Kok, J. | Search bioRxiv

bioRxiv2018

Homologous expression and characterization of gassericin T and gassericin S, a novel class IIb bacteriocin produced by Lactobacillus gasseri LA327

Lactobacillus gasseri LA327 isolated from the large intestine tissue in humans is a bacteriocinogenic strain and is predicted to produce two kinds of class IIb bacteriocins, i.e. gassericin T (GT) and acidocin LF221A (Acd LF221A). In this study, DNA sequencing of the genes for GT and Acd LF221A on Lb. gasseri LA327 revealed that the amino acid sequences for GT completely corresponded with those of gat except for GatK (histidine kinase). However, those for the Acd LF221A had analogues which differed in at least one amino acid residue to be a putative class IIb bacteriocin designated as gassericin S (GS). By deletion test of GT structural genes (gatAX), the LA327 strain retained the bacteriocin activity, and the LA327 mutant strain lacking the ABC-type transporter gene (gatT) completely lost the bacteriocin activity. This indicates that LA327 strain is a GS producer, and GS production is performed via gat with the inclusion of gatT. Homologous expression using deletion mutants for GS and GT containing each single peptide elucidated that GS (GasAX) and GT (GatAX) showed synergistic activity as class IIb bacteriocins, respectively, and no synergistic activity was observed between each peptide of GS and GT. The molecular mass of GS was estimated to be theoretical ca. 5,400 Da by in situ activity assay after SDS-PAGE, clarifying that GS was actually expressed as an active class IIb bacteriocin. Furthermore, stability of GS expressed against pH, heat and protease was determined.\n\nImportanceWe determined the complete DNA sequence for GS, a novel class IIb bacteriocin of Lb. gasseri, and succeeded to express GS as active bacteriocins. Our results clarified the interaction of each class IIb component peptide for GT in addition to GS via construction of homologous mutants which were not dependent on the purification. These data may demonstrate the characteristics of class IIb bacteriocins for Lb. gasseri.

microbiology↗

bioRxiv2017

Glucose limitation in Lactococcus shapes a single-peaked fitness landscape exposing membrane occupancy as a constraint

A central theme in biology is to understand the molecular basis of fitness: which strategies succeed under which conditions; how are they mechanistically implemented; and which constraints shape trade-offs between alternative strategies. We approached these questions with parallel bacterial evolution experiments in chemostats. Chemostats provide a constant environment with a defined resource limitation (glucose), in which the growth rate can be controlled. Using Lactococcus lactis, we found a single mutation in a global regulator of carbon metabolism, CcpA, to confer predictable fitness improvements across multiple growth rates. In silico protein structural analysis complemented with biochemical and phenotypic assays, show that the mutation reprograms the CcpA regulon, specifically targeting transporters. This supports that membrane occupancy, rather than biosynthetic capacity, is the dominant constraint for the observed fitness enhancement. It also demonstrates that cells can modulate a pleiotropic regulator to work around limiting constraints.

genomics↗