Cultivation of hard-to-culture subsurface mercury-resistant bacteria and discovery of new merA gene sequences

L D Rasmussen; C Zawadsky; S J Binnerup; Gunnar Øregaard; Søren Johannes Sørensen; N.  Kroer

doi:10.1128/AEM.00049-08

Cultivation of hard-to-culture subsurface mercury-resistant bacteria and discovery of new merA gene sequences

L D Rasmussen, C Zawadsky, S J Binnerup, Gunnar Øregaard, Søren Johannes Sørensen, N. Kroer

Microbiology

35 Citations (Scopus)

Abstract

Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments.

Original language	English
Journal	Applied and Environmental Microbiology
Volume	74
Issue number	12
Pages (from-to)	3795-803
Number of pages	8
ISSN	0099-2240
DOIs	https://doi.org/10.1128/AEM.00049-08
Publication status	Published - 2008

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10.1128/AEM.00049-08

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@article{878a5180f83f11ddb219000ea68e967b,

title = "Cultivation of hard-to-culture subsurface mercury-resistant bacteria and discovery of new merA gene sequences",

abstract = "Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments.",

author = "Rasmussen, {L D} and C Zawadsky and Binnerup, {S J} and Gunnar {\O}regaard and S{\o}rensen, {S{\o}ren Johannes} and N. Kroer",

note = "Keywords: Anti-Bacterial Agents; Bacteria; Biodiversity; Colony Count, Microbial; DNA Fingerprinting; DNA, Bacterial; DNA, Ribosomal; Drug Resistance, Bacterial; Electrophoresis, Polyacrylamide Gel; Genes, Bacterial; Mercury; Molecular Sequence Data; Nucleic Acid Denaturation; Phylogeny; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Random Amplified Polymorphic DNA Technique; Sequence Analysis, DNA; Sequence Homology; Soil Microbiology",

year = "2008",

doi = "10.1128/AEM.00049-08",

language = "English",

volume = "74",

pages = "3795--803",

journal = "Applied and Environmental Microbiology",

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publisher = "American Society for Microbiology",

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T1 - Cultivation of hard-to-culture subsurface mercury-resistant bacteria and discovery of new merA gene sequences

AU - Rasmussen, L D

AU - Zawadsky, C

AU - Binnerup, S J

AU - Øregaard, Gunnar

AU - Sørensen, Søren Johannes

AU - Kroer, N.

N1 - Keywords: Anti-Bacterial Agents; Bacteria; Biodiversity; Colony Count, Microbial; DNA Fingerprinting; DNA, Bacterial; DNA, Ribosomal; Drug Resistance, Bacterial; Electrophoresis, Polyacrylamide Gel; Genes, Bacterial; Mercury; Molecular Sequence Data; Nucleic Acid Denaturation; Phylogeny; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Random Amplified Polymorphic DNA Technique; Sequence Analysis, DNA; Sequence Homology; Soil Microbiology

PY - 2008

Y1 - 2008

N2 - Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments.

AB - Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments.

U2 - 10.1128/AEM.00049-08

DO - 10.1128/AEM.00049-08

M3 - Journal article

C2 - 18441111

SN - 0099-2240

VL - 74

SP - 3795

EP - 3803

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

IS - 12

ER -