Lakhasly

Microbial Physiology, a mandatory course for Biotechnology undergraduates, traditionally includes six lab practices over two months. Due to the pandemic, a revised approach using Pseudomonas aeruginosa PAO1 was developed to efficiently cover key concepts within a shorter timeframe. This approach uses P. aeruginosa PAO1, a Gram-negative opportunistic pathogen, whose virulence factors are regulated by its Quorum Sensing (QS) system, involving autoinducers like N-acyl homoserine lactones (AHLs). Three interconnected QS systems (LasI-LasR, RhlI-RhlR, and PQS) regulate gene expression through autoinduction loops. Quorum Quenching (QQ), the interference with QS systems, is demonstrated using a aiiA gene (encoding an AHL-degrading enzyme) expressed in P. aeruginosa PAO1. The reduction in AHL levels is observed using biosensor strains: Chromobacterium subtsugae CV026 (detects C4-C8 AHLs, producing violacein) and P. putida F117 (pKR-C12) (detects C10-C14 AHLs, producing green fluorescent protein). These experiments, detailed in Figure 1, integrate bacterial conjugation, bioactive molecules, signaling/regulation, and macromolecule degradation, offering a comprehensive and interconnected learning experience within the limited lab time.

‏INTRODUCTION
‏In our Faculty, Microbial Physiology is a mandatory course
‏for undergraduate students for the Bachelor’s degree in
‏Biotechnology. This course involves 6 weeks of practices in the lab,
‏during a two-month period with one lab practice per week,
‏meaning that they take, at most, six practice classes during the
‏course. An extensive list of concepts must be acquired by the
‏students throughout the Microbial Physiology course, including
‏regulation of microbial physiology, synthesis and degradation of
‏macromolecules, metabolic pathways, among others. Other
‏concepts (e.g., microbial growth, nutrition, and antibiosis) and
‏skills (e.g., aseptic techniques, preparation of culture media,
‏seeding and incubation) are acquired in General Microbiology
‏course in the previous semester.
‏The global pandemic situation that we have gone through in
‏the last 2 and a half years, has led us to rethink our traditional
‏laboratory practices. In our Faculty, the coming back to the in-
‏person classes occurred in the middle of the semester. With less
‏weeks for face-to-face teaching, teachers needed to achieve the
‏study goals without lowering the education quality. For students,
‏they had to acquire, in a short time, the corresponding skills
‏related to their degree.
‏Employing Pseudomonas aeruginosa strain PAO1, we propose a
‏series of lab practices in which students, in a short period of time,
‏learn different concepts of microbial physiology in an interrelated
‏way:
‏• Bacterial conjugation as a gene transfer mechanism
‏1. Bioactive molecules
‏2. Signaling and regulation of microbial physiology
‏through cell-to-cell communication
‏3. Degradation of macromolecules
‏Pseudomonas (P.) aeruginosa is a well know Gram-negative
‏strain; an opportunistic pathogen widely spread in soil and
‏aquatic environments (Qin et al. 2022). In P. aeruginosa PAO1,
‏several virulence factors, including extracellular enzymes,
‏production of biofilm, toxins, secondary metabolites, and
‏siderophores, are regulated by its Quorum Sensing (QS) system
‏(Chadha, Harjai, and Chhibber 2022).
‏QS is a well know cell density-dependent intercellular
‏communication system that allows individual cells to act as a
‏community. QS mechanisms are based on the production, release,
‏and detection of signal molecules called autoinducers (Fuqua and
‏Greenberg 2002). Similar to several other Alphaproteobacteria,
‏Betaproteobacteria and Gammaproteobacteria, in P. aeruginosa,
‏these bioactive molecules are N-acyl homoserine lactones (AHLs)
‏(Chadha, Harjai, and Chhibber 2022). To date, three QS systems
‏intimately linked among them and arranged in a hierarchical
‏regulatory cascade are known in P. aeruginosa. The autoinducer
‏synthase LasI and RhlI produces AHL molecules as autoinducers:
‏N-oxododecanoyl-L-homoserine lactone (3OC12-HSL) and N-
‏butyryl-L-homoserine lactone (C4-HSL), respectively. When these
‏autoinducers reach a critical threshold level by accumulation due
‏to bacterial population growth, they interact with their receptors
‏LasR and RhlR, respectively, which are also transcriptional
‏activators (Chadha, Harjai, and Chhibber 2022). LasR and RhlR
‏stimulates the lasI and rhlI expression, creating autoinduction
‏loops. A third QS system that relies on the production of 2-heptyl-
‏3-hydroxy-4-quinolone (Pseudomonas quinolone signal or PQS) is
‏interconnected with the LasI-LasR and RhlI-RhlR systems.
‏In nature, microorganisms interfere with the QS systems of
‏competitors mainly through the enzymatic inactivation of the QS
‏signals, or the production of metabolites that impede the normal
‏functioning of the signal receptors, a phenomenon known as
‏Quorum Quenching (QQ) (Grandclément et al. 2016). In the lab,
‏the cell-to-cell communication can be blocked with easy and fast
‏experiments to understand the functioning of the QS system. In
‏the approach chosen for the present work, a aiiA gene encoding
‏the AiiA lactonase from Bacillus sp. A24, an AHL-degrading
‏enzyme, is expressed under a constitutive plac promoter in P.
‏aeruginosa PAO1, reducing the accumulation of autoinducers and
‏the expression of several traits (Reimmann et al. 2002). The easiest
‏way to expose the QQ phenomenon is evidencing with biosensor
‏strains the decrease in the AHL levels. These strains have a genetic
‏deficiency on signal production, but detect and respond to
‏exogenous signals by means of a functional receptor coupled to a
‏reporter gene that produces an observable phenotype when
‏specific QS signals are present (Miller and Gilmore 2020; Steindler
‏and Venturi 2007). Chromobacterium (C.) subtsugae CV026 and P .
‏putida F117 (pKR-C12) are the biosensors used in these practical
‏lab works. C. subtsugae CV026 produce violacein, a purple
‏pigment, in presence of AHLs with acyl side chains from C4 to C8
‏in length (McClean et al. 1997). P . putida F117 (pKR-C12) produce
‏green fluorescent protein in presence of AHLs with acyl side
‏chains from C10 to C14 in length (Riedel et al. 2001).
‏The experimental workflow presented in this work is detailed
‏in Figure 1. Each individual assay is thought of as a piece of a
‏puzzle that fits together giving a single, complete, overall, and
‏multi-focused approach for the lab classes.