Stabilisation of the RirA [4Fe-4S] cluster results in loss of iron-sensing function.

RirA is a global iron regulator in diverse Alphaproteobacteria that belongs to the Rrf2 superfamily of transcriptional regulators, which can contain an iron-sulfur (Fe-S) cluster. Under iron-replete conditions, RirA contains a [4Fe-4S] cluster, enabling high-affinity binding to RirA-regulated operator sequences, thereby causing the repression of cellular iron uptake. Under iron deficiency, one of the cluster irons dissociates, generating an unstable [3Fe-4S] form that subsequently degrades to a [2Fe-2S] form and then to apo RirA, resulting in loss of high-affinity DNA-binding. The cluster is coordinated by three conserved cysteine residues and an unknown fourth ligand. Considering the lability of one of the irons and the resulting cluster fragility, we hypothesized that the fourth ligand may not be an amino acid residue. To investigate this, we considered that the introduction of an amino acid residue that could coordinate the cluster might stabilize it. A structural model of RirA, based on the Rrf2 family nitrosative stress response regulator NsrR, highlighted residue 8, an Asn in the RirA sequence, as being appropriately positioned to coordinate the cluster. Substitution of Asn8 with Asp, the equivalent, cluster-coordinating residue of NsrR, or with Cys, resulted in proteins that contained a [4Fe-4S] cluster, with N8D RirA exhibiting spectroscopic properties very similar to NsrR. The variant proteins retained the ability to bind RirA-regulated DNA, and could still act as repressors of RirA-regulated genes in vivo. However, they were significantly more stable than wild-type RirA when exposed to O2 and/or low iron. Importantly, they exhibited reduced capacity to respond to cellular iron levels, even abolished in the case of the N8D version, and thus were no longer iron sensing. This work demonstrates the importance of cluster fragility for the iron-sensing function of RirA, and more broadly, how a single residue substitution can alter cluster coordination and functional properties in the Rrf2 superfamily of regulators.

Oceanospirillales containing the DMSP lyase DddD are key utilisers of carbon from DMSP in coastal seawater.

BACKGROUND: Ubiquitous and diverse marine microorganisms utilise the abundant organosulfur molecule dimethylsulfoniopropionate (DMSP), the main precursor of the climate-active gas dimethylsulfide (DMS), as a source of carbon, sulfur and/or signalling molecules. However, it is currently difficult to discern which microbes actively catabolise DMSP in the environment, why they do so and the pathways used. RESULTS: Here, a novel DNA-stable isotope probing (SIP) approach, where only the propionate and not the DMS moiety of DMSP was 13C-labelled, was strategically applied to identify key microorganisms actively using DMSP and also likely DMS as a carbon source, and their catabolic enzymes, in North Sea water. Metagenomic analysis of natural seawater suggested that Rhodobacterales (Roseobacter group) and SAR11 bacteria were the major microorganisms degrading DMSP via demethylation and, to a lesser extent, DddP-driven DMSP lysis pathways. However, neither Rhodobacterales and SAR11 bacteria nor their DMSP catabolic genes were prominently labelled in DNA-SIP experiments, suggesting they use DMSP as a sulfur source and/or in signalling pathways, and not primarily for carbon requirements. Instead, DNA-SIP identified gammaproteobacterial Oceanospirillales, e.g. Amphritea, and their DMSP lyase DddD as the dominant microorganisms/enzymes using DMSP as a carbon source. Supporting this, most gammaproteobacterial (with DddD) but few alphaproteobacterial seawater isolates grew on DMSP as sole carbon source and produced DMS. Furthermore, our DNA-SIP strategy also identified Methylophaga and other Piscirickettsiaceae as key bacteria likely using the DMS, generated from DMSP lysis, as a carbon source. CONCLUSIONS: This is the first study to use DNA-SIP with 13C-labelled DMSP and, in a novel way, it identifies the dominant microbes utilising DMSP and DMS as carbon sources. It highlights that whilst metagenomic analyses of marine environments can predict microorganisms/genes that degrade DMSP and DMS based on their abundance, it cannot disentangle those using these important organosulfur compounds for their carbon requirements. Note, the most abundant DMSP degraders, e.g. Rhodobacterales with DmdA, are not always the key microorganisms using DMSP for carbon and releasing DMS, which in this coastal system were Oceanospirillales containing DddD. Video abstract.

Sensing iron availability via the fragile [4Fe-4S] cluster of the bacterial transcriptional repressor RirA.

Rhizobial iron regulator A (RirA) is a global regulator of iron homeostasis in many nitrogen-fixing Rhizobia and related species of α-proteobacteria. It belongs to the widespread Rrf2 super-family of transcriptional regulators and features three conserved Cys residues that characterise the binding of an iron-sulfur cluster in other Rrf2 family regulators. Here we report biophysical studies demonstrating that RirA contains a [4Fe-4S] cluster, and that this form of the protein binds RirA-regulated DNA, consistent with its function as a repressor of expression of many genes involved in iron uptake. Under low iron conditions, [4Fe-4S] RirA undergoes a cluster conversion reaction resulting in a [2Fe-2S] form, which exhibits much lower affinity for DNA. Under prolonged low iron conditions, the [2Fe-2S] cluster degrades to apo-RirA, which does not bind DNA and can no longer function as a repressor of the cell's iron-uptake machinery. [4Fe-4S] RirA was also found to be sensitive to O2, suggesting that both iron and O2 are important signals for iron metabolism. Consistent with this, in vivo data showed that expression of RirA-regulated genes is also affected by O2. These data lead us to propose a novel regulatory model for iron homeostasis, in which RirA senses iron via the incorporation of a fragile iron-sulfur cluster that is sensitive to iron and O2 concentrations.

The abundant marine bacterium Pelagibacter simultaneously catabolizes dimethylsulfoniopropionate to the gases dimethyl sulfide and methanethiol.

Marine phytoplankton produce ∼10(9) tonnes of dimethylsulfoniopropionate (DMSP) per year(1,2), an estimated 10% of which is catabolized by bacteria through the DMSP cleavage pathway to the climatically active gas dimethyl sulfide(3,4). SAR11 Alphaproteobacteria (order Pelagibacterales), the most abundant chemo-organotrophic bacteria in the oceans, have been shown to assimilate DMSP into biomass, thereby supplying this cell's unusual requirement for reduced sulfur(5,6). Here, we report that Pelagibacter HTCC1062 produces the gas methanethiol, and that a second DMSP catabolic pathway, mediated by a cupin-like DMSP lyase, DddK, simultaneously shunts as much as 59% of DMSP uptake to dimethyl sulfide production. We propose a model in which the allocation of DMSP between these pathways is kinetically controlled to release increasing amounts of dimethyl sulfide as the supply of DMSP exceeds cellular sulfur demands for biosynthesis.

Screening of metagenomic and genomic libraries reveals three classes of bacterial enzymes that overcome the toxicity of acrylate.

Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate resistance, likely, again, through the removal of the toxic product acrylyl-CoA.

Manganese uptake in marine bacteria; the novel MntX transporter is widespread in Roseobacters, Vibrios, Alteromonadales and the SAR11 and SAR116 clades.

We showed that two very different manganese transporters occur in various important genera of marine bacteria. The ABC transporter encoded by sitABCD of the model Roseobacter-clade bacterium Ruegeria pomeroyi DSS-3 is required for Mn(2+) import and was repressed by the Mur (Manganese uptake regulator) transcriptional regulator in Mn-replete media. Most genome-sequenced Roseobacter strains contain SitABCD, which are in at least two sub-groups, judged by their amino-acid sequences. However, a few Roseobacters, for example, Roseovarius nubinhibens, lack sitABCD, but these contain another gene, mntX, which encodes a predicted inner membrane polypeptide and is preceded by cis-acting Mur-responsive MRS sequences. It was confirmed directly that mntX of Roseovarius nubinhibens encodes a manganese transporter that was required for growth in Mn-depleted media and that its expression was repressed by Mur in Mn-replete conditions. MntX homologues occur in the deduced proteomes of several bacterial species. Strikingly, all of these live in marine habitats, but are in distantly related taxonomic groups, in the γ- and α-proteobacteria. Notably, MntX was prevalent in nearly all strains of Vibrionales, including the important pathogen, Vibrio cholerae. It also occurs in a strain of the hugely abundant Candidatus Pelagibacter (SAR11), and in another populous marine bacterium, Candidatus Puniceispirillum marinum (SAR116). Consistent with this, MntX was abundant in marine bacterial metagenomes, with one sub-type occurring in an as-yet unknown bacterial clade.

Complete genome sequence of the melanogenic marine bacterium Marinomonas mediterranea type strain (MMB-1(T)).

Marinomonas mediterranea MMB-1(T) Solano & Sanchez-Amat 1999 belongs to the family Oceanospirillaceae within the phylum Proteobacteria. This species is of interest because it is the only species described in the genus Marinomonas to date that can synthesize melanin pigments, which is mediated by the activity of a tyrosinase. M. mediterranea expresses other oxidases of biotechnological interest, such as a multicopper oxidase with laccase activity and a novel L-lysine-epsilon-oxidase. The 4,684,316 bp long genome harbors 4,228 protein-coding genes and 98 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

The Ruegeria pomeroyi acuI gene has a role in DMSP catabolism and resembles yhdH of E. coli and other bacteria in conferring resistance to acrylate.

The Escherichia coli YhdH polypeptide is in the MDR012 sub-group of medium chain reductase/dehydrogenases, but its biological function was unknown and no phenotypes of YhdH(-) mutants had been described. We found that an E. coli strain with an insertional mutation in yhdH was hyper-sensitive to inhibitory effects of acrylate, and, to a lesser extent, to those of 3-hydroxypropionate. Close homologues of YhdH occur in many Bacterial taxa and at least two animals. The acrylate sensitivity of YhdH(-) mutants was corrected by the corresponding, cloned homologues from several bacteria. One such homologue is acuI, which has a role in acrylate degradation in marine bacteria that catabolise dimethylsulfoniopropionate (DMSP) an abundant anti-stress compound made by marine phytoplankton. The acuI genes of such bacteria are often linked to ddd genes that encode enzymes that cleave DMSP into acrylate plus dimethyl sulfide (DMS), even though these are in different polypeptide families, in unrelated bacteria. Furthermore, most strains of Roseobacters, a clade of abundant marine bacteria, cleave DMSP into acrylate plus DMS, and can also demethylate it, using DMSP demethylase. In most Roseobacters, the corresponding gene, dmdA, lies immediately upstream of acuI and in the model Roseobacter strain Ruegeria pomeroyi DSS-3, dmdA-acuI were co-regulated in response to the co-inducer, acrylate. These observations, together with findings by others that AcuI has acryloyl-CoA reductase activity, lead us to suggest that YdhH/AcuI enzymes protect cells against damaging effects of intracellular acryloyl-CoA, formed endogenously, and/or via catabolising exogenous acrylate. To provide "added protection" for bacteria that form acrylate from DMSP, acuI was recruited into clusters of genes involved in this conversion and, in the case of acuI and dmdA in the Roseobacters, their co-expression may underpin an interaction between the two routes of DMSP catabolism, whereby the acrylate product of DMSP lyases is a co-inducer for the demethylation pathway.

Phylogenetic diversity of the dddP gene for dimethylsulfoniopropionate-dependent dimethyl sulfide synthesis in mangrove soils.

The dddP gene encodes an enzyme that cleaves dimethylsulfoniopropionate (DMSP) into dimethyl sulfide (DMS) plus acrylate and has been identified in various marine bacteria and some fungi. The diversity of dddP genes was investigated by culture-independent PCR-based analysis of metagenomic DNA extracted from 4 mangrove soils in Southern China. A phylogenetic tree of 144 cloned dddP sequences comprised 7 groups, 3 of which also included dddP genes from previously identified Ddd(+) (DMSP-dependent DMS production) bacteria. However, most (69%) of the DddP sequences from the mangroves were in 4 other subgroups that did not include sequences from known bacteria, demonstrating a high level of diversity of this gene in these environments. Each clade contained clones from all of the sample sites, suggesting that different dddP types are widespread in mangroves of different geographical locations. Furthermore, it was found the dddP genotype distribution was remarkably influenced by the soil properties pH, available sulfur, salt, and total nitrogen.

DddW, a third DMSP lyase in a model Roseobacter marine bacterium, Ruegeria pomeroyi DSS-3.

Ruegeria pomeroyi DSS-3 is a model Roseobacter marine bacterium, particularly regarding its catabolism of dimethylsulfoniopropionate (DMSP), an abundant anti-stress molecule made by marine phytoplankton. We found a novel gene, dddW, which encodes a DMSP lyase that cleaves DMSP into acrylate plus the environmentally important volatile dimethyl sulfide (DMS). Mutations in dddW reduced, but did not abolish DMS production. Transcription of dddW was greatly enhanced by pre-growth of cells with DMSP, via a LysR-type regulator. Close DddW homologs occur in only one other Roseobacter species, and there are no close homologs and only a few related sequences in metagenomes of marine bacteria. In addition to DddW, R. pomeroyi DSS-3 had been shown to have two other, different, DMSP lyases, DddP and DddQ, plus an enzyme that demethylates DMSP, emphasizing the importance of this substrate for this model bacterium.

Complete genome sequence of Marinomonas posidonica type strain (IVIA-Po-181(T)).

Marinomonas posidonica IVIA-Po-181(T) Lucas-Elío et al. 2011 belongs to the family Oceanospirillaceae within the phylum Proteobacteria. Different species of the genus Marinomonas can be readily isolated from the seagrass Posidonia oceanica. M. posidonica is among the most abundant species of the genus detected in the cultured microbiota of P. oceanica, suggesting a close relationship with this plant, which has a great ecological value in the Mediterranean Sea, covering an estimated surface of 38,000 Km(2). Here we describe the genomic features of M. posidonica. The 3,899,940 bp long genome harbors 3,544 protein-coding genes and 107 RNA genes and is a part of the GenomicEncyclopedia ofBacteriaandArchaea project.

Catabolism of dimethylsulphoniopropionate: microorganisms, enzymes and genes.

The compatible solute dimethylsulphoniopropionate (DMSP) has important roles in marine environments. It is an anti-stress compound made by many single-celled plankton, some seaweeds and a few land plants that live by the shore. Furthermore, in the oceans it is a major source of carbon and sulphur for marine bacteria that break it down to products such as dimethyl sulphide, which are important in their own right and have wide-ranging effects, from altering animal behaviour to seeding cloud formation. In this Review, we describe how recent genetic and genomic work on the ways in which several different bacteria, and some fungi, catabolize DMSP has provided new and surprising insights into the mechanisms, regulation and possible evolution of DMSP catabolism in microorganisms.

Heme binding to the second, lower-affinity site of the global iron regulator Irr from Rhizobium leguminosarum promotes oligomerization.

The iron responsive regulator Irr is found in a wide range of α-proteobacteria, where it regulates many genes in response to the essential but toxic metal iron. Unlike Fur, the transcriptional regulator that is used for iron homeostasis by almost all other bacterial lineages, Irr does not sense Fe(2+) directly, but, rather, interacts with a physiologically important form of iron, namely heme. Recent studies of Irr from the N(2)-fixing symbiont Rhizobium leguminosarum (Irr(Rl)) showed that it binds heme with submicromolar affinity at a His-Xxx-His (HxH) motif. This caused the protein to dissociate from its cognate DNA regulatory iron control element box sequences, thus allowing expression of its target genes under iron-replete conditions. In the present study, we report new insights into the mechanisms and consequences of heme binding to Irr. In addition to the HxH motif, Irr binds heme at a second, lower-affinity site. Spectroscopic studies of wild-type Irr and His variants show that His46 and probably His66 are involved in coordinating heme in a low-spin state at this second site. By contrast to the well-studied Irr from Bradyrhizobium japonicum, neither heme site of Irr(Rl) stabilizes ferrous heme. Furthermore, we show that heme-free Irr(Rl) exists as a mixture of dimeric and larger, likely hexameric, forms and that heme binding promotes Irr(Rl) oligomerization. Bioanalytical studies of Irr(Rl) variants showed that this property is not dependent on the HxH motif but is associated with heme binding at the second site. STRUCTURED DIGITAL ABSTRACT: • Irr binds to irr by molecular sieving (View Interaction 1, 2) • Irr binds to irr by cosedimentation in solution (View interaction).

DddY, a periplasmic dimethylsulfoniopropionate lyase found in taxonomically diverse species of Proteobacteria.

The abundant compatible solute dimethylsulfoniopropionate (DMSP) is made by many marine algae. Different marine bacteria catabolise DMSP by various mechanisms, some of which liberate the environmentally important gas dimethyl sulfide (DMS). We describe an enzyme, DddY, which cleaves DMSP into DMS plus acrylate and is located in the bacterial periplasm, unlike other DMSP lyases that catalyse this reaction. There are dddY-like genes in strains of Alcaligenes, Arcobacter and Shewanella, in the ß-, ɛ- and γ-proteobacteria, respectively. In Alcaligenes, dddY is in a cluster of ddd and acu genes that resemble, but also have significant differences to, those in other bacteria that catabolise both DMSP and acrylate. Although production of DMS and transcription of Alcaligenes dddY are both apparently inducible by pre-growth of cells with DMSP, this substrate must be catabolised to form acrylate, the bona fide coinducer.

Unusual regulation of a leaderless operon involved in the catabolism of dimethylsulfoniopropionate in Rhodobacter sphaeroides.

Rhodobacter sphaeroides strain 2.4.1 is a widely studied bacterium that has recently been shown to cleave the abundant marine anti-stress molecule dimethylsulfoniopropionate (DMSP) into acrylate plus gaseous dimethyl sulfide. It does so by using a lyase encoded by dddL, the promoter-distal gene of a three-gene operon, acuR-acuI-dddL. Transcription of the operon was enhanced when cells were pre-grown with the substrate DMSP, but this induction is indirect, and requires the conversion of DMSP to the product acrylate, the bona fide co-inducer. This regulation is mediated by the product of the promoter-proximal gene acuR, a transcriptional regulator in the TetR family. AcuR represses the operon in the absence of acrylate, but this is relieved by the presence of the co-inducer. Another unusual regulatory feature is that the acuR-acuI-dddL mRNA transcript is leaderless, such that acuR lacks a Shine-Dalgarno ribosomal binding site and 5'-UTR, and is translated at a lower level compared to the downstream genes. This regulatory unit may be quite widespread in bacteria, since several other taxonomically diverse lineages have adjacent acuR-like and acuI-like genes; these operons also have no 5' leader sequences or ribosomal binding sites and their predicted cis-acting regulatory sequences resemble those of R. sphaeroides acuR-acuI-dddL.

DddQ, a novel, cupin-containing, dimethylsulfoniopropionate lyase in marine roseobacters and in uncultured marine bacteria.

Ruegeria (previously Silicibacter) pomeroyi DSS-3, a marine roseobacter, can catabolize dimethylsulfoniopropionate (DMSP), a compatible solute that is made in large amounts by marine plankton and algae. This strain was known to demethylate DMSP via a demethylase, encoded by the dmdA gene, and it can also cleave DMSP, releasing the environmentally important volatile dimethyl sulfide (DMS) in the process. We found that this strain has two different genes, dddP and dddQ, which encode enzymes that cleave DMSP, generating DMS plus acrylate. DddP had earlier been found in other roseobacters and is a member of the M24 family of peptidases. The newly discovered DddQ polypeptide contains a predicted cupin metal-binding pocket, but has no other similarity to any other polypeptide with known function. DddP(-) and DddQ(-) mutants each produced DMS at significantly reduced levels compared with wild-type R. pomeroyi DSS-3, and transcription of the corresponding ddd genes was enhanced when cells were pre-grown with DMSP. Ruegeria pomeroyi DSS-3 also has a gene product that is homologous to DddD, a previously identified enzyme that cleaves DMSP, but which forms DMS plus 3-OH-propionate as the initial catabolites. However, mutations in this dddD-like gene did not affect DMS production, and it was not transcribed under our conditions. Another roseobacter strain, Roseovarius nubinhibens ISM, also contains dddP and has two functional copies of dddQ, encoded by adjacent genes. Judged by their frequencies in the Global Ocean Sampling metagenomic databases, DddP and DddQ are relatively abundant among marine bacteria compared with the previously identified DddL and DddD enzymes.

Wide host-range cloning for functional metagenomics.

We describe how wide host-range cloning vectors can lead to more flexible and effective procedures to isolate novel genes by screening metagenomic libraries in a range of bacterial hosts, not just the conventionally used Escherichia coli. We give examples of various wide host-range plasmid, cosmid, and BAC cloning vectors and the types of genes and activities that have been successfully obtained to date. We present a detailed protocol that involves the construction and screening of a metagenomic library comprising fragments of bacterial DNA, obtained from a wastewater treatment plant and cloned in a wide host-range cosmid. We also consider future prospects and how techniques and tools can be improved.

The dddP gene of Roseovarius nubinhibens encodes a novel lyase that cleaves dimethylsulfoniopropionate into acrylate plus dimethyl sulfide.

The cloned dddP gene of the marine bacterium Roseovarius nubinhibens allows Escherichia coli to form the volatile dimethyl sulfide (DMS) from dimethylsulfoniopropionate (DMSP), an abundant anti-stress compatible solute made by many marine plankton and macroalgae. Using purified DddP, we show here that this enzyme is a DMSP lyase that cleaves DMSP to DMS plus acrylate. DddP forms a functional homodimeric enzyme, has a pH optimum of 6.0 and was a K(m) of approximately 14 mM for the DMSP substrate. DddP belongs to the M24B family of peptidases, some members of which have metal cofactors. However, the metal chelators EDTA and bipyridyl did not affect DddP activity in vitro and the as-isolated enzyme did not contain metal ions. Thus, DddP resembles those members of the M24B family, such as creatinase, which also act on a non-peptide substrate and have no metal cofactor. Site-directed mutagenesis of the active-site region of DddP completely abolished its activity. Another enzyme, termed DddL, which occurs in other alphaproteobacteria, had also been shown to generate DMS plus acrylate from DMSP. However, DddL and DddP have no sequence similarity to each other, so DddP represents a second, wholly different class of DMSP lyase.