Microorganisms produce costly cooperative goods whose benefit is partially shared with nonproducers, called 'mixed' goods. The Black Queen Hypothesis predicts that partial privatization has two major ...evolutionary implications. First, to favor strains producing several types of mixed goods over nonproducing strains. Second, to favor the maintenance of cooperative traits through different strains instead of having all cooperative traits present in a single strain (metabolic specialization). Despite the importance of quorum sensing regulation of mixed goods, it is unclear how partial privatization affects quorum sensing evolution. Here, we studied the influence of partial privatization on the evolution of quorum sensing. We developed a mathematical population genetics model of an unstructured microbial population considering four strains that differ in their ability to produce an autoinducer (quorum sensing signaling molecule) and a mixed good. Our model assumes that the production of the autoinducers and the mixed goods is constitutive and/or depends on quorum sensing. Our results suggest that, unless autoinducers are costless, partial privatization cannot favor quorum sensing. This result occurs because with costly autoinducers: (1) a strain that produces both autoinducer and goods (fully producing strain) cannot persist in the population; (2) the strain only producing the autoinducer and the strain producing mixed goods in response to the autoinducers cannot coexist, i.e., metabolic specialization cannot be favored. Together, partial privatization might have been crucial to favor a primordial form of quorum sensing-where autoinducers were thought to be a metabolic byproduct (costless)-but not the transition to nowadays costly autoinducers.
•We reviewed the discovery and development process of broad-spectrum antiviral agents.•We summarized the information on 120 safe-in-man agents in a freely accessible database.•Further studies will ...increase the number of broad-spectrum antivirals, expand the spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections.
Viral diseases are one of the leading causes of morbidity and mortality in the world. Virus-specific vaccines and antiviral drugs are the most powerful tools to combat viral diseases. However, broad-spectrum antiviral agents (BSAAs, i.e. compounds targeting viruses belonging to two or more viral families) could provide additional protection of the general population from emerging and re-emerging viral diseases, reinforcing the arsenal of available antiviral options. Here, we review discovery and development of BSAAs and summarize the information on 120 safe-in-man agents in a freely accessible database (https://drugvirus.info/). Future and ongoing pre-clinical and clinical studies will increase the number of BSAAs, expand the spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections as well as co-infections.
Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide ...insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ∼2 weeks.
Summary
Extracellular polysaccharides comprise a major component of the biofilm matrix. Many species that are adept at biofilm formation have the capacity to produce multiple types of ...polysaccharides. Pseudomonas aeruginosa produces at least three extracellular polysaccharides, alginate, Pel and Psl, that have been implicated in biofilm development. Non‐mucoid strains can use either Pel or Psl as the primary matrix structural polysaccharide. In this study, we evaluated a range of clinical and environmental P. aeruginosa isolates for their dependence on Pel and Psl for biofilm development. Mutational analysis demonstrates that Psl plays an important role in surface attachment for most isolates. However, there was significant strain‐to‐strain variability in the contribution of Pel and Psl to mature biofilm structure. This analysis led us to propose four classes of strains based upon their Pel and Psl functional and expression profiles. Our data also suggest that Pel and Psl can serve redundant functions as structural scaffolds in mature biofilms. We propose that redundancy could help preserve the capacity to produce a biofilm when exopolysaccharide genes are subjected to mutation. To test this, we used PAO1, a common lab strain that primarily utilizes Psl in the matrix. As expected, a psl mutant strain initially produced a poor biofilm. After extended cultivation, we demonstrate that this strain acquired mutations that upregulated expression of the Pel polysaccharide, demonstrating the utility of having a redundant scaffold exopolysaccharide. Collectively, our studies revealed both unique and redundant roles for two distinct biofilm exopolysaccharides.
Bacteria have a tendency to attach to surfaces and grow as structured communities called biofilms. Chronic biofilm infections are a problem because they tend to resist antibiotic treatment and are ...difficult to eradicate. Bacterial biofilms have an extracellular matrix that is usually composed of a mixture of polysaccharides, proteins, and nucleic acids. This matrix has long been assumed to play a passive structural and protective role for resident biofilm cells. Here we show that this view is an oversimplification and that the biofilm matrix can play an active role in stimulating its own synthesis. Working with the model biofilm bacterium Pseudomonas aeruginosa , we found that Psl, a major biofilm matrix polysaccharide for this species, acts as a signal to stimulate two diguanylate cyclases, SiaD and SadC, to produce the intracellular secondary messenger molecule c-di-GMP. Elevated intracellular concentrations of c-di-GMP then lead to the increased production of Psl and other components of the biofilm. This mechanism represents a unique positive feedback regulatory circuit, where the expression of an extracellular polysaccharide promotes biofilm growth in a manner analogous to autocrine signaling in eukaryotes.
Extracellular polysaccharides are important components of biofilms. In non-mucoid Pseudomonas aeruginosa strains, the Pel and Psl polysaccharides are major structural components of the biofilm ...matrix. In this study, we demonstrate that the alternative σ-factor RpoS is a positive transcriptional regulator of psl gene expression. Furthermore, we show that psl mRNA has an extensive 5′ untranslated region, to which the post-transcriptional regulator RsmA binds and represses psl translation. Our observations suggest that upon binding RsmA, the region spanning the ribosome binding site of psl mRNA folds into a secondary stem-loop structure that blocks the Shine-Dalgarno sequence, preventing ribosome access and protein translation. This constitutes a novel mechanism for translational repression by this family of regulators.
In traditional models ofin vitrobiofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted ...to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development ofPseudomonas aeruginosabiofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation.
During the past decades, there has been a consensus around the model of development of a biofilm, involving attachment of single planktonic bacterial cells to a surface and the subsequent development of a mature biofilm. This study presents results that call for a modification of this rigorous model. We show how free floating biofilm aggregates can have a profound local effect on biofilm development when attaching to a surface. Our findings show that an aggregate landing on a surface will eventually outcompete the biofilm population arising from single cells attached around the aggregate and dominate the local biofilm development. These results point to a regime where preformed biofilm aggregates may have a fitness advantage over planktonic cells when it comes to accessing nutrients. Our findings add to the increasingly prominent comprehension that biofilm lifestyle is the default for bacteria and that planktonic single cells may be only a transition state at the most.
Bacterial biofilms are usually assumed to originate from individual cells deposited on a surface. However, many biofilm-forming bacteria tend to aggregate in the planktonic phase so that it is ...possible that many natural and infectious biofilms originate wholly or partially from pre-formed cell aggregates. Here, we use agent-based computer simulations to investigate the role of pre-formed aggregates in biofilm development. Focusing on the initial shape the aggregate forms on the surface, we find that the degree of spreading of an aggregate on a surface can play an important role in determining its eventual fate during biofilm development. Specifically, initially spread aggregates perform better when competition with surrounding unaggregated bacterial cells is low, while initially rounded aggregates perform better when competition with surrounding unaggregated cells is high. These contrasting outcomes are governed by a trade-off between aggregate surface area and height. Our results provide new insight into biofilm formation and development, and reveal new factors that may be at play in the social evolution of biofilm communities.
The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on ...gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for AmrZ regulation of biofilm formation and chronicity.
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