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

Abstract

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 Francisella origin (FnOri) or to the broad host origin RSF1010 and included a transfer origin (oriT) to facilitate transformation via conjugation. Moreover, we have created versions of each vector, which confer resistance to either kanamycin or chloramphenicol. Lastly, to enable promoter-swapping, we engineered the constitutive pJ23100 promoter element to be flanked by BsaI sites, thereby enabling promoter exchange by the Golden Gate assembly to evaluate CCP expression with different host promoters. To demonstrate the usability of the pKEK-Chrom plasmid series, we evaluated their expression in Escherichia coli, Shewanella oneidensis, and Vibrio alginolyticus. We further demonstrated the utility of promoter swapping in Francisella novicida and validated the functionality of the RSF1010 origin in Acinetobacter baumannii. In summary, the pKEK-Chrom plasmid series provides a palette of different CCPs that streamline their use in non-model gram-negative bacteria.

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 Escherichia coli, Vibrio alginolyticus, Shewanella oneidensis, Francisella novicida, and Acinetobacter baumannii.

Keywords: Acinetobacter baumannii; Francisella novicida; Golden Gate assembly; RSF1010; Vibrio alginolyticus; broad-host range; chromoproteins; non-model bacteria; plasmid; promoter exchange.

Fig 1

Schematic of the pKEK-Chrom plasmid series. This schematic demonstrates the critical components that enable CCP expression in diverse gram-negative bacteria, including (1) swappable promoter driving protein expression, (2) a variety of CCPs, (3) two different antibiotic resistance markers, and (4) two different options for broad host origins of replication. CCP = chromophore-containing protein, RBS = ribosome binding site, and pFN = pFN_1451 (18). Created in BioRender.

Fig 2

Expression of CCPs in diverse gram-negative bacteria. Expression of fluorescent proteins from the pKEK-Chrom-p15a-FnOri plasmid series in E. coli, V. alginolyticus, and S. oneidensis harboring expression vectors for eforCP (pKEK3193), DasherGFP (pKEK3194), tsPurple (pKEK3199), aeBlue (pKEK3198), YukonOFP (pKEK3197), sfGFP (pKEK3220), and sfCherry2 (pKEK3221). (A) Strains grown on agar plates and visualized under white (“W”), red (“UV-Red”), or green (“Blue-green”) filter. (B) Pellets of Escherichia coli strains from panel A grown in liquid media. (C) E. coli strains from panel A expressing sfGFP, DasherGFP, sfCherry2, and eforCP visualized via fluorescence microscopy. Created in BioRender.

Fig 3

Promoter swap enables CCP expression in Francisella. The promoter element pJ23100-BsaI in the pKEK-Chrom plasmid series enables promoter swapping in one-step Golden Gate reaction using circular pKEK-Chrom plasmids. (A) The schematic outlines how the pJ23100 promoter was swapped with the Francisella pgroEL via the Golden Gate assembly using BsaI. The location of BsaI recognition sites within pKEK-Chrom plasmids are indicated in red; the resulting overhangs that guide ligation are boxed; the universal primer sequence to be used to amplify alternative promoters is indicated; and a schematic with the resulting promoter-swapped plasmid product is shown. (B) F. novicida strains harboring either pJ23100- (top) or pgroEL (bottom)-driven expression of sfCherry2 (pKEK3236 or pKEK3377), sfGFP (pKEK3235 or pKEK3372), DasherGFP (pKEK3231 or pKEK3310), and tsPurple (pKEK3232 or pKEK3311) grown on agar plates and visualized under white (“W”), red (“UV-Red”), or green (“Blue-green”) filter. Created in BioRender.

Fig 4

Alternate ori RSF1010 broadens host range and facilitates CCP expression in A. baumannii. (A) Schematic showing the plasmid structure of the pKEK-Chrom-RSF1010-p15a plasmid set evaluated in A. baumannii. (B) A. baumannii strains harboring plasmids expressing eforCP (pKEK3273), DasherGFP (pKEK3225), tsPurple (pKEK3226), aeblue (pKEK3228), Yukon (pKEK3227), sfGFP (pKEK3237), and sfCherry2 (pKEK3238), as well as A. baumannii with no fluorescent protein (“−”) were grown on agar plates and visualized under white (“W”), red (“UV-Red”), or green (“Blue-green”) filter. FP = fluorescent protein. Created in BioRender.