2021 Joseph W. Richards Fellowship – Summary Report: Toward On-Chip Electronic Monitoring of Bacterial Conjugation

Intercellular conjugation is a primary communication method by which bacteria transfer genetic material from one cell to another. Because conjugation mediates the transfer of genetic material, it enables the spread of antibiotic resistance and other virulence factors within a population. Current techniques for its study have low throughput and require extensive sample preparation, making real-time monitoring of conjugation dynamics challenging.To address these shortcomings, we are exploring two-dimensional (2D) materials as a strain-sensitive platform for on-chip, electronic monitoring of conjugation events.

Chromosome scale assembly of allopolyploid genome of the diatom Fistulifera solaris

Microalgae including diatoms are of interest for environmentally-friendly manufacturing such as biofuel production. However, only a very few of their genomes have been elucidated owing to their diversified and complex evolutionary history. The genome of the marine oleaginous diatom Fistulifera solaris, an allopolyploid diatom possessing two subgenomes, has been analyzed previously by pyrosequencing. However, many unsolved regions and unconnected scaffolds remained. Here we report the entire chromosomal structure of the genome of F. solaris strain JPCC DA0580 using a long-read nanopore sequencing platform. From just one single run using a MinION flow-cell, the chromosome scale assembly with telomere-to-telomere resolution was achieved for 41 out of 44 chromosomes. Centromere regions were also predicted from the chromosomes, and we discovered conserved motifs in the predicted regions. The function of the motifs was experimentally confirmed by successful transformation of the diatom via bacterial conjugation. This discovery provides insights into chromosome replication, facilitating the rational design of artificial chromosomes for large-scale metabolic engineering of diatoms. The chromosome scale assembly also suggests the potential existence of multi-copy mini-chromosomes and tandemly repeated lipogenesis genes related to the oleaginous phenotype of F. solaris. The nanopore sequencing also solved the sequential arrangement of the repeat region in the F. solaris mitochondrial genome. Findings of this study will be useful to understand and further engineer the oleaginous phenotype of F. solaris.

Monitoring Bacterial Conjugation by Optical Microscopy

Bacterial conjugation is the main mechanism for horizontal gene transfer, conferring plasticity to the genome repertoire. This process is also the major instrument for the dissemination of antibiotic resistance genes. Hence, gathering primary information of the mechanism underlying this genetic transaction is of a capital interest. By using fluorescent protein fusions to the ATPases that power conjugation, we have been able to track the localization of these proteins in the presence and absence of recipient cells. Moreover, we have found that more than one copy of the conjugative plasmid is transferred during mating. Altogether, these findings provide new insights into the mechanism of such an important gene transfer device.

Love in the Laboratory

This chapter describes the marriage of two prodigies and how it represented a fruitful alliance of complementary research personalities: the brilliant theoretician and the skillful experimenter. Esther Zimmer and Joshua Lederberg were two of the youngest scientists to attend the 1946 summer symposium at Cold Spring Harbor. Edward Tatum arranged for his protégé, young Lederberg, to present his stupendous discovery of bacterial conjugation, showing that bacteria could mate and recombine their genes. Zimmer and Lederberg began a short romance and married five months later. The young couple moved near the campus of Yale University, where Joshua wrote up his thesis and Esther researched Neurospora genetics with Norman Giles. The following summer, Tatum negotiated with Yale to grant an accelerated PhD to Joshua. The University of Wisconsin offered him an assistant professorship, and Joshua and Esther moved to Madison in 1947. There they established the first research program in bacterial genetics.

The Anomaly of Bacterial Genetics

This chapter reviews the research that set the stage for Joshua Lederberg’s surprising discovery of bacterial conjugation. While the foundational research of Gregor Mendel and his principles of inheritance had been effectively combined with Darwinian evolution, producing the Modern Synthesis in the mid-forties, bacteria did not fit into this grand synthesis. Most biologists believed that bacteria were too primitive to have real genes. But Delbruck, Hershey and Luria organized the Phage School, leading a novel approach to discovering the molecular biology of the gene by studying bacteriophages. Microbiologists like Tracy Sonneborn and Carl Lindegren turned to alternative microorganisms—protists, fungi, and yeast—to develop new model systems that offered advantages over the classical genetics organisms of animals and plants. The research of Edward Tatum and Jacques Monod indicated that mutations seemed to explain variation in bacteria. For many years, however, bacteriologists had known that bacteria reproduced by fission. The lack of any genetic hybridization seemed to argue against using bacteria to study basic genetic processes.

Modeling bacterial resistance to antibiotics: bacterial conjugation and drug effects

Antibiotic resistance is a major burden in many hospital settings as it drastically reduces the successful probability of treating bacterial infections. Generally, resistance is associated with bacterial fitness reduction and selection pressure from antibiotic usage. Here, we investigate the effects of bacterial conjugation, plasmid loss, and drug responses on the population dynamics of sensitive and resistant bacteria by using a mathematical model. Two types of drugs are considered here: antibiotic M that kills only sensitive bacteria and antibiotic N that kills both bacteria. Our results highlight that larger dose and longer dosing interval of antibiotic M may result in the higher prevalence of resistant bacteria while they do the opposite for antibiotic N. When delays in administering initial and second doses are incorporated, the results demonstrate that the delays may lead to the higher prevalence of resistant bacteria when antibiotic M or N is administered with the longer time of bacteria remaining at the lower prevalence of the latter. Our results highlight that switching antibiotic agents during a treatment course and different bacterial strain characteristics result in a significant impact on the prevalence of resistant bacteria.

Molecular Mechanisms Influencing Bacterial Conjugation in the Intestinal Microbiota

Bacterial conjugation is a widespread and particularly efficient strategy to horizontally disseminate genes in microbial populations. With a rich and dense population of microorganisms, the intestinal microbiota is often considered a fertile environment for conjugative transfer and a major reservoir of antibiotic resistance genes. In this mini-review, we summarize recent findings suggesting that few conjugative plasmid families present in Enterobacteriaceae transfer at high rates in the gut microbiota. We discuss the importance of mating pair stabilization as well as additional factors influencing DNA transfer efficiency and conjugative host range in this environment. Finally, we examine the potential repurposing of bacterial conjugation for microbiome editing.

Phytochrome Mediated Responses in Agrobacterium fabrum: Growth, Motility and Plant Infection

The soil bacterium and plant pathogen Agrobacterium fabrum C58 has two phytochrome photoreceptors, Agp1 and Agp2. We found that plant infection and tumor induction by A. fabrum is down-regulated by light and that phytochrome knockout mutants of A. fabrum have diminished infection rates. The regulation pattern of infection matches with that of bacterial conjugation reported earlier, suggesting similar regulatory mechanisms. In the regulation of conjugation and plant infection, phytochromes are active in darkness. This is a major difference to plant phytochromes, which are typically active after irradiation. We also found that propagation and motility were affected in agp1− and agp2− knockout mutants, although propagation was not always affected by light. The regulatory patterns can partially but not completely be explained by modulated histidine kinase activities of Agp1 and Agp2. In a mass spectrometry-based proteomic study, 24 proteins were different between light and dark grown A. fabrum, whereas 382 proteins differed between wild type and phytochrome knockout mutants, pointing again to light independent roles of Agp1 and Agp2.

Multiple Layered Control of the Conjugation Process of the Bacillus subtilis Plasmid pLS20

Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter Pc located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum-signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the Pc promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed.