Conjugative transfer through type IV secretion multiprotein complexes is the most important means of spreading antimicrobial resistance. Plasmid pIP501, frequently found in clinical Enterococcus faecalis and Enterococcus faecium isolates, is the first Gram-positive (G+) conjugative plasmid for which self-transfer to Gram-negative (G-) bacteria has been demonstrated. The pIP501-encoded type IV secretion system (T4SS) protein TraN localizes to the cytoplasm and shows specific DNA binding. The specific DNA-binding site upstream of the pIP501 origin of transfer (oriT) was identified by a novel footprinting technique based on exonuclease digestion and sequencing, suggesting TraN to be an accessory protein of the pIP501 relaxase TraA. The structure of TraN was determined to 1.35 Å resolution. It revealed an internal dimer fold with antiparallel β-sheets in the centre and a helix-turn-helix (HTH) motif at both ends. Surprisingly, structurally related proteins (excisionases from T4SSs of G+ conjugative transposons and transcriptional regulators of the MerR family) resembling only one half of TraN were found. Thus, TraN may be involved in the early steps of pIP501 transfer, possibly triggering pIP501 TraA relaxase activity by recruiting the relaxosome to the assembled mating pore.

Bioprinting of engineered bacteria is of great interest for applications of synthetic biology in the context of living biomaterials, but so far, only a few viable approaches are available for the printing of gels hosting live Escherichia coli bacteria. Here, we develop a gentle extrusionbased bioprinting method based on an inexpensive alginate/agarose ink mixture that enables printing of E. coli into three-dimensional hydrogel structures up to 10 mm in height. We first characterize the rheological properties of the gel ink and then study the growth of the bacteria inside printed structures. We show that the maturation of fluorescent proteins deep within the printed structures can be facilitated by the addition of a calcium peroxide-based oxygen generation system. We then utilize the bioprinter to control different types of interactions between bacteria that depend on their spatial position. We next show quorum-sensing-based chemical communication between the engineered sender and receiver bacteria placed at different positions inside the bioprinted structure and finally demonstrate the fabrication of barrier structures defined by nonmotile bacteria that can guide the movement of chemotactic bacteria inside a gel. We anticipate that a combination of 3D bioprinting and synthetic biological approaches will lead to the development of living biomaterials containing engineered bacteria as dynamic functional units.

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