Mobilizable shuttle vectors with fluorescent markers functional across different species of bacteria.

Chromophore-containing proteins (CCPs), including fluorescent and non-fluorescent (chromoproteins), have been widely used in microbiological research. However, several roadblocks often limit their use in non-model bacterial species, including efficient transformation, suitable plasmid origins of replication, and optimal promoter choice. Here, we have engineered a set of 32 shuttle plasmids designed to overcome these roadblocks in an effort to streamline this process for future research. We have selected eight different CCPs: eforCP, YukonOFP, DasherGFP, tinsel Purple, aeBlue, FuGFP, super-folder GFP, and super-folder Cherry2. To broaden the potential host range, we utilized two distinct backbones with p15a either fused to a IMPORTANCE: Chromophore-containing proteins (CCPs), including both fluorescent proteins and pigment-producing (non-fluorescent) chromoproteins, have become invaluable tools for microbial research. However, their successful implementation in understudied bacterial species lacking established genetic tools often requires substantial time and resources. Our goal was to develop a set of plasmid-based vectors that could streamline CCP expression in gram-negative bacteria. To do so, we developed a set of 32 plasmid vectors, the pKEK-Chrom plasmid series, specifically designed to facilitate CCP expression across different bacteria, including