Publications by authors named "Veeranagouda Yaligara"

29 Publications

  • Page 1 of 1

High-Throughput Cellular RNA Sequencing (HiCAR-Seq): Cost-Effective, High-Throughput 3' mRNA-Seq Method Enabling Individual Sample Quality Control.

Curr Protoc Mol Biol 2020 09;132(1):e123

Molecular Biology and Genomics, Translational Sciences, Sanofi R&D, Chilly-Mazarin, France.

High-throughput screening is one of the pillars of drug development. Unbiased transcriptome profiling is now widely used for a deeper understanding of a drug's mechanisms of action, off target effects, and cytotoxicity. Although currently available high-throughput RNA-Seq (HT RNA-Seq) methods such as PLATE-Seq, DRUG-Seq, and BRB-Seq serve these purposes, the inherent nature of these methods does not allow sample-wise sequencing library quality control. Here, we describe an HTR method called High-throughput CellulAr RNA Sequencing (HiCAR-Seq). HiCAR-Seq was optimized to work directly on cultured cells (as little as 1,000 cells) or 10 ng of total RNA. HiCAR-Seq involves reverse transcription from cultured cells or total RNA using oligo-dT primers followed by the PCR amplification of full-length cDNAs using sample-specific barcode primers in individual plate wells. Amplification of cDNA from every sample can be verified using Bioanalyzer. This step not only reveals cDNA amplification but also provides greater precision for pooling equal concentrations of cDNA from different samples. A single pooled cDNA library is made suitable for sequencing on Illumina sequencers using a tagmentation kit. Because HiCAR-Seq targets a small region at the 3' of the mRNAs, as little as 3 to 4 million reads/sample are enough to infer changes in gene expression in human or mouse cells. We believe that HiCAR-Seq represents a robust and competitive addition to the existing set of transcriptome-based high-throughput screening methods. © 2020 Wiley Periodicals LLC. Basic Protocol 1: cDNA synthesis and barcoding/enrichment PCR Basic Protocol 2: Nextera tagmentation/amplification, quantification, and sequencing.
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http://dx.doi.org/10.1002/cpmb.123DOI Listing
September 2020

ABHD11, a new diacylglycerol lipase involved in weight gain regulation.

PLoS One 2020 24;15(6):e0234780. Epub 2020 Jun 24.

Sanofi Research and Development, Chilly-Mazarin, France.

Obesity epidemic continues to spread and obesity rates are increasing in the world. In addition to public health effort to reduce obesity, there is a need to better understand the underlying biology to enable more effective treatment and the discovery of new pharmacological agents. Abhydrolase domain-containing protein 11 (ABHD11) is a serine hydrolase enzyme, localized in mitochondria, that can synthesize the endocannabinoid 2-arachidonoyl glycerol (2AG) in vitro. In vivo preclinical studies demonstrated that knock-out ABHD11 mice have a similar 2AG level as WT mice and exhibit a lean metabolic phenotype. Such mice resist to weight gain in Diet Induced Obesity studies (DIO) and display normal biochemical plasma parameters. Metabolic and transcriptomic analyses on serum and tissues of ABHD11 KO mice from DIO studies show a modulation in bile salts associated with reduced fat intestinal absorption. These data suggest that modulating ABHD11 signaling pathway could be of therapeutic value for the treatment of metabolic disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234780PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313976PMC
September 2020

The RESOLUTE consortium: unlocking SLC transporters for drug discovery.

Authors:
Giulio Superti-Furga Daniel Lackner Tabea Wiedmer Alvaro Ingles-Prieto Barbara Barbosa Enrico Girardi Ulrich Goldmann Bettina Gürtl Kristaps Klavins Christoph Klimek Sabrina Lindinger Eva Liñeiro-Retes André C Müller Svenja Onstein Gregor Redinger Daniela Reil Vitaly Sedlyarov Gernot Wolf Matthew Crawford Robert Everley David Hepworth Shenping Liu Stephen Noell Mary Piotrowski Robert Stanton Hui Zhang Salvatore Corallino Andrea Faedo Maria Insidioso Giovanna Maresca Loredana Redaelli Francesca Sassone Lia Scarabottolo Michela Stucchi Paola Tarroni Sara Tremolada Helena Batoulis Andreas Becker Eckhard Bender Yung-Ning Chang Alexander Ehrmann Anke Müller-Fahrnow Vera Pütter Diana Zindel Bradford Hamilton Martin Lenter Diana Santacruz Coralie Viollet Charles Whitehurst Kai Johnsson Philipp Leippe Birgit Baumgarten Lena Chang Yvonne Ibig Martin Pfeifer Jürgen Reinhardt Julian Schönbett Paul Selzer Klaus Seuwen Charles Bettembourg Bruno Biton Jörg Czech Hélène de Foucauld Michel Didier Thomas Licher Vincent Mikol Antje Pommereau Frédéric Puech Veeranagouda Yaligara Aled Edwards Brandon J Bongers Laura H Heitman Ad P IJzerman Huub J Sijben Gerard J P van Westen Justine Grixti Douglas B Kell Farah Mughal Neil Swainston Marina Wright-Muelas Tina Bohstedt Nicola Burgess-Brown Liz Carpenter Katharina Dürr Jesper Hansen Andreea Scacioc Giulia Banci Claire Colas Daniela Digles Gerhard Ecker Barbara Füzi Viktoria Gamsjäger Melanie Grandits Riccardo Martini Florentina Troger Patrick Altermatt Cédric Doucerain Franz Dürrenberger Vania Manolova Anna-Lena Steck Hanna Sundström Maria Wilhelm Claire M Steppan

Nat Rev Drug Discov 2020 07;19(7):429-430

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http://dx.doi.org/10.1038/d41573-020-00056-6DOI Listing
July 2020

RNA Fragmentation and Sequencing (RF-Seq): Cost-Effective, Time-Efficient, and High-Throughput 3' mRNA Sequencing Library Construction in a Single Tube.

Curr Protoc Mol Biol 2019 12;129(1):e109

Molecular Biology and Genomics, Translational Sciences, Sanofi R&D, Chilly-Mazarin, France.

Over the past decade, transcriptomic studies using next-generation sequencing (NGS)-based RNA sequencing (RNA-Seq) have greatly contributed to characterizing biochemical and physiological changes in cells and tissues across organisms and experimental conditions. Critical steps in RNA-Seq include the preparation of the sequencing library from extracted RNA. Currently, a large panoply of RNA-Seq kits are commercially available. In these kits, conversion of RNA into a sequencing library involves multiple steps, which are labor-intensive, and cost per sample for library preparation may limit routine use of RNA-Seq. Here we describe a simple method for RNA-Seq library construction, referred to as RNA Fragmentation and Sequencing (RF-Seq). RF-Seq requires as little as 10 ng of total RNA and facilitates the sequencing of the 3' end of mRNAs. RF-Seq involves the fragmentation of total RNA followed by reverse transcription in presence of the oligo(dT) primer/template switch oligonucleotide and a sample barcoding/enrichment within a single PCR tube/well. The sample barcoding/enrichment step provides more flexibility for individual sample handling. The use of just twenty orthogonal Illumina TruSeq HT barcoding primers facilitates the preparation of 96 uniquely labeled RF-Seq libraries in a single 96-well PCR plate. Twelve RF-Seq libraries can be prepared within 4 hr, with an approximate cost of $10/sample. We provide an example of using RF-Seq to measure gene expression upon activation of an innate immune pathway using STING activator in human blood cells, highlighting the potential usefulness of the proposed method in routine transcriptomic applications such as high-throughput drug screening and/or preclinical toxicity assays. © 2019 by John Wiley & Sons, Inc. Basic Protocol: RNA fragmentation and sequencing (RF-Seq): Cost-effective, time-efficient, and high-throughput 3' mRNA sequencing library construction in a single tube.
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http://dx.doi.org/10.1002/cpmb.109DOI Listing
December 2019

Transposon Mutagenesis of Bacteroides fragilis.

Methods Mol Biol 2019 ;2016:105-116

Greater Los Angeles VA Health Care System, Los Angeles, CA, USA.

Bacteroides fragilis is Gram-negative obligatory anaerobe which usually resides in the gut of humans and animals. As an important member of the human gut microbiota it plays a vital role in digestion and absorption of nutrients as well as shaping of host immune system. B. fragilis is also infamous for causing serious infections. Treatment of B. fragilis infections caused emergence of multidrug-resistant strains. Molecular biology tools such as transposon mutagenesis help to decipher and understand commensal and pathogenic faces of B. fragilis. Using two mariner transposon vectors we describe the detailed methodology for the transposon mutagenesis of B. fragilis. We also describe two methods for the identification of transposon integration site (TIS) in transposon mutants. Transposon mutagenesis methods described in this chapter serve as a great tool for studying B. fragilis.
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http://dx.doi.org/10.1007/978-1-4939-9570-7_10DOI Listing
March 2020

Circular pellicles formed by Pseudomonas alkylphenolica KL28 are a sophisticated architecture principally designed by matrix substance.

J Microbiol 2018 Nov 24;56(11):790-797. Epub 2018 Oct 24.

Department of Bio Health Science, Changwon National University, Changwon, 51140, Republic of Korea.

The colonization of liquid surfaces as floating biofilms or pellicles is a bacterial adaptation to optimally occupy the airliquid (A-L) niche. In aerobic heterotrophs, pellicle formation is beneficial for the utilization of O and nonpolar organic compounds. Pseudomonas alkylphenolica KL28, an alkylphenol degrader, forms flat circular pellicles that are 0.3-0.5 mm in diameter. In this study, we first monitored the pellicle developmental patterns of multicellular organization from the initial settlement stage. The pellicles developed by clonal growth and mutants for flagella and pilus formation established normal pellicles. In contrast, the mutants of an epm gene cluster for biosynthesis of alginate-like polymer were incompetent in cell alignment for initial two-dimensional (2D) pellicle growth, suggesting the role of the Epm polymer as a structural scaffold for pellicle biofilms. Microscopic observation revealed that the initial 2D growth transited to multilayers by an accumulated self-produced extracellular polymeric substance that may exert a constraint force. Electron microscopy and confocal laser scanning microscopy revealed that the fully matured pellicle structures were densly packed with matrix-encased cells displaying distinct arrangements. The cells on the surface of the pellicle were relatively flat, and those inside were longitudinally cross-packed. The extracellular polysaccharide stained by Congo red was denser on the pellicle rim and a thin film was observed in the open spaces, indicative of its role in pellicle flotation. Our results demonstrate that P. alkylphenolica KL28 coordinately dictates the cell arrangements of pellicle biofilms by the controlled growth of constituent cells that accumulate extracellular polymeric substances.
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http://dx.doi.org/10.1007/s12275-018-8252-7DOI Listing
November 2018

CRISPR-Cas9-Edited Site Sequencing (CRES-Seq): An Efficient and High-Throughput Method for the Selection of CRISPR-Cas9-Edited Clones.

Curr Protoc Mol Biol 2018 01 16;121:31.14.1-31.14.11. Epub 2018 Jan 16.

Molecular Biology and Genomics, Translational Sciences, Sanofi R&D, Chilly-Mazarin, France.

The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmb.53DOI Listing
January 2018

Novel large-scale chromosomal transfer in Bacteroides fragilis contributes to its pan-genome and rapid environmental adaptation.

Microb Genom 2017 11;3(11)

6​Research, GLAVAHCS, 11301 Wilshire Blvd., 691/151J Bldg. 115, Room 312, Los Angeles, CA, USA.

Bacteroides fragilis, an important component of the human gastrointestinal microbiota, can cause lethal extra-intestinal infection upon escape from the gastrointestinal tract. We demonstrated transfer and recombination of large chromosomal segments from B. fragilis HMW615, a multidrug resistant clinical isolate, to B. fragilis 638R. In one example, the transfer of a segment of ~435 Kb/356 genes replaced ~413 Kb/326 genes of the B. fragilis 638R chromosome. In addition to transfer of antibiotic resistance genes, these transfers (1) replaced complete divergent polysaccharide biosynthesis loci; (2) replaced DNA inversion-controlled intergenic shufflons (that control expression of genes encoding starch utilization system outer membrane proteins) with more complex, divergent shufflons; and (3) introduced additional intergenic shufflons encoding divergent Type 1 restriction/modification systems. Conjugative transposon-like genes within a transferred segment and within a putative integrative conjugative element (ICE5) ~45 kb downstream from the transferred segment both encode proteins that may be involved in the observed transfer. These data indicate that chromosomal transfer is a driver of antigenic diversity and nutrient adaptation in Bacteroides that (1) contributes to the dissemination of the extensive B. fragilis pan-genome, (2) allows rapid adaptation to a changing environment and (3) can confer pathogenic characteristics to host symbionts.
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http://dx.doi.org/10.1099/mgen.0.000136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729914PMC
November 2017

Transposon Insertion Site Sequencing (TIS-Seq): An Efficient and High-Throughput Method for Determining Transposon Insertion Site(s) and Their Relative Abundances in a PiggyBac Transposon Mutant Pool by Next-Generation Sequencing.

Curr Protoc Mol Biol 2017 10 2;120:21.35.1-21.35.11. Epub 2017 Oct 2.

Molecular Biology and Genomics, Translational Sciences, Sanofi R&D, Chilly-Mazarin, France.

The PiggyBac (PB) transposon has emerged as a novel mutagenesis tool for understanding gene function and for phenotypic screening in eukaryotes. Successful screening of PB transposon mutants relies on efficient identification of transposon insertion site(s) (TIS) in mutant cells. However, currently available methods suffer from time-consuming steps. Here, we present the method for transposon insertion site sequencing (TIS-Seq) for high-throughput identification of TIS in transposon mutants. TIS-Seq provides qualitative and quantitative information on mutants present in a given PB transposon mutant library. TIS-Seq also facilitates identification of TIS in up to 96 individual/hand-picked mutants in a single MiniSeq/MiSeq run. TIS-Seq is a versatile method that can be easily modified to identify TIS from any kind of transposon mutant, as long as one end of the DNA sequence is known. Therefore, TIS-Seq is a promising method for transposon mutant screening. © 2017 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmb.47DOI Listing
October 2017

Next-Generation Sequencing to Investigate Urinary microRNAs from Macaca fascicularis (Cynomolgus Monkey).

Methods Mol Biol 2017 ;1641:349-378

Translational Medicine and Early Development, Biomarkers and Clinical Bioanalyses-Genomics, Sanofi R&D, Vitry-sur-Seine, France.

Advanced sequencing technologies like next-generation sequencing (NGS) not only detect microRNAs (miRNAs) in biological samples but also facilitate de novo identification of miRNAs. Using an Ion Torrent's Ion Proton System, here we described miRNAs sequencing of urine samples collected from Macaca fascicularis (Cynomolgus monkey) to investigate miRNAs as potential novel biomarkers of nephrotoxicity in this species. Urinary miRNA sequencing methodologies described here include (a) urinary exosomal RNA isolation, (b) sequencing library preparation, (c) sequencing template preparation, and (d) template library sequencing using Ion Proton System. The sequencing method presented in this study serves as a valuable resource in the identification of novel urinary miRNAs in M. fascicularis.
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http://dx.doi.org/10.1007/978-1-4939-7172-5_20DOI Listing
April 2018

A CHASE3/GAF sensor hybrid histidine kinase BmsA modulates biofilm formation and motility in Pseudomonas alkylphenolica.

Microbiology (Reading) 2016 11 15;162(11):1945-1954. Epub 2016 Sep 15.

Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea.

Pseudomonas alkylphenolica is an important strain in the biodegradation of toxic alkylphenols and mass production of bioactive polymannuronate polymers. This strain forms a diverse, 3D biofilm architecture, including mushroom-like aerial structures, circular pellicles and surface spreading, depending on culture conditions. A mutagenesis and complementation study showed that a predicted transmembrane kinase, PSAKL28_21690 (1164 aa), harbouring a periplasmic CHASE3 domain flanked by two transmembrane helices in addition to its cytoplasmic GAF, histidine kinase and three CheY-like response regulator domains, plays a positive role in the formation of the special biofilm architecture and a negative role in swimming activity. In addition, the gene, named here as bmsA, is co-transcribed with three genes encoding proteins with CheR (PSAKL28_21700) and CheB (PSAKL28_21710) domains and response regulator and histidine kinase domains (PSAKL28_21720). This gene cluster is thus named bmsABCD and is found widely distributed in pseudomonads and other bacteria. Deletion of the genes in the cluster, except forbmsA, did not result in changes in biofilm-related phenotypes. The RNA-seq analysis showed that the expression of genes coding for flagellar synthesis was increased when bmsA was mutated. In addition, the expression of rsmZ, which is one of final targets of the Gac regulon, was not significantly altered in the bmsA mutant, and overexpression of bmsA in the gacA mutant did not produce the WT phenotype. These results indicate that the sensory Bms regulon does not affect the upper cascade of the Gac signal transduction pathway for the biofilm-related phenotypes in P. alkylphenolica.
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http://dx.doi.org/10.1099/mic.0.000373DOI Listing
November 2016

Evaluation of novel biomarkers of nephrotoxicity in Cynomolgus monkeys treated with gentamicin.

Toxicol Appl Pharmacol 2016 07 19;303:1-10. Epub 2016 Apr 19.

National Center for Safety Evaluation of Drugs (NCSED), National Institutes for Food and Drug Control, Beijing, China. Electronic address:

Most studies to evaluate kidney safety biomarkers have been performed in rats. This study was conducted in Cynomolgus monkeys in order to evaluate the potential usefulness of novel biomarkers of nephrotoxicity in this species. Groups of 3 males were given daily intramuscular injections of gentamicin, a nephrotoxic agent known to produce lesions in proximal tubules, at dose-levels of 10, 25, or 50mg/kg/day for 10days. Blood and 16-h urine samples were collected on Days -7, -3, 2, 4, 7, and at the end of the dosing period. Several novel kidney safety biomarkers were evaluated, with single- and multiplex immunoassays and in immunoprecipitation-LC/MS assays, in parallel to histopathology and conventional clinical pathology parameters. Treatment with gentamicin induced a dose-dependent increase in kidney tubular cell degeneration/necrosis, ranging from minimal to mild severity at 10mg/kg/day, moderate at 25mg/kg/day, and to severe at 50mg/kg/day. The results showed that the novel urinary biomarkers, microalbumin, α1-microglobulin, clusterin, and osteopontin, together with the more traditional clinical pathology parameters, urinary total protein and N-acetyl-β-D-glucosaminidase (NAG), were more sensitive than blood urea nitrogen (BUN) and serum creatinine (sCr) to detect kidney injury in the monkeys given 10mg/kg/day gentamicin for 10days, a dose leading to an exposure which is slightly higher than the desired therapeutic exposure in clinics. Therefore, these urinary biomarkers represent non-invasive biomarkers of proximal tubule injury in Cynomolgus monkeys which may be potentially useful in humans.
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http://dx.doi.org/10.1016/j.taap.2016.04.012DOI Listing
July 2016

Identification of microRNAs in Macaca fascicularis (Cynomolgus Monkey) by Homology Search and Experimental Validation by Small RNA-Seq and RT-qPCR Using Kidney Cortex Tissues.

PLoS One 2015 12;10(11):e0142708. Epub 2015 Nov 12.

Sanofi R&D, Disposition Safety and animal Research, Vitry-sur-Seine, France.

MicroRNAs (miRNAs) present in tissues and biofluids are emerging as sensitive and specific safety biomarkers. MiRNAs have not been thoroughly described in M. fascicularis, an animal model used in pharmaceutical industry especially in drug safety evaluation. Here we investigated the miRNAs in M. fascicularis. For Macaca mulatta, a closely related species of M. fascicularis, 619 stem-loop precursor miRNAs (pre-miRNAs) and 914 mature miRNAs are available in miRBase version 21. Using M. mulatta miRNAs as a reference list and homology search tools, we identified 604 pre-miRNAs and 913 mature miRNAs in the genome of M. fascicularis. In order to validate the miRNAs identified by homology search we attempted to sequence miRNAs expressed in kidney cortex from M. fascicularis. MiRNAs expressed in kidney cortex may indeed be released in urine upon kidney cortex damage and be potentially used to monitor drug induced kidney injury. Hence small RNA sequencing libraries were prepared using kidney cortex tissues obtained from three naive M. fascicularis and sequenced. Analysis of sequencing data indicated that 432 out of 913 mature miRNAs were expressed in kidney cortex tissues. Assigning these 432 miRNAs to pre-miRNAs revealed that 273 were expressed from both the -5p and -3p arms of 150 pre-miRNAs and 159 miRNAs expressed from either the -5p or -3p arm of 176 pre-miRNAs. Mapping sequencing reads to pre-miRNAs also facilitated the detection of twenty-two new miRNAs. To substantiate miRNAs identified by small RNA sequencing, 313 miRNAs were examined by RT-qPCR. Expression of 262 miRNAs in kidney cortex tissues ware confirmed by TaqMan microRNA RT-qPCR assays. Analysis of kidney cortex miRNA targeted genes suggested that they play important role in kidney development and function. Data presented in this study may serve as a valuable resource to assess the renal safety biomarker potential of miRNAs in Cynomolgus monkeys.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142708PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642959PMC
June 2016

The Ellis Island Effect: A novel mobile element in a multi-drug resistant clinical isolate includes a mosaic of resistance genes from Gram-positive bacteria.

Mob Genet Elements 2014 14;4:e29801. Epub 2014 Jul 14.

GLAVAHCS; Los Angeles, CA USA ; David Geffen School of Medicine; University of California Los Angeles; Los Angeles, CA USA.

 , a Gram-negative anaerobic bacterium, is alternately a gut commensal or virulent pathogen and is an important reservoir for horizontal gene transfer (HGT) of bacterial resistance and virulence genes in the human gastrointestinal tract. We identified a unique conjugative transposon (CTn) in a multidrug resistant clinical isolate of (BF-HMW615); we named this element CTnHyb because it included a hybrid mosaic of foreign elements. This study reports the characterization of CTnHyb and discusses the potential impact on horizontal spread of resistance genes. CTnHyb contains several efflux pump genes and several genes that confer or may confer antibiotic resistance to tetracycline, kanamycin, metronidazole and spectinomycin (truncated gene). CTnHyb also contains a mosaic of mobile elements from Gram-positive organisms. CTnHyb is easily transferred from BF-HMW615 (the original isolate) to BF638R (lab strain) and integrated into the BF638R chromosome. The "foreign" (from Gram-positive bacteria) nucleotide sequences within CTnHyb were > 99% preserved indicating that the gene acquisition from the Gram-positive bacteria was very recent. CTnHyb is a novel CTn residing in a multidrug resistant strain of . The global nature and wide phylogenetic reach of HGT means that any gene in any bacterium can potentially be mobilized. Understanding the mechanisms that drive the formation and transfer of these elements and, potentially, ways to limit the transfer are necessary to prevent a devastating spread of resistance elements.
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http://dx.doi.org/10.4161/mge.29801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145004PMC
July 2014

Deficiency of the ferrous iron transporter FeoAB is linked with metronidazole resistance in Bacteroides fragilis.

J Antimicrob Chemother 2014 Oct 14;69(10):2634-43. Epub 2014 Jul 14.

GLAVAHCS, Los Angeles, CA, USA UCLA School of Medicine, Los Angeles, CA, USA

Background: Metronidazole is the most commonly used antimicrobial for Bacteroides fragilis infections and is recommended for prophylaxis of colorectal surgery. Metronidazole resistance is increasing and the mechanisms of resistance are not clear.

Methods: A transposon mutant library was generated in B. fragilis 638R (BF638R) to identify the genetic loci associated with resistance to metronidazole.

Results: Thirty-two independently isolated metronidazole-resistant mutants had a transposon insertion in BF638R_1421 that encodes the ferrous transport fusion protein (feoAB). Deletion of feoAB resulted in a 10-fold increased MIC of metronidazole for the strain. The metronidazole MIC for the feoAB mutant was similar to that for the parent strain when grown on media supplemented with excess iron, suggesting that the increase seen in the MIC of metronidazole was due to reduced cellular iron transport in the feoAB mutant. The furA gene repressed feoAB transcription in an iron-dependent manner and disruption of furA resulted in constitutive transcription of feoAB, regardless of whether or not iron was present. However, disruption of feoAB also diminished the capacity of BF638R to grow in a mouse intraperitoneal abscess model, suggesting that inorganic ferrous iron assimilation is essential for B. fragilis survival in vivo.

Conclusions: Selection for feoAB mutations as a result of metronidazole treatment will disable the pathogenic potential of B. fragilis and could contribute to the clinical efficacy of metronidazole. While mutations in feoAB are probably not a direct cause of clinical resistance, this study provides a key insight into intracellular metronidazole activity and the link with intracellular iron homeostasis.
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http://dx.doi.org/10.1093/jac/dku219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164142PMC
October 2014

Identification of genes required for the survival of B. fragilis using massive parallel sequencing of a saturated transposon mutant library.

BMC Genomics 2014 Jun 4;15:429. Epub 2014 Jun 4.

GLAVAHCS, Bldg, 115 Room 312 11301 Wilshire Blvd, Los Angeles, CA 90073, USA.

Background: Bacteroides fragilis is a Gram-negative anaerobe that is normally a human gut commensal; it comprises a small percentage of the gut Bacteroides but is the most frequently isolated Bacteroides from human infections. Identification of the essential genes necessary for the survival of B. fragilis provides novel information which can be exploited for the treatment of bacterial infections.

Results: Massive parallel sequencing of saturated transposon mutant libraries (two mutant pools of approximately 50,000 mutants each) was used to determine the essential genes for the growth of B. fragilis 638R on nutrient rich medium. Among the 4326 protein coding genes, 550 genes (12.7%) were found to be essential for the survival of B. fragilis 638R. Of the 550 essential genes, only 367 genes were assigned to a Cluster of Orthologous Genes, and about 290 genes had Kyoto Encyclopedia of Genes and Genomes orthologous members. Interestingly, genes with hypothetical functions accounted for 41.3% of essential genes (227 genes), indicating that the functions of a significant percentage of the genes used by B. fragilis 638R are still unknown. Global transcriptome analysis using RNA-Seq indicated that most of the essential genes (92%) are, in fact, transcribed in B. fragilis 638R including most of those coding for hypothetical proteins. Three hundred fifty of the 550 essential genes of B. fragilis 638R are present in Database of Essential Genes. 10.02 and 31% of those are genes included as essential genes for nine species (including Gram-positive pathogenic bacteria).

Conclusions: The essential gene data described in this investigation provides a valuable resource to study gene function and pathways involved in B. fragilis survival. Thorough examination of the B. fragilis-specific essential genes and genes that are shared between divergent organisms opens new research avenues that will lead to enhanced understanding of survival strategies used by bacteria in different microniches and under different stress situations.
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http://dx.doi.org/10.1186/1471-2164-15-429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072883PMC
June 2014

An alginate-like exopolysaccharide biosynthesis gene cluster involved in biofilm aerial structure formation by Pseudomonas alkylphenolia.

Appl Microbiol Biotechnol 2014 May 4;98(9):4137-48. Epub 2014 Feb 4.

Department of Microbiology and Biomedical Science Institute at CWNU, Changwon National University, Changwon-si, Kyongnam, 641-773, South Korea,

Pseudomonas alkylphenolia is known to form different types of multicellular structures depending on the environmental stimuli. Aerial structures formed during vapor p-cresol utilization are unique. Transposon mutants that showed a smooth colony phenotype failed to form a differentiated biofilm, including aerial structures and pellicles, and showed deficient surface spreading motility. The transposon insertion sites were located to a gene cluster designated epm (extracellular polymer matrix), which comprises 11 ORFs in the same transcriptional orientation. The putative proteins encoded by the genes in the epm cluster showed amino acid sequence homology to those found in the alginate biosynthesis gene clusters, e.g., in Pseudomonas aeruginosa at similarity levels of 32.3-86.4 %. This overall resemblance indicated that the epm gene cluster encodes proteins that mediate the synthesis of an exopolysaccharide composed of uronic acid(s) similar to alginate. Our preliminary results suggested that the epm-derived polymer is a substituted polymannuronic acid. Gene clusters homologous to the epm gene cluster are found in the genomes of a few species of the genera Pseudomonas, Alcanivorax, and Marinobacter. A mutational analysis showed that the epmJ and epmG genes encoding putative exopolysaccharide-modifying enzymes are required to form multicellular structures. An analysis of the activity of the promoter P epmD using a transcriptional fusion to the green fluorescence protein gene showed that the epm genes are strongly expressed at the tips of the specialized aerial structures. Our results suggested that the epm gene cluster is involved in the formation of a scaffold polysaccharide that is required to form multicellular structures in P. alkylphenolia.
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http://dx.doi.org/10.1007/s00253-014-5529-6DOI Listing
May 2014

Two multidrug-resistant clinical isolates of Bacteroides fragilis carry a novel metronidazole resistance nim gene (nimJ).

Antimicrob Agents Chemother 2013 Aug 28;57(8):3767-74. Epub 2013 May 28.

GLAVAHCS, Los Angeles, California, USA.

Two multidrug-resistant Bacteroides fragilis clinical isolates contain and express a novel nim gene, nimJ, that is not recognized by the "universal" nim primers and can confer increased resistance to metronidazole when introduced into a susceptible strain on a multicopy plasmid. HMW615, an appendiceal isolate, contains at least two copies of nimJ on its genome, while HMW616, an isolate from a patient with sepsis, contains one genomic copy of nimJ. B. fragilis NimJ is phylogenetically closer to Prevotella baroniae NimI and Clostridium botulinum NimA than to the other known Bacteroides Nim proteins. The predicted protein structure of NimJ, based on fold recognition analysis, is consistent with the crystal structures derived for known Nim proteins, and specific amino acid residues important for substrate binding in the active site are conserved. This study demonstrates that the "universal" nim primers will not detect all nim genes with the ability to confer metronidazole resistance, but nimJ alone cannot account for the very high metronidazole MICs of these resistant clinical isolates.
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http://dx.doi.org/10.1128/AAC.00386-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719759PMC
August 2013

Transposon mutagenesis of Bacteroides fragilis using a mariner transposon vector.

Anaerobe 2013 Aug 7;22:126-9. Epub 2013 May 7.

GLAVAHCS, Los Angeles, CA 90073, USA.

The mariner transposon vector pYV07 was tested for use in the mutagenesis of Bacteroides fragilis 638R. The transposon vector efficiently generated mutants in B. fragilis 638R. The transposon disrupted genes were scattered throughout the genome of B. fragilis 638R. This method serves as a powerful tool to study B. fragilis.
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http://dx.doi.org/10.1016/j.anaerobe.2013.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3729920PMC
August 2013

Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome.

FEMS Microbiol Lett 2012 Aug 18;333(2):94-100. Epub 2012 Jun 18.

GLAVAHCS, Los Angeles, CA, USA; UCLA School of Medicine, Los Angeles, CA 90073, USA.

Genetic analysis of Bacteroides fragilis (BF) is hindered because of the lack of efficient transposon mutagenesis methods. Here, we describe a simple method for transposon mutagenesis using EZ::TN5, a commercially available system that we optimized for use in BF638R. The modified EZ::TN5 transposon contains an Escherichia coli conditional origin of replication, a kanamycin resistance gene for E. coli, an erythromycin resistance gene for BF , and 19 basepair transposase recognition sequences on either ends. Electroporation of the transposome (transposon-transposase complex) into BF638R yielded 3.2 ± 0.35 × 10(3) CFU μg(-1) of transposon DNA. Modification of the transposon by the BF638R restriction/modification system increased transposition efficiency sixfold. Electroporation of the EZ::TN5 transposome results in a single-copy insertion of the transposon evenly distributed across the genome of BF638R and can be used to construct a BF638R transposon library. The transposon was also effective in mutating a BF clinical isolate and a strain of the related species, Bacteroides thetaiotaomicron. The EZ::TN5-based mutagenesis described here is more efficient than other transposon mutagenesis approaches previously reported for BF.
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http://dx.doi.org/10.1111/j.1574-6968.2012.02602.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500642PMC
August 2012

Identification of a p-cresol degradation pathway by a GFP-based transposon in Pseudomonas and its dominant expression in colonies.

J Microbiol Biotechnol 2011 Nov;21(11):1179-83

Department of Microbiology, Changwon National University, Changwon-si, Kyongnam 641-773, Korea.

In this study, the chromosome-encoded pcuRCAXB genes that are required for p-cresol degradation have been identified by using a newly constructed green fluorescent protein (GFP)-based promoter probe transposon in the long-chain alkylphenol degrader Pseudomonas alkylphenolia. The deduced amino acid sequences of the genes showed the highest identities at the levels of 65-93% compared with those in the databases. The transposon was identified to be inserted in the pcuA gene, with the promoterless gfp gene being under the control of the pcu catabolic gene promoter. The expression of GFP was positively induced by p-cresol and was about 10 times higher by cells grown on agar than those in liquid culture. In addition, phydroxybenzoic acid was detected during p-cresol degradation. These results indicate that P. alkylphenolia additionally possesses a protocatechuate ortho-cleavage route for pcresol degradation that is dominantly expressed in colonies.
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http://dx.doi.org/10.4014/jmb.1104.04006DOI Listing
November 2011

Ssg, a putative glycosyltransferase, functions in lipo- and exopolysaccharide biosynthesis and cell surface-related properties in Pseudomonas alkylphenolia.

FEMS Microbiol Lett 2011 Feb 17;315(1):38-45. Epub 2010 Dec 17.

Department of Microbiology, Changwon National University, Changwon-si, Kyongnam, Korea.

In the presence of vaporized p-cresol, Pseudomonas alkylphenolia KL28 forms specialized aerial structures (SAS). A transposon mutant of strain KL28 (C23) incapable of forming mature SAS was isolated. Genetic analysis of the C23 mutant revealed the transposon insertion in a gene (ssg) encoding a putative glycosyltransferase, which is homologous to the Pseudomonas aeruginosa PAO1 PA5001 gene. Deletion of ssg in KL28 caused the loss of lipopolysaccharide O antigen and altered the composition of the exopolysaccharide. Wild-type KL28 produced a fucose-, glucose- and mannose-rich exopolysaccharide, while the mutant exopolysaccharide completely lacked fucose and mannose, resulting in an exopolysaccharide with glucose as the major component. The mutant strain showed reduced surface spreading, pellicle and biofilm formation, probably due to the cumulative effect of lipopolysaccharide truncation and altered exopolysaccharide composition. Our results show that the ssg gene of KL28 is involved in both lipopolysaccharide and exopolysaccharide biosynthesis and thus plays an important role in cell surface properties and cell-cell interactions of P. alkylphenolia.
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http://dx.doi.org/10.1111/j.1574-6968.2010.02172.xDOI Listing
February 2011

alpha-Galactosidase from Bacillus megaterium VHM1 and its application in removal of flatulence-causing factors from soymilk.

J Microbiol Biotechnol 2010 Nov;20(11):1546-54

Department of Biochemistry, Gulbarga University, Gulbarga-585 106, Karnataka-585 106, India.

A bacterial strain capable of producing extracellular alpha-galactosidase was isolated from sugar cane industrial waste soil sample. Microbiological, physiological, and biochemical studies revealed that isolate belonged to Bacillus sp,. Furthermore, 16S rDNA sequence analysis of new isolates was identified as Bacillus megaterium VHM1. The production of alpha-galactosidase was optimized by various physical culture conditions. Guar gum and yeast extract acted as the best carbon and nitrogen source, respectively for the production of alpha-galactosidase. The enzyme showed an optimum pH at 7.5 and was stable over a pH between 5 and 9. The enzyme was optimally active in 55degreesC and the enzyme was thermostable with half life of 120 minutes at 55 degrees C and lost their 90%, residual activity in 120 minutes at 60 degrees C. alpha-Galactosidase was strongly inhibited by Ag2, Cu2, and Hg2+ at 1mM concentration. The metal ions Fe2, Mn2+, and Mg2+ had no effect on alpha-galactosidase activity, Zn2+,Ni2+, and Ca2+ reduced the enzyme activity slightly. The B megaterium VHM1 enzyme treatment completely hydrolyzed flatulence-causing sugars of soymilk within one and half hour.
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http://dx.doi.org/10.4014/jmb.0912.12012DOI Listing
November 2010

Purification and physiochemical characterization of melanin pigment from Klebsiella sp. GSK.

J Microbiol Biotechnol 2010 Nov;20(11):1513-20

Department of Biochemistry, Gulbarga University, Gulbarga - 585 106, Karnataka State, India.

The bacterium capable of producing melanin pigment in the presence of L-tyrosine was isolated from crop field soil sample and identified as Klebsiella sp. GSK based on morphological, biochemical and 16S rDNA sequencing. The polymerization of this pigment occurs outside the cell wall, which has granular structure as melanin ghosts. The chemical characterization of pigment particles showed acid resistant, alkali soluble, insoluble in most of the organic solvents and water. The pigment gets bleached when subjected to the action of oxidants as well as reductants. This pigment was precipitated with FeCl3, ammoniacal silver nitrate and potassium ferricynide. The pigment showed high absorbance in the UV region and decreased absorbance when shifted towards the visible region. The melanin pigment was further charecterized by FT-IR and EPR spectroscopy. A key enzyme 4-hydroxyphenylacetic acid hydroxylase catalyzes the formation of melanin pigment by hydroxylation of L-tyrosine was detected in this bacterium. Inhibition studies with specific inhibitor kojic acid and KCN proved that melanin is synthesized by DOPA-Melanin pathway.
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http://dx.doi.org/10.4014/jmb.1002.02006DOI Listing
November 2010

Formation of specialized aerial architectures by Rhodococcus during utilization of vaporized p-cresol.

Microbiology (Reading) 2009 Nov 23;155(Pt 11):3788-3796. Epub 2009 Jul 23.

Department of Microbiology, Changwon National University, Changwon-si, Kyongnam 641-773, Republic of Korea.

When grown with vaporized alkylphenols such as p-cresol as the sole carbon and energy source, several isolated Rhodococcus strains formed growth structures like miniature mushrooms, termed here specialized aerial architectures (SAA), that reached sizes of up to 0.8 mm in height. Microscopic examination allowed us to view the distinct developmental stages during the formation of SAA from a selected strain, Rhodococcus sp. KL96. Initially, mounds consisting of long rod cells arose from a lawn of cells, and then highly branched structures were formed from the mounds. During the secondary stage of development, branching began after long rod cells grew outward and twisted longitudinally, serving as growth points, and the cells at the base of the mound became short rods that supported upward growth. Cells in the highly fluffy structures were eventually converted, via reductive division, into structures that resembled cocci, with a diameter of approximately 0.5 microm, that were arranged in chains. Most cells inside the SAA underwent a phase variation in order to form wrinkled colonies from cells that originally formed smooth colonies. Approximately 2 months was needed for complete development of the SAA, and viable cells were recovered from SAA that were incubated for more than a year. An extracellular polymeric matrix layer and lipid bodies appeared to play an important role in structural integrity and as a metabolic energy source, respectively. To our knowledge, similar formation of aerial structures for the purpose of substrate utilization has not been reported previously for Gram-positive bacteria.
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http://dx.doi.org/10.1099/mic.0.029926-0DOI Listing
November 2009

Metabolism of acenaphthylene via 1,2-dihydroxynaphthalene and catechol by Stenotrophomonas sp. RMSK.

Biodegradation 2009 Nov 20;20(6):837-43. Epub 2009 Jun 20.

Department of Biochemistry, Gulbarga University, Gulbarga, Karnataka 585106, India.

Stenotrophomonas sp. RMSK capable of degrading acenaphthylene as a sole source of carbon and energy was isolated from coal sample. Metabolites produced were analyzed and characterized by TLC, HPLC and mass spectrometry. Identification of naphthalene-1,8-dicarboxylic acid, 1-naphthoic acid, 1,2-dihydroxynaphthalene, salicylate and detection of key enzymes namely 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-1,2-dioxygenase in the cell free extract suggest that acenaphthylene metabolized via 1,2-dihydroxynaphthalene, salicylate and catechol. The terminal metabolite, catechol was then metabolized by catechol-1,2-dioxygenase to cis,cis-muconic acid, ultimately forming TCA cycle intermediates. Based on these studies, the proposed metabolic pathway in strain RMSK is, acenaphthylene --> naphthalene-1,8-dicarboxylic acid --> 1-naphthoic acid --> 1,2-dihydroxynaphthalene --> salicylic acid --> catechol --> cis,cis-muconic acid.
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http://dx.doi.org/10.1007/s10532-009-9271-1DOI Listing
November 2009

Ultramicrocells form by reductive division in macroscopic Pseudomonas aerial structures.

Environ Microbiol 2009 May;11(5):1117-25

Department of Microbiology, Changwon National University, Changwon-si, Kyongnam, Korea.

Bacterial aerial growth with reductive cellular division and morphological development has not been reported from single-cell bacteria. Here we show that within 1 month of incubation in vaporized p-cresol, Pseudomonas sp. KL28 form shiny, highly branched specialized aerial structures of millimetre-scale diameter. The developmental process displayed spatially and temporally distinct stages; an initial sphere stage was followed by ramification, which led to highly branched tip formation. In this morphogenesis process, the extracellular matrix (ECM) played an important role for maintaining the integrity of sectional populations and the boundaries between adjacent sectors. In addition, cellular division, lysis and migration within the aerial structures were also accompanied. During prolonged incubation, clusters of short-rod cells covered by an outer layer of thick ECM underwent reductive transformation and then replicative reductive division to form oval ultramicrocells of < 0.4 microm in diameter. In addition, the aerial structures protected these rather fragile cells from desiccation and served as a selection pressure for wrinkly, spreading cell variants. The formation of aerial structures is affected positively and negatively by a GacA regulator and RpoS, respectively, and is linked to other phenotypes. Our results demonstrate that Pseudomonas has an ecological adaptation to form mushroom-like aerial structures, which can be a tool for studying cell-cell interactions and bacterial development.
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http://dx.doi.org/10.1111/j.1462-2920.2008.01841.xDOI Listing
May 2009

Enterobacter sp. VKGH12 growing with n-butanol as the sole carbon source and cells to which the alcohol is added as pure toxin show considerable differences in their adaptive responses.

FEMS Microbiol Lett 2006 Jan;254(1):48-54

Department of Biochemistry, Gulbarga University, Gulbarga, India.

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is able to grow in toxic concentrations of n-butanol up to 1.5 % (volume in volume) as the sole carbon and energy source. Morphology changes in the cells growing on increasing concentrations of n-butanol were observed. The size of the bacteria decreased with increasing concentrations of n-butanol, also leading to an enhanced ratio between the surface and volume of the cells. This is in complete contradiction to the reaction of glucose-grown cells to which n-butanol had been added as a toxin. Similar differences were found in typical adaptive responses to toxic organic compounds, namely changes in fatty acid composition of membrane lipids and the activity of catalase. In both cases, reactions depending on the n-butanol concentrations could be observed when the toxin was added to glucose-grown cells, whereas no reaction was observable when the cells were growing in n-butanol as the sole carbon and energy source. This is another proof for the observation that there are certain differences between the adaptive strategies of cells when adapting to high concentrations of a growth substrate and those when adapting to a toxin added to growing cells.
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http://dx.doi.org/10.1111/j.1574-6968.2005.00017.xDOI Listing
January 2006