scholarly journals Genetic Diversity of nifH Gene Sequences inPaenibacillus azotofixans Strains and Soil Samples Analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments

1998 ◽  
Vol 64 (8) ◽  
pp. 2770-2779 ◽  
Author(s):  
Alexandre S. Rosado ◽  
Gabriela F. Duarte ◽  
Lucy Seldin ◽  
Jan Dirk Van Elsas
Keyword(s):  
Genetic Diversity ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Nifh Gene ◽  
Sequence Divergence ◽  
Paenibacillus Polymyxa ◽  
Sequence Information ◽  
Gene Sequences ◽  
Degenerate Primers ◽  
Gradient Gel Electrophoresis

ABSTRACT The diversity of dinitrogenase reductase gene (nifH) fragments in Paenibacillus azotofixans strains was investigated by using molecular methods. The partial nifHgene sequences of eight P. azotofixans strains, as well as one strain each of the close relatives Paenibacillus durum,Paenibacillus polymyxa, and Paenibacillus macerans, were amplified by PCR by using degenerate primers and were characterized by DNA sequencing. We found that there are twonifH sequence clusters, designated clusters I and II, inP. azotofixans. The data further indicated that there was sequence divergence among the nifH genes of P. azotofixans strains at the DNA level. However, the gene products were more conserved at the protein level. Phylogenetic analysis showed that all nifH cluster II sequences were similar to the alternative (anf) nitrogenase sequence. A nested PCR assay for the detection of nifH (cluster I) of P. azotofixans was developed by using the degenerate primers as outer primers and two specific primers, designed on the basis of the sequence information obtained, as inner primers. The specificity of the inner primers was tested with several diazotrophic bacteria, and PCR revealed that these primers are specific for the P. azotofixans nifH gene. A GC clamp was attached to one inner primer, and a denaturing gradient gel electrophoresis (DGGE) protocol was developed to study the genetic diversity of this region of nifH inP. azotofixans strains, as well as in soil and rhizosphere samples. The results revealed sequence heterogeneity among differentnifH genes. Moreover, nifH is probably a multicopy gene in P. azotofixans. Both similarities and differences were detected in the P. azotofixans nifH DGGE profiles generated with soil and rhizosphere DNAs. The DGGE assay developed here is reproducible and provides a rapid way to assess the intraspecific genetic diversity of an important functional gene in pure cultures, as well as in environmental samples.

scholarly journals The effect of inoculum dose on the genetic diversity detected within Helicoverpa armigera nucleopolyhedrovirus populations

2013 ◽  
Vol 94 (11) ◽  
pp. 2524-2529 ◽  
Author(s):  
Vicky Lynne Baillie ◽  
Gustav Bouwer
Keyword(s):  
Genetic Diversity ◽  
Helicoverpa Armigera ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Model System ◽  
Inoculum Dose ◽  
Significant Difference ◽  
Helicoverpa Armígera ◽  
Gradient Gel Electrophoresis ◽  
Neonate Larvae

Environmental and infection variables may affect the genetic diversity of baculovirus populations. In this study, Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was used as a model system for studying the effects of a key infection variable, inoculum dose, on the genetic diversity within nucleopolyhedrovirus populations. Diversity and equitability indices were calculated from DNA polymerase-specific denaturing gradient gel electrophoresis profiles obtained from individual H. armigera neonate larvae inoculated with either an LD5 or LD95 of HearNPV. Although the genetic diversity detected in larvae treated with an LD95 was not statistically different from the diversity detected in the HearNPV inoculum samples, there was a statistically significant difference in the genetic diversity detected in the LD5-inoculated larvae compared with the genetic diversity detected in the HearNPV samples used for the inoculations. The study suggests that inoculum dose needs to be considered carefully in experiments that evaluate HearNPV genetic diversity or in studies where differences in genetic diversity may have phenotypic consequences.

scholarly journals Analysis of Bacterial Communities in Soil by Use of Denaturing Gradient Gel Electrophoresis and Clone Libraries, as Influenced by Different Reverse Primers

2008 ◽  
Vol 74 (9) ◽  
pp. 2717-2727 ◽  
Author(s):  
Jolanda K. Brons ◽  
Jan Dirk van Elsas
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Soil Analysis ◽  
Rrna Gene ◽  
Sequence Information ◽  
Soil Bacterial Diversity ◽  
Clone Libraries ◽  
Gradient Gel Electrophoresis ◽  
Candidate Division ◽  
Forward Primer

ABSTRACT To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel reverse primers was tested with the canonical forward primer as to the DGGE fingerprints obtained from grassland soil. Analysis of these DGGE profiles by GelCompar showed that they all fell into two main clusters separated by a G/A alteration at position 14 in the reverse primer used. To assess differences between the dominant bacteria amplified, we then produced four (100-membered) 16S rRNA gene clone libraries by using reverse primers with either an A or a G at position 14, designated R1401-1a, R1401-1b, R1401-2a, and R1401-2b. Subsequent sequence analysis revealed that, on the basis of the about 410-bp sequence information, all four primers amplified similar, as well as different (including novel), bacterial groups from soil. Most of the clones fell into two main phyla, Firmicutes and Proteobacteria. Within Firmicutes, the majority of the clones belonged to the genus Bacillus. Within Proteobacteria, the majority of the clones fell into the alpha or gamma subgroup whereas a few were delta and beta proteobacteria. The other phyla found were Actinobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, Gemmatimonadetes, Chlorobi, Bacteroidetes, Chlamydiae, candidate division TM7, Ferribacter, Cyanobacteria, and Deinococcus. Statistical analysis of the data revealed that reverse primers R1401-1b and R1401-1a both produced libraries with the highest diversities yet amplified different types. Their concomitant use is recommended.

Genetic Diversity of the Biofilm CoveringMontacuta ferruginosa (Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA†

1998 ◽  
Vol 64 (9) ◽  
pp. 3464-3472 ◽  
Author(s):  
David C. Gillan ◽  
Arjen G. C. L. Speksnijder ◽  
Gabriel Zwart ◽  
Chantal De Ridder
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
Dna Extraction ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gradient Gel Electrophoresis ◽  
Biofilm Bacteria ◽  
Sequence Types

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas,Colwellia, and Oceanospirillum (members of the γ-Proteobacteria lineage), as well as Flexibacter maritimus (a member of theCytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.

scholarly journals Application of molecular biological methods for studying probiotics and the gut flora

2002 ◽  
Vol 88 (S1) ◽  
pp. s29-s37 ◽  
Author(s):  
A. L. McCartney
Keyword(s):  
Molecular Biology ◽  
Gel Electrophoresis ◽  
Dietary Intervention ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Molecular Methods ◽  
Sequence Information ◽  
Gut Flora ◽  
Gradient Gel Electrophoresis ◽  
Probiotic Strains

Increasingly, the microbiological scientific community is relying on molecular biology to define the complexity of the gut flora and to distinguish one organism from the next. This is particularly pertinent in the field of probiotics, and probiotic therapy, where identifying probiotics from the commensal flora is often warranted. Current techniques, including genetic fingerprinting, gene sequencing, oligonucleotide probes and specific primer selection, discriminate closely related bacteria with varying degrees of success. Additional molecular methods being employed to determine the constituents of complex microbiota in this area of research are community analysis, denaturing gradient gel electrophoresis (DGGE)/temperature gradient gel electrophoresis (TGGE), fluorescentin situhybridisation (FISH) and probe grids. Certain approaches enable specific aetiological agents to be monitored, whereas others allow the effects of dietary intervention on bacterial populations to be studied. Other approaches demonstrate diversity, but may not always enable quantification of the population. At the heart of current molecular methods is sequence information gathered from culturable organisms. However, the diversity and novelty identified when applying these methods to the gut microflora demonstrates how little is known about this ecosystem. Of greater concern is the inherent bias associated with some molecular methods. As we understand more of the complexity and dynamics of this diverse microbiota we will be in a position to develop more robust molecular-based technologies to examine it. In addition to identification of the microbiota and discrimination of probiotic strains from commensal organisms, the future of molecular biology in the field of probiotics and the gut flora will, no doubt, stretch to investigations of functionality and activity of the microflora, and/or specific fractions. The quest will be to demonstrate the roles of probiotic strainsin vivoand not simply their presence or absence.

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