Meijer, W. J. J. | Search bioRxiv

bioRxiv2025

EXPRESSION OF CONJUGATION GENES IS CONTROLLED BY PROCESSIVE ANTITERMINATION AND A NOVEL ZIPPER-TYPE TRANSCRIPTIONAL ATTENUATION MECHANISM

Proper expression of genes clustered in operons, particularly large operons, can be complex, often consisting of multiple regulatory switches. The conjugation operon present on the Bacillus subtilis conjugative plasmid pLS20 is over 32 kb long. This operon starts with a 456 nt leader region, which is followed by the first two genes of the operon, encoding a two-component processive antitermination system. Here, we demonstrate that the long leader region encodes a transcriptional attenuator that we named cATTpLS20. In vivo and in vitro analyses showed that the attenuator is composed of three segments: a stem-loop structure with a long imperfect stem that is preceded by a sequence that may form a weak stem-loop and followed by a sequence that may form an intrinsic terminator. Sequences of the upstream stem loop and downstream terminator are complementary, allowing further extension of the long stem, thereby generating an antiterminator conformation. Based on similarity with a zipper we coined this the zipper-type attenuator. Similar zipper-type attenuators are present upstream of the first genes of conjugation operons of all pLS20 family plasmids, as well as on many other conjugative plasmids in Gram-positive bacteria, suggesting that a common attenuation mechanism regulates expression of many conjugative operons. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/689506v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@a86a7eorg.highwire.dtl.DTLVardef@e787f8org.highwire.dtl.DTLVardef@8fdb75org.highwire.dtl.DTLVardef@90f842_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Spatial dynamics and emergent properties of pLS20 conjugation on solid surfaces

Horizontal gene transfer (HGT) is a major evolutionary process in bacteria, driving the dissemination of genetic traits including antibiotic resistance (AR). In this study, we employ a hybrid modeling approach, combining agent-based simulations and Ordinary Differential Equation (ODE) models, to investigate bacterial conjugation--a key HGT mechanism whose dynamics remain poorly understood. Our agent-based simulations of the transfer dynamics of the conjugative plasmid pLS20 from Bacillus subtilis reveal that spatial organization, colony growth dynamics, and quorum-sensing regulation significantly influence plasmid dissemination. Increased donor-recipient mixing enhances plasmid transmission by reducing quorum-induced repression, while colony growth-driven displacement of donor cells alters the local distribution of quorum-sensing signals, enabling sustained conjugation activity at the colony periphery. Complementary ODE modeling captures macroscopic trends in plasmid transmission, providing insights into the interplay between spatial factors and regulatory mechanisms. By bridging single-cell regulatory dynamics with population-level behaviors, this study advances our understanding of bacterial conjugation on solid surfaces, offering potential strategies for mitigating the spread of antibiotic resistance. Author summaryBacteria can exchange genetic material through a process called horizontal gene transfer, which helps them adapt to new environments but also develop traits like antibiotic resistance. One of the most important ways bacteria share genes is through conjugation--a mechanism where a conjugative element transfers from a donor to a recipient via a channel connecting both cells. Although considerable knowledge has been gathered over the last decades concerning regulation of the conjugation genes and the structure of the transferosome responsible for transfer of the conjugative element, far less is known about the dynamics of conjugative transfers within populations of cells, especially on solid medium. Our study focuses on the conjugative plasmid pLS20 from Bacillus subtilis, a model bacterium related to several pathogens. Using computer simulations, we modeled how this plasmid spreads within bacterial colonies growing on solid surfaces. We found that the spatial organization of bacteria plays a large role: well-mixed populations allow the plasmid to spread more effectively, while certain growth patterns disrupt the signaling processes that regulate gene transfer. This study helps us better understand how bacteria transfer genes in complex environments and could guide new strategies to combat antibiotic resistance.

systems biology↗