Deep mutational scanning reveals the molecular determinants of RNA polymerase-mediated adaptation and tradeoffs.

RNA polymerase (RNAP) is emblematic of complex biological systems that control multiple traits involving trade-offs such as growth versus maintenance. Laboratory evolution has revealed that mutations in RNAP subunits, including RpoB, are frequently selected. However, we lack a systems view of how mutations alter the RNAP molecular functions to promote adaptation. We, therefore, measured the fitness of thousands of mutations within a region of rpoB under multiple conditions and genetic backgrounds, to find that adaptive mutations cluster in two modules. Mutations in one module favor growth over maintenance through a partial loss of an interaction associated with faster elongation. Mutations in the other favor maintenance over growth through a destabilized RNAP-DNA complex. The two molecular handles capture the versatile RNAP-mediated adaptations. Combining both interaction losses simultaneously improved maintenance and growth, challenging the idea that growth-maintenance tradeoff resorts only from limited resources, and revealing how compensatory evolution operates within RNAP.

Genetic and Phenotypic Study of the Pectobacterium versatile Beta-Lactamase, the Enzyme Most Similar to the Plasmid-Encoded TEM-1.

Genus Pectobacterium bacteria include important agricultural pathogens. Pectobacterium versatile isolates contain a chromosome-borne beta-lactamase, PEC-1. This enzyme is the closest relative of TEM-1, a plasmid-borne beta-lactamase widespread in the Enterobacterales. We performed bioinformatics and phenotypic analyses to investigate the genetic and phenotypic features of PEC-1 and its frequency and ability to spread within genus Pectobacterium. We also compared the characteristics of PEC-1 and TEM-1 and evaluated the likelihood of transfer. We found that blaPEC-1 was present principally in a small number of genetic environments in P. versatile. Identical blaPEC-1 genetic environments were present in closely related species, consistent with the high frequency of genetic exchange within the genus Pectobacterium. Despite the similarities between PEC-1 and TEM-1, their genetic environments displayed no significant identity, suggesting an absence of recent transfer. Phenotypic analyses on clonal constructs revealed similar hydrolysis spectra. Our results suggest that P. versatile is the main reservoir of PEC-1, which seems to transfer to closely related species. The genetic distance between PEC-1 and TEM-1, and the lack of conserved elements in their genetic environments, suggest that any transfer that may have occurred must have taken place well before the antibiotic era. IMPORTANCE This study aimed to compare the chromosomal beta-lactamase from Pectobacterium versatile, PEC-1, with the well-known and globally distributed TEM-1 in terms of genetic and functional properties. Despite the similarities between the enzymes, we obtained no definitive proof of gene transfer for the emergence of blaPEC-1 from blaTEM-1. Indeed, given the limited degree of sequence identity and the absence of a common genetic environment, it seems unlikely that any transfer of this gene has occurred recently. However, although blaPEC-1 was found mostly in one specific clade of the P. versatile species, certain isolates from other closely related species, such as Pectobacterium brasiliense and Pectobacterium polaris, may also carry this gene inserted into common genetic environments. This observation suggests that genetic exchanges are frequent, accounting for the diffusion of blaPEC-1 between isolates from different Pectobacterium species and, potentially, to exogenous mobile genetic elements.