Goldlust, K. | bioRxiv Science

bioRxiv2026

Calcium signals natural transformation in Acinetobacter baumannii

The acquisition of resistance to antibiotics in the opportunistic pathogen Acinetobacter baumannii may be linked to its capacity to undergo natural transformation. This mode of horizontal gene transfer relies on the import of extracellular DNA and its chromosomal integration by homologous recombination. Type IV pilus activity initiates the capture of extracellular DNA, which is then transported to the cytoplasm for recombination in the chromosome. While most Acinetobacter baumannii strains are transformable, the conditions allowing the expression of type IV pilus and other transformation genes remain largely unexplored. By investigating transformation-permissive conditions, we uncovered that calcium is a potent inducer of natural transformation. Type IV pilus genes and other transformation-specific genes (comEA, dprA) are upregulated by submillimolar concentrations of calcium ions, in a growth phase-dependent manner. In contrast, sodium chloride represses expression of pilA, counteracting the calcium-dependent induction, explaining the reported absence of transformation in NaCl-containing medium (such as LB). Independently of transcriptional induction, calcium ions also directly bind the type IV pilus through the calcium-dependent adhesin PilY1. Our data support a model in which calcium strengthens the interaction of PilY1 with the minor pilin complex, increasing pilus dynamics and subsequent pilus-dependent DNA capture. Hence, calcium signals natural transformation through both transcriptional and structural activation of type IV pilus. In addition to providing new insights into the regulation of natural transformation in A. baumannii this work led us to establish a protocol for genetic engineering of A. baumannii by natural transformation. ImportanceAcinetobacter baumannii is a nosocomial pathogen considered a critical research priority due to its resistance to last resort antibiotics. Understanding how A. baumannii evolves and acquires resistance to antibiotics is thus of prime importance. This species is capable of natural transformation, a means to acquire and spread genetic information, including antibiotic resistance genes. However, the conditions under which this process is active in this species remain elusive. We identify calcium ions as potent inducers of natural transformation and propose a model of the signaling of natural transformation by calcium ions. This opens the way for further investigations into the contribution of natural transformation to acquisition of antibiotic resistance. In addition, it provides an efficient way to genetically manipulate most A. baumannii strains.

microbiology↗

bioRxiv2023

The F pilus serves as a conduit for the DNA during conjugation between physically distant bacteria

Horizontal transfer of F-like plasmids by bacterial conjugation is responsible for disseminating antibiotic resistance and virulence determinants among pathogenic Enterobacteriaceae species, a growing health concern worldwide. Central to this process is the conjugative F pilus, a long extracellular filamentous polymer that extends from the surface of plasmid donor cells, allowing it to probe the environment and make contact with the recipient cell. It is well established the F pilus can retract to bring mating pair cells in tight contact before DNA transfer. However, whether DNA transfer can occur through the extended pilus has been a subject of active debate. In this study, we use live-cell microscopy to show that the F pilus can indeed serve as a conduit for the DNA during transfer between physically distant cells. Our findings enable us to propose a new model for conjugation that revises our understanding of the DNA transport mechanism and the dissemination of drug resistance in complex bacterial communities. One-Sentence SummaryPlasmid DNA passes through the F pilus during conjugational transfer between physically distant bacteria.

microbiology↗

bioRxiv2020

MiSiC, a general deep learning-based method for the high-throughput cell segmentation of complex bacterial communities

Studies of microbial communities by live imaging require new tools for the robust identification of bacterial cells in dense and often inter-species populations, sometimes over very large scales. Here, we developed MiSiC, a general deep-learning-based segmentation method that automatically segments a wide range of spatially structured bacterial communities with very little parameter adjustment, independent of the imaging modality. Using a bacterial predator-prey interaction model, we demonstrate that MiSiC enables the analysis of interspecies interactions, resolving processes at subcellular scales and discriminating between species in millimeter size datasets. The simple implementation of MiSiC and the relatively low need in computing power make its use broadly accessible to fields interested in bacterial interactions and cell biology.

microbiology↗