Cyanobacteria are suitable for sustainable, solar-powered biotechnological applications. Synthetic biology connects biology with computational design and an engineering perspective, but requires efficient tools and information about the function of biological parts and systems. To enable the development of cyanobacterial Synthetic Biology, several molecular tools were developed and characterized: (i) a broad-host-range BioBrick shuttle vector, pPMQAK1, was constructed and confirmed to replicate in Escherichia coli and three different cyanobacterial strains. (ii) The fluorescent proteins Cerulean, GFPmut3B and EYFP have been demonstrated to work as reporter proteins in cyanobacteria, in spite of the strong background of photosynthetic pigments. (iii) Several promoters, like PrnpB and variants of PrbcL, and a version of the promoter Ptrc with two operators for enhanced repression, were developed and characterized in Synechocystis sp. strain PCC6803. (iv) It was shown that a system for targeted protein degradation, which is needed to enable dynamic expression studies, is working in Synechocystis sp. strain PCC6803. The pPMQAK1 shuttle vector allows the use of the growing numbers of BioBrick parts in many prokaryotes, and the other tools herein implemented facilitate the development of new parts and systems in cyanobacteria.

Cyanobacteria may possess two distinct nickel-iron (NiFe)-hydrogenases: an uptake enzyme found in N(2)-fixing strains, and a bidirectional one present in both non-N(2)-fixing and N(2)-fixing strains. The uptake hydrogenase (encoded by hupSL) catalyzes the consumption of the H(2) produced during N(2) fixation, while the bidirectional enzyme (hoxEFUYH) probably plays a role in fermentation and/or acts as an electron valve during photosynthesis. hupSL constitute a transcriptional unit, and are essentially transcribed under N(2)-fixing conditions. The bidirectional hydrogenase consists of a hydrogenase and a diaphorase part, and the corresponding five hox genes are not always clustered or cotranscribed. The biosynthesis/maturation of NiFe-hydrogenases is highly complex, requiring several core proteins. In cyanobacteria, the genes that are thought to affect hydrogenases pleiotropically (hyp), as well as the genes presumably encoding the hydrogenase-specific endopeptidases (hupW and hoxW) have been identified and characterized. Furthermore, NtcA and LexA have been implicated in the transcriptional regulation of the uptake and the bidirectional enzyme respectively. Recently, the phylogenetic origin of cyanobacterial and algal hydrogenases was analyzed, and it was proposed that the current distribution in cyanobacteria reflects a differential loss of genes according to their ecological needs or constraints. In addition, the possibilities and challenges of cyanobacterial-based H(2) production are addressed.

A strain affiliated with the Roseobacter clade and producing a new antibiotic named tropodithietic acid (L. Liang, Ph.D. thesis, University of Göttingen, Göttingen, Germany, 2003) was isolated from the German Wadden Sea. The compound showed strong inhibiting properties with respect to marine bacteria of various taxa and marine algae. Antibiotic production was found to occur during the complete growth phase. Strain mutants without antagonistic properties appeared several times spontaneously.Since the first antibiotic from a marine bacterium was described in 1966 (6), the number of new compounds has increased constantly during the years. Even though only a few compounds from marine organisms might be interesting for the pharmaceutical market today (7), some bacterial species are already used as biocontrols and are added to aquaculture stocks. Most secondary metabolites from marine bacteria found so far were isolated from Streptomyces and Alteromonas species (24). In recent studies wide arrays of marine bacteria were tested for bacterial antagonistic effects, and it was demonstrated that this trait appears to be a widespread feature in marine habitats and present in many bacterial groups (5, 9, 15). Production of secondary metabolites by bacteria of the Roseobacter group has been reported previously (8, 13), and some organisms of this group are thought to be either probiotic or antibiotic in different aquacultures (3,19). Here we describe the antagonistic activity of a new strain of the Roseobacter clade against marine bacteria and algae.A water sample was taken above an intertidal mud flat of the German Wadden Sea (53°42Ј20ЉN, 07°43Ј11ЉE) on 25 October 1999 (water temperature, 9.6°C; pH, 7.9; O 2 saturation, 94%; salinity, 34‰). The sample was taken directly to the laboratory for further processing. Tubes containing 9 ml of marine broth 2216 (Difco) were inoculated with 1 ml of seawater, and the contents were vigorously mixed. Using these tubes, we prepared dilution series with 1:10 steps. The cultures were incubated in the dark at 4, 15, 20, and 28°C (with shaking) for 4 weeks. Growth was determined microscopically and by monitoring turbidity. Aliquots (100 l) from the highest and lowest most-probable-number dilutions of each parallel experiment in which growth was obtained were spread on agar plates with marine agar 2216 (Difco). Plates were incubated at 4, 15, 20, or 28°C in the dark. Different types of colonies obtained were streaked out on fresh plates and transferred at least three times for purification. Bacterial strains were compared by colony morphology and color and by denaturing gradient gel electrophoresis as previously described (23). For a screening of the production of new antimicrobial substances, strains were selected which showed pigmentation or for which no closely related organisms were found by BLAST analysis of their 16S rRNA gene fragments.To detect antimicrobial activity, agar diffusion tests with standard test organisms were performed using Bacillus subtilis and Staphylococcus aureus (bot...

A heterotrophic, antibiotic-producing bacterium, strain T5T, was isolated from the German Wadden Sea, located in the southern region of the North Sea. Sequence analysis of the 16S rRNA gene of this strain demonstrated close affiliation with Roseobacter gallaeciensis BS107T (99 % similarity), but the results of genotypic (DNA–DNA hybridization and DNA G+C content) and phenotypic characterization revealed that strain T5T represents a novel species. The novel organism is strictly aerobic, Gram-negative, rod-shaped, motile and forms brown-pigmented colonies. Strain T5T produces the antibiotic tropodithietic acid throughout the exponential phase which inhibits the growth of bacteria from different taxa, as well as marine algae. Strain T5T requires sodium ions and utilizes a wide range of substrates, including oligosaccharides, sugar alcohols, organic acids and amino acids. The DNA G+C content is 55.7 mol%. Comparative 16S rRNA gene sequence analysis revealed that strains T5T and Roseobacter gallaeciensis BS107T group with Leisingera methylohalidivorans as their closest described relative within the Roseobacter clade (97.9 and 97.6 % sequence similarity, respectively) and with Ruegeria algicola (96.6 and 96.5 % similarity, respectively) of the Alphaproteobacteria. Comparison of strains T5T and Roseobacter gallaeciensis BS107T with Roseobacter denitrificans and Roseobacter litoralis showed striking differences in 16S rRNA gene sequence similarities, chemical composition, pigmentation, presence of bacteriochlorophyll a and antibiotic production. On the basis of these results, it is proposed that Roseobacter gallaeciensis is reclassified as the type species of a new genus, Phaeobacter, as Phaeobacter gallaeciensis comb. nov. (type strain BS107T=CIP 105210T=ATCC 700781T=NBRC 16654T=DSM 17395T). Strain T5T (=LMG 22475T=DSM 16374T) is proposed as the type strain of a novel species of this genus, Phaeobacter inhibens sp. nov. At the same time, emended descriptions are provided of the genera Roseobacter, Ruegeria and Leisingera, as well as reclassifying Ruegeria algicola as the type species of a new genus, Marinovum, with the name Marinovum algicola comb. nov.

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