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Veeranagouda Y.,GLAVAHCS | Veeranagouda Y.,University of California at Los Angeles | Husain F.,GLAVAHCS | Wexler H.M.,GLAVAHCS | Wexler H.M.,University of California at Los Angeles
FEMS Microbiology Letters | Year: 2012

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 × 103 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 Published 2012. This article is a U.S. Government work and is in the public domain in the USA.3332 August 2012 10.1111/j.1574-6968.2012.02602.x Research Letter Research Letters Published 2012. This article is a U.S. Government work and is in the public domain in the USA.. Source


Veeranagouda Y.,GLAVAHCS | Veeranagouda Y.,University of California at Los Angeles | Husain F.,GLAVAHCS | Wexler H.M.,GLAVAHCS | Wexler H.M.,University of California at Los Angeles
Anaerobe | Year: 2013

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. © 2013 . Source


Veeranagouda Y.,GLAVAHCS | Veeranagouda Y.,University of California at Los Angeles | Husain F.,GLAVAHCS | Boente R.,GLAVAHCS | And 7 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2014

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 mutantwas 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. Source


Veeranagouda Y.,GLAVAHCS | Veeranagouda Y.,University of California at Los Angeles | Husain F.,GLAVAHCS | Tenorio E.L.,Tufts Medical Center | And 2 more authors.
BMC Genomics | Year: 2014

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. © 2014 Veeranagouda et al.; licensee BioMed Central Ltd. Source


Husain F.,GLAVAHCS | Veeranagouda Y.,GLAVAHCS | Veeranagouda Y.,University of California at Los Angeles | Hsi J.,University of California at Los Angeles | And 4 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2013

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. Copyright © 2013, American Society for Microbiology. All Rights Reserved. Source

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