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Vibrio cholerae FeoB hydrolyzes ATP and GTP in vitro in the absence of stimulatory factors.

ABSTRACT: Feo is the most widely conserved system for ferrous iron transport in prokaryotes, and it is important for virulence in some pathogens. However, its mechanism of iron transport is not fully understood. In this study, we used full-length Vibrio cholerae FeoB (VcFeoB) as a model system to study whether its enzymatic activity is affected by regulatory factors commonly associated with FeoB proteins from other species or with G-proteins that have homology to FeoB. VcFeoB showed a higher rate of hydrolysis of both ATP and GTP than its N-terminal domain alone; likewise, ions such as K+ and Fe2+ did not modulate its nucleotide hydrolysis. We also showed that the three V. cholerae Feo proteins (FeoA, FeoB, and FeoC) work in a 1 : 1 : 1 molar ratio in vivo. Although both FeoA and FeoC are required for Feo-mediated iron transport, neither of these proteins affected the VcFeoB NTPase rate. These results are consistent with an active transport mechanism independent of stimulatory factors and highlight the importance of using full-length FeoB proteins as a reliable proxy to study Feo-mediated iron transport in vitro.

SUBMITTER: Gomez-Garzon C

PROVIDER: S-EPMC7769900 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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<i>Vibrio cholerae</i> FeoB hydrolyzes ATP and GTP <i>in vitro</i> in the absence of stimulatory factors.

Gómez-Garzón Camilo C Payne Shelley M SM

Metallomics : integrated biometal science 20201201 12

Feo is the most widely conserved system for ferrous iron transport in prokaryotes, and it is important for virulence in some pathogens. However, its mechanism of iron transport is not fully understood. In this study, we used full-length Vibrio cholerae FeoB (VcFeoB) as a model system to study whether its enzymatic activity is affected by regulatory factors commonly associated with FeoB proteins from other species or with G-proteins that have homology to FeoB. VcFeoB showed a higher rate of hydro ...[more]

PMID: 33174898

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Project description:The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relA(VCH)) was cloned and sequenced. The relA(VCH) gene encodes a 738-amino-acid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A deltarelA::kan allele was generated by replacing approximately 31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.

Dataset Information

Vibrio cholerae FeoB hydrolyzes ATP and GTP in vitro in the absence of stimulatory factors.

Publications

<i>Vibrio cholerae</i> FeoB hydrolyzes ATP and GTP <i>in vitro</i> in the absence of stimulatory factors.

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