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

RirA is a global iron regulator in diverse 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.

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 C-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.

Sensing iron availability 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

Unlabelled: 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.

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.

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.

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.

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.

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.

Heme-responsive DNA binding by the global iron regulator Irr from Rhizobium leguminosarum.

Heme, a physiologically crucial form of iron, is a cofactor for a very wide range of proteins and enzymes. These include DNA regulatory proteins in which heme is a sensor to which an analyte molecule binds, effecting a change in the DNA binding affinity of the regulator. Given that heme, and more generally iron, must be carefully regulated, it is surprising that there are no examples yet in bacteria in which heme itself is sensed directly by a reversibly binding DNA regulatory protein.

Molecular dissection of bacterial acrylate catabolism--unexpected links with dimethylsulfoniopropionate catabolism and dimethyl sulfide production.

A bacterium in the genus Halomonas that grew on dimethylsulfoniopropionate (DMSP) or acrylate as sole carbon sources and that liberated the climate-changing gas dimethyl sulfide in media containing DMSP was obtained from the phylloplane of the macroalga Ulva. We identified a cluster that contains genes specifically involved in DMSP catabolism (dddD, dddT) or in degrading acrylate (acuN, acuK) or that are required to break down both substrates (dddC, dddA). Using NMR and HPLC analyses to trace 13C- or 14C-labelled acrylate and DMSP in strains of Escherichia coli with various combinations of cloned ddd and/or acu genes, we deduced that DMSP is imported by the BCCT-type transporter DddT, then converted by DddD to 3-OH-propionate (3HP), liberating dimethyl sulfide in the process.

The opportunistic coral pathogen Aspergillus sydowii contains dddP and makes dimethyl sulfide from dimethylsulfoniopropionate.

The ascomycete Aspergillus sydowii is associated with a serious epizootic of sea fan corals in the Caribbean. Corals are rich in the compatible solute, dimethylsulfoniopropionate (DMSP), produced by their symbionts, the dinoflagellate Symbiodinium. As other Aspergillus species can catabolize DMSP, liberating dimethyl sulfide (DMS) in the process, we tested A.

Identification of genes for dimethyl sulfide production in bacteria in the gut of Atlantic Herring (Clupea harengus).

Phytoplankton are the primary producers of the sulfur-containing compatible solute dimethylsulfoniopropionate (DMSP). These cells are consumed by mesozooplankton, which, in turn, may be eaten by marine vertebrates. From the gut of one such animal, the Atlantic Herring Clupea harengus, we isolated strains of the gamma-proteobacteria Pseudomonas and Psychrobacter that grew on DMSP as sole carbon source, and which produced the environmentally important sulfurous volatile dimethyl sulfide (DMS).

A common genomic framework for a diverse assembly of plasmids in the symbiotic nitrogen fixing bacteria.

This work centres on the genomic comparisons of two closely-related nitrogen-fixing symbiotic bacteria, Rhizobium leguminosarum biovar viciae 3841 and Rhizobium etli CFN42. These strains maintain a stable genomic core that is also common to other rhizobia species plus a very variable and significant accessory component. The chromosomes are highly syntenic, whereas plasmids are related by fewer syntenic blocks and have mosaic structures.