Pseudomonas fluorescens mutants affected in the bacteriocin production

GAMBETTA, C; ASCONAPE, J; LOPEZ RAMIREZ, V; BARRIONUEVO, L; TRAVAGLIA, C; FISCHER, S

Mendoza

Congreso; LVIII Reunión Anual de SAIB; 2022

SAIB

The bacteriocins are part of a diverse family of protein compounds characterized by their narrow killing spectrum; being toxic only to bacteria closely related to the producing strain. The rhizospheric strain P. fluorescens SF4c is known to produce phage tail-like bacteriocins, called tailocins. These bacteriocins are high molecular weight bactericidal protein complexes that resemble and are evolutionarily related to bacteriophage tails. SF4ctailocins SF4 tailocins have activity against phytopathogenic strains of the genera Pseudomonas and Xanthomonas. At present, the scientific interest is focused on the potential application of these antimicrobials in health, food, and agriculture. The SF4c tailocins regulation pathway is currently unknown. Recently, we have found a gene that is involved in the activation of these bacteriocins; however, the synthesis repressor gene is not yet known. Taking into account the aforementioned, we hypothesize that a mutation in the repressor gene of tailocins increases bacteriocin titers in cultures of P. fluorescens SF4c. To evaluate it, mini-Tn5-based transposon mutagenesis was used to generate random mutations. In our laboratory, we have a transcriptional fusion (pPROBE::Ptail) of the SF4c tailocin promoter to the green fluorescent protein (GFP) reporter gene. UV light-induced P. fluorescens SF4c (pPROBE::Ptail) strain cultures express the GFP protein, observing fluorescent colonies under blue light; this indicates that the promoter is being expressed, while under normal conditions, colonies do not fluoresce. A triparental conjugation was performed among a donor strain (E. coli CC118 λpir) containing the pUT::mini-Tn5 Km1 plasmid, a helper strain (E. coli HB101), and the receptor strain P. fluorescens SF4c. After, the mutants were picked and grown in minimum culture media supplemented with kanamycin (25 μg/ml) and tetracycline (10 μg/ml). Those mutants that exhibited enhanced fluorescence (increased expression of GFP) were selected. Promoter activity was quantified by fluorescence spectrometry using the proper positive and negative controls. Next, the presence of the mini-Tn5 Km1 transposon in the genome of the mutants was confirmed by PCR amplification of the transposon's kanamycin resistance gene. Finally, growth curve patterns and the tailocin production of the mutant strains were analyzed and compared with those of P. fluorescens SF4c (pPROBE::Ptail). The mutants 55, 174, 175, 233 y 253 showed statistically significant enhanced fluorescence; this means that there is an increase in the expression of tailocin promoter. Up to now, the results are promising in terms of obtaining information on the gene regulation system for P. fluorescens bacteriocins production.