Evolution of the bacterial Cu resistome as revealed by comparative genomics

Yanan Qin

Evolution of the bacterial Cu resistome as revealed by comparative genomics

Yanan Qin

Abstract

Copper is found widely in the environment. It acts as a double-edged sword because of its chemicalcharacteristics. If the amount of copper in the organism is too low, copper-containing enzymes willnot be able to catalyze important reactions in diverse life processes. However, in excess, copper istoxic to microorganisms. The release of large amounts of bioavailable copper from geochemicalprocesses notably after the Great Oxidation Event and lately various anthropogenic activities led toincreased accumulation of copper in the environment. Hence, to ensure their own survival, bacteriahave developed mechanisms of maintaining cellular copper homeostasis or mechanisms ofdetoxification and resistance to copper such as copper export, copper sequestration and copperoxidation via multi-copper oxidases. Copper as a metal pollutant constitutes a significant threat tosome ecosystems and human health. Hence, it is of interest to understand how copper contaminationaffects the bacterial copper resistome. The aim of this PhD thesis was to investigate the role of Cucontamination in shaping the soil bacterial Cu resistome using Pseudomonas spp. as a modelorganism and also characterize the Cu resistance determinants in S. Typhimurium strains isolatedfrom copper-fed pig feces. Further, I reviewed the role of protozoan predation on the presence ofbacterial Cu resistance determinants.Pseudomonas spp. was selected as a model organism to investigate the evolution of the soilbacterial copper resistome. Comparative genomic analysis was performed to identify and analyzethe copper resistance determinants in the genomes of different Pseudomonas spp. strains.Pseudomonas spp. strains were isolated from a field site contaminated with CuSO4 between 1912and 1926 (long-term Cu contaminated soil), the adjacent control site which was located just outsidethe contaminated area (non-Cu contaminated soil) and laboratory spiked Cu soil (short-term Cuspiked soil), respectively. Then, 36 of Pseudomonas spp. isolates were whole genome sequencedand analyzed (Manuscript 1 and Manuscript 2).Manuscript 1 is a short genome report, where we only analyzed two of the 36 Pseudomonas spp.strains. P. jessenii C2, which was isolated from non-Cu contaminated soil, comprised 6,420,113 bp,with 5814 protein-coding genes and 67 RNA genes on the genome. P. jessenii H16 was isolatedfrom long-term Cu contaminated soil and comprised 6,807,788 bp, with 5995 protein-coding genesand 70 RNA genes on the genome. P. jessenii H16 displayed significantly higher resistance tocopper when compared to P. jessenii C2. Of special interest was a specific adaptation to this harshcopper contaminated environment as P. jessenii H16 contained a copper resistance island of around50,000 bp.Manuscript 2 is an extended study, in which we analyzed all 36 sequenced genomes. The resultshowed a Pseudomonas spp. population that was diverse and resilient towards elevated copperconcentrations after more than 90 years of adaptation to Cu. Comparative genomics for allsequenced Pseudomonas spp. identified several Cu resistance determinants such as cueO, cueR,copABCDGZ and cusRSF present in all sequenced genomes. Moreover, all Pseudomonas spp.strains isolated from long-term Cu contaminated soil contained a long Cu resistance island(copABCDGZ, cueO, cusFRS and cusABC) that most likely had been acquired via horizontal genetransfer. In addition, the number of Cu resistance determinats within the Cu resistance islands waspositively correlated to phenotypic Cu resistance among Pseudomonas spp. By contrast, the totalnumbert of Cu resistance determinants was a rather poor predictor of phenotypic Cu resistance. Theresults also suggest that the amount of copBRGZ and cusFABC Cu resistance determinants could beidentified as promising functional gene markers for ecological studies of Cu resistance inPseudomonas spp.Manuscript 3 used copper-fed pig feces as inoculum for the isolation of copper resistant entericbacteria. Salmonella Typhimurium S7, S15 and S23 strains were isolated and their whole genomeswere sequenced. All three S. Typhimurium genomes generally contained copper resistance genesincluding: the copA, cueO, golT, golBS, gesABC, MerR-family transcriptional regulator cueR, RNDcusCFBA type system and cueP. In addition, a mobile 20-gene copper resistance determinantswhich previously was described as individual pco and sil determinants were identified in all threegenomes. Moreover, S. Typhimurium S7, S15 and S23 also revealed a six-gene cluster merEDAPTRencoding proteins conferring mercury resistance and the five-gene ars operon arsRDABCconferring resistance to arsenic.Manuscript 4 is a review. Here we proposed that bacterial metal resistance determinants mayprotect bacteria against protozoan grazing and macrophage killing and discuss the evidence for thishypothesis. Hence, bacterial Cu resistance is ancient and cannot only be attributed to geological oranthropogenic metal contamination in different environments.In conclusion, this PhD study demonstrates that copper contamination not only select for Curesistance among already existing strains, but that it also drives de novo eveolution of novel Curesistant strains containing extensive Cu resistance islands in their genomes. In addition, protozoanpredation and macrophage killing of bacteria represent ancient selection pressures for Cu resistancein bacteria.

Original language	English

Publisher	Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen
Publication status	Published - 2018

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Cite this

@phdthesis{7eba1438ef12466f81fbfc4650eea384,

title = "Evolution of the bacterial Cu resistome as revealed by comparative genomics",

abstract = "Copper is found widely in the environment. It acts as a double-edged sword because of its chemicalcharacteristics. If the amount of copper in the organism is too low, copper-containing enzymes willnot be able to catalyze important reactions in diverse life processes. However, in excess, copper istoxic to microorganisms. The release of large amounts of bioavailable copper from geochemicalprocesses notably after the Great Oxidation Event and lately various anthropogenic activities led toincreased accumulation of copper in the environment. Hence, to ensure their own survival, bacteriahave developed mechanisms of maintaining cellular copper homeostasis or mechanisms ofdetoxification and resistance to copper such as copper export, copper sequestration and copperoxidation via multi-copper oxidases. Copper as a metal pollutant constitutes a significant threat tosome ecosystems and human health. Hence, it is of interest to understand how copper contaminationaffects the bacterial copper resistome. The aim of this PhD thesis was to investigate the role of Cucontamination in shaping the soil bacterial Cu resistome using Pseudomonas spp. as a modelorganism and also characterize the Cu resistance determinants in S. Typhimurium strains isolatedfrom copper-fed pig feces. Further, I reviewed the role of protozoan predation on the presence ofbacterial Cu resistance determinants.Pseudomonas spp. was selected as a model organism to investigate the evolution of the soilbacterial copper resistome. Comparative genomic analysis was performed to identify and analyzethe copper resistance determinants in the genomes of different Pseudomonas spp. strains.Pseudomonas spp. strains were isolated from a field site contaminated with CuSO4 between 1912and 1926 (long-term Cu contaminated soil), the adjacent control site which was located just outsidethe contaminated area (non-Cu contaminated soil) and laboratory spiked Cu soil (short-term Cuspiked soil), respectively. Then, 36 of Pseudomonas spp. isolates were whole genome sequencedand analyzed (Manuscript 1 and Manuscript 2).Manuscript 1 is a short genome report, where we only analyzed two of the 36 Pseudomonas spp.strains. P. jessenii C2, which was isolated from non-Cu contaminated soil, comprised 6,420,113 bp,with 5814 protein-coding genes and 67 RNA genes on the genome. P. jessenii H16 was isolatedfrom long-term Cu contaminated soil and comprised 6,807,788 bp, with 5995 protein-coding genesand 70 RNA genes on the genome. P. jessenii H16 displayed significantly higher resistance tocopper when compared to P. jessenii C2. Of special interest was a specific adaptation to this harshcopper contaminated environment as P. jessenii H16 contained a copper resistance island of around50,000 bp.Manuscript 2 is an extended study, in which we analyzed all 36 sequenced genomes. The resultshowed a Pseudomonas spp. population that was diverse and resilient towards elevated copperconcentrations after more than 90 years of adaptation to Cu. Comparative genomics for allsequenced Pseudomonas spp. identified several Cu resistance determinants such as cueO, cueR,copABCDGZ and cusRSF present in all sequenced genomes. Moreover, all Pseudomonas spp.strains isolated from long-term Cu contaminated soil contained a long Cu resistance island(copABCDGZ, cueO, cusFRS and cusABC) that most likely had been acquired via horizontal genetransfer. In addition, the number of Cu resistance determinats within the Cu resistance islands waspositively correlated to phenotypic Cu resistance among Pseudomonas spp. By contrast, the totalnumbert of Cu resistance determinants was a rather poor predictor of phenotypic Cu resistance. Theresults also suggest that the amount of copBRGZ and cusFABC Cu resistance determinants could beidentified as promising functional gene markers for ecological studies of Cu resistance inPseudomonas spp.Manuscript 3 used copper-fed pig feces as inoculum for the isolation of copper resistant entericbacteria. Salmonella Typhimurium S7, S15 and S23 strains were isolated and their whole genomeswere sequenced. All three S. Typhimurium genomes generally contained copper resistance genesincluding: the copA, cueO, golT, golBS, gesABC, MerR-family transcriptional regulator cueR, RNDcusCFBA type system and cueP. In addition, a mobile 20-gene copper resistance determinantswhich previously was described as individual pco and sil determinants were identified in all threegenomes. Moreover, S. Typhimurium S7, S15 and S23 also revealed a six-gene cluster merEDAPTRencoding proteins conferring mercury resistance and the five-gene ars operon arsRDABCconferring resistance to arsenic.Manuscript 4 is a review. Here we proposed that bacterial metal resistance determinants mayprotect bacteria against protozoan grazing and macrophage killing and discuss the evidence for thishypothesis. Hence, bacterial Cu resistance is ancient and cannot only be attributed to geological oranthropogenic metal contamination in different environments.In conclusion, this PhD study demonstrates that copper contamination not only select for Curesistance among already existing strains, but that it also drives de novo eveolution of novel Curesistant strains containing extensive Cu resistance islands in their genomes. In addition, protozoanpredation and macrophage killing of bacteria represent ancient selection pressures for Cu resistancein bacteria.",

author = "Yanan Qin",

year = "2018",

language = "English",

publisher = "Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen",

}

TY - BOOK

T1 - Evolution of the bacterial Cu resistome as revealed by comparative genomics

AU - Qin, Yanan

PY - 2018

Y1 - 2018

N2 - Copper is found widely in the environment. It acts as a double-edged sword because of its chemicalcharacteristics. If the amount of copper in the organism is too low, copper-containing enzymes willnot be able to catalyze important reactions in diverse life processes. However, in excess, copper istoxic to microorganisms. The release of large amounts of bioavailable copper from geochemicalprocesses notably after the Great Oxidation Event and lately various anthropogenic activities led toincreased accumulation of copper in the environment. Hence, to ensure their own survival, bacteriahave developed mechanisms of maintaining cellular copper homeostasis or mechanisms ofdetoxification and resistance to copper such as copper export, copper sequestration and copperoxidation via multi-copper oxidases. Copper as a metal pollutant constitutes a significant threat tosome ecosystems and human health. Hence, it is of interest to understand how copper contaminationaffects the bacterial copper resistome. The aim of this PhD thesis was to investigate the role of Cucontamination in shaping the soil bacterial Cu resistome using Pseudomonas spp. as a modelorganism and also characterize the Cu resistance determinants in S. Typhimurium strains isolatedfrom copper-fed pig feces. Further, I reviewed the role of protozoan predation on the presence ofbacterial Cu resistance determinants.Pseudomonas spp. was selected as a model organism to investigate the evolution of the soilbacterial copper resistome. Comparative genomic analysis was performed to identify and analyzethe copper resistance determinants in the genomes of different Pseudomonas spp. strains.Pseudomonas spp. strains were isolated from a field site contaminated with CuSO4 between 1912and 1926 (long-term Cu contaminated soil), the adjacent control site which was located just outsidethe contaminated area (non-Cu contaminated soil) and laboratory spiked Cu soil (short-term Cuspiked soil), respectively. Then, 36 of Pseudomonas spp. isolates were whole genome sequencedand analyzed (Manuscript 1 and Manuscript 2).Manuscript 1 is a short genome report, where we only analyzed two of the 36 Pseudomonas spp.strains. P. jessenii C2, which was isolated from non-Cu contaminated soil, comprised 6,420,113 bp,with 5814 protein-coding genes and 67 RNA genes on the genome. P. jessenii H16 was isolatedfrom long-term Cu contaminated soil and comprised 6,807,788 bp, with 5995 protein-coding genesand 70 RNA genes on the genome. P. jessenii H16 displayed significantly higher resistance tocopper when compared to P. jessenii C2. Of special interest was a specific adaptation to this harshcopper contaminated environment as P. jessenii H16 contained a copper resistance island of around50,000 bp.Manuscript 2 is an extended study, in which we analyzed all 36 sequenced genomes. The resultshowed a Pseudomonas spp. population that was diverse and resilient towards elevated copperconcentrations after more than 90 years of adaptation to Cu. Comparative genomics for allsequenced Pseudomonas spp. identified several Cu resistance determinants such as cueO, cueR,copABCDGZ and cusRSF present in all sequenced genomes. Moreover, all Pseudomonas spp.strains isolated from long-term Cu contaminated soil contained a long Cu resistance island(copABCDGZ, cueO, cusFRS and cusABC) that most likely had been acquired via horizontal genetransfer. In addition, the number of Cu resistance determinats within the Cu resistance islands waspositively correlated to phenotypic Cu resistance among Pseudomonas spp. By contrast, the totalnumbert of Cu resistance determinants was a rather poor predictor of phenotypic Cu resistance. Theresults also suggest that the amount of copBRGZ and cusFABC Cu resistance determinants could beidentified as promising functional gene markers for ecological studies of Cu resistance inPseudomonas spp.Manuscript 3 used copper-fed pig feces as inoculum for the isolation of copper resistant entericbacteria. Salmonella Typhimurium S7, S15 and S23 strains were isolated and their whole genomeswere sequenced. All three S. Typhimurium genomes generally contained copper resistance genesincluding: the copA, cueO, golT, golBS, gesABC, MerR-family transcriptional regulator cueR, RNDcusCFBA type system and cueP. In addition, a mobile 20-gene copper resistance determinantswhich previously was described as individual pco and sil determinants were identified in all threegenomes. Moreover, S. Typhimurium S7, S15 and S23 also revealed a six-gene cluster merEDAPTRencoding proteins conferring mercury resistance and the five-gene ars operon arsRDABCconferring resistance to arsenic.Manuscript 4 is a review. Here we proposed that bacterial metal resistance determinants mayprotect bacteria against protozoan grazing and macrophage killing and discuss the evidence for thishypothesis. Hence, bacterial Cu resistance is ancient and cannot only be attributed to geological oranthropogenic metal contamination in different environments.In conclusion, this PhD study demonstrates that copper contamination not only select for Curesistance among already existing strains, but that it also drives de novo eveolution of novel Curesistant strains containing extensive Cu resistance islands in their genomes. In addition, protozoanpredation and macrophage killing of bacteria represent ancient selection pressures for Cu resistancein bacteria.

AB - Copper is found widely in the environment. It acts as a double-edged sword because of its chemicalcharacteristics. If the amount of copper in the organism is too low, copper-containing enzymes willnot be able to catalyze important reactions in diverse life processes. However, in excess, copper istoxic to microorganisms. The release of large amounts of bioavailable copper from geochemicalprocesses notably after the Great Oxidation Event and lately various anthropogenic activities led toincreased accumulation of copper in the environment. Hence, to ensure their own survival, bacteriahave developed mechanisms of maintaining cellular copper homeostasis or mechanisms ofdetoxification and resistance to copper such as copper export, copper sequestration and copperoxidation via multi-copper oxidases. Copper as a metal pollutant constitutes a significant threat tosome ecosystems and human health. Hence, it is of interest to understand how copper contaminationaffects the bacterial copper resistome. The aim of this PhD thesis was to investigate the role of Cucontamination in shaping the soil bacterial Cu resistome using Pseudomonas spp. as a modelorganism and also characterize the Cu resistance determinants in S. Typhimurium strains isolatedfrom copper-fed pig feces. Further, I reviewed the role of protozoan predation on the presence ofbacterial Cu resistance determinants.Pseudomonas spp. was selected as a model organism to investigate the evolution of the soilbacterial copper resistome. Comparative genomic analysis was performed to identify and analyzethe copper resistance determinants in the genomes of different Pseudomonas spp. strains.Pseudomonas spp. strains were isolated from a field site contaminated with CuSO4 between 1912and 1926 (long-term Cu contaminated soil), the adjacent control site which was located just outsidethe contaminated area (non-Cu contaminated soil) and laboratory spiked Cu soil (short-term Cuspiked soil), respectively. Then, 36 of Pseudomonas spp. isolates were whole genome sequencedand analyzed (Manuscript 1 and Manuscript 2).Manuscript 1 is a short genome report, where we only analyzed two of the 36 Pseudomonas spp.strains. P. jessenii C2, which was isolated from non-Cu contaminated soil, comprised 6,420,113 bp,with 5814 protein-coding genes and 67 RNA genes on the genome. P. jessenii H16 was isolatedfrom long-term Cu contaminated soil and comprised 6,807,788 bp, with 5995 protein-coding genesand 70 RNA genes on the genome. P. jessenii H16 displayed significantly higher resistance tocopper when compared to P. jessenii C2. Of special interest was a specific adaptation to this harshcopper contaminated environment as P. jessenii H16 contained a copper resistance island of around50,000 bp.Manuscript 2 is an extended study, in which we analyzed all 36 sequenced genomes. The resultshowed a Pseudomonas spp. population that was diverse and resilient towards elevated copperconcentrations after more than 90 years of adaptation to Cu. Comparative genomics for allsequenced Pseudomonas spp. identified several Cu resistance determinants such as cueO, cueR,copABCDGZ and cusRSF present in all sequenced genomes. Moreover, all Pseudomonas spp.strains isolated from long-term Cu contaminated soil contained a long Cu resistance island(copABCDGZ, cueO, cusFRS and cusABC) that most likely had been acquired via horizontal genetransfer. In addition, the number of Cu resistance determinats within the Cu resistance islands waspositively correlated to phenotypic Cu resistance among Pseudomonas spp. By contrast, the totalnumbert of Cu resistance determinants was a rather poor predictor of phenotypic Cu resistance. Theresults also suggest that the amount of copBRGZ and cusFABC Cu resistance determinants could beidentified as promising functional gene markers for ecological studies of Cu resistance inPseudomonas spp.Manuscript 3 used copper-fed pig feces as inoculum for the isolation of copper resistant entericbacteria. Salmonella Typhimurium S7, S15 and S23 strains were isolated and their whole genomeswere sequenced. All three S. Typhimurium genomes generally contained copper resistance genesincluding: the copA, cueO, golT, golBS, gesABC, MerR-family transcriptional regulator cueR, RNDcusCFBA type system and cueP. In addition, a mobile 20-gene copper resistance determinantswhich previously was described as individual pco and sil determinants were identified in all threegenomes. Moreover, S. Typhimurium S7, S15 and S23 also revealed a six-gene cluster merEDAPTRencoding proteins conferring mercury resistance and the five-gene ars operon arsRDABCconferring resistance to arsenic.Manuscript 4 is a review. Here we proposed that bacterial metal resistance determinants mayprotect bacteria against protozoan grazing and macrophage killing and discuss the evidence for thishypothesis. Hence, bacterial Cu resistance is ancient and cannot only be attributed to geological oranthropogenic metal contamination in different environments.In conclusion, this PhD study demonstrates that copper contamination not only select for Curesistance among already existing strains, but that it also drives de novo eveolution of novel Curesistant strains containing extensive Cu resistance islands in their genomes. In addition, protozoanpredation and macrophage killing of bacteria represent ancient selection pressures for Cu resistancein bacteria.

UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01011006830&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK

M3 - Ph.D. thesis

BT - Evolution of the bacterial Cu resistome as revealed by comparative genomics

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -

Evolution of the bacterial Cu resistome as revealed by comparative genomics

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