scholarly journals Shifts in Abundance and Diversity of Mobile Genetic Elements after the Introduction of Diverse Pesticides into an On-Farm Biopurification System over the Course of a Year

2014 ◽  
Vol 80 (13) ◽  
pp. 4012-4020 ◽  
Author(s):  
Simone Dealtry ◽  
Peter N. Holmsgaard ◽  
Vincent Dunon ◽  
Sven Jechalke ◽  
Guo-Chun Ding ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Mobile Genetic Elements ◽  
Replication Initiation ◽  
Genetic Elements ◽  
Class 1 ◽  
Genes Encoding ◽  
Abundance And Diversity ◽  
And Shifts

ABSTRACTBiopurification systems (BPS) are used on farms to control pollution by treating pesticide-contaminated water. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticides. Therefore, the composition and shifts of MGEs, in particular, of IncP-1 plasmids carried by BPS bacterial communities exposed to various pesticides, were monitored over the course of an agricultural season. PCR amplification of total community DNA using primers targeting genes specific to different plasmid groups combined with Southern blot hybridization indicated a high abundance of plasmids belonging to IncP-1, IncP-7, IncP-9, IncQ, and IncW, while IncU and IncN plasmids were less abundant or not detected. Furthermore, the integrase genes of class 1 and 2 integrons (intI1,intI2) and genes encoding resistance to sulfonamides (sul1,sul2) and streptomycin (aadA) were detected and seasonality was revealed. Amplicon pyrosequencing of the IncP-1trfAgene coding for the replication initiation protein revealed high IncP-1 plasmid diversity and an increase in the abundance of IncP-1β and a decrease in the abundance of IncP-1ε over time. The data of the chemical analysis showed increasing concentrations of various pesticides over the course of the agricultural season. As an increase in the relative abundances of bacteria carrying IncP-1β plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides.

Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 967-978 ◽  
Author(s):  
C. Viana-Niero ◽  
P. E. de Haas ◽  
D. van Soolingen ◽  
S. C. Leão
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Phospholipase C ◽  
Genetic Polymorphisms ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Clinical Isolates ◽  
Significant Similarity ◽  
Genomic Deletions ◽  
Genes Encoding ◽  
Mycobacterium Africanum

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and ‘Mycobacterium canettii’. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

scholarly journals Mobile Genetic Elements Related to the Diffusion of Plasmid-Mediated AmpC β-Lactamases or Carbapenemases from Enterobacteriaceae: Findings from a Multicenter Study in Spain

2015 ◽  
Vol 59 (9) ◽  
pp. 5260-5266 ◽  
Author(s):  
L. Zamorano ◽  
E. Miró ◽  
C. Juan ◽  
L. Gómez ◽  
G. Bou ◽  
...  
Keyword(s):  
Southern Hybridization ◽  
Mobile Genetic Elements ◽  
Pulsed Field Gel Electrophoresis ◽  
Content Type ◽  
Genetic Elements ◽  
Chromosomal Origin ◽  
Class 1 ◽  
Rapid Dissemination ◽  
Large Plasmids ◽  
Genetic Context

ABSTRACTWe examined the genetic context of 74 acquiredampCgenes and 17 carbapenemase genes from 85 of 640Enterobacteriaceaeisolates collected in 2009. Using S1 pulsed-field gel electrophoresis and Southern hybridization, 37 of 74blaAmpCgenes were located on large plasmids of different sizes belonging to six incompatibility groups. We used sequencing and PCR mapping to investigate the regions flanking the acquiredampCgenes. TheblaCMY-2-like genes were associated with ISEcp1; the surroundingblaDHAgenes were similar toKlebsiella pneumoniaeplasmid pTN60013 associated with IS26and thepspandsapoperons; and theblaACC-1genes were associated with IS26elements inserted into ISEcp1. All of the carbapenemase genes (blaVIM-1,blaIMP-22, andblaIMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination ofampCgenes amongEnterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons, or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons (In846 to In848) are described in this study.

scholarly journals Properties of genes encoding transfer RNAs as integration sites for genomic islands and prophages in Klebsiella pneumoniae

2020 ◽  
Author(s):  
Camilo Berríos-Pastén ◽  
Rodolfo Acevedo ◽  
Patricio Arros ◽  
Macarena A. Varas ◽  
Kelly L. Wyres ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Integration Site ◽  
Phylogenetic Analyses ◽  
Bacterial Genome ◽  
Mobile Genetic Elements ◽  
Genomic Islands ◽  
Genomic Context ◽  
Genetic Elements ◽  
Genes Encoding ◽  
Integration Sites

ABSTRACTThe evolution of traits including antibiotic resistance, virulence, and increased fitness in Klebsiella pneumoniae and related species has been linked to the acquisition of mobile genetic elements through horizontal transfer. Among them, genomic islands (GIs) preferentially integrating at genes encoding tRNAs and the tmRNA (t(m)DNAs) would be significant in promoting chromosomal diversity. Here, we studied the whole set of t(m)DNAs present in 66 Klebsiella chromosomes, investigating their usage as integration sites and the properties of the integrated GIs. A total of 5,624 t(m)DNAs were classified based on their sequence conservation, genomic context, and prevalence. 161 different GIs and prophages were found at these sites, hosting 3,540 gene families including various related to virulence and drug resistance. Phylogenetic analyses supported the acquisition of several of these elements through horizontal gene transfer, likely mediated by a highly diverse set of encoded integrases targeting specific t(m)DNAs and sublocations inside them. Only a subset of the t(m)DNAs had integrated GIs and even identical tDNA copies showed dissimilar usage frequencies, suggesting that the genomic context would influence the integration site selection. This usage bias, likely towards avoiding disruption of polycistronic transcriptional units, would be conserved across Gammaproteobacteria. The systematic comparison of the t(m)DNAs across different strains allowed us to discover an unprecedented number of K. pneumoniae GIs and prophages and to raise important questions and clues regarding the fundamental properties of t(m)DNAs as targets for the integration of mobile genetic elements and drivers of bacterial genome evolution and pathogen emergence.

scholarly journals Influence of industrial contamination on mobile genetic elements: class 1 integron abundance and gene cassette structure in aquatic bacterial communities

2008 ◽  
Vol 2 (4) ◽  
pp. 417-428 ◽  
Author(s):  
Meredith S Wright ◽  
Craig Baker-Austin ◽  
Angela H Lindell ◽  
Ramunas Stepanauskas ◽  
Hatch W Stokes ◽  
...  
Keyword(s):  
Bacterial Communities ◽  
Gene Cassette ◽  
Mobile Genetic Elements ◽  
Class 1 Integron ◽  
Industrial Contamination ◽  
Genetic Elements ◽  
Class 1

scholarly journals Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA

2002 ◽  
Vol 68 (10) ◽  
pp. 5017-5025 ◽  
Author(s):  
Balbina Nogales ◽  
Kenneth N. Timmis ◽  
David B. Nedwell ◽  
A. Mark Osborn
Keyword(s):  
Reverse Transcription ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Estuarine Sediments ◽  
Rt Pcr ◽  
Sediment Samples ◽  
Reverse Transcription Pcr ◽  
Nitrogen Inputs ◽  
Denitrification Genes

ABSTRACT The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization. This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling. Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments. A high diversity of nirS sequences was observed. Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.

scholarly journals Specific and Sensitive Detection of Ralstonia solanacearum in Soil on the Basis of PCR Amplification of fliC Fragments

2003 ◽  
Vol 69 (12) ◽  
pp. 7248-7256 ◽  
Author(s):  
J. Schönfeld ◽  
H. Heuer ◽  
J. D. van Elsas ◽  
K. Smalla
Keyword(s):  
Ralstonia Solanacearum ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Environmental Isolates ◽  
System A ◽  
Gene Coding ◽  
Pcr Product ◽  
Geographic Origins ◽  
Pcr Products

ABSTRACT Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 103 CFU g of bulk soil−1. The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.

Pediococcus parvulus gtf Gene Encoding the GTF Glycosyltransferase and Its Application for Specific PCR Detection of β-d-Glucan–Producing Bacteria in Foods and Beverages

2006 ◽  
Vol 69 (1) ◽  
pp. 161-169 ◽  
Author(s):  
MARIA LAURA WERNING ◽  
IDOIA IBARBURU ◽  
MARIA TERESA DUEÑAS ◽  
ANA IRASTORZA ◽  
JESÚS NAVAS ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Oenococcus Oeni ◽  
Alcoholic Beverages ◽  
Gene Encoding ◽  
Specific Pcr ◽  
Genomic Locations

Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, β-D-glucan–producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize β-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the β-d-glucan producers Lacto-bacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of β-d-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 × 102 CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.

scholarly journals Prevalence of Integrons and Insertion Sequences in ESBL-Producing E. coli Isolated from Different Sources in Navarra, Spain

2018 ◽  
Vol 15 (10) ◽  
pp. 2308 ◽  
Author(s):  
Lara Pérez-Etayo ◽  
Melibea Berzosa ◽  
David González ◽  
Ana Vitas
Keyword(s):  
Mobile Genetic Elements ◽  
Resistant Bacteria ◽  
Insertion Sequences ◽  
Northern Spain ◽  
Antibiotic Resistant ◽  
Genetic Elements ◽  
Antimicrobial Resistance Genes ◽  
Class 1 ◽  
Wide Variability ◽  
Different Sources

Mobile genetic elements play an important role in the dissemination of antibiotic resistant bacteria among human and environmental sources. Therefore, the aim of this study was to determine the occurrence and patterns of integrons and insertion sequences of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from different sources in Navarra, northern Spain. A total of 150 isolates coming from food products, farms and feeds, aquatic environments, and humans (healthy people and hospital inpatients), were analyzed. PCRs were applied for the study of class 1, 2, and 3 integrons (intI1, intI2, and intI3), as well as for the determination of insertion sequences (IS26, ISEcp1, ISCR1, and IS903). Results show the wide presence and dissemination of intI1 (92%), while intI3 was not detected. It is remarkable, the prevalence of intI2 among food isolates, as well as the co-existence of class 1 and class 2 (8% of isolates). The majority of isolates have two or three IS elements, with the most common being IS26 (99.4%). The genetic pattern IS26–ISEcp1 (related with the pathogen clone ST131) was present in the 22% of isolates (including human isolates). In addition, the combination ISEcp1–IS26–IS903–ISCR1 was detected in 11 isolates being, to our knowledge, the first study that describes this genetic complex. Due to the wide variability observed, no relationship was determined among these mobile genetic elements and β-lactam resistance. More investigations regarding the genetic composition of these elements are needed to understand the role of multiple types of integrons and insertion sequences on the dissemination of antimicrobial resistance genes among different environments.

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