The production of N-acyl homoserine lactones (AHLs) is widely distributed within the marine Roseobacter clade, and it was proposed that AHL-mediated quorum sensing (QS) is one of the most common cell-to-cell communication mechanisms in roseobacters. The traits regulated by AHL-mediated QS are yet not known for members of the Roseobacter clade, but production of the antibiotic tropodithietic acid (TDA) was supposed to be controlled by AHL-mediated QS in Phaeobacter spp. We describe here for the first time the functional role of luxR and luxI homologous genes of an organism of the Roseobacter clade, i.e., pgaR and pgaI in Phaeobacter gallaeciensis. Our results demonstrate that the AHL synthase gene pgaI is responsible for production of N-3-hydroxydecanoylhomoserine lactone (3OHC 10 -HSL). Insertion mutants of pgaI and pgaR are both deficient in TDA biosynthesis and the formation of a yellow-brown pigment when grown in liquid marine broth medium. This indicates that in P. gallaeciensis the production of both secondary metabolites is controlled by AHLmediated QS. Quantitative real-time PCR showed that the transcription level of tdaA, which encodes an essential transcriptional regulator for TDA biosynthesis, decreased 28-and 51-fold in pgaI and pgaR genetic backgrounds, respectively. These results suggest that both the response regulator PgaR and the 3OHC 10 -HSL produced by PgaI induce expression of tdaA, which in turn positively regulates expression of the tda genes. Moreover, we confirmed that TDA can also act as autoinducer in P. gallaeciensis, as previously described for Silicibacter sp. strain TM1040, but only in the presence of the response regulator PgaR.

Dinoroseobacter shibae, a member of the Roseobacter clade abundant in marine environments, is characterized by a pronounced pleomorphism. Cell shapes range from variable-sized ovoid rods to long filaments with a high copy number of chromosomes. Time-lapse microscopy shows cells dividing either by binary fission or by budding from the cell poles. Here we demonstrate that this morphological heterogeneity is induced by quorum sensing (QS). D. shibae utilizes three acylated homoserine lactone (AHL) synthases (luxI 1-3 ) to produce AHLs with unsaturated C18 side chains. A DluxI 1 -knockout strain completely lacking AHL biosynthesis was uniform in morphology and divided by binary fission only. Transcriptome analysis revealed that expression of genes responsible for control of cell division was reduced in this strain, providing the link between QS and the observed phenotype. In addition, flagellar biosynthesis and type IV secretion system (T4SS) were downregulated. The wild-type phenotype and gene expression could be restored through addition of synthetic C18-AHLs. Their effectiveness was dependent on the number of double bonds in the acyl side chain and the regulated trait. The wild-type expression level of T4SS genes was fully restored even by an AHL with a saturated C18 side chain that has not been detected in D. shibae. QS induces phenotypic individualization of D. shibae cells rather than coordinating the population. This strategy might be beneficial in unpredictably changing environments, for example, during algal blooms when resource competition and grazing exert fluctuating selective pressures. A specific response towards non-native AHLs might provide D. shibae with the capacity for complex interspecies communication.

Bacteria of the Roseobacter clade are widespread in the ocean and occur in many different habitats. In the genome of Dinoroseobacter shibae DFL-12, luxI homologous genes that encode synthases responsible for the formation of N-acylhomoserine lactones (AHLs) have been described. These compounds are known autoinducers that regulate several biological traits-namely, flagella formation and cell differentiation-in D. shibae through quorum sensing. The AHLs produced by D. shibae mainly consisted of N-octadecadienoylhomoserine lactone (C18:2-AHL) and N-octadecenoylhomoserine lactone (C18:1-HSL). In the wild type these AHLs are synthesized only in low abundance. The luxI genes were therefore expressed in Escherichia coli; this resulted in the formation of AHLs mostly different from those found in the D. shibae wild type. A luxI1 -deficient mutant of D. shibae was then reprovided with an overexpressed luxI1 gene. This strain produced large amounts of C18:2-AHL and C18:1-AHL, allowing full characterization of these compounds by mass spectrometric techniques and derivatization. Synthesis of the proposed structures confirmed that the major compound is (2E,11Z)-N-octadeca-2,11-dienoylhomoserine lactone (6, C18:2-HSL), accompanied by (Z)-N-octadec-11-enoylhomoserine lactone (5, C18:1-HSL). AHL 6 has not been reported before from other organisms and contains an unusual 2E double bond.

In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called “classical” heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD from Methanosarcina barkeri were functionally characterized. Using an in vivo enzyme activity assay it was shown that AhbA and AhbB (Mbar_A1459 and Mbar_A1460) together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar_A1793) was shown to catalyze the formation of iron-coproporphyrin III in vivo. Finally, recombinant AhbD (Mbar_A1458) was produced in E. coli and purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using an in vitro enzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme.

A homology model of the nucleotide-activated P2YR was created based on the X-ray structures of the P2Y receptor. Docking studies were performed, and receptor mutants were created to probe the identified binding interactions. Mutation of residues predicted to interact with the ribose (Arg110) and the phosphates of the nucleotide agonists (Arg265, Arg292) or that contribute indirectly to binding (Tyr288) abolished activity. The Y114F, R194A, and F261A mutations led to inactivity of diadenosine tetraphosphate and to a reduced response of UTP. Significant reduction in agonist potency was observed for all other receptor mutants (Phe111, His184, Ser193, Phe261, Tyr268, Tyr269) predicted to be involved in agonist recognition. An ionic lock between Asp185 and Arg292 that is probably involved in receptor activation interacts with the phosphate groups. The antagonist AR-C118925 and anthraquinones likely bind to the orthosteric site. The updated homology models will be useful for virtual screening and drug design.