All organisms must regulate the cellular uptake, efflux, and intracellular trafficking of essential elements, including d-block metal ions. In bacteria, such regulation is achieved by the action of metal-responsive transcriptional regulators. Among several families of zinc-responsive transcription factors, the ‘zinc uptake regulator’ Zur is the most widespread. Zur normally represses transcription in its zinc-bound form, in which DNA-binding affinity is enhanced allosterically. Experimental and bioinformatic searches for Zur-regulated genes have revealed that in many cases, Zur proteins govern zinc homeostasis in a much more profound way than merely through the expression of uptake systems. Zur regulons also comprise biosynthetic clusters for metallophore synthesis, ribosomal proteins, enzymes, and virulence factors. In recognition of the importance of zinc homeostasis at the host–pathogen interface, studying Zur regulons of pathogenic bacteria is a particularly active current research area.

Zinc is a recognized essential element for the majority of organisms, and is indispensable for the correct function of hundreds of enzymes and thousands of regulatory proteins. In aquatic photoautotrophs including cyanobacteria, zinc is thought to be required for carbonic anhydrase and alkaline phosphatase, although there is evidence that at least some carbonic anhydrases can be cambialistic, i.e., are able to acquire in vivo and function with different metal cofactors such as Co2+ and Cd2+. Given the global importance of marine phytoplankton, zinc availability in the oceans is likely to have an impact on both carbon and phosphorus cycles. Zinc concentrations in seawater vary over several orders of magnitude, and in the open oceans adopt a nutrient-like profile. Most studies on zinc handling by cyanobacteria have focused on freshwater strains and zinc toxicity; much less information is available on marine strains and zinc limitation. Several systems for zinc homeostasis have been characterized in the freshwater species Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803, but little is known about zinc requirements or zinc handling by marine species. Comparative metallo-genomics has begun to explore not only the putative zinc proteome, but also specific protein families predicted to have an involvement in zinc homeostasis, including sensors for excess and limitation (SmtB and its homologs as well as Zur), uptake systems (ZnuABC), putative intracellular zinc chaperones (COG0523) and metallothioneins (BmtA), and efflux pumps (ZiaA and its homologs).

Marine cyanobacteria are critical players in global nutrient cycles that crucially depend on trace metals in metalloenzymes, including zinc for CO2 fixation and phosphorus acquisition. How strains proliferating in the vast oligotrophic ocean gyres thrive at ultra-low zinc concentrations is currently unknown. Using Synechococcus sp. WH8102 as a model we show that its zinc-sensor protein Zur differs from all other known bacterial Zur proteins in overall structure and the location of its sensory zinc site. Uniquely, Synechococcus Zur activates metallothionein gene expression, which supports cellular zinc quotas spanning two orders of magnitude. Thus, a single zinc sensor facilitates growth across pico- to micromolar zinc concentrations with the bonus of banking this precious resource. The resultant ability to grow well at both ultra-low and excess zinc, together with overall lower zinc requirements, likely contribute to the broad ecological distribution of Synechococcus across the global oceans.

Bacterial trimethylamine N-oxide (TMAO) demethylase, Tdm, carries out an unusual oxygen-independent demethylation reaction, resulting in the formation of dimethylamine and formaldehyde. In this study, site-directed mutagenesis, homology modelling and metal analyses by inorganic mass spectrometry have been applied to gain insight into metal stoichiometry and underlying catalytic mechanism of Tdm of Methylocella silvestris BL2. Herein, we demonstrate that active Tdm has 1 molar equivalent of Zn 2+ and 1 molar equivalent of non-haem Fe 2+ . We further investigated Zn 2+ -and Fe 2+ -binding sites through homology modelling and site-directed mutagenesis and found that Zn 2+ is coordinated by a 3-sulfur-1-O motif. An aspartate residue (D198) likely bridges Fe 2+ and Zn 2+ centres, either directly or indirectly via H-bonding through a neighbouring H 2 O molecule. H276 contributes to Fe 2+ binding, mutation of which results in an inactive enzyme, and the loss of iron, but not zinc. Site-directed mutagenesis of Tdm also led to the identification of three hydrophobic aromatic residues likely involved in substrate coordination (F259, Y305, W321), potentially through a cation-p interaction. Furthermore, a crossover experiment using a substrate analogue gave direct evidence that a trimethylamine-alike intermediate was produced during the Tdm catalytic cycle, suggesting TMAO has a dual role of being both a substrate and an oxygen donor for formaldehyde formation. Together, our results provide novel insight into the role of Zn 2+ and Fe 2+ in the catalysis of TMAO demethylation by this unique oxygen-independent enzyme. IntroductionBacterial trimethylamine N-oxide (TMAO) demethylase (Tdm) is a key enzyme involved in bacterial degradation of trimethylamine (TMA) and TMAO [1][2][3]. The enzyme was first proposed in the 1970s and has been partially purified from Bacillus sp. PM6 [4] and Pseudomonas aminovorans (now Aminobacter aminovorans [5]). Despite being purified from aerobic hosts, Tdm can convert TMAO anaerobically to equimolar amounts of dimethylamine (DMA) and formaldehyde (HCHO) (1 TMAO ? 1 DMA + 1 HCHO) [2,3,5]. The gene encoding Tdm has only been identified very recently and it is now known that tdm is widely Abbreviations ADH, alcohol dehydrogenase; Asc, ascorbic acid; BS-DMA, benzenesulfonyl dimethylamine adduct; BS-MEA, benzenesulfonyl methylethylamine adduct; DMEA, dimethyethylamine; EDTA, ethylenediaminetetraacetic acid; GCV_T, glycine cleavage T protein; HCHO, formaldehyde; HMS, hydroxymethanesulfonate; ICP, inductively coupled plasma-mass spectrometry; MS, mass spectrometer; OES, optical emission spectrometer; Tdm, trimethylamine N-oxide demethylase; THF, tetrahydrofolate; TMAO, trimethylamine N-oxide. 3979The Tdm is constituted of two domains, an uncharacterized DUF1989-containing domain at its N terminus and a tetrahydrofolate (THF)-binding domain (GCV_T) at its C-terminus. DUF1989 in Tdm shows modest sequence similarity (< 30%) to urea-carboxylase-associated proteins, whose functions in urea catabolism a...