP domain and 163 had two USP domains. The 488 single USP domain sequences were further categorized into 340 sequences with ATP-binding motif and 148 sequences without ATP-binding motif. The 163 double USP domain sequences were categorized into (1) both USP domains with ATP-binding motif (3 sequences); (2) both USP domains without ATP-binding motif (138 sequences); and (3) one USP domain with ATP-binding motif (21 sequences). We developed visual analytics resources to facilitate the investigation of these categories of datasets in the presence or absence of the mercury-methylating gene pair (hgcAB). Future research could utilize these functional categories to investigate the participation of universal stress proteins in the bacterial cellular uptake of inorganic mercury and methylmercury production, especially in anaerobic aquatic environments.

The diversity of unique 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid coding common genes and Universal stress protein in Ectoine TRAP cluster (UspA) in 32 Halomonas species

Objectives To decipher the diversity of unique ectoine-coding housekeeping genes in the genus Halomonas. Results In Halomonas, 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid has a crucial role as a stress-tolerant chaperone, a compatible solute, a cell membrane stabilizer, and a reduction in cell damage under stressful conditions. Apart from the current 16S rRNA biomarker, it serves as a blueprint for identifying Halomonas species. Halomonas elongata 1H9 was found to have 11 ectoine-coding genes. The presence of a superfamily of conserved ectoine-coding among members of the genus Halomonas was discovered after genome annotations of 93 Halomonas spp. As a result of the inclusion of 11 single copy ectoine coding genes in 32 Halomonas spp., genome-wide evaluations of ectoine coding genes indicate that 32 Halomonas spp. have a very strong association with H. elongata 1H9, which has been proven evidence-based approach to elucidate phylogenetic relatedness of ectoine-coding child taxa in the genus Halomonas. Total 32 Halomonas species have a single copy number of 11 distinct ectoine-coding genes that help Halomonas spp., produce ectoine under stressful conditions. Furthermore, the existence of the Universal stress protein (UspA) gene suggests that Halomonas species developed directly from primitive bacteria, highlighting its role during the progression of microbial evolution.

The diversity of unique 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid coding common genes and novel Universal stress protein in Ectoine TRAP cluster (UspA) in 32 Halomonas species

ObjectivesTo decipher the diversity of unique ectoine-coding housekeeping genes in the genus Halomonas.ResultsIn Halomonas, 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid has a crucial role as a stress-tolerant chaperone, a compatible solute, a cell membrane stabilizer, and a reduction in cell damage under stressful conditions. Apart from the current 16S rRNA biomarker, it serves as a blueprint for identifying Halomonas species. Halomonas elongate 1H9 was found to have 11 ectoine-coding genes. The presence of a superfamily of conserved ectoine-coding among members of the genus Halomonas was discovered after genome annotations of 93 Halomonas spp. As a result of the inclusion of 11 single copy ectoine coding genes in 32 Halomonas spp., genome-wide evaluations of ectoine coding genes indicate that 32 Halomonas spp. have a very strong association with Halomonas elongata 1H9, which has been proven evidence-based approach to elucidate phylogenetic relatedness of ectoine-coding child taxa in the genus Halomonas. Total 32 Halomonas species have a single copy number of 11 distinct ectoine-coding genes that help Halomonas spp., that produce ectoine under stressful conditions. Furthermore, the existence of the Universal stress protein (UspA) gene suggests that Halomonas species developed directly from primitive bacteria, highlighting the role of Halomonas species in evolutionary terms.

GRUSP, an Universal Stress Protein, Is Involved in Gibberellin-dependent Induction of Flowering in Arabidopsis thaliana

The effect of T-DNA insertion in the 3'-UTR region of Arabidopsis thaliana At3g58450 gene encoding the Germination-Related Universal Stress Protein (GRUSP) was studied. It was found that under a long-day condition this mutation delays transition to flowering of grusp-115 transgenic line that due to a reduced content of endogenous bioactive gibberellins GA1 and GA3 in comparison to the wild-type plants (Col-0). Exogenous GA accelerated flowering of both lines but did not change the time of difference in the onset of flowering between Col-0 and grusp-115. In addition to changes in GA metabolism, grusp-115 evidently has disturbances in realization of the signal that induces flowering. This is confirmed by the results of gene expression of the floral integrator FLOWERING LOCUS T (FT) and the floral repressor FLOWERING LOCUS C (FLC), which are key flowering regulators and acting opposite. We hypothesize that the formation of grusp-115 phenotype can also be affected by a low expression level of FT due to up-regulated FLC expression.

The diversity of unique 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid coding common genes and novel Universal stress protein in Ectoine TRAP cluster (UspA) in 32 Halomonas species

Objectives To decipher the diversity of unique ectoine-coding housekeeping genes in the genus Halomonas.Results In Halomonas, 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid has a crucial role as a stress-tolerant chaperone, a compatible solute, a cell membrane stabilizer, and a reduction in cell damage under stressful conditions. Apart from the current 16S rRNA biomarker, it serves as a blueprint for identifying Halomonas species. Halomonas elongate 1H9 was found to have 11 ectoine-coding genes. The presence of a superfamily of conserved ectoine-coding among members of the genus Halomonas was discovered after genome annotations of 93 Halomonas spp. As a result of the inclusion of 11 single copy ectoine coding genes in 32 Halomonas spp., genome-wide evaluations of ectoine coding genes indicate that 32 Halomonas spp. have a very strong association with Halomonas elongata 1H9, which has been proven evidence-based approach to elucidate phylogenetic relatedness of ectoine-coding child taxa in the genus Halomonas. Total 32 Halomonas species have a single copy number of 11 distinct ectoine-coding genes that help Halomonas spp. produce ectoine under stressful conditions. Furthermore, the existence of the Universal stress protein (UspA) gene suggests that Halomonas species developed directly from primitive bacteria, highlighting the role of Halomonas species in evolutionary terms.

Identification of Potential Binders of Mtb Universal Stress Protein (Rv1636) Through an in silico Approach and Insights Into Compound Selection for Experimental Validation

Millions of deaths caused by Mycobacterium tuberculosis (Mtb) are reported worldwide every year. Treatment of tuberculosis (TB) involves the use of multiple antibiotics over a prolonged period. However, the emergence of resistance leading to multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) is the most challenging aspect of TB treatment. Therefore, there is a constant need to search for novel therapeutic strategies that could tackle the growing problem of drug resistance. One such strategy could be perturbing the functions of novel targets in Mtb, such as universal stress protein (USP, Rv1636), which binds to cAMP with a higher affinity than ATP. Orthologs of these proteins are conserved in all mycobacteria and act as “sink” for cAMP, facilitating the availability of this second messenger for signaling when required. Here, we have used the cAMP-bound crystal structure of USP from Mycobacterium smegmatis, a closely related homolog of Mtb, to conduct a structure-guided hunt for potential binders of Rv1636, primarily employing molecular docking approach. A library of 1.9 million compounds was subjected to virtual screening to obtain an initial set of ~2,000 hits. An integrative strategy that uses the available experimental data and consensus indications from other computational analyses has been employed to prioritize 22 potential binders of Rv1636 for experimental validations. Binding affinities of a few compounds among the 22 prioritized compounds were tested through microscale thermophoresis assays, and two compounds of natural origin showed promising binding affinities with Rv1636. We believe that this study provides an important initial guidance to medicinal chemists and biochemists to synthesize and test an enriched set of compounds that have the potential to inhibit Mtb USP (Rv1636), thereby aiding the development of novel antitubercular lead candidates.