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

Abstract

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.

Figure 1

Maps of sitABCD genes of Ruegeria pomeroyi and their genetically modified derivatives. The open arrows in the top section show the dimensions of the individual genes in the sitABCD operon, the corresponding gene tags being SPO3366, SPO3365, SPO3364 and SPO3363, respectively. Numbers of base pairs in intergenic spaces are shown, ‘−4' denoting overlap between sitC and sitD. The approximate locations of the two MRS regulatory sequences are indicated. The middle section shows the sequence upstream of the ATG start of sitA, the two MRS motifs being boxed. The conserved ‘TG' base pairs in MRS1 and in MRS2 that were changed by site-directed mutagenesis are highlighted. The dimensions of the 1086-bp fragment used to construct the sitA-lacZ transcriptional fusion plasmid pBIO2050 are shown.

Figure 2

Effects of sitA and of the mntX genes of Roseovarius nubinhibens, Ca. Pelagibacter ubique and Vibrio cholerae on growth of Ruegeria pomeroyi in Mn-depleted minimal media. Cultures of Ruegeria pomeroyi wild type, or the SitA^- mutant J529 or J529 corrected with cloned mntX of Roseovarius nubinhibens (pBIO2066), Ca. Pelagibacter ubique (pBIO2088) or V. cholerae (pBIO2150) were diluted into RSS minimal medium either lacking any added MnCl₂ (white squares), or supplemented with 10 μℳ MnCl₂ (black squares). Cultures were grown, with shaking, at 28 °C, and growth was measured by absorbance at 600 nm.

Figure 3

Neighbour-joining tree of MntX polypeptides in known bacteria and in the GOS marine metagenome. Using Mega5, a tree of the MntX homologues in the bacteria described in the text and in the GOS marine metagenome was constructed, bootstrap values being presented. These homologues were of five sub-classes as indicated, each corresponding to those found in the bacterial clade that is shown. Group ‘X' contains no sequences from any known bacterium. The number of GOS sequences found in each of the five groups are indicated. Accession numbers of the individual GOS sequences are in Supplementary Table S2 and blow-up versions of the five branches are shown in Supplementary Figure S3, A–E.