Ok, K. | bioRxiv Science

bioRxiv2025

A horizontally acquired cyclic di-GMP phosphodiesterase regulated by zinc and quorum sensing

The second messenger cyclic di-GMP (cdG) in V. cholerae is indispensable for the regulation of biofilm formation, motility, and a variety of other important bacterial behaviors in the majority of bacteria. The human pathogen Vibrio cholerae has a diverse repertoire of diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that control the intracellular cdG concentration depending on local environmental cues and its physiological state. Determining the transcriptional regulation of these enzymes and the respective environmental signals that control their activity is important to understand how and when V. cholerae switches between motile and sessile lifestyles in different environments. In some strains of the current V. cholerae 7th pandemic El Tor biotype, the horizontally acquired Vibrio Seventh Pandemic 2 (VSP-2) island encodes an uncharacterized PDE at the gene locus vc0515 which we named zpdA (Zinc-inhibited Phosphodiesterase-A). We show here that zpdA transcription is repressed by Zur when Zn2+ is abundant, as well as by the quorum sensing regulator HapR when cells grow to high density. Furthermore, we find that the PDE activity of the purified ZpdA protein is inhibited by Zn2+ but is dependent on alternative divalent cations such as Mn2+, which we find is elevated in V. cholerae cells grown under zinc limiting conditions. We conclude that ZpdA is an active metal-dependent PDE that is regulated by Zn2+ availability at both the level of transcription and post-translation leading to elevated cdG levels when Zn2+ is abundant. Our results demonstrate the important role of metal availability in modulating cdG signaling in bacteria. ImportanceV. cholerae colonizes estuarine environments and human host where it transitions between motile to sessile states which are controlled by cdG levels. cdG levels change in response to a variety of signals and are controlled by the activity of DGCs and PDEs, enzymes that make and degrade cdG. In this work, we show that Zn2+ and the cell density regulator HapR repress ZpdA, the PDE present in the VSP-2 island, at the level of transcription, and that Zn2+unexpectedly alters the PDE activity of ZpdA protein itself. Our study highlights the role of metal availability as an important signaling cue that controls V. cholerae biology.

microbiology↗

bioRxiv2024

A co-conserved gene pair supports Caulobacter iron homeostasis during chelation stress

Synthetic metal chelators are widely used in industrial, clinical, and agricultural settings, leading to their accumulation in the environment. We measured the growth of Caulobacter crescentus, a soil and aquatic bacterium, in the presence of the ubiquitous chelator ethylenediaminetetraacetic acid (EDTA) and found that it restricts growth by lowering intracellular iron levels. Using barcoded transposon sequencing, we identified an operonic gene pair, cciT-cciO, that is required to maintain iron homeostasis in laboratory media during EDTA challenge. cciT encodes one of four TonB-dependent transporters that are regulated by the ferric uptake repressor (Fur) and stands out among this group of genes in its ability to support Caulobacter growth across diverse media conditions. The function of CciT strictly requires cciO, which encodes a cytoplasmic FeII dioxygenase-family protein. Our results thus define a functional partnership between an outer membrane iron receptor and a cytoplasmic dioxygenase that are broadly co-conserved in Proteobacteria. We expanded our analysis to natural environments by examining the growth of mutant strains in freshwater from two lakes, each with biochemical and geochemical profiles that differ markedly from standard laboratory media. In lake water, Caulobacter growth did not require cciT or cciO and was less affected by EDTA treatment. This result aligns with our observation that EDTA toxicity is influenced by common forms of biologically chelated iron and the spectrum of free cations present in the medium. Our study defines a conserved iron acquisition system in Proteobacteria and bridges laboratory-based physiology studies with real-world conditions. IMPORTANCEMetal-chelating chemicals are widely used across industries, including as preservatives in the food sector, but their full impact on microbial physiology is not well understood. We identified two genes, cciT and cciO, that function together to support Caulobacter crescentus iron balance when cells are exposed to the common synthetic chelator, EDTA. CciT is an outer membrane transporter and CciO is a dioxygenase-family protein that are mutually conserved in many bacteria, including several human pathogens, where mutations in cciT homologs are linked to clinical resistance to the siderophore antibiotic, cefiderocol. This study identifies a conserved genetic system that supports iron homeostasis during chelation stress and illuminates the iron acquisition versatility and stress resilience of Caulobacter in freshwater environments.

microbiology↗