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Characterization of the ?7 Mutant of Cupriavidus metallidurans with Deletions of Seven Secondary Metal Uptake Systems.

ABSTRACT: Central to the ability of Cupriavidus metallidurans to maintain its metal homoeostasis is the metal transportome, composed of uptake and efflux systems. Seven secondary metal import systems, ZupT, PitA, CorA1, CorA2, CorA3, ZntB, and HoxN, interact and are at the core of the metal uptake transportome. The 7-fold deletion mutant ?7 (?zupT ?pitA ?corA1?corA2?corA3?zntB ?hoxN) of parent strain AE104 is still able to maintain its cellular metal content, although at the cost of reduced fitness (M. Herzberg, L. Bauer, A. Kirsten, and D. H. Nies, Metallomics, in press, http://dx.doi.org/10.1039/C5MT00295H). Strain ?7 does not express genes for backup importers, and so ?7 should use metal uptake systems also produced in the AE104 parent cells. These systems should be activated in ?7 by posttranscriptional regulatory processes. The decreased fitness of ?7 correlated with a zinc-dependent downregulation of the overall metabolic backbone of the cells even at nontoxic external zinc concentrations. Responsible for this decreased fitness of ?7 was a negative interference of the activity of two P-type ATPases, MgtA and MgtB, which, on the other hand, kept ?7 at a fitness level higher than that of the ?9 (?7 ?mgtA::kan ?mgtB) mutant strain. This revealed a complicated interplay of the metal uptake transportome of C. metallidurans, which is composed of the seven secondary uptake systems, MgtA, MgtB, and yet-unknown components, with cytoplasmic transition metal pools and posttranscriptional regulatory processes. IMPORTANCE Bacteria, including pathogenic strains, need to make use of the metal composition and speciation of their environment to fulfill the requirement of the cytoplasmic metal content and composition. This task is performed by the bacterial metal transportome, composed of uptake and efflux systems. Seven interacting secondary metal uptake systems are at the core of the metal transportome in C. metallidurans. This publication verifies that posttranscriptional events are responsible for activation of even more, yet-unknown, metal import systems in the 7-fold deletion mutant ?7. Two P-type ATPases were identified as new members of the metal uptake transportome. This publication demonstrates the complexity of the metal transportome and the regulatory processes involved.

SUBMITTER: Große C

PROVIDER: S-EPMC5069749 | biostudies-literature | 2016 Jan-Feb

REPOSITORIES: biostudies-literature

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Characterization of the Δ7 Mutant of Cupriavidus metallidurans with Deletions of Seven Secondary Metal Uptake Systems.

Große Cornelia C Herzberg Martin M Schüttau Marcel M Wiesemann Nicole N Hause Gerd G Nies Dietrich H DH

mSystems 20160101 1

Central to the ability of Cupriavidus metallidurans to maintain its metal homoeostasis is the metal transportome, composed of uptake and efflux systems. Seven secondary metal import systems, ZupT, PitA, CorA1, CorA2, CorA3, ZntB, and HoxN, interact and are at the core of the metal uptake transportome. The 7-fold deletion mutant Δ7 (ΔzupT ΔpitA ΔcorA1ΔcorA2ΔcorA3ΔzntB ΔhoxN) of parent strain AE104 ...[more]

PMID: 27822513

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Project description:The Zur regulon is central to zinc homeostasis in the zinc-resistant bacterium Cupriavidus metallidurans It comprises the transcription regulator Zur, the zinc importer ZupT, and three members of the COG0523 family of metal-chaperoning G3E-type GTPases, annotated as CobW1, CobW2, and CobW3. The operon structures of the zur and cobW1 loci were determined. To analyze the interplay between the Zur regulon components and metal resistance, deletion mutants were constructed from the wild-type strain CH34 and various other strains. The Zur regulon components interacted with the plasmid-encoded and chromosomally encoded metal resistance factors to acquire metals from complexes of EDTA and for homeostasis of and resistance to zinc, nickel, cobalt, and cadmium. The three G3E-type GTPases were characterized in more detail. CobW1 bound only 1 Zn atom per mol of protein with a stability constant slightly above that of 2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene (Zincon) and an additional 0.5 Zn with low affinity. The CobW1 system was necessary to obtain metals from EDTA complexes. The GTPase CobW2 is a zinc storage compound and bound 0.5 to 1.5 Zn atoms tightly and up to 6 more with lower affinity. The presence of MgGTP unfolded the protein partially. CobW3 had no GTPase activity and equilibrated metal import by ZupT with that of the other metal transport systems. It sequestered 8 Zn atoms per mol with decreasing affinity. The three CobWs bound to the metal-dependent protein FolEIB2, which is encoded directly downstream of cobW1 This demonstrated an important contribution of the Zur regulon components to metal homeostasis in C. metalliduransIMPORTANCE Zinc is an important transition metal cation and is present as an essential component in many enzymes, such as RNA polymerase. As with other transition metals, zinc is also toxic at higher concentrations so that living cells have to maintain strict control of their zinc homeostasis. Members of the COG0523 family of metal-chaperoning GE3-type GTPases exist in archaea, bacteria, and eucaryotes, including humans, and they may be involved in delivery of zinc to thousands of different proteins. We used a combination of molecular, physiological, and biochemical methods to demonstrate the important but diverse functions of COG0523 proteins in C. metallidurans, which are produced as part of the Zur-controlled zinc starvation response in this bacterium.

Project description:Background: The high number of heavy metal resistance genes in the soil bacterium Cupriavidus metallidurans CH34 makes it an interesting model organism to study microbial responses to heavy metals. Results: In this study the transcriptional response of this bacterium was measured after challenging it to a wide range of sub-lethal concentrations of various essential or toxic metals. Considering the global transcriptional responses for each challenge as well as by identifying the overlap in upregulated genes between different metal responses, the sixteen metals could be clustered in three different groups. Additionally, next to the assessment of the transcriptional response of already known metal resistance genes, new metal response gene clusters were identified. The majority of the metal response loci showed similar expression profiles when cells were exposed to different metals, suggesting complex cross-talk at transcriptional level between the different metal responses. The highly redundant nature of these metal resistant regions – illustrated by the large number of paralogous genes – combined with the phylogenetic distribution of these metal response regions within evolutionary related and other metal resistant bacteria, provides important insights on the recent evolution of this naturally soil dwelling bacterium towards a highly metal-resistant strain found in harsh and anthropogenic environments. Conclusions: The metal-resistant soil bacterium Cupriavidus metallidurans CH34 displays myriads of gene expression patterns when exposed to a wide range of heavy metals at non-lethal concentrations. The interplay between the different gene expression clusters points towards a complex cross-regulated regulatory network governing heavy metal resistance in C. metallidurans CH34. Keywords: Cupriavidus metallidurans CH34, transcriptional regulation, heavy metal resistance Two-condition experiments. Comparing samples after induction with heavy metals versus non-induced samples. Biological duplicate or triplicate. Each array contains 3 or 4 technical replicates.

Dataset Information

Characterization of the ?7 Mutant of Cupriavidus metallidurans with Deletions of Seven Secondary Metal Uptake Systems.

Publications

Characterization of the Δ7 Mutant of <i>Cupriavidus metallidurans</i> with Deletions of Seven Secondary Metal Uptake Systems.

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