Chromatic Bacteria – A Broad Host-Range Plasmid and Chromosomal Insertion Toolbox for Fluorescent Protein Expression in Bacteria

Differential fluorescent labeling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behavior in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green–yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5 transposon delivery plasmid, and a Tn7 transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5 transposon delivery system. The present study enables labeling bacteria with a set of plasmids available to the community. One potential application of fluorescently-tagged bacterial species is the study of bacteria–bacteria, bacteria–host, and bacteria–environment interactions

Surfactants produced by epiphytic bacteria and their role in diesel degradation

Hydrocarbon contaminations cause serious harm in the environment. Bacterial bioremediation is an environmentally friendly method to reduce such contaminants. To increase the bioavailability of hydrocarbons, bacteria produce surfactants. Many leaf colonising bacteria are surfactant producers but have not been tested for their potential to remediate hydrocarbon contaminations. The aim of this thesis was to investigate if surfactants produced by leaf colonising bacteria enhance the degradation of hydrocarbons in terrestrial environments. A high proportion of the bacterial strains investigated in this thesis was found to produce surfactants and degrade diesel in liquid culture. Furthermore, four leaf isolates of Pseudomonas and their surfactant mutants were used to study the effect of surfactants on diesel degradation and colonisation on Arabidopsis thaliana leaves. No differences in the ability to colonise leaves were observed when comparing the wild type with its mutant derivatives. However, when tested in liquid cultures, all mutants were found to grow slower on minimal medium supplemented with diesel. This effect was complemented by the addition of wild type or synthetic surfactants to the growth medium. This evidences the role of surfactants in enhancing diesel bioavailability in liquid environments. By contrast, the same bacterial strains did not exhibit significant growth differences in diesel contaminated soil microcosms. Wild types and mutants were equally able to grow and degrade diesel as determined by gas chromatography coupled with flame ionisation detection. In conclusion, many epiphytic bacteria are hydrocarbon degraders and should be investigated further as potential candidates for hydrocarbon degradation in soil. Furthermore, in Pseudomonads, surfactant production does impact diesel degradation in liquid but not in soil

Chromatic Bacteria – A Broad Host-Range Plasmid and Chromosomal Insertion Toolbox for Fluorescent Protein Expression in Bacteria

Differential fluorescent labeling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behavior in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green-yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5 transposon delivery plasmid, and a Tn7 transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5 transposon delivery system. The present study enables labeling bacteria with a set of plasmids available to the community. One potential application of fluorescently-tagged bacterial species is the study of bacteria-bacteria, bacteria-host, and bacteria-environment interactions