When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing

ABSTRACT Geobacter sulfurreducens strain KN400 was recovered in previous studies in which a culture of the DL1 strain of G. sulfurreducens served as the inoculum in investigations of microbial current production at low anode potentials (−400 mV versus Ag/AgCl). Differences in the genome sequences of...

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Autores principales:	Pravin Malla Shrestha, Kelly P. Nevin, Minita Shrestha, Derek R. Lovley
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2013
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9405072f697740edaca6acebf998565b
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spelling	oai:doaj.org-article:9405072f697740edaca6acebf998565b2021-11-15T15:40:29ZWhen Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing10.1128/mBio.00591-122150-7511https://doaj.org/article/9405072f697740edaca6acebf998565b2013-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00591-12https://doaj.org/toc/2150-7511ABSTRACT Geobacter sulfurreducens strain KN400 was recovered in previous studies in which a culture of the DL1 strain of G. sulfurreducens served as the inoculum in investigations of microbial current production at low anode potentials (−400 mV versus Ag/AgCl). Differences in the genome sequences of KN400 and DL1 were too great to have arisen from adaptive evolution during growth on the anode. Previous deep sequencing (80-fold coverage) of the DL1 culture failed to detect sequences specific to KN400, suggesting that KN400 was an external contaminant inadvertently introduced into the anode culturing system. In order to evaluate this further, a portion of the gene for OmcS, a c-type cytochrome that both KN400 and DL1 possess, was amplified from the DL1 culture. HiSeq-2000 Illumina sequencing of the PCR product detected the KN400 sequence, which differs from the DL1 sequence at 14 bp, at a frequency of ca. 1 in 105 copies of the DL1 sequence. A similar low frequency of KN400 was detected with quantitative PCR of a KN400-specific gene. KN400 persisted at this frequency after intensive restreaking of isolated colonies from the DL1 culture. However, a culture in which KN400 could no longer be detected was obtained by serial dilution to extinction in liquid medium. The KN400-free culture could not grow on an anode poised at −400 mV. Thus, KN400 cryptically persisted in the culture dominated by DL1 for more than a decade, undetected by even deep whole-genome sequencing, and was only fortuitously uncovered by the unnatural selection pressure of growth on a low-potential electrode. IMPORTANCE Repeated streaking of isolated colonies on solidified medium remains a common strategy for obtaining pure cultures, especially of difficult-to-cultivate microorganisms such as strict anaerobes. The results presented here demonstrate that verifying the purity of cultures obtained in this manner may be difficult because extremely rare variants can persist, undetectable with even deep genomic DNA sequencing. The only way to ensure that a culture is pure is to cultivate it from an initial single cell, which may be technically difficult for many environmentally significant microbes.Pravin Malla ShresthaKelly P. NevinMinita ShresthaDerek R. LovleyAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 4, Iss 2 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Pravin Malla Shrestha Kelly P. Nevin Minita Shrestha Derek R. Lovley When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing
description	ABSTRACT Geobacter sulfurreducens strain KN400 was recovered in previous studies in which a culture of the DL1 strain of G. sulfurreducens served as the inoculum in investigations of microbial current production at low anode potentials (−400 mV versus Ag/AgCl). Differences in the genome sequences of KN400 and DL1 were too great to have arisen from adaptive evolution during growth on the anode. Previous deep sequencing (80-fold coverage) of the DL1 culture failed to detect sequences specific to KN400, suggesting that KN400 was an external contaminant inadvertently introduced into the anode culturing system. In order to evaluate this further, a portion of the gene for OmcS, a c-type cytochrome that both KN400 and DL1 possess, was amplified from the DL1 culture. HiSeq-2000 Illumina sequencing of the PCR product detected the KN400 sequence, which differs from the DL1 sequence at 14 bp, at a frequency of ca. 1 in 105 copies of the DL1 sequence. A similar low frequency of KN400 was detected with quantitative PCR of a KN400-specific gene. KN400 persisted at this frequency after intensive restreaking of isolated colonies from the DL1 culture. However, a culture in which KN400 could no longer be detected was obtained by serial dilution to extinction in liquid medium. The KN400-free culture could not grow on an anode poised at −400 mV. Thus, KN400 cryptically persisted in the culture dominated by DL1 for more than a decade, undetected by even deep whole-genome sequencing, and was only fortuitously uncovered by the unnatural selection pressure of growth on a low-potential electrode. IMPORTANCE Repeated streaking of isolated colonies on solidified medium remains a common strategy for obtaining pure cultures, especially of difficult-to-cultivate microorganisms such as strict anaerobes. The results presented here demonstrate that verifying the purity of cultures obtained in this manner may be difficult because extremely rare variants can persist, undetectable with even deep genomic DNA sequencing. The only way to ensure that a culture is pure is to cultivate it from an initial single cell, which may be technically difficult for many environmentally significant microbes.
format	article
author	Pravin Malla Shrestha Kelly P. Nevin Minita Shrestha Derek R. Lovley
author_facet	Pravin Malla Shrestha Kelly P. Nevin Minita Shrestha Derek R. Lovley
author_sort	Pravin Malla Shrestha
title	When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing
title_short	When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing
title_full	When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing
title_fullStr	When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing
title_full_unstemmed	When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing
title_sort	when is a microbial culture “pure”? persistent cryptic contaminant escapes detection even with deep genome sequencing
publisher	American Society for Microbiology
publishDate	2013
url	https://doaj.org/article/9405072f697740edaca6acebf998565b
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When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing

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