Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis.

Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.

Recent advances in understanding Streptomyces.

About 2,500 papers dated 2014-2016 were recovered by searching the PubMed database for Streptomyces, which are the richest known source of antibiotics. This review integrates around 100 of these papers in sections dealing with evolution, ecology, pathogenicity, growth and development, stress responses and secondary metabolism, gene expression, and technical advances. Genomic approaches have greatly accelerated progress. For example, it has been definitively shown that interspecies recombination of conserved genes has occurred during evolution, in addition to exchanges of some of the tens of thousands of non-conserved accessory genes. The closeness of the association of Streptomyces with plants, fungi, and insects has become clear and is reflected in the importance of regulators of cellulose and chitin utilisation in overall Streptomyces biology. Interestingly, endogenous cellulose-like glycans are also proving important in hyphal growth and in the clumping that affects industrial fermentations. Nucleotide secondary messengers, including cyclic di-GMP, have been shown to provide key input into developmental processes such as germination and reproductive growth, while late morphological changes during sporulation involve control by phosphorylation. The discovery that nitric oxide is produced endogenously puts a new face on speculative models in which regulatory Wbl proteins (peculiar to actinobacteria) respond to nitric oxide produced in stressful physiological transitions. Some dramatic insights have come from a new model system for Streptomyces developmental biology, Streptomyces venezuelae, including molecular evidence of very close interplay in each of two pairs of regulatory proteins. An extra dimension has been added to the many complexities of the regulation of secondary metabolism by findings of regulatory crosstalk within and between pathways, and even between species, mediated by end products. Among many outcomes from the application of chromosome immunoprecipitation sequencing (ChIP-seq) analysis and other methods based on "next-generation sequencing" has been the finding that 21% of Streptomyces mRNA species lack leader sequences and conventional ribosome binding sites. Further technical advances now emerging should lead to continued acceleration of knowledge, and more effective exploitation, of these astonishing and critically important organisms.

Specialised metabolites regulating antibiotic biosynthesis in Streptomyces spp.

Streptomyces bacteria are the major source of antibiotics and other secondary metabolites. Various environmental and physiological conditions affect the onset and level of production of each antibiotic by influencing concentrations of the ligands for conserved global regulatory proteins. In addition, as reviewed here, well-known autoregulators such as γ-butyrolactones, themselves products of secondary metabolism, accumulate late in growth to concentrations allowing their effective interaction with cognate binding proteins, in a necessary prelude to antibiotic biosynthesis. Most autoregulator binding proteins target the conserved global regulatory gene adpA, and/or regulatory genes for 'cluster-situated regulators' (CSRs) linked to antibiotic biosynthetic gene clusters. It now appears that some CSRs bind intermediates and end products of antibiotic biosynthesis, with regulatory effects interwoven with those of autoregulators. These ligands can exert cross-pathway effects within producers of more than one antibiotic, and when excreted into the extracellular environment may have population-wide effects on production, and mediate interactions with neighbouring microorganisms in natural communities, influencing speciation. Greater understanding of these autoregulatory and cross-regulatory activities may aid the discovery of new signalling molecules and their use in activating cryptic antibiotic biosynthetic pathways.

Developmental biology of Streptomyces from the perspective of 100 actinobacterial genome sequences.

To illuminate the evolution and mechanisms of actinobacterial complexity, we evaluate the distribution and origins of known Streptomyces developmental genes and the developmental significance of actinobacteria-specific genes. As an aid, we developed the Actinoblast database of reciprocal blastp best hits between the Streptomyces coelicolor genome and more than 100 other actinobacterial genomes (http://streptomyces.org.uk/actinoblast/). We suggest that the emergence of morphological complexity was underpinned by special features of early actinobacteria, such as polar growth and the coupled participation of regulatory Wbl proteins and the redox-protecting thiol mycothiol in transducing a transient nitric oxide signal generated during physiologically stressful growth transitions. It seems that some cell growth and division proteins of early actinobacteria have acquired greater importance for sporulation of complex actinobacteria than for mycelial growth, in which septa are infrequent and not associated with complete cell separation. The acquisition of extracellular proteins with structural roles, a highly regulated extracellular protease cascade, and additional regulatory genes allowed early actinobacterial stationary phase processes to be redeployed in the emergence of aerial hyphae from mycelial mats and in the formation of spore chains. These extracellular proteins may have contributed to speciation. Simpler members of morphologically diverse clades have lost some developmental genes.

Curing baldness activates antibiotic production.

Kalan and colleagues describe an unusual way of activating a "silent" gene cluster for the biosynthesis of a new antibiotic by analyzing and curing the aerial growth defect of an old Streptomyces isolate, Streptomyces calvus. This commentary addresses the broad scientific and historical context and practical and biological significance of this finding.

Molecular regulation of antibiotic biosynthesis in streptomyces.

Streptomycetes are the most abundant source of antibiotics. Typically, each species produces several antibiotics, with the profile being species specific. Streptomyces coelicolor, the model species, produces at least five different antibiotics. We review the regulation of antibiotic biosynthesis in S. coelicolor and other, nonmodel streptomycetes in the light of recent studies. The biosynthesis of each antibiotic is specified by a large gene cluster, usually including regulatory genes (cluster-situated regulators [CSRs]). These are the main point of connection with a plethora of generally conserved regulatory systems that monitor the organism's physiology, developmental state, population density, and environment to determine the onset and level of production of each antibiotic. Some CSRs may also be sensitive to the levels of different kinds of ligands, including products of the pathway itself, products of other antibiotic pathways in the same organism, and specialized regulatory small molecules such as gamma-butyrolactones. These interactions can result in self-reinforcing feed-forward circuitry and complex cross talk between pathways. The physiological signals and regulatory mechanisms may be of practical importance for the activation of the many cryptic secondary metabolic gene cluster pathways revealed by recent sequencing of numerous Streptomyces genomes.

Molecular and cytological analysis of the expression of Streptomyces sporulation regulatory gene whiH.

The whiH gene is required for the orderly sporulation septation that divides aerial hyphae into spores in Streptomyces coelicolor. Here, we use a whiHp-mCherry transcriptional reporter construct to show that whiHp is active specifically in aerial hyphae, fluorescence being dependent on sporulation sigma factor WhiG. The results show that the promoter is active before the septation event that separates the subapical compartment from the tip compartment destined to become a spore chain. We conclude that WhiG-directed RNA polymerase activity, which is required for whiH transcription, must precede this septation event and is not restricted to apical sporogenic compartment of the aerial hyphae. Further, it is demonstrated that WhiH, a predicted member of the GntR family of transcription factors, is able to bind specifically to a sequence in its own promoter, strongly suggesting that it acts as an autoregulatory transcription factor. Finally, we show by site-directed mutagenesis and a genetic complementation test that whiH is translated from a start codon overlapping with the previously identified transcription start point, implying leaderless transcription.

Importance and regulation of inositol biosynthesis during growth and differentiation of Streptomyces.

Unusually among bacteria, actinobacteria possess myo-inositol 1-phosphate synthase (mIPS). In the developmentally complex Streptomyces coelicolor, the mIPS-encoding gene (inoA) is cotranscribed with a putative regulatory gene (inoR). The inoRA transcript was more abundant in an inoR in-frame deletion mutant, and InoR formed different complexes in vitro with an extensive region around the inoRA promoter. Binding was relieved by adding glucose 6-phosphate. Thus, InoR is a metabolite-sensitive autorepressor that influences inoA expression, and hence the level of inositol, by controlling transcription from P(inoRA) . Disruption of inoA resulted in inositol-dependent growth and development, with full phenotypic correction at 0.1 mM inositol: at lower inositol concentrations differentiation was arrested at intermediate stages. This pattern may partly reflect increased demand for membrane phospholipids during sporulation septation. A corresponding sharp upregulation of inoRA transcription coincident with sporulation was dependent on a developmental regulator, WhiI. A truncated form of WhiI could bind two sites downstream of P(inoRA) , and one of the WhiI-binding sites overlapped the InoR-binding site. The combined action of a metabolic regulator and a developmental regulator at the simple P(inoRA) promoter is a previously undescribed strategy for the differential provision of developmentally appropriate levels of a substance required during the formation of spore chains.

Structure of Streptomyces maltosyltransferase GlgE, a homologue of a genetically validated anti-tuberculosis target.

GlgE is a recently identified (1→4)-α-d-glucan:phosphate α-d-maltosyltransferase involved in α-glucan biosynthesis in bacteria and is a genetically validated anti-tuberculosis drug target. It is a member of the GH13_3 CAZy subfamily for which no structures were previously known. We have solved the structure of GlgE isoform I from Streptomyces coelicolor and shown that this enzyme has the same catalytic and very similar kinetic properties to GlgE from Mycobacterium tuberculosis. The S. coelicolor enzyme forms a homodimer with each subunit comprising five domains, including a core catalytic α-amylase-type domain A with a (ß/α)(8) fold. This domain is elaborated with domain B and two inserts that are specifically configured to define a well conserved donor pocket capable of binding maltose. Domain A, together with domain N from the neighboring subunit, forms a hydrophobic patch that is close to the maltose-binding site and capable of binding cyclodextrins. Cyclodextrins competitively inhibit the binding of maltooligosaccharides to the S. coelicolor enzyme, showing that the hydrophobic patch overlaps with the acceptor binding site. This patch is incompletely conserved in the M. tuberculosis enzyme such that cyclodextrins do not inhibit this enzyme, despite acceptor length specificity being conserved. The crystal structure reveals two further domains, C and S, the latter being a helix bundle not previously reported in GH13 members. The structure provides a framework for understanding how GlgE functions and will help guide the development of inhibitors with therapeutic potential.

Unexpected and widespread connections between bacterial glycogen and trehalose metabolism.

Glycogen, a large α-glucan, is a ubiquitous energy storage molecule among bacteria, and its biosynthesis by the classical GlgC-GlgA pathway and its degradation have long been well understood - or so we thought. A second pathway of α-glucan synthesis, the four-step GlgE pathway, was recently discovered in mycobacteria. It requires trehalose as a precursor, and has been genetically validated as a novel anti-tuberculosis drug target. The ability to convert glycogen into trehalose was already known, so the GlgE pathway provides a complementary way of cycling these two metabolites. As well as containing cytosolic storage glycogen, mycobacteria possess an outer capsule containing a glycogen-like α-glucan that is implicated in immune system evasion, so the GlgE pathway might be linked to capsular α-glucan biosynthesis. Another pathway (the Rv3032 pathway) for α-glucan biosynthesis in mycobacteria generates a methylglucose lipopolysaccharide thought to be associated with fatty acid metabolism. A comparative genomic analysis was carried out to evaluate the occurrence and role of the classical pathway, the new GlgE pathway and the Rv3032 pathway across bacteria occupying very different ecological niches. The GlgE pathway is represented in 14 % of sequenced genomes from diverse bacteria (about half as common as the classical pathway), while the Rv3032 pathway is restricted with few exceptions to mycobacteria, and the GlgB branching enzyme, usually presumed to be associated with the classical pathway, correlates more strongly with the new GlgE pathway. The microbiological implications of recent discoveries in the light of the comparative genomic analysis are discussed.

The actinobacteria-specific gene wblA controls major developmental transitions in Streptomyces coelicolor A3(2).

The Streptomyces coelicolor A3(2) sporulation gene whiB is the paradigm of a family of genes (wbl, whiB-like) that are confined to actinobacteria. The chromosome of S. coelicolor contains 11 wbl genes, among which five are conserved in many actinobacteria: whiB itself; whiD, a sporulation gene; wblC, which is required for multi-drug resistance; and wblA and wblE, whose roles had previously been little studied. We succeeded in disrupting wblA and the six non-conserved genes, but could not disrupt wblE. Although mutations in the six non-conserved wbl genes (including some multiple wbl mutants) produced no readily detectable phenotype, mutations in wblA had novel and complex effects. The aerial mycelium of wblA mutants was coloured red, because of the ectopic presence of pigmented antibiotics (actinorhodin and undecylprodigiosin) normally confined to lower parts of wild-type colonies, and consisted almost entirely of non-sporulating, thin, straight filaments, often bundled together in a fibrillar matrix. Rare spore chains were also formed, which exhibited wild-type properties but were genetically still wblA mutants. A wblA mutant achieved higher biomass than the wild-type. Microarray analysis indicated major transcriptional changes in a wblA mutant: using a relatively stringent cut-off, 183 genes were overexpressed, including genes for assimilative primary metabolism and actinorhodin biosynthesis, and 103 were underexpressed, including genes associated with stages of aerial hyphal growth. We suggest that WblA is important in both the slow-down of biomass accumulation and the change from aerial hyphal initial cells to the subapical stem and apical compartments that precede sporulation; and that the mutant aerial mycelium consists of recapitulated defective aerial hyphal initial cells.

The actinobacterial signature protein ParJ (SCO1662) regulates ParA polymerization and affects chromosome segregation and cell division during Streptomyces sporulation.

Bacterial chromosome segregation usually involves cytoskeletal ParA proteins, ATPases which can form dynamic filaments. In aerial hyphae of the mycelial bacterium Streptomyces coelicolor, ParA filaments extend over tens of microns and are responsible for segregation of dozens of chromosomes. We have identified a novel interaction partner of S. coelicolor ParA, ParJ. ParJ negatively regulates ParA polymerization in vitro and is important for efficient chromosome segregation in sporulating aerial hyphae. ParJ-EGFP formed foci along aerial hyphae even in the absence of ParA. ParJ, which is encoded by sco1662, turned out to be one of the five actinobacterial signature proteins, and another of the five is a ParJ paralogue. We hypothesize that polar growth, which is characteristic not only of streptomycetes, but even of simple Actinobacteria, may be interlinked with ParA polymer assembly and its specific regulation by ParJ.

The complex whiJ locus mediates environmentally sensitive repression of development of Streptomyces coelicolor A3(2).

A segment of DNA was isolated that complemented several poorly characterised sporulation-defective white-colony mutants of Streptomyces coelicolor A3(2) from an early collection (Hopwood et al., J Gen Microbiol 61: 397-408, 1970). Complementation was attributable to a gene, SCO4543, named whiJ, encoding a likely DNA-binding protein. Surprisingly, although some mutations in whiJ had a white colony phenotype, complete deletion of the wild-type or mutant gene gave a wild-type morphology. The whiJ gene is a member of a large paralogous set of S. coelicolor genes including abaAorfA, which regulates antibiotic production; and genes flanking whiJ are paralogues of other gene classes that are often associated with whiJ-like genes (Gehring et al., Proc Natl Acad Sci USA 97: 9642-9647, 2000). Thus, the small gene SCO4542 encodes a paralogue of the abaAorfD gene product, and SCO4544 encodes a paralogue of a family of likely anti-sigma factors (including the product of abaAorfB). Deletion of SCO4542 resulted in a medium-dependent bald- or white-colony phenotype, which could be completely suppressed by the simultaneous deletion of whiJ. A model is proposed in which WhiJ binds to operator sequences to repress developmental genes, with repression being released by interaction with the WhiJ-associated SCO4542 protein. It is suggested that this activity of SCO4542 protein is prevented by an unknown signal.

The complex extracellular biology of Streptomyces.

Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.

Genome sequence of the Fleming strain of Micrococcus luteus, a simple free-living actinobacterium.

Micrococcus luteus (NCTC2665, "Fleming strain") has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to beta-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.

Extracellular signalling, translational control, two repressors and an activator all contribute to the regulation of methylenomycin production in Streptomyces coelicolor.

Bioinformatic analysis of the plasmid-linked gene cluster associated with biosynthesis of methylenomycin (Mm) suggested that part of the cluster directs synthesis of a gamma-butyrolactone-like autoregulator. Autoregulator activity could be extracted from culture fluids, but differed from gamma-butyrolactones in being alkali resistant. The activity has recently been shown to comprise a series of novel autoregulator molecules, the methylenomycin furans (termed MMF). MMF autoregulator activity is shown to account for the ability of certain Mm non-producing mutants to act as 'secretors' in cosynthesis with other 'convertor' mutants. Three genes implicated in MMF biosynthesis are flanked by two regulatory genes, which are related to genes for gamma-butyrolactone-binding proteins. Genetic evidence suggests that these two genes encode components of a hetero-oligomeric repressor of MMF and Mm biosynthesis. The Mm biosynthetic genes themselves depend on the activator gene mmyB, which appears to be repressed by the putative MmyR/MmfR complex until enough MMF accumulates to release repression. The presence of TTA codons in mmyB and the main MMF biosynthetic gene causes Mm production to be dependent on the pleiotropically acting bldA gene, which encodes the tRNA for the rarely used UUA codon.

2-Alkyl-4-hydroxymethylfuran-3-carboxylic acids, antibiotic production inducers discovered by Streptomyces coelicolor genome mining.

All of the genetic elements necessary for the production of the antibiotic methylenomycin (Mm) and its regulation are contained within the 22-kb mmy-mmf gene cluster, which is located on the 356-kb linear plasmid SCP1 of Streptomyces coelicolor A3(2). A putative operon of 3 genes within this gene cluster, mmfLHP, was proposed to direct the biosynthesis of an A-factor-like signaling molecule, which could play a role in the regulation of Mm biosynthesis. The mmfLHP operon was expressed under the control of its native promoter in S. coelicolor M512, a host lacking the SCP1 plasmid, and the ability to produce prodiginine and actinorhodin antibiotics. Comparative metabolic profiling led to the identification and structure elucidation of a family of 5 new 2-alkyl-4-hydroxymethylfuran-3-carboxylic acids (AHFCAs), collectively termed Mm furans (MMFs), as the products of the mmfLHP genes. MMFs specifically induce the production of the Mm antibiotics in S. coelicolor. Comparative genomics analyses and searches of the natural product chemistry literature indicated that other streptomycetes may produce AHFCAs, suggesting that they could form a general class of antibiotic biosynthesis inducers in Streptomyces species, with analogous functions to the better known gamma-butyrolactone regulatory molecules.

Complex extracellular interactions of proteases and a protease inhibitor influence multicellular development of Streptomyces coelicolor.

Streptomyces coelicolor produces an extracellular protease inhibitor protein, STI (Streptomyces trypsin inhibitor). We show that post-growth elimination of STI is needed for colonies to develop aerial mycelium efficiently. Inactivation of STI, and thus the normal progression of colony development, at least partly involves an extracellular protease specified by gene SCO5913. Two-hybrid analysis identified two possible targets of STI inhibition (the products of SCO1355 and SCO5447), both extracellular proteases containing a domain homologous with the P-domain of eukaryotic convertases, proteases that mediate the processing of many precursors with important cellular or developmental roles. At least the SCO1355 protease is needed for the normal progression of development. Two components of the proposed cascade are dependent on the tRNA for the rare UUA (leucine) codon, which is specified by the developmental gene bldA. A model is presented that links intracellular regulatory events with an extracellular protease cascade to facilitate normal development.

A possible extended family of regulators of sigma factor activity in Streptomyces coelicolor.

SCO4677 is one of a large number of similar genes in Streptomyces coelicolor that encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB of Bacillus subtilis. However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific sigma(F), suggesting that it is an antagonistic regulator of sigma(F). Two other proteins, encoded by SCO0781 and SCO0869, were found to interact with the SCO4677 anti-sigma(F) during a subsequent global yeast two-hybrid screen, and the SCO0869-SCO4677 protein-protein interaction was confirmed by coimmunoprecipitation. The SCO0781 and SCO0869 proteins resemble well-known anti-anti-sigma factors such as SpoIIAA of B. subtilis. It appears that streptomycetes may possess an extraordinary abundance of anti-sigma factors, some of which may influence diverse processes through interactions with multiple partners: a novel feature for such regulatory proteins.