PCR-based detection of mobile genetic elements in total community DNA

Smalla, Kornelia; Krögerrecklenfort, Ellen; Heuer, Holger; Dejonghe, Winnie; Top, Eva M.; Osborn, Mark J.; Niewint, Jessica; Tebbe, Christoph C.; Barr, Michelle; Bailey, Mark; Greated, Alicia; Thomas, Christopher M.; Turner, Sarah L.; Young, P.; Nikolakopoulou, Dora; Karagouni, Amalia D.; Wolters, A.C.; Elsas, Jan Dirk van; Drønen, Karine; Sandaa, Ruth Anne; Borin, Sara; Brabhu, Julia; Grohmann, Elisabeth; Sobecky, Patricia A.

doi:10.1099/00221287-146-6-1256

Cited by 50 publications

(56 citation statements)

References 2 publications

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“…Traditionally, most attention has focused on plasmids and transposons. This is particularly true for environmental microbiology (24). However, since integrons have recently been demonstrated to occur in the chromosomes of diverse bacterial species (22) and integron integrases are recoverable from environmental samples (18), we reasoned that integrons are widespread in natural environments.…”

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confidence: 99%

Gene Cassette PCR: Sequence-Independent Recovery of Entire Genes from Environmental DNA

Stokes¹,

Holmes²,

Nield³

et al. 2001

Appl Environ Microbiol

169

207

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The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes.

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mentioning

confidence: 99%

Gene Cassette PCR: Sequence-Independent Recovery of Entire Genes from Environmental DNA

Stokes¹,

Holmes²,

Nield³

et al. 2001

Appl Environ Microbiol

169

207

View full text Add to dashboard Cite

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“…PCR amplification of DNA which was directly extracted from environmental samples with primers targeting replicon or resistance gene sequences, when combined with Southern blot hybridization, enables their rapid and specific detection. While PCR-based detection of MGE and resistance genes in community DNA is an ideal method to analyze many samples for the presence and abundance of the sequence targeted (23), no information is obtained on its genomic context. For this, the isolation of the respective MGE is essential.…”

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confidence: 99%

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element

Smalla¹,

Haines

Jones

et al. 2006

Appl Environ Microbiol

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100

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Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1␤ replicon-specific sequences detected in total community DNA. The isolation of IncP-1␤ plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1␤ plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1␤ plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRT⌬P probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1␤ plasmids R751 and pB8. However, this is the first example of an IncP-1␤ plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.

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“…In contrast, several studies have previously indicated widespread dissemination of the clinical IncP plasmid RK2 (Gotz et al, 1996 ;Smalla et al, 2000 ;Sobecky et al, 1997). Results from our comparative analysis of the putative Rep proteins in this study, together with those from previously characterized plasmid replicons, indicates that both RCR-and theta-replicating plasmids are to be found in marine isolates.…”

Section: Discussionmentioning

confidence: 94%

Endogenous isolation of replicon probes for assessing plasmid ecology of marine sediment microbial communities The GenBank accession numbers for the 16S rRNA sequences determined in this work are AF249334–AF249338 and AF284226–AF284230.

et al. 2001

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Six functional replication origins (repGA14, repGA33, repGA70, repSD41, repSD164 and repSD172), obtained from endogenously isolated, broad-hostrange (BHR) marine plasmids ranging in size from 5 to 60 kb, were used to determine plasmid occurrence in three coastal marine sediment sites (in California, Georgia and South Carolina, USA). The plasmid-specific replicons were isolated from plasmid-bearing marine sediment bacteria belonging to the α and γ subclasses of the Proteobacteria. The plasmid sources of the endogenous replicons were considered to be cryptic due to a lack of identifiable phenotypic traits. The putative Rep proteins from a number of these replicons showed similarity to replicons of two recognized families : RCR group III (repSD164) and the FIA family of theta group A (repSD41, repSD121, repGA33 and repGA14). Plasmids isolated from marine bacteria belonging to the genera Pseudoalteromonas, Shewanella and Vibrio cultivated from geographically different coastal sites exhibited homology to two of the marine plasmid replicons, repSD41 and repGA70, obtained from a Vibrio sp. The repGA33 plasmid origin, obtained from a Shewanella sp. isolated from coastal Georgia, was detected in 7 % of the Georgia marine sediment Shewanella sp. isolates. Microbial community DNA extracted from marine sediments was also screened for the presence of the plasmid replication sequences. Community DNA samples amplified by PCR yielded a positive signal for the repSD172 and repGA14 replication sequences. The replication origin of BHR plasmid RK2 (IncP) was also detected in marine Vibrio sp. and microbial community DNA extracted from the three coastal sites. These findings provide molecular evidence that marine sediment bacteria harbour an untapped population of BHR plasmids.

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PCR-based detection of mobile genetic elements in total community DNA

Cited by 50 publications

References 2 publications

Gene Cassette PCR: Sequence-Independent Recovery of Entire Genes from Environmental DNA

Gene Cassette PCR: Sequence-Independent Recovery of Entire Genes from Environmental DNA

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element

Endogenous isolation of replicon probes for assessing plasmid ecology of marine sediment microbial communities The GenBank accession numbers for the 16S rRNA sequences determined in this work are AF249334–AF249338 and AF284226–AF284230.

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