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A co-conserved gene pair supports Caulobacter iron homeostasis and resistance to chelation stress

ABSTRACT: Bacteria assimilate a diverse set of metals that enable metabolic functions essential for growth and survival, but metal homeostasis can be disrupted by natural and synthetic chelators in the environment. We have identified genes that influence fitness of the common soil and aquatic bacterium, Caulobacter crescentus, in the presence of a growth-limiting concentration of the synthetic chelator ethylenediaminetetraacetic acid (EDTA). Among the identified genes important in EDTAacclimationthere are several controlled by the metal-responsive regulators Fur, Zur, and UzcRS. However, a subset of genes in the iron-responsive Fur regulon emerged as the primary fitness determinants during EDTA challenge. EDTA treatment reduced intracellular iron levels and a syntenic gene pair, cciT and cciO, that supports Caulobacter iron homeostasis is necessary for growth in EDTA. cciT encodes one of four Fur-regulated TonB-dependent transporters (TBDTs) and – of these regulators – uniquely supports wild-type levels of growth on defined and complex lab media. The iron acquisition and EDTA resistance functions of the CciT transporter specifically require the presence of cciO, which encodes a 2-oxoglutarate-Fe(II) dioxygenase. Thus, there is a critical functional partnership between an outer membrane transporter and a cytoplasmic dioxygenase that are broadly co-conserved in the phylum Proteobacteria. cciT and cciO mutants cultivated in natural freshwater were not impacted by an equivalent EDTA treatment, demonstrating robustness of the highly redundant set of Caulobacter iron acquisition systems in an ecosystem context. This study advances our understanding of genes involved in metal acquisition and chelation stress survival, and illuminates the complex interactions between the chemical environment and the processes that regulate intracellular metal homeostasis.

ORGANISM(S): Caulobacter vibrioides

PROVIDER: GSE274268 | GEO | 2024/08/14

REPOSITORIES: GEO

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Project description:In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater Î±-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identify 46 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB dependent receptor gene clusters, feoAB), riboflavin biosynthesis and some genes encoding hypothetical proteins. Most of these genes are associated with Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by Î²-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 50 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, are also included in this group many genes encoding TonB dependent receptors and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses performed in the promoters of these genes suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of more 103 genes, including upregulation of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as downregulation of genes implicated in amino acid metabolism, chemotaxis and motility. Altogether, our results showed that adaptation of C. crescentus to iron limitation involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins as a general strategy Two experimental procedures, each of them performed in two replicates. A total of four independent biological samples were used

Project description:Porphyromonas gingivalis is a major pathogen associated with the microbial biofilm-mediated disease chronic periodontitis. P. gingivalis has an obligate requirement for iron and protoporphyrin IX which it satisfies by transporting heme and iron liberated from the human host. The level of cellular iron in P. gingivalis affects the expression of a distinct iron-associated regulon of 64 genes and low iron invokes an iron sparing response. Iron homeostasis is usually mediated in Gram-negative bacteria at the transcriptional level by the Ferric Uptake Regulator (Fur). There is a single predicted P. gingivalis Fur superfamily orthologue named Har (heme associated regulator) that lacks the conserved metal binding residues found in other Fur orthologues. We show that Har binds both heme and ferrous iron resulting in a conformational change in the protein. Har was unable to complement the Escherichia coli H1780 fur mutant and there was no change in cellular metal content in a P. gingivalis Har mutant compared with the wild-type. The Har regulon of 44 genes is not predicted to play a role in iron homeostasis. Together these data indicated that Har does not regulate iron homeostasis in P. gingivalis. However, Har was required for heme-responsive biofilm development and its regulon overlapped P. gingivalis regulons previously identified after growth in heme limitation or as a homotypic biofilm. P. gingivalis is unique as an iron-dependent Gram-negative bacterium with a single heme-binding Fur superfamily orthologue, Har, that does not regulate iron homeostasis. Paired samples were compared on the same microarray using a two-colour system. A total of 6 paired microarray hybridizations were performed representing 6 biological replicates, where a balanced dye design was used, with the overall analysis including three microarrays where P. gingivalis 33277 samples were labeled with Cy3 and the paired ECR455 samples were labeled with Cy5 and three other microarrays where samples were labeled with the opposite combination of fluorophores.

Project description:In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identify 46 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB dependent receptor gene clusters, feoAB), riboflavin biosynthesis and some genes encoding hypothetical proteins. Most of these genes are associated with Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 50 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, are also included in this group many genes encoding TonB dependent receptors and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses performed in the promoters of these genes suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of more 103 genes, including upregulation of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as downregulation of genes implicated in amino acid metabolism, chemotaxis and motility. Altogether, our results showed that adaptation of C. crescentus to iron limitation involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins as a general strategy

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A co-conserved gene pair supports Caulobacter iron homeostasis and resistance to chelation stress

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