Biotechnological production based on bacterial biofilms

Genetic studies for glycerol conversion to bacterial celluloseBiofilms can be described as microbial communities embedded in an extracellular polymeric matrix that attach to both biological and non-biological surfaces. Biofilms are considered an evolutive adaptation to adverse environments that allows bacteria to outlast. This feature makes biofilm forming bacteria attractive for the industrial production of toxic compounds, for example indigo dye used for jeans staining.In this Master degree Final Project, we are going to research the ability of Pseudomonas putida KT2442, a well-known biofilm producer, for the biosynthesis of cellulose (one of the main component of biofilm matrix) through the utilization of glycerol, a by-product from biodiesel industry as carbon source. Previous studies have shown that P. putida displays a prolonged lag phase when glycerol is the only carbon source in the growth media. This long lag phase is mediated by GlpR, the negative repressor of glp gene cluster2. The glp operon encodes for GlpF facilitator, which allows glycerol to diffuse inside the cell; GlpK, the glycerol kinase that phosphorylates glycerol to glycerol-3-phosphate; and GlpD, a glycerol-3-phosphate (G3P) dehydrogenase that transform G3P into dihydroxyacetone-phosphate. Therefore, this Master Degree Final Project has two main objectives: 1) Reduce the P. putida KT2442 glycerol growth lag phase through the deletion of glpR gen by homologous recombination; and 2) the deletion of exopolysaccharides genes of biofilm matrix, in order to only produce cellulose. This is an example of grey biotechnology and white biotechnology, because of the elimination of glycerol which is an environmental pollutant and the design of a process to produce a molecule of interest, respectively

Development of a reporter system for screening anti-biofilm activities

Biofilm formation is responsible for increasing antibiotic tolerance in pathogenic bacteria. It is estimated that approximately 80% of chronic infections are associated with this phenomenon. Therefore, the search for therapeutic agents with specific biofilm targets has become of vital importance. One of the main strategies is the search for enzymes that degrade the different components of the extracellular matrix. However, since the composition of the matrix varies among the different microorganisms, an alternative would be to interfere with the signaling cascades that lead to the formation of the biofilm or stimulate its dispersion

Evaluation of the efficiency of CRISPR-Cas systems in Gram-negative bacteria

CRISPR-Cas is a genetic tool based on a prokaryotic defense system. This is a technology that involves the simple design of an RNA molecule (crRNA) and its binding to a Cas nuclease, forming a complex capable of breaking DNA. This system, in the presence of a repair system, also allows the introduction of mutations in a targeted manner.  CRISPR-Cas has been widely used in eukaryotic cell genome editing, but not so much in prokaryotes [1]. The absence of efficient repair systems in bacteria could be the main reason, but this could be supplemented by the exogenous introduction of a bacterial non-homologous repair system, or NHEJ. Most bacterial NHEJs have only two proteins: Ku, which binds to and protects the broken ends; and LigD, which ligates them [2]. In this work a CRISPR-Cas system has been implemented and studied in Pseudomonas putida, a Gram-negative bacterium of great biotechnological interest, which lacks efficient NHEJ repair systems [3]. For this, two plasmids for the expression of Cas12a and NHEJ from Sphingopyxis granuli TFA have been introduced, in addition to a reporter plasmid that includes the necessary spacer and the target. The efficacy of Cas12 and the NHEJ in double-strand break repair in P. putida has been verified by fluorescence, PCR, and sequencing. However, when the repaired plasmids were analyzed, an unexpected homologous recombination repair (HDR) mechanism was found. The characterization of this phenomenon is currently being carried out

Functional dissection of the large adhesion protein

There are numerous microorganisms which have the ability to switch between being in planktonic stateor form biofilms, these biofilms are complex communities of microorganisms attached to surfaces orassociated with interfaces. This concept has a lot of relevance in ecology due to the fact that thesemicrobial communities are often composed of multiple species that interact with each other and theirenvironment. The longest gene in the Gram-negative bacterium Pseudomonas putida genome encodesLapA, a >9000 amino-acid surface adhesin essential to surface adhesion and biofilm formation. LapA isa complex protein, containing numerous functional domains and a large array of repeated sequences.However, the exact function of any of these elements in LapA is unknown.The strategy of our project is the construction of different lapA variants containing internal deletions ofthe putative functional domains, and study the role of each of these in LapA-dependent phenotypes .Each version will be inserted in the chromosome of a Δ lapA mutant using a Tn 7 -based delivery system.An Initial synthetic construct containing the complete N-terminal and C-terminal domains and a 3xHAtag for immunodetection, but lacking all repeated sequences is already available, and constructsbearing progressively shorter N-terminal domains are underway. Addition of different numbers ofrepeats will be tested afterwards.Phenotypic assays will include swimming and adhesion assays using different surfaces, biofilmformation curves and microscopic assessment of biofilm morphology under different conditions. Weexpect that this approach will provide useful insight into the functions of the different domains of LapAand the dynamics of biofilm development in P. putida

Development of tools for the widespread implementation of CRISPR-Cas technologies in Gram-negative bacteria

CRISPR-Cas technology has made a huge impact due to its potential for editing, regulating and targeting genomes overthe past years. It has been proven a very powerful and useful tool with lot of potential and a wide range of applications within the biotechnological, pharmaceutical and food industry, among others. Nevertheless, one of the biggestshortcomings of this technology is that, despite the multiple of applications already available, there is potential for many moreuses yet to be discovered.According to that, the Bacteria domain holds the promise of an entire world of opportunities and possibilities, as thistechnology has not been implemented, with the exception of Escherichia coli and a few other species. Many examples ofhighly interesting organisms are Gram-negative bacteria, such as the symbiotic nitrogen-fixing Rhizobium and Shinorizobium spp. Developing the CRISPR-Cas system to be able to modify genetically these organisms, establishingspecific protocols for directed genome or gene expression manipulation, would be a game changer and set a baseline forfurther research. The aim of our project is to develop CRISPR-Cas-based genome editing tools of widespread use in Gram-negative bacteria and test their fucntion in three members of the Rhizobial grou, namely Rhizobium legumirosarum, Shinorhizobium melilotiand Sinorhizobium fredii. The strategy designed consists on the construction two broad host-range plasmids: one expressing the nuclease Cas12a, and a second one expressing a Cas12a gRNA, and containing the gRNA target sequence and GFP (Green Fluorescent Protein) as a reproter gene. The ability of Cas12a to induce loss of this reporter plasmid (and the GFP reporter encoded therein) will be assessed, and the efficiency by which expression of different repair systems enable persistance of the plasmid by introducing mutations at the cleaved target calculated. The combination of efficient target cleavage and efficient mutation-inducing repair will provide a suitable toolkit for genome engineering in these organisms. As we progress with this project we expect to be able to offer the scientific community a complete toolbox of Cas nucleases, repair systems and testing procedures that will allow the identification of the optimal tool combination for each organism

Phenotypic heterogeneity in Pseudomonas syringae

The notion of isogenic bacterial populations displaying phenotypic differences is widely accepted today. A particular case of phenotypic heterogeneity is bistability. Bistability occurs when bacterial population splits into two subpopulations showing distinct phenotypes. Phenotypic heterogeneity can allow some individuals to survive sudden environmental changes (risk-spreading) and can also lead to the cooperation (division of labour) between individuals. The relevance of this process has been highlighted in some animal pathogens, nevertheless, little is known about the occurrence or impact of these processes in the adaptation of bacteria to non-animal hosts. Pseudomonas syringae is a plant-pathogenic bacterium whose virulence depend of the T3SS expression. We have reported that T3SS expression is bistable in hrp-induction medium. This bistability generates two subpopulations, that show differences in virulence. Flagella is also an important virulence determinant for Pseudomonas syringae colonization. Here, we show how flagella expression also displays markedly phenotypic heterogeneity during growth within the plant. Although subpopulations displaying flagONT3SSON and flagOFFT3SSOFF can be identified within the plant, we provide evidence of cross regulation between T3SS and flagella expression at the individual cell level and propose phenotypic heterogeneity as an adaptative value for Pseudomonas adaptation to the plant host.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Development of a novel and rapid molecular diagnostic system for Hepatitis C virus (HCV) detection.

Hepatitis C virus (HCV) is an enveloped, single-stranded positive sense RNA [ssRNA(+)] virus which causes cirrhosis, hepatocellular carcinoma and liver failure in infected patients [1]. The Global Hepatitis Report posted in 2017, indicates that around 71 million people were living with HCV in 2015 [2]. Moreover, the most recent data collected in 2019 by WHO was able to estimate that 58 million people live with chronic hepatitis C infection and about 1.5 million new infections occur per year. Due to the increasing number of infections, one of the recommendations postulated in the 2022 guidelines ("Updated recommendations on HCV simplified service delivery and HCV diagnostics: policy brief") was to achieve more efficient and simplified hepatitis diagnosis [3]. The diagnosis of an active HCV infection requieres the detection of HCV RNA, and nowadays it is performed by the gold standard real-time reverse transcription polymerase chain reaction (RT-qPCR). Nevertheless, it involves specific laboratory facilities, well-trained personnel and high-cost equipment, maintenance and reagents [4]. Consequently, this project was born on the need of developing a new diagnostic system for direct HCV molecular detection based on the RT-LAMP (reverse transcription loop-mediated isothermal amplification) technique that could be implemented in resource-limited settings. This strategy is focused on the nucleic acid-based amplification method using from four to six primers under isothermal conditions to amplify specifically and effectively the target sequence [4]. The aim of the project is to validate the system and integrate it in a future point-of-care (POC) diagnostic device

Contribution of Phenotypic Heterogeneity to plant colonization by Pseudomonas syringae

In bacterial clonal populations, cell to cell differences can be originated by the response to different stimuli present in the environment. However, the sources of variation may not always be directly correlated with stimuli. In some cases, these differences are merely a consequence of the noise in gene expression or in others, a programmed event under genetic or epigenetic control. The presence of different phenotypes can allow some individuals to survive sudden environmental changes (risk-spreading) and can also lead to the division of labour between individuals. The relevance of this process has been demonstrated in Salmonella and other human pathogens for the expression of virulence genes and has been linked to the establishment of a successful infection. However, little is known about the importance of this process in the colonization of the plant tissue. In the phytopathogenic bacteria Pseudomonas syringae we have demonstrated that the T3SS show phenotypic heterogeneity during the colonization of the plant. We have also established that flagella is expressed and displays phenotypic heterogeneity during colonization of the apoplast. These processes are counter-regulated. Nonetheless, all possible combinations for T3SS and flagella expression are formed within the apoplastic population, including T3SSON/FlagellaON and T3SSOFF/FlagellaOFF bacteria. We show that expression and function of these virulence-relevant loci impact bacterial fitness and describe how plant defences modulate their expression at the population level. All these observations support the notion that the phenotypic heterogeneity is a relevant process for the adaptation of P. syringae to the plant host.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Phenotypic heterogeneity during plant colonization

Isogenic bacterial populations can display phenotypic differences. Cell-to-cell phenotypic differences can be a consequence of noisy gene expression, or a programmed event under genetic or epigenetic control. Bistability occurs when populations splits into subpopulations showing distinct phenotypes. This heterogeneity can allow individuals to survive environmental changes or can lead to cooperation between individuals. This is highly relevant for some animal pathogens, but little is known about it for plant colonizing bacteria. We have reported that T3SS expression in Pseudomonas syringae is bistable in hrp-induction medium and displays phenotypic heterogeneity during plant colonization. This bistability generates two subpopulations that show differences in virulence. Flagella is also an important virulence determinant for Pseudomonas syringae colonization that displays a degree of counter-regulation with the T3SS. Salmonella enterica serovar typhimurium SPI-1 T3SS is also expressed bistably and counter-regulates with flagella. SPI-1 bistable expression is a important asset during mouse infection as it leads to cooperative virulence. But although S. enterica colonize plants as alternative hosts and requires SPI-1 to do so efficiently, little is know about the expression of this system during plant colonization We present our newest findings regarding phenotypic heterogeneity of virulence relevant traits of P. syringae and S. enterica during plant colonization.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Expression of flagellar and type III secretion systems is under stochastic and deterministic regulation in Pseudomonas syringae.

We previously described bistability and phenotypic heterogeneity of the type III secretion system (T3SS) of Pseudomonas syringae (Rufián et al., 2016). First example of such phenomenon in a plant pathogen. Here, we describe heterogenous flagellar expression leads to phenotypic heterogeneity within P. syringae populations. We find that although as reported flagellin is downregulated inside the plant, it is still expressed by a part of the bacterial population that maintains high expression levels during colonization of the plant apoplast. We demonstrate that expression of the T3SS and flagellar systems undergo counter regulation that is displayed at a single-cell level as T3SSON/FlagellaOFF and T3SSOFF/FlagellaON subpopulations. Despite this counter regulation, T3SSON/FlagellaON and T3SSOFF/FlagellaOFF bacteria can also be found within the apoplast at significant levels. Genetic analysis of the elements involved shows that counter-regulation is reciprocal: altered levels of T3SS transcriptional activator HrpL affect flagellar expression and altered levels of flagellar master regulator FleQ affect T3SS gene expression. But it also shows that the heterogeneity of each of these systems arises through independent mechanisms and display different dynamics. The regulatory loops involved in establishing T3SS and flagellar heterogeneity in P. syringae are different to those described for these systems in animal pathogen, suggesting convergent evolution of heterogeneity. Finally, we analyze the biological implications of heterogeneity and propose that, through a division of labor strategy, heterogeneity may provide adaptive value to this pathogen. This is one of the few examples where phenotypic heterogeneity is analyzed in natural conditions within the context of host colonization.Proyecto PID2021-127245OB-I00; MCIN/ AEI/10.13039/501100011033/ Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech