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.

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.

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.

Metagenomic Analysis of Microbial Community Affiliated with Termitarium Reveals High Lignocellulolytic Potential.

Termitarium (nest of termites) is a rich source of microbial populations whose resources remain untapped to date. Using the metagenomic sequencing approach, we generated 38 GB sequences comprising 808,386 contigs (896 MB) with a maximum contig size of 470 kb. The taxonomic profile obtained by BLAST against the NCBI NR database and annotation by MEGAN showed that the termitarium microbial community was dominated by Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Functional annotation using the CAZY database revealed a huge diversity of glycosyl hydrolase genes from 104 families, some of which appeared to be part of polysaccharide utilization systems (PUL). Strikingly, Actinobacteria was the main contributor of the cellulolytic and hemicellulolytic GHs. Genes involving in lignin degradation were also abundantly identified in this metagenome. Comparative analysis of COG profiles of termitarium with those of other lignocellulolytic microbial communities showed a distant clustering pattern resulting from the dietary differences in carbohydrate compositions. Altogether, this study revealed that termitarium hosts a unique microbial community, which can efficiently degrade lignocelluloses.

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.

Functional analysis of membrane vesicles of Listeria monocytogenes suggests a possible role in virulence and physiological stress response.

Membrane vesicles (MVs) are naturally secreted by many pathogenic organisms and have various functions that include the release of microbial virulence factors that contributes to pathogenesis. However, very little is known regarding the function of Gram-positive bacteria membrane vesicles. Here, we investigated the functional role of membrane vesicles of Listeria monocytogenes. We found that L. monocytogenes secreted MVs are spherical and diameter size around 192.3 nm. Here, we investigated the role of L. monocytogenes membrane vesicles in interbacterial communication to cope with antibiotic stress. We found that MVs are protecting the bacteria against the antibiotics trimethoprim and streptomycin. These MVs enabled streptomycin-susceptible L. monocytogenes 1143 to survive in the presence of streptomycin. The zeta potential, dynamic light scattering (DLS) and 1-Nphenylnapthylamine (NPN)-uptake assay reveals that MVs protect the bacterium from active antibiotics by different strategies. Exposure to environmental stressors was shown to increase the level of MV production in L. monocytogenes. The biological activity of MV-associated listeriolysin O, internalin B, and phosphatidylinositol-specific phospholipase C (PI-PLC) was investigated using epithelial cell cytotoxicity. The reduced cytotoxicity was observed in Δhly MVs on Caco-2 cells suggesting that MVs are biologically active. It is shown that a potent toxin LLO contributes to the MV mediated pathogenesis of L. monocytogenes.

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.

Comprehensive proteomic analysis and pathogenic role of membrane vesicles of Listeria monocytogenes serotype 4b reveals proteins associated with virulence and their possible interaction with host.

Membrane vesicles (MVs) are produced by various Gram positive and Gram negative pathogenic bacteria and play an important role in virulence. In this study, the membrane vesicles (MVs) of L. monocytogenes were isolated from the culture supernatant. High-resolution electron microscopy and dynamic light scattering analysis revealed that L. monocytogenes MVs are spherical with a diameter of 200 to 300 nm in size. Further, comprehensive proteomic analyses of MVs and whole cells of L. monocytogenes were performed using LC/MS/MS. A total of 1355 and 312 proteins were identified in the L. monocytogenes cells and MVs, respectively. We identified that 296 proteins are found in both whole cells, and MV proteome and 16 proteins were identified only in the MVs. Also, we have identified the virulence factors such as listeriolysin O (LLO), internalin B (InlB), autolysin, p60, NLP/P60 family protein, UPF0356 protein, and PLC-A in MVs. Computational prediction of host-MV interactions revealed a total of 1841 possible interactions with the host involving 99 MV proteins and 1513 host proteins. We elucidated the possible pathway that mediates internalization of L. monocytogenes MV to host cells and the subsequent pathogenesis mechanisms. The in vitro infection assays showed that the purified MVs could induce cytotoxicity in Caco-2 cells. Using endocytosis inhibitors, we demonstrated that MVs are internalized via actin-mediated endocytosis. These results suggest that L. monocytogenes MVs can interact with host cell and contribute to the pathogenesis of L. monocytogenes during infection.

Genome Investigation of a Cariogenic Pathogen with Implications in Cardiovascular Diseases.

The proportion of people suffering from cardiovascular diseases has risen by 34% in the last 15 years in India. Cardiomyopathy is among the many forms of CVD s present. Infection of heart muscles is the suspected etiological agent for the same. Oral pathogens gaining entry into the bloodstream are responsible for such infections. Streptococcus mutans is an oral pathogen with implications in cardiovascular diseases. Previous studies have shown certain strains of S. mutans are found predominantly within atherosclerotic plaques and extirpated valves. To decipher the genetic differences responsible for endothelial cell invasion, we have sequenced the genome of Streptococcus mutans B14. Pan-genome analysis, search for adhesion proteins through a special algorithm, and protein-protein interactions search through HPIDB have been done. Pan-genome analysis of 187 whole genomes, assemblies revealed 6965 genes in total and 918 genes forming the core gene cluster. Adhesion to the endothelial cell is a critical virulence factor distinguishing virulent and non-virulent strains. Overall, 4% of the total proteins in S. mutans B14 were categorized as adhesion proteins. Protein-protein interaction between putative adhesion proteins and Human extracellular matrix components was predicted, revealing novel interactions. A conserved gene catalyzing the synthesis of branched-chain amino acids in S. mutans B14 shows possible interaction with isoforms of cathepsin protein of the ECM. This genome sequence analysis indicates towards other proteins in the S. mutans genome, which might have a specific role to play in host cell interaction.

Cardiac mitochondrial dynamics: miR-mediated regulation during cardiac injury.

Mitochondrial integrity is indispensable for cardiac health. With the advent of modern imaging technologies, mitochondrial motility and dynamics within the cell are extensively studied. Terminally differentiated and well-structured cardiomyocytes depict little mitochondrial division and fusion, questioning the contribution of mitochondrial fusion proteins (Mitofusin 1/2 and Optic Atrophy 1 protein) and fission factors (Dynamin-like protein 1 and mitochondrial fission 1 protein) in cardiomyocyte homeostasis. Emerging evidences suggest that alterations in mitochondrial morphology from globular, elongated network to punctate fragmented disconnected structures are a pathological response to ensuing cardiac stress and cardiomyocyte cell death, bringing forth the following question, "what maintains this balance between fusion and fission?" The answer hinges upon the classical "junk" DNA: microRNAs, the endogenous non-coding RNAs. Because of their essential role in numerous signaling pathways, microRNAs are considered to play major roles in the pathogenesis of various diseases. Mitochondria are not exempted from microRNA-mediated regulation. This review defines the importance of mitochondrial structural integrity and the microRNA-mitochondrial dynamics tandem, an imminent dimension of the cardiac homeostasis network. Elucidating their coordinated interaction could spur RNA-based therapeutics for resuscitating functional mitochondrial population during cardiovascular disorders.

Critical Evaluation and Compilation of Physicochemical Determinants and Membrane Interactions of MMGP1 Antifungal Peptide.

A growing issue of pathogen resistance to antibiotics has fostered the development of innovative approaches for novel drug development. Here, we report the physicochemical and biological properties of an antifungal peptide, MMGP1, based on computational analysis. Computation of physicochemical properties has revealed that the natural biological activities of MMGP1 are coordinated by its intrinsic properties such as net positive charge (+5.04), amphipathicity, high hydrophobicity, low hydrophobic moment, and higher isoelectric point (11.915). Prediction of aggregation hot spots in MMGP1 had revealed the presence of potentially aggregation-prone segments that can nucleate in vivo aggregation (on the membrane), whereas no aggregating regions were predicted for in vitro aggregation (in solutions) of MMGP1. This ability of MMGP1 to form oligomeric aggregates on membrane further substantiates its direct-cell penetrating potency. Monte Carlo simulation of the interactions of MMGP1 in the aqueous phase and different membrane environments revealed that increasing the proportion of acidic lipids on membrane had led to increase in the peptide helicity. Furthermore, the peptide adopts energetically favorable transmembrane configuration, by inserting peptide loop and helix termini into the membrane containing >60% of anionic lipids. The charged lipid-based insertion of MMGP1 into membrane might be responsible for the selectivity of peptide toward fungal cells. Additionally, MMGP1 possessed DNA-binding property. Computational docking has identified DNA-binding residues (TRP3, SER4, MET7, ARG8, PHE10, ALA11, GLY20, THR21, ARG22, MET23, TRP34, and LYS36) in MMGP1 crucial for its DNA-binding property. Furthermore, computational mutation analysis revealed that aromatic amino acids are crucial for in vivo aggregation, membrane insertion, and DNA-binding property of MMGP1. These data provide new insight into the molecular determinants of MMGP1 antifungal activity and also serves as the template for the design of novel peptide antibiotics.

Comparative Analysis of Microbial Diversity in Termite Gut and Termite Nest Using Ion Sequencing.

Termite gut and termite nest possess complex microbial communities. However, only limited information is available on the comparative investigation of termite gut- and nest-associated microbial communities. In the present study, we examined and compared the bacterial diversity of termite gut and their respective nest by high-throughput sequencing of V3 hypervariable region of 16S rDNA. A total of 14 barcoded libraries were generated from seven termite gut samples and their respective nest samples, and sequenced using Ion Torrent platform. The sequences of each group were pooled, which yielded 170,644 and 132,000 reads from termite gut and termite nest samples, respectively. Phylogenetic analysis revealed significant differences in the bacterial diversity and community structure between termite gut and termite nest samples. Phyla Verrucomicrobia and Acidobacteria were observed only in termite gut, whereas Synergistetes and Chlorobi were observed only in termite nest samples. These variations in microbial structure and composition could be attributed with the differences in physiological conditions prevailing in the termite gut (anoxic and alkaline) and termite nest (oxic, slightly acidic and rich in organic matter) environment. Overall, this study unmasked the complexity of bacterial population in the respective niche. Interestingly, majority of the sequence reads could be classified only up to the domain level indicating the presence of a huge number of uncultivable or unidentified novel bacterial species in both termite gut and nest samples. Whole metagenome sequencing and assessing the metabolic potential of these samples will be useful for biotechnological applications.

Molecular cloning and expression of thermostable glucose-tolerant ß-glucosidase of Penicillium funiculosum NCL1 in Pichia pastoris and its characterization.

A partial peptide sequence of ß-glucosidase isoform (Bgl4) of Penicillium funiculosum NCL1 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cDNA (bgl4) encoding Bgl4 protein was cloned from P. funiculosum NCL1 RNA by consensus RT-PCR. The bgl4 gene encoded 857 amino acids that contained catalytic domains specific for glycoside hydrolase family 3. The cDNA was over-expressed in Pichia pastoris KM71H and the recombinant protein (rBgl4) was purified with the specific activity of 1,354.3 U/mg. The rBgl4 was a glycoprotein with the molecular weight of ~130 kDa and showed optimal activity at pH 5.0 and 60 °C. The enzyme was thermo-tolerant up to 60 °C for 60 min. The rBgl4 was highly active on aryl substrates with ß-glucosidic, ß-xylosidic linkages and moderately active on cellobiose and salicin. It showed remarkably high substrate conversion rate of 3,332 and 2,083 µmol/min/mg with the substrates p-nitrophenyl ß-glucoside and cellobiose respectively. In addition, the rBgl4 showed tolerance to glucose concentration up to 400 mM. It exhibited twofold increase in glucose yield when supplemented with crude cellulase of Trichoderma reesei Rut-C30 in cellulose hydrolysis. These results suggested that rBgl4 is a thermo- and glucose-tolerant ß-glucosidase and is a potential supplement for commercial cellulase in cellulose hydrolysis and thereby assures profitability in bioethanol production.

Molecular Serotyping and Pathogenic Potential of Listeria monocytogenes Isolated from Milk and Milk Products in Tamil Nadu, India.

Listeria monocytogenes, an important bacterial pathogen, is responsible for foodborne illnesses worldwide. Examination of food samples for the presence of L. monocytogenes and assessment of their pathogenicity is usually an effective strategy in the prevention of listeriosis. In the present study, we have tested 307 samples of milk and milk products from various places in Tamil Nadu, India for the presence of L. monocytogenes using ISO 11290 and U.S. Food and Drug Administration Bacteriological Analytical Manual methods. 16S rDNA sequencing and duplex polymerase chain reaction (PCR) analysis for prs and iap genes were used to identify L. monocytogenes at the species level. Fifteen of the 307 samples screen tested positive for L. monocytogenes. Molecular serotyping of the L. monocytogenes isolates by multiplex PCR revealed the predominance of the serogroups 1/2a and 4b. Fourteen of the 15 isolates contained all the virulence genes (inlA, inlB, hlyA, and plcA) screened for using multiplex PCR. Only one isolate of L. monocytogenes was negative for the plcA gene and in vitro phosphatidylinositol-phospholipase C activity. L. monocytogenes strains that belong to the serogroup 4b exhibited higher nematocidal activity against Caenorhabditis elegans than the serogroup 1/2a. Worms infected with L. monocytogenes were symptomatic with aberrant contraction of body muscles, loss of pharyngeal pumping, and decreased locomotion, which highlights the pathogenic potential of the L. monocytogenes isolates.

Pseudomonas aeruginosa PAO1 induces distinct cell death mechanisms in H9C2 cells and its differentiated form.

Bacterial infections in myocardium may lead to the myocardial damage, which may progress to dilated cardiomyopathy and cardiac arrest. Pseudomonas aeruginosa has been reported to cause myocarditis and other systemic infections especially in immunocompromised patients. To understand the cellular responses during the establishment of infection in myocardium, we challenged differentiated H9C2 cells with P. aeruginosa PAO1. We also did comparison studies with infected undifferentiated form of H9C2 cells. Invasion studies revealed that PAO1 can invade both forms of cells and is able to survive and replicate within the host. Internalization of PAO1 was confirmed by live cell imaging and flow cytometry analysis. Though invasion of the pathogen triggered an increased ROS production in the host cells at earlier post-infection periods, it was decreased at later post-infection periods. Invasion of PAO1 induced cell death through apoptosis in differentiated H9C2 cells. Significant decrease in cell size, formation of polarized mitochondria, and nuclear fragmentation were observed in the infected differentiated cells. On the contrary, cell death preceded by multinucleation was observed in infected undifferentiated H9C2 cells. Morphological markers such as multinuclei and micro nuclei were observed. Cell cycle arrest in G2/M phase corroborates that the undifferentiated H9C2 cells experienced cell death preceded by multinucleation.

Identification of Recombination and Positively Selected Genes in Brucella.

Brucella is a facultative intracellular bacterium belongs to the class alpha proteobacteria. It causes zoonotic disease brucellosis to wide range of animals. Brucella species are highly conserved in nucleotide level. Here, we employed a comparative genomics approach to examine the role of homologous recombination and positive selection in the evolution of Brucella. For the analysis, we have selected 19 complete genomes from 8 species of Brucella. Among the 1599 core genome predicted, 24 genes were showing signals of recombination but no significant breakpoint was found. The analysis revealed that recombination events are less frequent and the impact of recombination occurred is negligible on the evolution of Brucella. This leads to the view that Brucella is clonally evolved. On other hand, 56 genes (3.5 % of core genome) were showing signals of positive selection. Results suggest that natural selection plays an important role in the evolution of Brucella. Some of the genes that are responsible for the pathogenesis of Brucella were found positively selected, presumably due to their role in avoidance of the host immune system.

Cloning, expression and characterization of a lipase encoding gene from human oral metagenome.

The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6-7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.

Massively parallel mutant selection identifies genetic determinants of Pseudomonas aeruginosa colonization of Drosophila melanogaster.

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing [Tn-Seq, also known as insertion sequencing (INSeq)] to identify genes in P. aeruginosa that contribute to fitness during the colonization of Drosophila melanogaster. Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies on P. aeruginosa identified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen in Drosophila, demonstrate the power of Tn-Seq in the fly model system and advance the existing knowledge of intestinal pathogenesis by D. melanogaster, revealing bacterial colonization determinants that contribute to a comprehensive portrait of P. aeruginosa lifestyles across habitats.IMPORTANCEDrosophila melanogaster is a powerful model for understanding host-pathogen interactions. Research with this system has yielded notable insights into mechanisms of host immunity and defense, many of which emerged from the analysis of bacterial mutants defective for well-characterized virulence factors. These foundational studies-and advances in high-throughput sequencing of transposon mutants-support unbiased screens of bacterial mutants in the fly. To investigate mechanisms of host-pathogen interplay and exploit the tractability of this model host, we used a high-throughput, genome-wide mutant analysis to find genes that enable the pathogen P. aeruginosa to colonize the fly. Our analysis reveals critical mediators of P. aeruginosa establishment in its host, some of which are required across fly and mouse systems. These findings demonstrate the utility of massively parallel mutant analysis and provide a platform for aligning the fly toolkit with comprehensive bacterial genomics.

Inactivation of the transcriptional regulator-encoding gene sdiA enhances rice root colonization and biofilm formation in Enterobacter cloacae GS1.

Enterobacter cloacae GS1 is a plant growth-promoting bacterium which colonizes rice roots. In the rhizosphere environment, N-acyl homoserine lactone (NAHL)-like quorum-sensing signals are known to be produced by host plants and other microbial inhabitants. E. cloacae GS1 was unable to synthesize NAHL quorum-sensing signals but had the NAHL-dependent transcriptional regulator-encoding gene sdiA. This study was aimed at understanding the effects of SdiA and NAHL-dependent cross talk in rice root colonization by E. cloacae GS1. Pleiotropic effects of sdiA inactivation included substantial increases in root colonization and biofilm formation, suggesting a negative role for SdiA in bacterial adhesion. We provide evidence that sdiA inactivation leads to elevated levels of biosynthesis of curli, which is involved in cellular adhesion. Extraneous addition of NAHLs had a negative effect on root colonization and biofilm formation. However, the sdiA mutant of E. cloacae GS1 was insensitive to NAHLs, suggesting that this NAHL-induced inhibition of root colonization and biofilm formation is SdiA dependent. Therefore, it is proposed that NAHLs produced by both plant and microbes in the rice rhizosphere act as cross-kingdom and interspecies signals to negatively impact cellular adhesion and, thereby, root colonization in E. cloacae GS1.

Influence of periplasmic oxidation of glucose on pyoverdine synthesis in Pseudomonas putida S11.

Fluorescent pseudomonads catabolize glucose simultaneously by two different pathways, namely, the oxidative pathway in periplasm and the phosphorylative pathway in cytoplasm. This study provides evidence for the role of glucose metabolism in the regulation of pyoverdine synthesis in Pseudomonas putida S11. We have characterized the influence of direct oxidation of glucose in periplasm on pyoverdine synthesis in P. putida S11. We identified a Tn5 transposon mutant of P. putida S11 showing increased pyoverdine production in minimal glucose medium (MGM). This mutant designated as IST1 had Tn5 insertion in glucose dehydrogenase (gcd) gene. To verify the role of periplasmic oxidation of glucose on pyoverdine synthesis, we constructed mutants S11 Gcd(-) and S11 PqqF(-) by antibiotic cassette mutagenesis. These mutants of P. putida S11 with loss of glucose dehydrogenase gene (gcd) or cofactor pyrroloquinoline quinone biosynthesis gene (pqqF) showed increased pyoverdine synthesis and impaired acid production in MGM. In minimal gluconate medium, the pyoverdine production of wild-type strain S11 and mutants S11 Gcd(-) and S11 PqqF(-) was higher than in MGM indicating that gluconate did not affect pyoverdine synthesis. In MGM containing PIPES-NaOH (pH 7.5) buffer which prevent pH changes due to gluconic acid production, strain S11 produced higher amount of pyoverdine similar to mutants S11 Gcd(-) and S11 PqqF(-). Therefore, it is proposed that periplasmic oxidation of glucose to gluconic acid decreases the pH of MGM and thereby influences pyoverdine synthesis of strain S11. The increased pyoverdine synthesis enhanced biotic and abiotic surface colonization of the strain S11.