Analysis of bio-optical active constituents for lentic ecosystem through spectral-spatial and in-vitro observation.

The neural network algorithm approach was adopted in Kolavai Lake to retrieve the inherent optical properties (IOP) of active constituents. The retrieval of IOP by absorption and the scattering of optically active constituents (OAC) through employing Sentinel-2 MSI reflectance and field measured the salinity and temperature. The result illustrates the relationship between the IOP and measured OAC's concentrations and its sensitivity towards spectral wavelength. It shows that the phytoplankton absorption ap is highly related with chlorophyll-a concentration and has an R2 value of 0.808. Furthermore, at the total absorption of water has high correlation with chl-a which indicates the significant dominance in the lentic water. Also, the pigment constituents are showing an R2 value of 0.754. The total backscattering of water (btot) is strongly related to the total suspended matter with R value > 0.73. The spatial distribution of OAC in Kolavai Lake helps monitor the lake water quality. This approach is well-performed in estimating the inherent optical properties of optically active constituents that gives insight for assessing the relationship between IOP and water quality. The research has proved to be a good potential for monitoring lentic water quality through Sentinel-2 MSI.

Genome sequencing and comparative genomic analysis of bovine mastitis-associated Staphylococcus aureus strains from India.

BACKGROUND: Bovine mastitis accounts for significant economic losses to the dairy industry worldwide. Staphylococcus aureus is the most common causative agent of bovine mastitis. Investigating the prevalence of virulence factors and antimicrobial resistance would provide insight into the molecular epidemiology of mastitis-associated S. aureus strains. The present study is focused on the whole genome sequencing and comparative genomic analysis of 41 mastitis-associated S. aureus strains isolated from India. RESULTS: The results elucidate explicit knowledge of 15 diverse sequence types (STs) and five clonal complexes (CCs). The clonal complexes CC8 and CC97 were found to be the predominant genotypes comprising 21 and 10 isolates, respectively. The mean genome size was 2.7 Mbp with a 32.7% average GC content. The pan-genome of the Indian strains of mastitis-associated S. aureus is almost closed. The genome-wide SNP-based phylogenetic analysis differentiated 41 strains into six major clades. Sixteen different spa types were identified, and eight isolates were untypeable. The cgMLST analysis of all S. aureus genome sequences reported from India revealed that S. aureus strain MUF256, isolated from wound fluids of a diabetic patient, was the common ancestor. Further, we observed that all the Indian mastitis-associated S. aureus isolates belonging to the CC97 are mastitis-associated. We identified 17 different antimicrobial resistance (AMR) genes among these isolates, and all the isolates used in this study were susceptible to methicillin. We also identified 108 virulence-associated genes and discuss their associations with different genotypes. CONCLUSION: This is the first study presenting a comprehensive whole genome analysis of bovine mastitis-associated S. aureus isolates from India. Comparative genomic analysis revealed the genome diversity, major genotypes, antimicrobial resistome, and virulome of clinical and subclinical mastitis-associated S. aureus strains.

Remote Sensing and Nonlinear Auto-regressive Neural Network (NARNET) Based Surface Water Chemical Quality Study: A Spatio-Temporal Hybrid Novel Technique (STHNT).

In recent days, the quality of water in inland water bodies has been threatened by various natural and anthropogenic activities. Henceforth, the continuous monitoring of water quality is mandatory to control the pollution level in surface water bodies. In this work, remote sensing technology integrated with an Artificial Intelligence (AI) algorithm, a new technique called Spatio-Temporal Hybrid Novel Technique (STHNT), was used to predict, and monitor the chemical water quality pollution level through the Water Quality Index (WQI). The Two Bands Regression Empirical (TBRE) water quality model has been used to retrieve water quality parameters from multi-resolution satellite imagery (Sentinel-2 MSI). The Nonlinear Auto-regressive Neural Network (NARNET), which is an Artificial Neural Network (ANN), was set up to predict the water quality index. Based on the model performed on the remote sensing water quality data, it is inferred that NARNET (Coefficient of determination-R2:0.9911, Root Mean Square Error-RMSE:1.693 and Sum of Squares of Error-SSE:14.33) provides significant results in predicting WQI. Therefore, the combined remote sensing technology with artificial intelligence plays a pivotal role in water resource management, which helps in reducing the pollution level in surface water bodies.

Extracytoplasmic sigma factor AlgU contributes to fitness of Pseudomonas aeruginosa PGPR2 during corn root colonization.

In bacteria, sigma factors are crucial in determining the plasticity of core RNA polymerase (RNAP) while promoter recognition during transcription initiation. This process is modulated through an intricate regulatory network in response to environmental cues. Previously, an extracytoplasmic function (ECF) sigma factor, AlgU, was identified to positively influence the fitness of Pseudomonas aeruginosa PGPR2 during corn root colonization. In this study, we report that the inactivation of the algU gene encoded by PGPR2_23995 hampers the root colonization ability of PGPR2. An insertion mutant in the algU gene was constructed by allele exchange mutagenesis. The mutant strains displayed threefold decreased root colonization efficiency compared with the wild-type strain when inoculated individually and in the competition assay. The mutant strain was more sensitive to osmotic and antibiotic stresses and showed higher resistance to oxidative stress. On the other hand, the mutant strain showed increased biofilm formation on the abiotic surface, and the expression of the pelB and pslA genes involved in the biofilm matrix formation were up-regulated. In contrast, the expression of algD, responsible for alginate production, was significantly down-regulated in the mutant strain, which is directly regulated by the AlgU sigma factor. The mutant strain also displayed altered motility. The expression of RNA binding protein RsmA was also impeded in the mutant strain. Further, the transcript levels of genes associated with the type III secretion system (T3SS) were analyzed, which revealed a significant down-regulation in the mutant strain. These results collectively provide evidence for the regulatory role of the AlgU sigma factor in modulating gene expression during root colonization.

Genome-wide identification of genetic requirements of Pseudomonas aeruginosa PAO1 for rat cardiomyocyte (H9C2) infection by insertion sequencing.

Pseudomonas aeruginosa is a major infectious agent among Gram-negative bacteria, which causes both acute and chronic infections. Infections due to P. aeruginosa are hard to treat, as it entails various strategies like virulence factors synthesis, drug efflux systems & resistance and protein secretion systems during pathogenesis. Despite extensive research in Pseudomonas pathogenesis, novel drug targets and potential therapeutic strategies are urgently needed. In this study, we investigated the genetic requirements of P. aeruginosa PAO1 for rat cardiomyocyte (H9C2) infection by insertion sequencing (INSeq). A mutant library comprising ~70,000 mutants of PAO1 was generated and the differentiated form of H9C2 cells (d-H9C2) was infected with the library. The infected d-H9C2 cells were maintained with antibiotic-protection and without any antibiotics in the growth media for 24 h. Subsequently, DNA library for INSeq was prepared, sequenced and fitness analysis was performed. One hundred and thirteen mutants were negatively selected in the infection condition with antibiotic-protection, whereas 143 mutants were negatively selected in antibiotic-free condition. Surprisingly, a higher number of mutants showed enriched fitness than the mutants of reduced fitness during the infection. We demonstrated that the genes associated with flagella and T3SS are important for adhesion and invasion of cardiomyocytes, while pili and proteases are conditionally essential during host cell lysis. Hence, our findings highlight the essential genes for cardiomyocyte infection, particularly during the intracellular phase. The aerotaxis receptor Aer, plays a critical role during intracellular life. Genes such as flgE, flgF, flhA, flhB, fliA, fliC, fliF, motA, aotJ, aer, wbpJ, ponA, fleQ, PA5205, hmgA, trkH and pslH are essential for infection.

Inactivation of CbrAB two-component system hampers root colonization in rhizospheric strain of Pseudomonas aeruginosa PGPR2.

Two-component systems (TCS) are one of the signal transduction mechanisms, which sense physiological/biological restraints and respond to changing environmental conditions by regulating the gene expression. Previously, by employing a forward genetic screen (INSeq), we identified that cbrA gene is essential for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. Here, we report the functional characterization of cbrAB TCS in PGPR2 during root colonization. We constructed insertion mutants in cbrA and its cognate response regulator cbrB. Genetic characterization revealed drastic down-regultion of sRNA crcZ gene in both mutant strains which play a critical role in carbon catabolite repression (CCR). The mutant strains displayed 10-fold decreased root colonization efficiency when compared to the wild-type strain. On the other hand, mutant strains formed higher biofilm on the abiotic surface, and the expression of pelB and pslA genes involved in biofilm matrix formation was up-regulated. In contrast, the expression of algD, responsible for alginate production, and its associated sigma factor algU was significantly down-regulated in mutant strains. We further analyzed the transcript levels of rsmA, controlled by the algU sigma factor, and found that the expression of rsmA was hampered in both mutants. The ability of mutant strains to swim and swarm was significantly hindered. Also, the expression of genes associated with type III secretion system (T3SS) was dysregulated in mutant strains. Taken together, regulation of gene expression by CbrAB TCS is intricate, and we confirm its role beyond carbon and nitrogen assimilation.

Genome-wide identification of probiotic Escherichia coli Nissle 1917 (EcN) fitness genes during adhesion to the intestinal epithelial cells Caco-2.

Escherichia coli Nissle 1917 (EcN) is an efficient probiotic strain extensively used worldwide because of its several health benefits. Adhesion to the intestinal cells is one of the prerequisites for a probiotic strain. To identify the genes essential for the adhesion of EcN on the intestinal cells, we utilized a quantitative genetic footprinting approach called transposon insertion sequencing (INSeq). A transposon insertion mutant library of EcN comprising of ~17,000 mutants was used to screen the adherence to the intestinal epithelial cells, Caco-2. The transposon insertion sites were identified from the input and output population by employing next-generation sequencing using the Ion torrent platform. Based on the relative abundance of reads in the input and output pools, we identified 113 candidate genes that are essential for the fitness of EcN during the adhesion and colonization on the Caco-2 cells. Functional categorization revealed that these fitness genes are associated with carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, post-translational modification, stress response, motility and adhesion, and signal transduction. To further validate the genes identified in our INSeq analysis, we constructed individual knock-out mutants in five genes (cyclic di-GMP phosphodiesterase (gmp), hda, uidC, leuO, and hypothetical protein-coding gene). We investigated their ability to adhere to Caco-2 cells. Evaluation of these mutants showed reduced adhesion on Caco-2 cells, confirming their role in adhesion. Understanding the functions of these genes may provide novel insights into molecular regulation during colonization of probiotic bacteria to the intestinal cells, and useful to develop designer probiotic strains.

Whole-Genome Sequence Analysis of Paenibacillus alvei JR949 Revealed Biosynthetic Gene Clusters Coding for Novel Antimicrobials.

The increased prevalence of multidrug-resistant pathogens poses a significant clinical threat, and hence, the discovery of novel antibiotics is the need of the hour. Several attempts are being made worldwide to screen and identify newer antibiotics from various microbial sources. The genus Paenibacillus is known for its biosynthetic potential and metabolic versatility in producing several secondary metabolites. In this study, we isolated Paenibacillus alvei strain JR949 from the soil, which exhibited antimicrobial activity against Enteropathogenic Escherichia coli (EPEC), Pseudomonas aeruginosa (PAO1), and methicillin-resistant Staphylococcus aureus (MRSA). The whole genome of this strain was sequenced using the Illumina platform. The genome mining of the draft genome sequence revealed a total of 31 biological gene clusters (BGCs) responsible for the synthesis of secondary metabolites. The construction of the similarity network of the BGCs and the comparative analysis with the genetically related strains aided the identification of metabolites produced by this strain. We identified BGCs coding for paenibactin, paenibacterin, anabaenopeptin NZ857, icosalide A/B, polymyxin, and bicornutinA1/A2 with 100% similarity. The BGCs with lower sequence similarity to paenibacterin, polymyxin B, colistin A/B, pellasoren, tridecaptin, pelgipeptin, and marthiapeptide were also identified. Furthermore, 13 putative NRPS BGCs, 3 NRPS-T1PKS hybrid clusters, a T1PKS, and a bacteriocin BGC were identified with very low similarity (≤ 25%) or no similarity with known antibiotics. Further experimental investigations may result in the discovery of novel antimicrobial drugs.

Whole-genome Sequencing and Mining of Protease Coding Genes in Bacillus paralicheniformis MKU3, and its Degradomics in Feather Meal Medium.

Bacillus paralicheniformis MKU3 produces commercially important keratinolytic proteases by utilizing chicken feather. To unravel the genetics of these degrading keratinolytic proteases in B. paralicheniformis MKU3, we sequenced the genome of this bacterium and studied the protease distribution and their characteristics using bioinformatics tools. Also, a proteomic analysis was performed to identify the consortium of proteases involved in feather hydrolysis. A total of 2,531,755 quality reads were obtained in whole genome sequencing with an approximate coverage fold of 105. The draft genome consists of 4,370,039 bp with 45 contigs. The draft genome codes for 4874 protein-coding genes. Furthermore, 109 genes coding for RNA, including 26 rRNA and 83 tRNA, were identified. Phylogenetic analysis of B. paralicheniformis MKU3 showed closest homolog to B. paralicheniformis F47. Genes coding for proteases belonging to five families were identified with the following proportions 37%, 36%, 9%, 14%, 2%, and 2% of serine-, metallo-, cysteine-, mixed-, and uncharacterized proteases, respectively. Metallo- and serine-protease represented more than 70% of the total proteases. Major protease families distributed in the genome were S8, S9, S33, M20, M50, C26, and C40. Most of the proteases showed significant similarity with the conserved domain database and also identified conserved catalytic sites and domains. SDS-PAGE and zymogram analysis of concentrated feather hydrolysis revealed the active proteases ranging from 10 to 250 kD in size. Proteomic analysis on the feather hydrolysis of B. paralicheniformis MKU3 identified two proteases belonging to serine proteases (S8) and other two as metalloproteases.

Functional characterization of asnC family transcriptional regulator in Pseudomonas aeruginosa PGPR2 during root colonization.

Transcriptional regulators in bacteria are the crucial players in mediating communication between environmental cues and DNA transcription through a complex network process. Pseudomonas aeruginosa PGPR2 is an efficient root colonizer and a biocontrol strain. Previously, we identified that the transcriptional regulator, asnC, negatively regulates the corn root colonization of P. aeruginosa PGPR2. In a transposon insertion sequencing (INSeq) screen, the asnC insertion mutant was positively selected during root colonization, meaning the disruption of asnC improves the fitness of the P. aeruginosa PGPR2 strain for the root colonization. In this study, we constructed isogenic mutant of asnC family transcriptional regulator encoded by PGPR2_17510 by allele exchange mutagenesis. The ΔasnC mutant was able to efficiently colonize corn roots with a twofold increase in population when compared to the wild-type strain. Similarly, the mutant strain outcompeted the wild-type strain in a competition assay, where the mutant strain represented 90% of the total population recovered from the root. We compared the whole transcriptome of the wild-type and the ΔasnC mutant of P. aeruginosa PGPR2 when exposed to the corn root exudates. The RNA-Seq revealed that a total of 360 genes were differentially expressed in the ΔasnC strain of P. aeruginosa PGPR2. Inactivation of asnC transcriptional regulator resulted in the up-regulation of several genetic factors implicated in metabolism, uptake of nutrients, motility, stress response, and signal transduction, which could play crucial roles in root colonization. This notion was further validated by phenotypic characterization and quantification of transcription pattern of selected genes associated with metabolism, motility, and carbon catabolite repression between wild type and mutant strain, which was in agreement with transcriptome data. Similarly, ΔasnC strain formed increased biofilm on abiotic surface validating our RNA-seq analysis, where transcript levels of several genes associated with biofilm formation were up-regulated in the mutant strain. We report that the inactivation of an asnC family transcriptional regulator encoded by PGPR2_17510 enhances the root colonization and biofilm-forming ability of P. aeruginosa PGPR2. Together, our results provide evidence for the molecular adaptations that enable ΔasnC mutant strain to colonize on the corn roots and to form a biofilm.

Evaluation of INSeq To Identify Genes Essential for Pseudomonas aeruginosa PGPR2 Corn Root Colonization.

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the INSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5- to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in Paeruginosa PGPR2 for root colonization.

Elevated levels of circulating DNA in cardiovascular disease patients: metagenomic profiling of microbiome in the circulation.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. An expanding body of evidence supports the role of human microbiome in the establishment of CVDs and, this has gained much attention recently. This work was aimed to study the circulating human microbiome in CVD patients and healthy subjects. The levels of circulating cell free DNA (circDNA) was higher in CVD patients (n = 80) than in healthy controls (n = 40). More specifically, the relative levels of circulating bacterial DNA and the ratio of 16S rRNA/ß-globin gene copy numbers were higher in the circulation of CVD patients than healthy individuals. In addition, we found a higher circulating microbial diversity in CVD patients (n = 3) in comparison to healthy individuals (n = 3) by deep shotgun sequencing. At the phylum level, we observed a dominance of Actinobacteria in CVD patients, followed by Proteobacteria, in contrast to that in healthy controls, where Proteobacteria was predominantly enriched, followed by Actinobacteria. The circulating virome in CVD patients was enriched with bacteriophages with a preponderance of Propionibacterium phages, followed by Pseudomonas phages and Rhizobium phages in contrast to that in healthy individuals, where a relatively greater abundance of eukaryotic viruses dominated by Lymphocystis virus (LCV) and Torque Teno viruses (TTV) was observed. Thus, the release of bacterial and viral DNA elements in the circulation could play a major role leading to elevated circDNA levels in CVD patients. The increased circDNA levels could be either the cause or consequence of CVD incidence, which needs to be explored further.