The euryhaline cyanobacterium Synechococcus sp. strain PCC 7002 has an obligate requirement for exogenous vitamin B 12 (cobalamin), but little is known about the roles of this compound in cyanobacteria. Bioinformatic analyses suggest that only the terminal enzyme in methionine biosynthesis, methionine synthase, requires cobalamin as a coenzyme in Synechococcus sp. strain PCC 7002. Methionine synthase (MetH) catalyzes the transfer of a methyl group from N 5 -methyl-5,6,7,8-tetrahydrofolate to L-homocysteine during L-methionine synthesis and uses methylcobalamin as an intermediate methyl donor. Numerous bacteria and plants alternatively employ a cobalamin-independent methionine synthase isozyme, MetE, that catalyzes the same methyl transfer reaction as MetH but uses N 5 -methyl-5,6,7,8-tetrahydrofolate directly as the methyl donor. The cobalamin auxotrophy of Synechococcus sp. strain PCC 7002 was complemented by using the metE gene from the closely related cyanobacterium Synechococcus sp. strain PCC 73109, which possesses genes for both methionine synthases. This result suggests that methionine biosynthesis is probably the sole use of cobalamin in Synechococcus sp. strain PCC 7002. Furthermore, a cobalaminrepressible gene expression system was developed in Synechococcus sp. strain PCC 7002 that was used to validate the presence of a cobalamin riboswitch in the promoter region of metE from Synechococcus sp. strain PCC 73109. This riboswitch acts as a cobalamin-dependent transcriptional attenuator for metE in that organism. IMPORTANCESynechococcus sp. strain PCC 7002 is a cobalamin auxotroph because, like eukaryotic marine algae, it uses a cobalamin-dependent methionine synthase (MetH) for the final step of L-methionine biosynthesis but cannot synthesize cobalamin de novo. Heterologous expression of metE, encoding cobalamin-independent methionine synthase, from Synechococcus sp. strain PCC 73109, relieved this auxotrophy and enabled the construction of a truly autotrophic Synechococcus sp. strain PCC 7002 more suitable for large-scale industrial applications. Characterization of a cobalamin riboswitch expands the genetic toolbox for Synechococcus sp. strain PCC 7002 by providing a cobalamin-repressible expression system. S ynechococcus sp. strain PCC 7002 is a euryhaline, unicellular cyanobacterium that tolerates high light intensities and a wide range of sodium chloride concentrations (1, 2). This organism has one of the highest growth rates known among cyanobacteria (3), has a fully sequenced genome, and is naturally transformable (2, 4). A versatile system for genetic complementation and overexpression has been developed for this organism (5). Despite generally being considered to be photoautotrophic, Synechococcus sp. strain PCC 7002 has an obligate requirement for exogenous cobalamin (6), a large and structurally complex cobalt-chelating tetrapyrrole compound. Although cobalamin can only be synthesized de novo by some eubacteria and archaea, it is widely used as a coenzyme by many organisms, including e...

The cyanobacterium Synechococcus sp. strain PCC 7002 is a cobalamin auxotroph and utilizes this coenzyme solely for the synthesis of L-methionine by methionine synthase (MetH). Synechococcus sp. strain PCC 7002 is unable to synthesize cobalamin de novo, and because of the large size of this tetrapyrrole, an active-transport system must exist for cobalamin uptake. Surprisingly, no cobalamin transport system was identified in the initial annotation of the genome of this organism. With more sophisticated in silico prediction tools, a btuB-cpdA-btuC-btuF operon encoding components putatively required for a B 12 uptake (btu) system was identified. The expression of these genes was predicted to be controlled by a cobalamin riboswitch. ) developed a cobalamin-dependent yellow fluorescent protein reporter system in a Synechococcus sp. strain PCC 7002 variant that had been genetically modified to allow cobalamin-independent growth. This reporter system was exploited to validate components of the btu uptake system by assessing the ability of targeted mutants to transport cobalamin. The btuB promoter and a variant counterpart mutated in an essential element of the predicted cobalamin riboswitch were fused to a yfp reporter. The combined data indicate that the btuB-cpdA-btuF-btuC operon in this cyanobacterium is transcriptionally regulated by a cobalamin riboswitch. IMPORTANCEWith a cobalamin-regulated reporter system for expression of yellow fluorescent protein, genes previously misidentified as encoding subunits of a siderophore transporter were shown to encode components of cobalamin uptake in the cyanobacterium Synechococcus sp. strain PCC 7002. This study demonstrates the importance of experimental validation of in silico predictions and provides a general scheme for in vivo verification of similar cobalamin transport systems. A putative cobalamin riboswitch was identified in Synechococcus sp. strain PCC 7002. This riboswitch acts as a potential transcriptional attenuator of the btu operon that encodes the components of the cobalamin active-transport system. S ynechococcus sp. strain PCC 7002 is a euryhaline, unicellular cyanobacterium that tolerates high light intensities and a wide range of NaCl concentrations (1, 2). This organism has one of the highest growth rates among cyanobacteria (1, 3) and is naturally transformable (4). Furthermore, the genome of Synechococcus sp. strain PCC 7002 has been sequenced (http://www.ncbi.nlm.nih .gov/), and a versatile system for genetic complementation and overexpression exists for this organism (5). Although it is generally considered to be a photoautotroph, Synechococcus sp. strain PCC 7002 actually has an obligate requirement for exogenous vitamin B 12 (cobalamin) (6). The average reported concentration of cobalamin in seawater is around 3 ng liter Ϫ1 (or 0.003 g liter Ϫ1 ) (7) but exhibits variable vertical distribution (8). As a marine organism incapable of synthesizing cobalamin de novo (9, 10), Synechococcus sp. strain PCC 7002 thus needs a specific and effective m...

Synechococcus sp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn 2ϩ -inducible expression platform that is coupled with a zurA mutation, providing enhanced Zn 2ϩ uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA 7942 ) and its cognate SmtB 7942 repressor from Synechococcus elongatus strain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA 6803 ) promoter of Synechocystis sp. strain PCC 6803. This metal-inducible expression system in Synechococcus sp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of the Drosophila melanogaster ␤-carotene 15,15=-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter in Synechococcus sp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology in Synechococcus sp. strain PCC 7002.IMPORTANCE This is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn 2ϩ -inducible system, which is based on the smtA 7942 operator/promoter and smtB 7942 repressor, as a versatile gene expression platform that expands the genetic toolbox of Synechococcus sp. strain PCC 7002.KEYWORDS Zn 2ϩ -inducible promoter, cyanobacteria, gene expression platform, metallothionein, ninaB, photosynthesis S ynechococcus sp. strain PCC 7002 is a euryhaline cyanobacterial model organism with a very high growth rate, natural transformability, tolerance to high-light irradiance, a fully sequenced genome, and an expanding genetic toolbox. Over the past 30 years, this cyanobacterium has been extensively used in the study of photosynthesis and for synthetic biology applications (1-4). Despite its importance as an industrially relevant organism, genetic tools in Synechococcus sp. strain PCC 7002 were rather

The One Environmental Health research approach, a subspecialty of the One Health initiative, focuses on toxic chemicals. Distinct disciplines work together to give a holistic perspective of a health concern through discrete disciplines, including, but not limited to, public health and the medical and veterinary sciences. In this article, we illustrate the concept of One Environmental Health with two case studies. One case study focuses on alligators and contributions to the field of endocrine disruption. The other case study focuses on whales and contributions to understanding carcinogenic metals. Both studies illustrate how the health of sentinel organisms has the potential to inform about the health of humans and the ecosystem.

A sustainable society will have to largely refrain from the use of fossil carbon deposits. In such a regime, renewable electricity can be harvested as a primary source of energy. However, as for the synthesis of carbon‐based materials from bulk chemicals, an alternative is required. A sustainable approach towards this is the synthesis of commodity chemicals from CO2, water and sunlight. Multiple paths to achieve this have been designed and tested in the domains of chemistry and biology. In the latter, the use of both chemotrophic and phototrophic organisms has been advocated. ‘Direct conversion’ of CO2 and H2O, catalyzed by an oxyphototroph, has excellent prospects to become the most economically competitive of these transformations, because of the relative ease of scale‐up of this process. Significantly, for a wide range of energy and commodity products, a proof of principle via engineering of the corresponding production organism has been provided. In the optimization of a cyanobacterial production organism, a wide range of aspects has to be addressed. Of these, here we will put our focus on: (1) optimizing the (carbon) flux to the desired product; (2) increasing the genetic stability of the producing organism and (3) maximizing its energy conversion efficiency. Significant advances have been made on all these three aspects during the past 2 years and these will be discussed: (1) increasing the carbon partitioning to >50%; (2) aligning product formation with the growth of the cells and (3) expanding the photosynthetically active radiation region for oxygenic photosynthesis.