Biology subjects

Halte, M.

Publications and source records attributed to Halte, M..

5 recordsLinked to original sources

bioRxiv2024

Fluorescent tools for the standardized work in Gram-negative bacteria

Standardized and thoroughly characterized genetic tools are a prerequisite for studying cellular processes to ensure the reusability and consistency of experimental results. The discovery of fluorescent proteins (FPs) represents a milestone in the development of genetic reporters for monitoring transcription or protein localization in vivo. FPs have revolutionized our understanding of cellular dynamics by enabling the real-time visualization and tracking of biological processes. Despite these advancements, challenges remain in the appropriate use of FPs, specifically regarding their proper application, protein turnover dynamics, and the undesired disruption of cellular functions. Here, we systematically compared a comprehensive set of 16 FPs and assessed their performance in vivo by focusing on key parameters, such as signal over background ratios and protein stability rates, using the gram-negative model organism Salmonella enterica as a representative host. We evaluated four protein degradation tags in both plasmid- and genome-based systems and our findings highlight the necessity of introducing degradation tags to analyze time-sensitive cellular processes. We demonstrate that the gain of dynamics mediated by the addition of degradation tags impacts the cell-to-cell heterogeneity of plasmid-based but not genome-based reporters. Finally, we probe the applicability of FPs for protein localization studies in living cells using super-resolution microscopy. In summary, our study underscores the importance of careful FP selection and paves the way for the development of improved genetic reporters to enhance the reproducibility and reliability of fluorescence-based research in gram- negative bacteria and beyond.

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↗

bioRxiv2022

Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest

Initiation of bacterial DNA replication takes place at the origin of replication, a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. The absence or failure of DNA replication can result in bacterial cell growth arrest or death. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. For this purpose, DNA replication was blocked using a CRISPRi approach specifically targeting DnaA boxes 6 and 7, which are essential for replication initiation. We characterized the phenotype of these cells and analyzed the overall changes in the proteome using quantitative mass spectrometry. Cells with arrested replication were elongating and not dividing but showed no evidence of DNA damage response. Moreover, these cells did not cease translation over time. This study sets the ground for future research on non-replicating but translationally active B. subtilis, which might be a valuable tool for biotechnological applications. ImportanceEven though bacteria are constantly replicating under laboratory conditions, natural environments expose them to various stresses like lack of nutrients, high salinity, and pH changes, which can keep them in non-replicating states. Non-replicating states can allow bacteria to become less sensitive or tolerant to antibiotics (persisters), remain inactive in specific niches for an extended period (dormancy), and adapt to some hostile ecosystems. Non-replicating states have been studied due to the possibility of repurposing energy to produce additional metabolites or proteins. Using CRISPRi targeting bacterial replication initiation sequences, we successfully arrested the replication of B. subtilis. We observed that non-replicating cells continued growing but not dividing, and the initial arrest did not induce global stress conditions such as SOS or stringent response. Notably, these cells continued their metabolic activity and translation. This study provides comprehensive insights into the physiological response of replication initiation blockage in B. subtilis.

microbiology↗

bioRxiv2022

A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes

Transmembrane potential is one of the main bioenergetic parameters of bacterial cells, and is directly involved in energising key cellular processes such as transport, ATP synthesis, and motility. The most common approach to measure membrane potential levels is through use of voltage-sensitive fluorescent dyes. Such dyes either accumulate or are excluded from the cell in a voltage-dependent manner, which can be followed by means of fluorescence microscopy, flow cytometry, or fluorometry. Since the cells ability to maintain transmembrane potential relies upon low membrane ion conductivity, voltage-sensitive dyes are also highly sensitive reporters for the activity of membrane-targeting antibacterials. However, the presence of an additional membrane layer in Gram-negative (diderm) bacteria significantly complicates their use. In this manuscript, we provide guidance on how membrane potential and its changes can be reliably monitored in Gram-negatives using the voltage-sensitive dye DiSC3(5). We also discuss the confounding effects caused by the presence of the outer membrane, or by measurements performed in buffers rather than growth medium. We hope that the discussed methods and protocols provide an easily accessible basis for the use of voltage-sensitive dyes in Gram-negative organisms, and raise awareness of potential experimental pitfalls associated with their use.

microbiology↗

bioRxiv2021

BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP

The widespread bacterial second messenger bis-(3-5)-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins. As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups. ImportanceThe second messenger c-di-GMP controls various bacterial functions including development of resistant biofilm communities and transition into dormant spores. In vivo detection of c-di-GMP levels is therefore crucial for a better understanding of how intracellular c-di-GMP levels induce changes of bacterial physiology. Here, we describe the design of a novel c-di-GMP biosensor and demonstrate its effective application in investigating fluctuations in intracellular c-di-GMP levels in Escherichia coli and Salmonella enterica serovar Typhimurium on a population-based and single-cell level.

microbiology↗