Background: Adjuvant therapies have been approved for patients with AJCC (American Joint Committee on Cancer) stage III and stage IV cutaneous melanoma (CM) after complete resection. These therapies might also be indicated for patients with high-risk stage II CM. Material and methods: We included patients diagnosed with stage II melanoma between 2000 and 2016 and for which primary tumour tissue was available. The prognostic value of the 11gene expression profiling score (GEPS) was evaluated as a dichotomized parameter (GEPS 0 vs. >0). Endpoints of the analysis were melanoma specific survival (MSS), distant metastasis-free survival (DMFS) and relapse-free survival (RFS). Results: GEPS was determined in 245 patients ranging between À0.7 and 3.53. A total of 111 females and 134 males were included; the median follow-up was 41 months. Kaplan Meier analyses showed statistically significant survival differences between patients with high GEPS (n Z 154) and low GEPS (n Z 91) for MSS (p Z 0.018), DMFS (p Z 0.005) and RFS (p Z 0.009). The 5-year and 10-year MSS was 92% in the low-GEPS and 82% and 67% in

Many enzymes require the molybdenum cofactor, Moco. Under Mo-limiting conditions, the high-affinity ABC transporter ModABC permits molybdate uptake and Moco biosynthesis in bacteria. Under Mo-replete conditions, Escherichia coli represses modABC transcription by the one-component regulator, ModE, consisting of a DNA-binding and a molybdate-sensing domain. Instead of a full-length ModE protein, many bacteria have a shorter ModE protein, ModE(S) , consisting of a DNA-binding domain only. Here, we asked how such proteins sense the intracellular molybdenum status. We show that the Agrobacterium tumefaciens ModE(S) protein Atu2564 is essential for modABC repression. ModE(S) binds two Mo-boxes in the modA promoter as shown by electrophoretic mobility shift assays. Northern analysis revealed cotranscription of modE(S) with the upstream gene, atu2565, which was dispensable for ModE(S) activity. To identify genes controlling ModE(S) function, we performed transposon mutagenesis. Tn5 insertions resulting in derepressed modA transcription mapped to the atu2565-modE(S) operon and several Moco biosynthesis genes. We conclude that A. tumefaciens ModE(S) activity responds to Moco availability rather than to molybdate concentration directly, as is the case for E. coli ModE. Similar results in Sinorhizobium meliloti suggest that Moco dependence is a common feature of ModE(S) regulators.

Rhodobacter capsulatus is able to grow with N 2 as the sole nitrogen source using either a molybdenum-dependent or a molybdenum-free iron-only nitrogenase whose expression is strictly inhibited by ammonium. Disruption of the fdxD gene, which is located directly upstream of the Mo-nitrogenase genes, nifHDK, abolished diazotrophic growth via Mo-nitrogenase at oxygen concentrations still tolerated by the wild type, thus demonstrating the importance of FdxD under semiaerobic conditions. In contrast, FdxD was not beneficial for diazotrophic growth depending on Fe-nitrogenase. These findings suggest that the 2Fe2S ferredoxin FdxD specifically supports the Mo-nitrogenase system, probably by protecting Mo-nitrogenase against oxygen, as previously shown for its Azotobacter vinelandii counterpart, FeSII. Expression of fdxD occurred under nitrogen-fixing conditions, but not in the presence of ammonium. Expression of fdxD strictly required NifA1 and NifA2, the transcriptional activators of the Mo-nitrogenase genes, but not AnfA, the transcriptional activator of the Fe-nitrogenase genes. Expression of the fdxD and nifH genes, as well as the FdxD and NifH protein levels, increased with increasing molybdate concentrations. Molybdate induction of fdxD was independent of the molybdate-sensing regulators MopA and MopB, which repress anfA transcription at micromolar molybdate concentrations. In this report, we demonstrate the physiological relevance of an fesII-like gene, fdxD, and show that the cellular nitrogen and molybdenum statuses are integrated to control its expression.

Rhodobacter capsulatus fixes atmospheric dinitrogen via two nitrogenases, Mo-and Fe-nitrogenase, which operate under different conditions. Here, we describe the functions in nitrogen fixation and regulation of the rcc00574 (cooA) and rcc00575 (cowN) genes, which are located upstream of the structural genes of Mo-nitrogenase, nifHDK. Disruption of cooA or cowN specifically impaired Mo-nitrogenase-dependent growth at carbon monoxide (CO) concentrations still tolerated by the wild type. The cooA gene was shown to belong to the Mo-nitrogenase regulon, which is exclusively expressed when ammonium is limiting. Its expression was activated by NifA1 and NifA2, the transcriptional activators of nifHDK. AnfA, the transcriptional activator of Fe-nitrogenase genes, repressed cooA, thereby counteracting NifA activation. CooA activated cowN expression in response to increasing CO concentrations. Base substitutions in the presumed CooA binding site located upstream of the cowN transcription start site abolished cowN expression, indicating that cowN regulation by CooA is direct. In conclusion, a transcription factor-based network controls cowN expression to protect Mo-nitrogenase (but not Fe-nitrogenase) under appropriate conditions.

Rhodobacter capsulatus is capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N 2 ) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, and lacZ reporter fusions. Biological nitrogen fixation (BNF) is a central process in the global nitrogen cycle, which converts chemically inert atmospheric dinitrogen to ammonia, a bioavailable form of nitrogen. BNF is catalyzed by complex metalloenzymes called nitrogenases, which are exclusively found in diazotrophic bacteria and archaea but not in eukaryotes (1). All diazotrophs synthesize a molybdenum-dependent nitrogenase containing an iron-molybdenum cofactor, FeMoco. In addition to Mo-nitrogenases, some bacteria synthesize alternative Mo-free nitrogenases, which contain either an iron-vanadium cofactor, FeVco, or an iron-only cofactor, FeFeco (2). Alternative nitrogenases are less efficient than Monitrogenases in terms of consumption of reducing power and ATP per molecule of N 2 fixed (3, 4). Consequently, diazotrophs preferentially utilize Mo-nitrogenase as long as sufficient molybdate, the only bioavailable form of molybdenum, is available. To support Mo-nitrogenase activity under Mo-limiting conditions, most diazotrophs synthesize a high-affinity molybdate transporter, ModABC (1).The purple nonsulfur alphaproteobacterium Rhodobacter capsulatus is known for its metabolic versatility, and it has been used for decades as a model organism to study photosynthesis, hydrogen production, and nitrogen fixation (5-9). In particular, it is capable of using light energy to generate the ATP required for the energetically demanding nitrogen fixation process. R. capsulatus synthesizes two nitrogenases, namely, a Mo-nitrogenase and a Fenitrogenase but no V-nitrogenase (10, 11). The synthesis and activity of the two nitrogenases are controlled at the transcriptional, translational, and posttranslational levels by a regulatory cascade responding to ammonium and Mo availability (8, 9). Upon ammonium limitation, the nitrogen regulatory protein NtrC becomes activated by phosphorylation. In turn, NtrC-P activates transcription of nifA and anfA, which encode the transcription activators of Mo-nitrogenase and Fe-nitrogenase genes, respectively. At high Mo concentrations, anfA transcription is repressed by two structurally and functionally related Mo-responsive regu-

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Rhodobacter capsulatus synthesizes the high-affinity ABC transporters CysTWA and ModABC to specifically import the chemically related oxyanions sulfate and molybdate, respectively. In addition, R. capsulatus has the low-affinity permease PerO acting as a general oxyanion transporter, whose elimination increases tolerance to molybdate and tungstate. Although PerO-like permeases are widespread in bacteria, their function has not been examined in any other species to date. Here, we present evidence that PerO permeases from the alphaproteobacteria Agrobacterium tumefaciens, Dinoroseobacter shibae, Rhodobacter sphaeroides, and Sinorhizobium meliloti and the gammaproteobacterium Pseudomonas stutzeri functionally substitute for R. capsulatus PerO in sulfate uptake and sulfate-dependent growth, as shown by assimilation of radioactively labeled sulfate and heterologous complementation. Disruption of perO genes in A. tumefaciens, R. sphaeroides, and S. meliloti increased tolerance to tungstate and, in the case of R. sphaeroides, to molybdate, suggesting that heterometal oxyanions are common substrates of PerO permeases. This study supports the view that bacterial PerO permeases typically transport sulfate and related oxyanions and, hence, form a functionally conserved permease family.IMPORTANCE Despite the widespread distribution of PerO-like permeases in bacteria, our knowledge about PerO function until now was limited to one species, Rhodobacter capsulatus. In this study, we showed that PerO proteins from diverse bacteria are functionally similar to the R. capsulatus prototype, suggesting that PerO permeases form a conserved family whose members transport sulfate and related oxyanions.KEYWORDS transport, sulfate, molybdate, tungstate, oxyanions, permease, PerO, Rhodobacter, ABC transporters T he oxyanions sulfate (SO 4 2-) and molybdate (MoO 4 2-) are the preferred sources for sulfur and molybdenum, respectively, in bacteria. To specifically import these structurally similar oxyanions, Escherichia coli synthesizes the ATP-binding cassette (ABC) transporters CysPTWA and ModABC (1, 2). ABC uptake systems consist of a periplasmic substrate binding protein, a transmembrane channel protein, and a cytoplasmic ATP-binding protein, which provides the energy for active substrate transport across the cytoplasmic membrane against a concentration gradient (3). In addition to molybdate, ModABC imports tungstate (WO 4 2-) but not sulfate (4, 5). In addition to sulfate, CysPTWA imports molybdate, albeit less efficiently than its primary substrate sulfate (6, 7).Recently, we described the permease PerO mediating transport of sulfate, molybdate, and tungstate in the photosynthetic purple nonsulfur bacterium Rhodobacter capsulatus (8). PerO belongs to the ArsB/NhaD ion transporter superfamily, whose members encompass 10 to 13 transmembrane domains and have diverse functions such as arsenite export, citrate/succinate antiport, and Na ϩ /H ϩ antiport. PerO contains two central TrkA_C domains, making this permease considerably larg...

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