A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition.

Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.

Marinomonas mediterranea synthesizes an R-type bacteriocin.

Prophages integrated into bacterial genomes can become cryptic or defective prophages, which may evolve to provide various traits to bacterial cells. Previous research on Marinomonas mediterranea MMB-1 demonstrated the production of defective particles. In this study, an analysis of the genomes of three different strains (MMB-1, MMB-2, and MMB-3) revealed the presence of a region named MEDPRO1, spanning approximately 52 kb, coding for a defective prophage in strains MMB-1 and MMB-2. This prophage seems to have been lost in strain MMB-3, possibly due to the presence of spacers recognizing this region in an I-F CRISPR array in this strain. However, all three strains produce remarkably similar defective particles. Using strain MMB-1 as a model, mass spectrometry analyses indicated that the structural proteins of the defective particles are encoded by a second defective prophage situated within the MEDPRO2 region, spanning approximately 13 kb. This finding was further validated through the deletion of this second defective prophage. Genomic region analyses and the detection of antimicrobial activity of the defective prophage against other Marinomonas species suggest that it is an R-type bacteriocin. Marinomonas mediterranea synthesizes antimicrobial proteins with lysine oxidase activity, and the synthesis of an R-type bacteriocin constitutes an additional mechanism in microbial competition for the colonization of habitats such as the surface of marine plants.IMPORTANCEThe interactions between bacterial strains inhabiting the same environment determine the final composition of the microbiome. In this study, it is shown that some extracellular defective phage particles previously observed in Marinomonas mediterranea are in fact R-type bacteriocins showing antimicrobial activity against other Marinomonas strains. The operon coding for the R-type bacteriocin has been identified.

Characterization of recombinant biosynthetic precursors of the cysteine tryptophylquinone cofactors of l-lysine-epsilon-oxidase and glycine oxidase from Marinomonas mediterranea.

The lysine-ε-oxidase, LodA, and glycine oxidase, GoxA, from Marinomonas mediteranea each possesses a cysteine tryptophylquinone (CTQ) cofactor. This cofactor is derived from posttranslational modifications which are covalent crosslinking of tryptophan and cysteine residues and incorporation of two oxygen atoms into the indole ring of Trp. In this manuscript, it is shown that the recombinant synthesis of LodA and GoxA containing a fully synthesized CTQ cofactor requires coexpression of a partner flavoprotein, LodB for LodA and GoxB for GoxA, which are not interchangeable. An inactive precursor of LodA or GoxA which contained a monohydroxylated Trp residue and no crosslink to the Cys was isolated from the soluble fraction when they were expressed alone. The structure of LodA revealed an Asp residue close to the cofactor which is conserved in quinohemoprotein amine dehydrogenase (QHNDH), containing CTQ, and methylamine dehydrogenase (MADH) containing tryptophan tryptophylquinone (TTQ) as cofactor. To study the role of this residue in the synthesis of the LodA precursor, Asp-512 was mutated to Ala. When the mutant protein was coexpressed with LodB an inactive protein was isolated which was soluble and contained no modifications at all, suggesting a role for this Asp in the initial LodB-independent hydroxylation of Trp. A similar role had been proposed for this conserved Asp residue in MADH. It is noteworthy that the formation of TTQ in MADH from the precursor also requires an accessory enzyme for its biosynthesis but it is a diheme enzyme MauG and not a flavoprotein. The results presented reveal novel mechanisms of post-translational modification involved in the generation of protein-derived cofactors. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Distribution in microbial genomes of genes similar to lodA and goxA which encode a novel family of quinoproteins with amino acid oxidase activity.

BACKGROUND: L-Amino acid oxidases (LAOs) have been generally described as flavoproteins that oxidize amino acids releasing the corresponding ketoacid, ammonium and hydrogen peroxide. The generation of hydrogen peroxide gives to these enzymes antimicrobial characteristics. They are involved in processes such as biofilm development and microbial competition. LAOs are of great biotechnological interest in different applications such as the design of biosensors, biotransformations and biomedicine. The marine bacterium Marinomonas mediterranea synthesizes LodA, the first known LAO that contains a quinone cofactor. LodA is encoded in an operon that contains a second gene coding for LodB, a protein required for the post-translational modification generating the cofactor. Recently, GoxA, a quinoprotein with sequence similarity to LodA but with a different enzymatic activity (glycine oxidase instead of lysine-ε-oxidase) has been described. The aim of this work has been to study the distribution of genes similar to lodA and/or goxA in sequenced microbial genomes and to get insight into the evolution of this novel family of proteins through phylogenetic analysis. RESULTS: Genes encoding LodA-like proteins have been detected in several bacterial classes. However, they are absent in Archaea and detected only in a small group of fungi of the class Agaromycetes. The vast majority of the genes detected are in a genome region with a nearby lodB-like gene suggesting a specific interaction between both partner proteins. Sequence alignment of the LodA-like proteins allowed the detection of several conserved residues. All of them showed a Cys and a Trp that aligned with the residues that are forming part of the cysteine tryptophilquinone (CTQ) cofactor in LodA. Phylogenetic analysis revealed that LodA-like proteins can be clustered in different groups. Interestingly, LodA and GoxA are in different groups, indicating that those groups are related to the enzymatic activity of the proteins detected. CONCLUSIONS: Genome mining has revealed for the first time the broad distribution of LodA-like proteins containing a CTQ cofactor in many different microbial groups. This study provides a platform to explore the potentially novel enzymatic activities of the proteins detected, the mechanisms of post-translational modifications involved in their synthesis, as well as their biological relevance.

Distribution in Different Organisms of Amino Acid Oxidases with FAD or a Quinone As Cofactor and Their Role as Antimicrobial Proteins in Marine Bacteria.

Amino acid oxidases (AAOs) catalyze the oxidative deamination of amino acids releasing ammonium and hydrogen peroxide. Several kinds of these enzymes have been reported. Depending on the amino acid isomer used as a substrate, it is possible to differentiate between l-amino acid oxidases and d-amino acid oxidases. Both use FAD as cofactor and oxidize the amino acid in the alpha position releasing the corresponding keto acid. Recently, a novel class of AAOs has been described that does not contain FAD as cofactor, but a quinone generated by post-translational modification of residues in the same protein. These proteins are named as LodA-like proteins, after the first member of this group described, LodA, a lysine epsilon oxidase synthesized by the marine bacterium Marinomonas mediterranea. In this review, a phylogenetic analysis of all the enzymes described with AAO activity has been performed. It is shown that it is possible to recognize different groups of these enzymes and those containing the quinone cofactor are clearly differentiated. In marine bacteria, particularly in the genus Pseudoalteromonas, most of the proteins described as antimicrobial because of their capacity to generate hydrogen peroxide belong to the group of LodA-like proteins.

LodB is required for the recombinant synthesis of the quinoprotein L-lysine-ε-oxidase from Marinomonas mediterranea.

Marinomonas mediterranea is a marine gamma-proteobacterium that synthesizes LodA, a novel L-lysine-ε-oxidase (E.C. 1.4.3.20). This enzyme oxidizes L-lysine generating 2-aminoadipate 6-semialdehyde, ammonium, and hydrogen peroxide. Unlike other L-amino acid oxidases, LodA is not a flavoprotein but contains a quinone cofactor. LodA is encoded by an operon with two genes, lodA and lodB. In the native system, LodB is required for the synthesis of a functional LodA. In this study, we report the recombinant expression of LodA in Escherichia coli using vectors that allow its expression and accumulation in the cytoplasm. To reveal the L-lysine-ε-oxidase activity using the Amplex Red method for hydrogen peroxide detection, it is necessary to first remove the E. coli cytoplasmic catalases. The flavoprotein LodB is the only M. mediterranea protein required in the recombinant system for the generation of the cofactor of LodA. In the absence of LodB, LodA does not contain the quinone cofactor and remains in an inactive form. The results presented indicate that LodB participates in the posttranslational modification of LodA that generates the quinone cofactor.

Both genes in the Marinomonas mediterranea lodAB operon are required for the expression of the antimicrobial protein lysine oxidase.

The melanogenic marine bacterium Marinomonas mediterranea synthesizes a novel antimicrobial protein (LodA) with lysine-epsilon oxidase activity (EC 1.4.3.20). Homologues to LodA have been detected in several Gram-negative bacteria, where they are involved in biofilm development. Adjacent to lodA is located a second gene, lodB, of unknown function. This genomic organization is maintained in all the microorganisms containing homologues to these genes. In this work we show that lodA and lodB constitute an operon. Western blot analysis and enzymatic determinations revealed that LodA is secreted to the external medium when the culture reaches the stationary phase. LodB, on the other hand, has only been detected inside cells, but it is not secreted. The expression of the lysine-epsilon oxidase (LOD) activity in M. mediterranea requires functional copies of both genes since mutants lacking either lodA or lodB do not show any LOD activity. The active form of LodA containing the quinonic cofactor is intracellularly generated in a process that takes place only in the presence of LodB, suggesting that the latter is involved in this process. Moreover, in the absence of one of the proteins, the stability of the partner protein is compromised leading to a marked decrease in its cellular levels.

Marinomonas alcarazii sp. nov., M. rhizomae sp. nov., M. foliarum sp. nov., M. posidonica sp. nov. and M. aquiplantarum sp. nov., isolated from the microbiota of the seagrass Posidonia oceanica.

Five novel Gram-reaction-negative aerobic marine bacterial strains with DNA G+C contents <50 mol% were isolated from the seagrass Posidonia oceanica. 16S rRNA sequence analysis indicated that they belonged to the genus Marinomonas. Major fatty acid compositions, comprising C10:0 3-OH, C16:0, C16:1ω7c and C18:1ω7c, supported the affiliation of these strains to the genus Marinomonas. Strains IVIA-Po-14b(T), IVIA-Po-145(T) and IVIA-Po-155(T) were closely related to Marinomonas pontica 46-16(T), according to phylogenetic analysis. However, DNA-DNA hybridization values <35 % among these strains revealed that they represented different species. Further differences in the phenotypes and minor fatty acid compositions were also found among the strains. Another two strains, designated IVIA-Po-181(T) and IVIA-Po-159(T), were found to be closely related to M. dokdonensis DSW10-10(T) but DNA-DNA relatedness levels <40 % in pairwise comparisons, as well as some additional differences in phenotypes and fatty acid compositions supported the creation of two novel species. Accordingly, strains IVIA-Po-14b(T )( = CECT 7730(T)  = NCIMB 14671(T)), IVIA-Po-145(T) ( = CECT 7377(T)  = NCIMB 14431(T)), IVIA-Po-155(T) ( = CECT 7731(T)  = NCIMB 14672(T)), IVIA-Po-181(T) ( = CECT 7376(T)  = NCIMB 14433(T)) and IVIA-Po-159(T) ( = CECT 7732(T)  = NCIMB 14673(T)) represent novel species, for which the names Marinomonas alcarazii sp. nov., Marinomonas rhizomae sp. nov., Marinomonas foliarum sp. nov., Marinomonas posidonica sp. nov. and Marinomonas aquiplantarum sp. nov. are proposed, respectively.

A histidine kinase and a response regulator provide phage resistance to Marinomonas mediterranea via CRISPR-Cas regulation.

CRISPR-Cas systems are used by many prokaryotes to defend against invading genetic elements. In many cases, more than one CRISPR-Cas system co-exist in the same cell. Marinomonas mediterranea MMB-1 possesses two CRISPR-Cas systems, of type I-F and III-B respectively, which collaborate in phage resistance raising questions on how their expression is regulated. This study shows that the expression of both systems is controlled by the histidine kinase PpoS and a response regulator, PpoR, identified and cloned in this study. These proteins show similarity to the global regulators BarA/UvrY. In addition, homologues to the sRNAs CsrB and CsrC and the gene coding for the post-transcriptional repressor CsrA have been also identified indicating the conservation of the elements of the BarA/UvrY regulatory cascade in M. mediterranea. RNA-Seq analyses have revealed that all these genetics elements are regulated by PpoS/R supporting their participation in the regulatory cascade. The regulation by PpoS and PpoR of the CRISPR-Cas systems plays a role in phage defense since mutants in these proteins show an increase in phage sensitivity.

Regulation of the Marinomonas mediterranea antimicrobial protein lysine oxidase by L-lysine and the sensor histidine kinase PpoS.

Some Gram-negative bacteria express a novel enzyme with lysine-epsilon-oxidase (LOD) activity (EC 1.4.3.20). The oxidation of l-Lys generates, among other products, hydrogen peroxide, which confers antimicrobial properties to this kind of enzyme and has been shown to be involved in cell death during biofilm development and differentiation. In addition to LOD, the melanogenic marine bacterium Marinomonas mediterranea, which forms part of the microbiota of the marine plant Posidonia oceanica, expresses two other oxidases of biotechnological interest, a multicopper oxidase, PpoA, with laccase activity and a tyrosinase named PpoB, which is responsible for melanin synthesis. By using both lacZ fusions with the lodAB promoter and quantitative reverse transcription-PCR (qRT-PCR), this study shows that the hybrid sensor histidine kinase PpoS regulates LOD activity at the transcriptional level. Although PpoS also regulates PpoA and PpoB, in this case, the regulatory effect cannot be attributed only to a transcriptional regulation. Further studies indicate that LOD activity is induced at the posttranscriptional level by l-Lys as well as by two structurally similar compounds, l-Arg and meso-2,6-diaminopimelic acid (DAP), neither of which is a substrate of the enzyme. The inducing effect of these compounds is specific for LOD activity since PpoA and PpoB are not affected by them. This study offers, for the first time, insights into the mechanisms regulating the synthesis of the antimicrobial protein lysine-epsilon-oxidase in M. mediterranea, which could be important in the microbial colonization of the seagrass P. oceanica.

Finding new enzymes from bacterial physiology: a successful approach illustrated by the detection of novel oxidases in Marinomonas mediterranea.

The identification and study of marine microorganisms with unique physiological traits can be a very powerful tool discovering novel enzymes of possible biotechnological interest. This approach can complement the enormous amount of data concerning gene diversity in marine environments offered by metagenomic analysis, and can help to place the activities associated with those sequences in the context of microbial cellular metabolism and physiology. Accordingly, the detection and isolation of microorganisms that may be a good source of enzymes is of great importance. Marinomonas mediterranea, for example, has proven to be one such useful microorganism. This Gram-negative marine bacterium was first selected because of the unusually high amounts of melanins synthesized in media containing the amino acid L-tyrosine. The study of its molecular biology has allowed the cloning of several genes encoding oxidases of biotechnological interest, particularly in white and red biotechnology. Characterization of the operon encoding the tyrosinase responsible for melanin synthesis revealed that a second gene in that operon encodes a protein, PpoB2, which is involved in copper transfer to tyrosinase. This finding made PpoB2 the first protein in the COG5486 group to which a physiological role has been assigned. Another enzyme of interest described in M. mediterranea is a multicopper oxidase encoding a membrane-associated enzyme that shows oxidative activity on a wide range of substrates typical of both laccases and tyrosinases. Finally, an enzyme very specific for L-lysine, which oxidises this amino acid in epsilon position and that has received a new EC number (1.4.3.20), has also been described for M. mediterranea. Overall, the studies carried out on this bacterium illustrate the power of exploring the physiology of selected microorganisms to discover novel enzymes of biotechnological relevance.

Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several gram-negative bacteria.

The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria.

A novel type of lysine oxidase: L-lysine-epsilon-oxidase.

The melanogenic marine bacterium M. mediterranea synthesizes marinocine, a protein with antibacterial activity. We cloned the gene coding for this protein and named it lodA [P. Lucas-Elío, P. Hernández, A. Sanchez-Amat, F. Solano, Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea. Biochim. Biophys. Acta 1721 (2005) 193-203; P. Lucas-Elío, D. Gómez, F. Solano, A. Sanchez-Amat, The antimicrobial activity of marinocine, synthesized by M. mediterranea, is due to the hydrogen peroxide generated by its lysine oxidase activity. J. Bacteriol. 188 (2006) 2493-2501]. Now, we show that this protein is a new type of lysine oxidase which catalyzes the oxidative deamination of free L-lysine into 6-semialdehyde 2-aminoadipic acid, ammonia and hydrogen peroxide. This new enzyme is compared to other enzymes related to lysine transformation. Two different groups have been used for comparison. Enzymes in the first group lead to 2-aminoadipic acid as a final product. The second one would be enzymes catalyzing the oxidative deamination of lysine releasing H2O2, namely lysine-alpha-oxidase (LalphaO) and lysyl oxidase (Lox). Kinetic properties, substrate specificity and inhibition pattern show clear differences with all above mentioned lysine-related enzymes. Thus, we propose to rename this enzyme lysine-epsilon-oxidase (lod for the gene) instead of marinocine. Lod shows high stereospecificity for free L-lysine, it is inhibited by substrate analogues, such as cadaverine and 6-aminocaproic acid, and also by beta-aminopropionitrile, suggesting the existence of a tyrosine-derived quinone cofactor at its active site.

Type III CRISPR-Cas systems can provide redundancy to counteract viral escape from type I systems.

CRISPR-Cas-mediated defense utilizes information stored as spacers in CRISPR arrays to defend against genetic invaders. We define the mode of target interference and role in antiviral defense for two CRISPR-Cas systems in Marinomonas mediterranea. One system (type I-F) targets DNA. A second system (type III-B) is broadly capable of acquiring spacers in either orientation from RNA and DNA, and exhibits transcription-dependent DNA interference. Examining resistance to phages isolated from Mediterranean seagrass meadows, we found that the type III-B machinery co-opts type I-F CRISPR-RNAs. Sequencing and infectivity assessments of related bacterial and phage strains suggests an 'arms race' in which phage escape from the type I-F system can be overcome through use of type I-F spacers by a horizontally-acquired type III-B system. We propose that the phage-host arms race can drive selection for horizontal uptake and maintenance of promiscuous type III interference modules that supplement existing host type I CRISPR-Cas systems.

Purification and partial characterization of marinocine, a new broad-spectrum antibacterial protein produced by Marinomonas mediterranea.

This work describes the purification and partial characterization of a novel antibacterial compound, here named marinocine, produced by Marinomonas mediterranea, a melanogenic marine bacterium with rich secondary metabolism. The antibacterial compound is a protein detected in the medium at death phase of growth. It has been purified to apparent homogeneity from the supernatants of cultures by means of ethanol precipitation followed by column chromatographies on DEAE-Sephadex and Sephacryl HR-200. The protein has an apparent molecular mass of 140-170 kDa according to gel permeation chromatography and non-denaturing SDS-PAGE, although in denaturing SDS-PAGE two mayor bands of 97 and 185 kDa appear. Marinocine is relatively heat-stable and shows a great resistance against many hydrolytic enzymes such as glycosidases, lipase, and proteases. The antibacterial range of the molecule includes Gram-positive and Gram-negative microorganisms, as well as some nosocomial isolates, Staphylococcus aureus and Pseudomonas sp., highly resistant to classical antibiotics. By contrast, marinocine did not show any effect on the eukaryotic microorganisms tested. Regarding eukaryotic CHO cells, the decrease on viability was much lower than the one observed on bacterial cells.

Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity.

A novel enzyme with lysine-epsilon oxidase activity was previously described in the marine bacterium Marinomonas mediterranea. This enzyme differs from other l-amino acid oxidases in not being a flavoprotein but containing a quinone cofactor. It is encoded by an operon with two genes lodA and lodB. The first one codes for the oxidase, while the second one encodes a protein required for the expression of the former. Genome sequencing of M. mediterranea has revealed that it contains two additional operons encoding proteins with sequence similarity to LodA. In this study, it is shown that the product of one of such genes, Marme_1655, encodes a protein with glycine oxidase activity. This activity shows important differences in terms of substrate range and sensitivity to inhibitors to other glycine oxidases previously described which are flavoproteins synthesized by Bacillus. The results presented in this study indicate that the products of the genes with different degrees of similarity to lodA detected in bacterial genomes could constitute a reservoir of different oxidases.

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.

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.

Taxonomic study of Marinomonas strains isolated from the seagrass Posidonia oceanica, with descriptions of Marinomonas balearica sp. nov. and Marinomonas pollencensis sp. nov.

Novel aerobic, Gram-negative bacteria with DNA G+C contents below 50 mol% were isolated from the culturable microbiota associated with the Mediterranean seagrass Posidonia oceanica. 16S rRNA gene sequence analyses revealed that they belong to the genus Marinomonas. Strain IVIA-Po-186 is a strain of the species Marinomonas mediterranea, showing 99.77 % 16S rRNA gene sequence similarity with the type strain, MMB-1(T), and sharing all phenotypic characteristics studied. This is the first description of this species forming part of the microbiota of a marine plant. A second strain, designated IVIA-Po-101(T), was closely related to M. mediterranea based on phylogenetic studies. However, it differed in characteristics such as melanin synthesis and tyrosinase, laccase and antimicrobial activities. In addition, strain IVIA-Po-101(T) was auxotrophic and unable to use acetate. IVIA-Po-101(T) shared 97.86 % 16S rRNA gene sequence similarity with M. mediterranea MMB-1(T), but the level of DNA-DNA relatedness between the two strains was only 10.3 %. On the basis of these data, strain IVIA-Po-101(T) is considered to represent a novel species of the genus Marinomonas, for which the name Marinomonas balearica sp. nov. is proposed. The type strain is IVIA-Po-101(T) (=CECT 7378(T) =NCIMB 14432(T)). A third novel strain, IVIA-Po-185(T), was phylogenetically distant from all recognized Marinomonas species. It shared the highest 16S rRNA gene sequence similarity (97.4 %) with the type strain of Marinomonas pontica, but the level of DNA-DNA relatedness between the two strains was only 14.5 %. A differential chemotaxonomic marker of this strain in the genus Marinomonas is the presence of the fatty acid C(17 : 0) cyclo. Strain IVIA-Po-185(T) is thus considered to represent a second novel species of the genus, for which the name Marinomonas pollencensis sp. nov. is proposed. The type strain is IVIA-Po-185(T) (=CECT 7375(T) =NCIMB 14435(T)). An emended description of the genus Marinomonas is given based on the description of these two novel species, as well as other Marinomonas species described after the original description of the genus.