Summary 14Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter 15 revisiting, mutated viruses and plasmids. Here we have determined how new spacers are produced and 16 selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference 17 to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step,

Bacteria resist phage infection using multiple strategies, including CRISPR-Cas and abortive infection (Abi) systems. Abi systems provide population-level protection from phage predation, via "altruistic" cell suicide. It has recently been shown that some Abi systems function via a toxin-antitoxin mechanism, such as the widespread AbiE family. The Streptococcus agalactiae AbiE system consists of a bicistronic operon encoding the AbiEi antitoxin and AbiEii toxin, which function as a Type IV toxin-antitoxin system. Here we examine the AbiEi antitoxin, which belongs to a large family of transcriptional regulators with a conserved N-terminal winged helix-turn-helix domain. This winged helix-turn-helix is essential for transcriptional repression of the abiE operon. The function of the AbiEi C-terminal domain is poorly characterized, but it contributes to transcriptional repression and is sufficient for toxin neutralization. We demonstrate that a conserved charged surface on one face of the C-terminal domain assists sequence-specific DNA binding and negative autoregulation, without influencing antitoxicity. Furthermore, AbiEi binds cooperatively to two inverted repeats within the abiE promoter and bends the DNA by 72°. These findings demonstrate that the mechanism of DNA binding by the widespread family of AbiEi antitoxins and transcriptional regulators can contribute to negative autoregulation.

Background Synechococcus sp. PCC 7002 (henceforth Synechococcus) is developing into a powerful synthetic biology chassis. In order to streamline the integration of genes into the Synechococcus chromosome, validation of neutral integration sites with optimization of the DNA transformation protocol parameters is necessary. Availability of BioBrick-compatible integration modules is desirable to further simplifying chromosomal integrations.ResultsWe designed three BioBrick-compatible genetic modules, each targeting a separate neutral integration site, A2842, A0935, and A0159, with varying length of homologous region, spanning from 100 to 800 nt. The performance of the different modules for achieving DNA integration were tested. Our results demonstrate that 100 nt homologous regions are sufficient for inserting a 1 kb DNA fragment into the Synechococcus chromosome. By adapting a transformation protocol from a related cyanobacterium, we shortened the transformation procedure for Synechococcus significantly.ConclusionsThe optimized transformation protocol reported in this study provides an efficient way to perform genetic engineering in Synechococcus. We demonstrated that homologous regions of 100 nt are sufficient for inserting a 1 kb DNA fragment into the three tested neutral integration sites. Integration at A2842, A0935 and A0159 results in only a minimal fitness cost for the chassis. This study contributes to developing Synechococcus as the prominent chassis for future synthetic biology applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-017-0061-8) contains supplementary material, which is available to authorized users.

Iron is an essential, yet scarce, nutrient in marine environments. Phytoplankton, and especially cyanobacteria, have developed a wide range of mechanisms to acquire iron and maintain their iron-rich photosynthetic machinery. Iron limitation studies often utilize either oceanographic methods to understand large scale processes, or laboratory-based, molecular experiments to identify underlying molecular mechanisms on a cellular level. Here, we aim to highlight the benefits of both approaches to encourage interdisciplinary understanding of the effects of iron limitation on cyanobacteria with a focus on avoiding pitfalls in the initial phases of collaboration. In particular, we discuss the use of trace metal clean methods in combination with sterile techniques, and the challenges faced when a new collaboration is set up to combine interdisciplinary techniques. Methods necessary for producing reliable data, such as High Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS), Flow Injection Analysis Chemiluminescence (FIA-CL), and 77K fluorescence emission spectroscopy are discussed and evaluated and a technical manual, including the preparation of the artificial seawater medium Aquil, cleaning procedures, and a sampling scheme for an iron limitation experiment is included. This paper provides a reference point for researchers to implement different techniques into interdisciplinary iron studies that span cyanobacteria physiology, molecular biology, and biogeochemistry.

Cyanobacteria have high iron requirements due to iron-rich photosynthetic machineries. Despite the high concentrations of iron in the Earth’s crust, iron is limiting in many marine environments due to iron’s low solubility. Oxic conditions leave a large portion of the ocean’s iron pool unavailable for biotic uptake, and so the physiochemical properties of iron are hugely important for iron’s bioavailability. Our study is the first to investigate the effect of iron source on iron internalization and extracellular reduction by Synechococcus sp. PCC 7002. The results indicated that the amorphous iron hydrolysis species produced by FeCl3 better support growth in Synechococcus through more efficient iron internalization and a larger degree of extracellular reduction of iron than the crystalline FeO(OH). An analysis of dissolved iron (II) indicated that biogenic reduction took place in cultures of Synechococcus grown on both FeCl3 and FeO(OH).