Biology subjects

Vukotic, G.

Publications and source records attributed to Vukotic, G..

3 recordsLinked to original sources

bioRxiv2026

Conserved emergent traits enable biobank-scale prediction of community function

Predicting the function of a microbial community from the identity of its members is a key goal of ecology and biotechnology. Here, we show that the systematic variation in how strains contribute to community function is phylogenetically conserved. This allows us to predict the collective function of communities solely from their member's taxonomic identity. Using soymilk fermentation as a model, we measured three industrially relevant functions - acidification, texture, sensory grade - across 307 synthetic communities combining 33 phylogenetically diverse lactic acid bacteria strains. The functional effect of a strain scaled linearly with the function of the receiving community, allowing us to summarize each strain's contribution across communities with two parameters, intercept and slope. These parameters were phylogenetically conserved, allowing us to impute their values for 17 previously unassayed strains, and use them to predict the three functions in 14 newly assembled communities. By redefining community function in terms of conserved emergent species traits, our results enable predictive engineering of microbial consortia at scale, and open a path towards genomic and evolutionary dissection of complex community phenotypes.

microbiology↗

bioRxiv2021

Construction of redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides

Many protein expression and purification systems are commercially available to provide a sufficient amount of pure, soluble and active native protein, such as the pMAL system based on E. coli maltose binding protein tag (MBP). Adding specific amino acid tags to the N- or C-terminus of the protein increases solubility and facilitates affinity purification of proteins. However, many of expressed tagged proteins consequently lose functionality, particularly small peptides such as antimicrobial peptides (AMPs). Objective of this study was to redesign the pMAL expression vector in order to increase the efficacy of MBP tag separation from native peptides. Redesign of the pMAL expression vector included introduction of the His6 tag and the enterokinase cleavage site downstream from the original MBP tag and Xa cleavage site enabling purification of native and active peptide (P) following two-step affinity chromatography. In the first step the entire MBP-His6-P fusion protein is purified through binding to Ni-NTA agarose. In the second step, the purification was performed by adding mixture of amylose and Ni-NTA agarose resins following cleavage of the fusion protein with active His6 tagged enterokinase. This removes MBP-His6 and His6-enterokinase leaving pure native protein in solution. The redesigned pMAL vectors were optimized for cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides expression. Two-step purification protocol was successfully applied in purification of active native AMPs, lactococcin A and human {beta}-defensin. Taken together, we established the optimal conditions and pipeline for overexpression and purification of large amount of native peptides, that can be implemented in any laboratory.

bioengineering↗

bioRxiv2018

Lactococcin B is inactivated by intrinsic proteinase PrtP digestion in Lactococcus lactis subsp. lactis BGMN1-501

In our previous study, we showed that PrtP is able to impair bacteriocin LcnB activity despite being produced by the same organism, and even if they were encoded by the same plasmid. However, exact cleavage site within LcnB bacteriocin, as well as the activity of the resulting peptides remained unknown. Here we further explored the interplay between these two proteins and defined, using mass spectrometry, that the hydrolysis occurs between the sixth and seventh amino acid on the N terminus of LcnB. Although it was suspected that the cleaved form of LcnB could retain some level of activity, both chemically synthesized and recombinant variant of truncated LcnB exhibited no antimicrobial activity. Wild type form of LcnB was recombinantly overexpressed using the same expression system, its antimicrobial activity was tested before and after the treatment with PrtP proteinase, and the degradation products were analyzed with reverse-phase high-pressure liquid chromatography. The results confirmed the inactivity of the truncated LcnB and additionally corroborated the PrtP cleavage site in LcnB bacteriocin.\n\nImportanceLactococcal enzyme PrtP, considered as a growth promoting factor, is involved in casein breakdown and enabling bacteria efficient growth in amino acids-poor, but protein-rich media. However, its interaction with bacteriocins was not known until recently. Bacteriocin LcnB can also be considered as growth promoting factor, since its known physiological role mirrors in preventing competing bacteria of reaching high growth densities. In this manuscript, we define the exact peptide bond inside the bacteriocin LcnB which is recognized by PrtP. This N-terminal removal of six amino acids completely inactivates the bacteriocin. The biological function of such action remains elusive. It is unexpected that in the same strain one enzyme inactivates a protein important for survival, unless it is some type of regulation.

microbiology↗