Transcriptomes of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula Exposed to Metal “Shock” Reveal Generic and Specific Metal Responses

ABSTRACTThe extremely thermoacidophilic archaeonMetallosphaera sedulamobilizes metals by novel membrane-associated oxidase clusters and, consequently, requires metal resistance strategies. This issue was examined by “shocking”M. sedulawith representative metals (Co2+, Cu2+, Ni2+, UO22+, Zn2+) at inhibitory and subinhibitory levels. Collectively, one-quarter of the genome (554 open reading frames [ORFs]) responded to inhibitory levels, and two-thirds (354) of the ORFs were responsive to a single metal. Cu2+(259 ORFs, 106 Cu2+-specific ORFs) and Zn2+(262 ORFs, 131 Zn2+-specific ORFs) triggered the largest responses, followed by UO22+(187 ORFs, 91 UO22+-specific ORFs), Ni2+(93 ORFs, 25 Ni2+-specific ORFs), and Co2+(61 ORFs, 1 Co2+-specific ORF). While one-third of the metal-responsive ORFs are annotated as encoding hypothetical proteins, metal challenge also impacted ORFs responsible for identifiable processes related to the cell cycle, DNA repair, and oxidative stress. Surprisingly, there were only 30 ORFs that responded to at least four metals, and 10 of these responded to all five metals. This core transcriptome indicated induction of Fe-S cluster assembly (Msed_1656-Msed_1657), tungsten/molybdenum transport (Msed_1780-Msed_1781), and decreased central metabolism. Not surprisingly, a metal-translocating P-type ATPase (Msed_0490) associated with a copper resistance system (Cop) was upregulated in response to Cu2+(6-fold) but also in response to UO22+(4-fold) and Zn2+(9-fold). Cu2+challenge uniquely induced assimilatory sulfur metabolism for cysteine biosynthesis, suggesting a role for this amino acid in Cu2+resistance or issues in sulfur metabolism. The results indicate thatM. sedulaemploys a range of physiological and biochemical responses to metal challenge, many of which are specific to a single metal and involve proteins with yet unassigned or definitive functions.IMPORTANCEThe mechanisms by which extremely thermoacidophilic archaea resist and are negatively impacted by metals encountered in their natural environments are important to understand so that technologies such as bioleaching, which leverage microbially based conversion of insoluble metal sulfides to soluble species, can be improved. Transcriptomic analysis of the cellular response to metal challenge provided both global and specific insights into how these novel microorganisms negotiate metal toxicity in natural and technological settings. As genetics tools are further developed and implemented for extreme thermoacidophiles, information about metal toxicity and resistance can be leveraged to create metabolically engineered strains with improved bioleaching characteristics.

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Inorganic Polyphosphate, Exopolyphosphatase, andPho84-Like Transporters May Be Involved in Copper Resistance inMetallosphaera sedulaDSM 5348T

Archaea ◽

10.1155/2018/5251061 ◽

2018 ◽

Vol 2018 ◽

pp. 1-12 ◽

Cited By ~ 5

Author(s):

Matías Rivero ◽

Constanza Torres-Paris ◽

Rodrigo Muñoz ◽

Ricardo Cabrera ◽

Claudio A. Navarro ◽

...

Keyword(s):

Present Report ◽

Resistance Mechanism ◽

Metal Resistance ◽

Copper Resistance ◽

Site Directed Mutagenesis ◽

Pcr Analysis ◽

Linear Polymers ◽

Inorganic Polyphosphate ◽

Metallosphaera Sedula

Polyphosphates (PolyP) are linear polymers of orthophosphate residues that have been proposed to participate in metal resistance in bacteria and archaea. In addition of having a CopA/CopB copper efflux system, the thermoacidophilic archaeonMetallosphaera sedulacontains electron-dense PolyP-like granules and a putative exopolyphosphatase (PPXMsed,Msed_0891) and four presumedpho84-like phosphate transporters (Msed_0846,Msed_0866,Msed_1094, andMsed_1512) encoded in its genome. In the present report, the existence of a possible PolyP-based copper-resistance mechanism inM. sedulaDSM 5348Twas evaluated.M. sedulaDSM 5348Taccumulated high levels of phosphorous in the form of granules, and its growth was affected in the presence of 16 mM copper. PolyP levels were highly reduced after the archaeon was subjected to an 8 mM CuSO4shift. PPXMsedwas purified, and the enzyme was found to hydrolyze PolyPin vitro. Essential residues for catalysis of PPXMsedwere E111 and E113 as shown by a site-directed mutagenesis of the implied residues. Furthermore,M. sedula ppx,pho84-like, andcopTMAgenes were upregulated upon copper exposure, as determined by qRT-PCR analysis. The results obtained support the existence of a PolyP-dependent copper-resistance system that may be of great importance in the adaptation of this thermoacidophilic archaeon to its harsh environment.

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Mechanism of Attenuation of Uranyl Toxicity by Glutathione in Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.00538-16 ◽

2016 ◽

Vol 82 (12) ◽

pp. 3563-3571 ◽

Cited By ~ 6

Author(s):

Muhammad H. Obeid ◽

Jana Oertel ◽

Marc Solioz ◽

Karim Fahmy

Keyword(s):

Heavy Metals ◽

Lactococcus Lactis ◽

Metal Toxicity ◽

Molecular Mechanisms ◽

Model Organism ◽

Cell Number ◽

Metal Resistance ◽

Titration Calorimetry ◽

Content Type

ABSTRACTBoth prokaryotic and eukaryotic organisms possess mechanisms for the detoxification of heavy metals, and these mechanisms are found among distantly related species. We investigated the role of intracellular glutathione (GSH), which, in a large number of taxa, plays a role in protection against the toxicity of common heavy metals. Anaerobically grownLactococcus lactiscontaining an inducible GSH synthesis pathway was used as a model organism. Its physiological condition allowed study of putative GSH-dependent uranyl detoxification mechanisms without interference from additional reactive oxygen species. By microcalorimetric measurements of metabolic heat during cultivation, it was shown that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10 to 150 μM. In this concentration range, no effect was observed with copper, which was used as a reference for redox metal toxicity. At higher copper concentrations, GSH aggravated metal toxicity. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH rather than to the reducing thiol group involved in copper interactions. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH. The opposite effects on uranyl and on copper toxicity can be related to the difference in coordination chemistry of the respective metal-GSH complexes, which cause distinct growth phase-specific effects on enzyme-metal interactions.IMPORTANCEUnderstanding microbial metal resistance is of particular importance for bioremediation, where microorganisms are employed for the removal of heavy metals from the environment. This strategy is increasingly being considered for uranium. However, little is known about the molecular mechanisms of uranyl detoxification. Existing studies of different taxa show little systematics but hint at a role of glutathione (GSH). Previous work could not unequivocally demonstrate a GSH function in decreasing the presumed uranyl-induced oxidative stress, nor could a redox-independent detoxifying action of GSH be identified. Combining metabolic calorimetry with cell number-based assays and genetics analysis enables a novel and general approach to quantify toxicity and relate it to molecular mechanisms. The results show that GSH-expressing microorganisms appear advantageous for uranyl bioremediation.

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Molecular Characterization of Copper Resistance Genes from Xanthomonas citri subsp.citriand Xanthomonas alfalfae subsp. citrumelonis

Applied and Environmental Microbiology ◽

10.1128/aem.03043-10 ◽

2011 ◽

Vol 77 (12) ◽

pp. 4089-4096 ◽

Cited By ~ 73

Author(s):

Franklin Behlau ◽

Blanca I. Canteros ◽

Gerald V. Minsavage ◽

Jeffrey B. Jones ◽

James H. Graham

Keyword(s):

Citrus Canker ◽

Open Reading Frames ◽

Copper Resistance ◽

Protective Measure ◽

Xanthomonas Citri ◽

Content Type ◽

Resistance Determinants ◽

Florida Citrus ◽

First Time

ABSTRACTCopper sprays have been widely used for control of endemic citrus canker caused byXanthomonas citrisubsp.citriin citrus-growing areas for more than 2 decades.Xanthomonas alfalfaesubsp.citrumelonispopulations were also exposed to frequent sprays of copper for several years as a protective measure against citrus bacterial spot (CBS) in Florida citrus nurseries. Long-term use of these bactericides has led to the development of copper-resistant (Cur) strains in bothX. citrisubsp.citriandX. alfalfaesubsp.citrumelonis, resulting in a reduction of disease control. The objectives of this study were to characterize for the first time the genetics of copper resistance inX. citrisubsp.citriandX. alfalfaesubsp.citrumelonisand to compare these organisms to other Curbacteria. Copper resistance determinants fromX. citrisubsp.citristrain A44(pXccCu2) from Argentina andX. alfalfaesubsp.citrumelonisstrain 1381(pXacCu2) from Florida were cloned and sequenced. Open reading frames (ORFs) related to the genescopL,copA,copB,copM,copG,copC,copD, andcopFwere identified inX. citrisubsp.citriA44. The same ORFs, exceptcopCandcopD, were also present inX. alfalfaesubsp.citrumelonis1381. Transposon mutagenesis of the cloned copper resistance determinants in pXccCu2 revealed that copper resistance inX. citrisubsp.citristrain A44 is mostly due tocopL,copA, andcopB, which are the genes in the cloned cluster with the highest nucleotide homology (≥92%) among different Curbacteria.

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Multiple Megaplasmids Confer Extremely High Levels of Metal Tolerance in Alteromonas Strains

Applied and Environmental Microbiology ◽

10.1128/aem.01831-19 ◽

2019 ◽

Vol 86 (3) ◽

Author(s):

Kathleen D. Cusick ◽

Shawn W. Polson ◽

Gabriel Duran ◽

Russell T. Hill

Keyword(s):

Gene Transfer ◽

Pseudomonas Syringae ◽

Molecular Mechanisms ◽

Metal Tolerance ◽

Bioinformatic Analysis ◽

Metal Resistance ◽

Copper Resistance ◽

The Other ◽

Copper Tolerance ◽

Content Type

ABSTRACT Alteromonas is a widely distributed genus of marine Gammaproteobacteria, with representatives shown to be key players in diverse processes, including biogeochemical cycling and biofouling of marine substrata. While Alteromonas spp. are early colonizers of copper-based antifouling paints on marine vessels, their mechanism of tolerance is poorly understood. PacBio whole-genome sequencing of Alteromonas macleodii strains CUKW and KCC02, isolated from Cu/Ni alloy test coupons submerged in oligotrophic coastal waters, indicated the presence of multiple megaplasmids (ca. 200 kb) in both. A pulsed-field gel electrophoresis method was developed and used to confirm the presence of multiple megaplasmids in these two strains; it was then used to screen additional Alteromonas strains for which little to no sequencing data exist. Plasmids were not detected in any of the other strains. Bioinformatic analysis of the CUKW and KCC02 plasmids identified numerous genes associated with metal resistance. Copper resistance orthologs from both the Escherichia coli Cue and Cus and Pseudomonas syringae Cop systems were present, at times as multiple copies. Metal growth assays in the presence of copper, cobalt, manganese, and zinc performed with 10 Alteromonas strains demonstrated the ability of CUKW and KCC02 to grow at metal concentrations inhibitory to all the other strains tested. This study reports multiple megaplasmids in Alteromonas strains. Bioinformatic analysis of the CUKW and KCC02 plasmids indicate that they harbor elements of the Tra system conjugation apparatus, although their type of mobility remains to be experimentally verified. IMPORTANCE Copper is commonly used as an antifouling agent on ship hulls. Alteromonas spp. are early colonizers of copper-based antifouling paint, but their mechanism of tolerance is poorly understood. Sequencing of A. macleodii strains isolated from copper test materials for marine ships indicated the presence of multiple megaplasmids. Plasmids serve as key vectors in horizontal gene transfer and confer traits such as metal resistance, detoxification, ecological interaction, and antibiotic resistance. Bioinformatic analysis identified many metal resistance genes and genes associated with mobility. Understanding the molecular mechanisms and capacity for gene transfer within marine biofilms provides a platform for the development of novel antifouling solutions targeting genes involved in copper tolerance and biofilm formation.

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The Transcriptomic Landscape of Cupriavidus metallidurans CH34 Acutely Exposed to Copper

Genes ◽

10.3390/genes11091049 ◽

2020 ◽

Vol 11 (9) ◽

pp. 1049

Author(s):

Laurens Maertens ◽

Natalie Leys ◽

Jean-Yves Matroule ◽

Rob Van Houdt

Keyword(s):

Sulfur Metabolism ◽

Model Organism ◽

Gene Clusters ◽

Resistance Mechanisms ◽

Metal Resistance ◽

Copper Resistance ◽

Messenger Rnas ◽

Antisense Transcripts ◽

Cupriavidus Metallidurans Ch34 ◽

Cupriavidus Metallidurans

Bacteria are increasingly used for biotechnological applications such as bioremediation, biorecovery, bioproduction, and biosensing. The development of strains suited for such applications requires a thorough understanding of their behavior, with a key role for their transcriptomic landscape. We present a thorough analysis of the transcriptome of Cupriavidus metallidurans CH34 cells acutely exposed to copper by tagRNA-sequencing. C. metallidurans CH34 is a model organism for metal resistance, and its potential as a biosensor and candidate for metal bioremediation has been demonstrated in multiple studies. Several metabolic pathways were impacted by Cu exposure, and a broad spectrum of metal resistance mechanisms, not limited to copper-specific clusters, was overexpressed. In addition, several gene clusters involved in the oxidative stress response and the cysteine-sulfur metabolism were induced. In total, 7500 transcription start sites (TSSs) were annotated and classified with respect to their location relative to coding sequences (CDSs). Predicted TSSs were used to re-annotate 182 CDSs. The TSSs of 2422 CDSs were detected, and consensus promotor logos were derived. Interestingly, many leaderless messenger RNAs (mRNAs) were found. In addition, many mRNAs were transcribed from multiple alternative TSSs. We observed pervasive intragenic TSSs both in sense and antisense to CDSs. Antisense transcripts were enriched near the 5′ end of mRNAs, indicating a functional role in post-transcriptional regulation. In total, 578 TSSs were detected in intergenic regions, of which 35 were identified as putative small regulatory RNAs. Finally, we provide a detailed analysis of the main copper resistance clusters in CH34, which include many intragenic and antisense transcripts. These results clearly highlight the ubiquity of noncoding transcripts in the CH34 transcriptome, many of which are putatively involved in the regulation of metal resistance.

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Comparative Genomic Analysis of Acinetobacter oleivorans DR1 To Determine Strain-Specific Genomic Regions and Gentisate Biodegradation

Applied and Environmental Microbiology ◽

10.1128/aem.05231-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7418-7424 ◽

Cited By ~ 20

Author(s):

Jaejoon Jung ◽

Eugene L. Madsen ◽

Che Ok Jeon ◽

Woojun Park

Keyword(s):

Metabolic Pathways ◽

Genomic Analysis ◽

Metal Resistance ◽

Genomic Region ◽

Open Reading Frames ◽

Comparative Genomic ◽

Content Type ◽

Large Size ◽

Genomic Regions ◽

Reading Frames

ABSTRACTThe comparative genomics ofAcinetobacter oleivoransDR1 assayed withA. baylyiADP1,A. calcoaceticusPHEA-2, andA. baumanniiATCC 17978 revealed that the incorporation of phage-related genomic regions and the absence of transposable elements have contributed to the large size (4.15 Mb) of the DR1 genome. A horizontally transferred genomic region and a higher proportion of transcriptional regulator- and signal peptide-coding genes were identified as characteristics of the DR1 genome. Incomplete glucose metabolism, metabolic pathways of aromatic compounds, biofilm formation, antibiotics and metal resistance, and natural competence genes were conserved in four compared genomes. Interestingly, only strain DR1 possesses gentisate 1,2-dioxygenase (nagI) and grows on gentisate, whereas other species cannot. Expression of thenagIgene was upregulated during gentisate utilization, and four downstream open reading frames (ORFs) were cotranscribed, supporting the notion that gentisate metabolism is a unique characteristic of strain DR1. The genomic analysis of strain DR1 provides additional insights into the function, ecology, and evolution ofAcinetobacterspecies.

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Cytoplasmic CopZ-Like Protein and Periplasmic Rusticyanin and AcoP Proteins as Possible Copper Resistance Determinants in Acidithiobacillus ferrooxidans ATCC 23270

Applied and Environmental Microbiology ◽

10.1128/aem.02810-15 ◽

2015 ◽

Vol 82 (4) ◽

pp. 1015-1022 ◽

Cited By ~ 18

Author(s):

Claudio A. Navarro ◽

Diego von Bernath ◽

Cristóbal Martínez-Bussenius ◽

Rodrigo A. Castillo ◽

Carlos A. Jerez

Keyword(s):

Acidithiobacillus Ferrooxidans ◽

Binding Proteins ◽

Metal Tolerance ◽

Metal Resistance ◽

Copper Resistance ◽

Copper Binding ◽

Content Type ◽

Resistance Determinants ◽

High Level ◽

Copper Binding Proteins

ABSTRACTAcidophilic organisms, such asAcidithiobacillus ferrooxidans, possess high-level resistance to copper and other metals.A. ferrooxidanscontains canonical copper resistance determinants present in other bacteria, such as CopA ATPases and RND efflux pumps, but these components do not entirely explain its high metal tolerance. The aim of this study was to find other possible copper resistance determinants in this bacterium. Transcriptional expression ofA. ferrooxidansgenes coding for a cytoplasmic CopZ-like copper-binding chaperone and the periplasmic copper-binding proteins rusticyanin and AcoP, which form part of an iron-oxidizing supercomplex, was found to increase when the microorganism was grown in the presence of copper. All of these proteins conferred more resistance to copper when expressed heterologously in a copper-sensitiveEscherichia colistrain. This effect was absent when site-directed-mutation mutants of these proteins with altered copper-binding sites were used in this metal sensitivity assay. These results strongly suggest that the three copper-binding proteins analyzed here are copper resistance determinants in this extremophile and contribute to the high-level metal resistance of this industrially important biomining bacterium.

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Complete Sequence of pOZ176, a 500-Kilobase IncP-2 Plasmid Encoding IMP-9-Mediated Carbapenem Resistance, from Outbreak Isolate Pseudomonas aeruginosa 96

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00423-13 ◽

2013 ◽

Vol 57 (8) ◽

pp. 3775-3782 ◽

Cited By ~ 48

Author(s):

Jianhui Xiong ◽

David C. Alexander ◽

Jennifer H. Ma ◽

Maxime Déraspe ◽

Donald E. Low ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Carbapenem Resistance ◽

Complete Sequence ◽

Open Reading Frames ◽

Content Type ◽

Conserved Sequence ◽

Carbapenem Resistant ◽

Class 1 ◽

Usage Analysis ◽

Type 3

ABSTRACTPseudomonas aeruginosa96 (PA96) was isolated during a multicenter surveillance study in Guangzhou, China, in 2000. Whole-genome sequencing of this outbreak strain facilitated analysis of its IncP-2 carbapenem-resistant plasmid, pOZ176. The plasmid had a length of 500,839 bp and an average percent G+C content of 57%. Of the 618 predicted open reading frames, 65% encode hypothetical proteins. The pOZ176 backbone is not closely related to any plasmids thus far sequenced, but some similarity to pQBR103 ofPseudomonas fluorescensSBW25 was observed. Two multiresistant class 1 integrons and several insertion sequences were identified. TheblaIMP-9-carrying integron containedaacA4→blaIMP-9→aacA4, flanked upstream by Tn21 tnpMRAand downstream by a completetnioperon of Tn402and amermodule, named Tn6016. The second integron carriedaacA4→catB8a→blaOXA-10and was flanked by Tn1403-liketnpRAand asul1-type 3′ conserved sequence (3′-CS), named Tn6217. Other features include three resistance genes similar to those of Tn5, a tellurite resistance operon, and twopiloperons. The replication and maintenance systems exhibit similarity to a genomic island ofRalstonia solanacearumGM1000. Codon usage analysis suggests the recent acquisition ofblaIMP-9. The origins of the integrons on pOZ176 indicated separate horizontal gene transfer events driven by antibiotic selection. The novel mosaic structure of pOZ176 suggests that it is derived from environmental bacteria.

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Death by Cystine: an Adverse Emergent Property from a Beneficial Series of Reactions

Journal of Bacteriology ◽

10.1128/jb.00546-15 ◽

2015 ◽

Vol 197 (23) ◽

pp. 3626-3628 ◽

Cited By ~ 4

Author(s):

Larry Reitzer

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Sulfur Metabolism ◽

Emergent Property ◽

Futile Cycle ◽

Content Type ◽

Link Type

In this issue of theJournal of Bacteriology, Chonoles Imlay et al. (K. R. Chonoles Imlay, S. Korshunov, and J. A. Imlay, J Bacteriol 197:3629–3644, 2015,http://dx.doi.org/10.1128/JB.00277-15) show that oxidative stress kills sulfur-restrictedEscherichia coligrown with sublethal H2O2when challenged with cystine. Killing requires rapid and seemingly unregulated cystine transport and equally rapid cystine reduction to cysteine. Cysteine export completes an energy-depleting futile cycle. Each reaction of the cycle could be beneficial. Together, a cystine-mediated vulnerability emerges during the transition from a sulfur-restricted to a sulfur-replete environment, perhaps because of complexities of sulfur metabolism.

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High Diversity of Magnetotactic Deltaproteobacteria in a Freshwater Niche

Applied and Environmental Microbiology ◽

10.1128/aem.03635-12 ◽

2013 ◽

Vol 79 (8) ◽

pp. 2813-2817 ◽

Cited By ~ 32

Author(s):

Yinzhao Wang ◽

Wei Lin ◽

Jinhua Li ◽

Yongxin Pan

Keyword(s):

Magnetotactic Bacteria ◽

Natural Environments ◽

Content Type ◽

Operational Taxonomic Units ◽

Geological Processes ◽

Freshwater Site ◽

High Diversity

ABSTRACTKnowledge of the diversity of magnetotactic bacteria in natural environments is crucial for understanding their contribution to various biological and geological processes. Here we report a high diversity of magnetotactic bacteria in a freshwater site. Ten out of 18 operational taxonomic units (OTUs) were affiliated with theDeltaproteobacteria. Some rod-shaped bacteria simultaneously synthesized greigite and magnetite magnetosomes.

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