Producing gene deletions in Escherichia coli by P1 transduction with excisable antibiotic resistance cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion

Type V secretion systems: an overview of passenger domain functions

Bacteria secrete proteins for different purposes such as communication, virulence functions, adhesion to surfaces, nutrient acquisition, or growth inhibition of competing bacteria. For secretion of proteins, Gram-negative bacteria have evolved different secretion systems, classified as secretion systems I through IX to date. While some of these systems consist of multiple proteins building a complex spanning the cell envelope, the type V secretion system, the subject of this review, is rather minimal. Proteins of the Type V secretion system are often called autotransporters (ATs). In the simplest case, a type V secretion system consists of only one polypeptide chain with a β-barrel translocator domain in the membrane, and an extracellular passenger or effector region. Depending on the exact domain architecture of the protein, type V secretion systems can be further separated into sub-groups termed type Va through e, and possibly another recently identified subtype termed Vf. While this classification works well when it comes to the architecture of the proteins, this is not the case for the function(s) of the secreted passenger. In this review, we will give an overview of the functions of the passengers of the different AT classes, shedding more light on the variety of functions carried out by type V secretion systems

Aptasensor for the detection of Moraxella catarrhalis adhesin UspA2

Innovative point-of-care (PoC) diagnostic platforms are desirable to surpass the deficiencies of conventional laboratory diagnostic methods for bacterial infections and to tackle the growing antimicrobial resistance crisis. In this study, a workflow was implemented, comprising the identification of new aptamers with high affinity for the ubiquitous surface protein A2 (UspA2) of the bacterial pathogen Moraxella catarrhalis and the development of an electrochemical biosensor functionalized with the best-performing aptamer as a bioreceptor to detect UspA2. After cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) was performed, next-generation sequencing was used to sequence the final aptamer pool. The most frequent aptamer sequences were further evaluated using bioinformatic tools. The two most promising aptamer candidates, Apt1 and Apt1_RC (Apt1 reverse complement), had Kd values of 214.4 and 3.4 nM, respectively. Finally, a simple and label-free electrochemical biosensor was functionalized with Apt1_RC. The aptasensor surface modifications were confirmed by impedance spectroscopy and cyclic voltammetry. The ability to detect UspA2 was evaluated by square wave voltammetry, exhibiting a linear detection range of 4.0 × 104–7.0 × 107 CFU mL−1, a square correlation coefficient superior to 0.99 and a limit of detection of 4.0 × 104 CFU mL−1 at pH 5.0. The workflow described has the potential to be part of a sensitive PoC diagnostic platform to detect and quantify M. catarrhalis from biological samples.The study received financial support from the ViBrANT project, which received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska Curie, grant agreement no. 765042. In addition, the authors acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and of LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. D.L. and A.S. received additional funding from the Research Council of Norway (grant 294605, Center for Digital Life). L.D.R.M. acknowledges funding from the FCT through the Scientific Employment Stimulus Program (2021.00221.CEECIND).info:eu-repo/semantics/publishedVersio

The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence

Yersinia ruckeri causes enteric redmouth disease (ERM) that mainly affects salmonid fishes and leads to significant economic losses in the aquaculture industry. An increasing number of outbreaks and the lack of effective vaccines against some serotypes necessitates novel measures to control ERM. Importantly, Y. ruckeri survives in the environment for long periods, presumably by forming biofilms. How the pathogen forms biofilms and which molecular factors are involved in this process, remains unclear. Yersinia ruckeri produces two surface‐exposed adhesins, belonging to the inverse autotransporters (IATs), called Y. ruckeri invasin (YrInv) and Y. ruckeri invasin‐like molecule (YrIlm). Here, we investigated whether YrInv and YrIlm play a role in biofilm formation and virulence. Functional assays revealed that YrInv and YrIlm promote biofilm formation on different abiotic substrates. Confocal microscopy revealed that they are involved in microcolony interaction and formation, respectively. The effect of both IATs on biofilm formation correlated with the presence of different biopolymers in the biofilm matrix, including extracellular DNA, RNA and proteins. Moreover, YrInv and YrIlm contributed to virulence in the Galleria mellonella infection model. Taken together, we propose that both IATs are possible targets for the development of novel diagnostic and preventative strategies to control ERM

Insights into the autotransport process of a trimeric autotransporter, Yersinia Adhesin A (YadA)

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram‐negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid‐state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N‐termini facing the extracellular surface, and a second with the N‐termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Conditional gene expression using ribozymes : Post-transcriptional control of amino acid identity in protein synthesis and temperature-dependent gene expression

During the main research study we wanted to demonstrate that it is possible to control the amino acid identity in vivo on a post-transcriptional level by switching designer tRNAs via the ligand-dependent regulation of ribozyme activity.For this purpose we designed and generated various modular constructs that are composed of distinct parts: an aptamer, a catalytic ribozyme core (hammerhead ribozyme) and of course the appropriate amber suppressor tRNA. Our design included the connection of aptamer (theophylline or thiamine pyrophosphate) to ribozyme via a small connecting sequence, which was randomized. With the help of in vivo screening process we managed to identify functional riboswitches that possess a desired activity (ON or OFF). This activity resulted in the conditional release or not of a suppressor tRNA which in turn serves as tool used for decoding of the amber stop codon in the mRNA of our reporter gene, a fluorescent protein.We managed to obtain novel, individual tRNA riboswitches that we successfully combined in a dual fashion, in order to switch one or the other tRNA, therefore the protein synthesis of one or the other protein variant.Considering the fact that suppression efficiency is inherently low due to release factor competition to the generated suppressor tRNA, this methodology is not quantitative. However, we demonstrated that the protein of interest can be selectively generated in relatively high yields with the use of desired small molecule trigger.This idea can be further developed and exploited in ways that benefit the field of protein chemistry. Considering the high modularity of our constructs, one can envision the multitude of possibilities arising from substituting or expanding by adding one or more parts to the construct. For example, use of different aptamer sensor in combination with a tRNA will unlock the potential generation of more than two different protein variants, using all the while, the same message RNA containing one amber stop codon for which all suppressor tRNAs compete for. Of course optimization of the current system by utilizing more stringent expression control systems could improve the overall performance of the riboswitching system. Another approach which can enhance the performance of our current system is the use of release factor knockout strains which is documented to double the efficiency of suppression systems. Moreover, the applicability of our already versatile tool can be exponentially increased when combined with the fascinating and thriving field of unnatural amino acids. Considering the fact that the central dogma is highly conserved throughout all domains of life, our approach should be able to be realized in higher organisms as well.Finally, we demonstrated that it is possible to design and synthesize ribozyme-based gene regulation elements that are able to respond to temperature changes. This can be achieved with the aid of a thermosensing hairpin that melts with increased temperatures which in turn modulates the function of the hammerhead ribozyme. By replacing the ligand responsive aptamer in the hammerhead ribozyme construct with a Salmonella RNA thermometer we expanded the concept of hammerhead ribozyme regulated gene expression. In this new fusion construct the hammerhead ribozyme functions by exposing a ribosome-binding site upon self-cleavage, while an RNA hairpin undergoes a conformational change caused by an increase in temperature. After in vivo screening of several temperature-sensitive clones for gene regulation activity, we identified two of such thermozymes. The designed tool acts in reverse manner to the natural thermo-sensitive RNA hairpin, which is used by Salmonella to increase gene expression. New tools were developed in order to regulate gene expression in response to temperature changes.On the whole, we presented methodologies that include the use of RNA-only toolboxes which can be adapted, modified and applied in the field of protein chemistry and protein engineering. Further development of the existing tools, along with their potential application in higher organisms, is possible to shed light in investigation of protein synthesis pathways, mutant generation and their mechanisms and functions

Assay development for the discovery of small-molecule inhibitors of bacterial adhesion to collagen

We set out to develop scalable assays to measure bacterial adhesion to mammalian extracellular matrix proteins, with the aim to perform high-throughput screening for inhibitors. Our model system is the trimeric autotransporter adhesin YadA from Yersinia enterocolitica that binds to collagen. Using bacterial cells expressing GFP under an inducible promotor, and co-expressing the adhesin of choice, we were able to establish a 384-well plate-based assay that allowed us to screen 28,000 compounds in 8 days (3520 compounds per day). We have collected all parameters that were essential in assay development, and describe how they can be tuned for improved performance. Out of 28,000 compounds, 5 compounds showed significant inhibitory activity, measured as loss of fluorescence compared to control wells. Our assay is easy to scale up, and can be adopted to different ECM component/Adhesin combinations. Alternatively, bacterial pathogens (harboring deletion mutants of adhesins compared to wildtype) could be used directly in the same assay if they express GFP as a reporter at high levels