Mature Pseudomonas aeruginosa biofilms undergo specific developmental events. Using a bacteriophage mutant, generated by deletion of the entire filamentous Pf4 prophage, we show that the phage is essential for several stages of the biofilm life cycle and that it significantly contributes to the virulence of P. aeruginosa in vivo. Here, we show for the first time that biofilms of the Pf4 phage-deficient mutant did not develop hollow centres or undergo cell death, typical of the differentiation process of wild-type (WT) P. aeruginosa PAO1 biofilms. Furthermore, microcolonies of the Pf4 mutant were significantly smaller in size and less stable compared with the WT biofilm. Small colony variants (SCVs) were detectable in the dispersal population of the WT biofilm at the time of dispersal and cell death, whereas no SCVs were detected in the effluent of the Pf4 mutant biofilm. This study shows that at the time when cell death occurs in biofilms of the WT, the Pf4 phage converts into a superinfective form, which correlates with the appearance of variants in the dispersal population. Unexpectedly, mice infected with the Pf4 mutant survived significantly longer than those infected with its isogenic WT strain, showing that Pf4 contributes to the virulence of P. aeruginosa. Hence, a filamentous prophage is a major contributor to the life cycle and adaptive behaviour of P. aeruginosa and offers an explanation for the prevalence of phage in this organism.
Generation of genetic diversity is a prerequisite for bacterial evolution and adaptation. Short-term diversification and selection within populations is, however, largely uncharacterised, as existing studies typically focus on fixed substitutions. Here, we use whole-genome deep-sequencing to capture the spectrum of mutations arising during biofilm development for two Pseudomonas aeruginosa strains. This approach identified single nucleotide variants with frequencies from 0.5% to 98.0% and showed that the clinical strain 18A exhibits greater genetic diversification than the type strain PA01, despite its lower per base mutation rate. Mutations were found to be strain specific: the mucoid strain 18A experienced mutations in alginate production genes and a c-di-GMP regulator gene; while PA01 acquired mutations in PilT and PilY1, possibly in response to a rapid expansion of a lytic Pf4 bacteriophage, which may use type IV pili for infection. The Pf4 population diversified with an evolutionary rate of 2.43 × 10 −3 substitutions per site per day, which is comparable to single-stranded RNA viruses. Extensive within-strain parallel evolution, often involving identical nucleotides, was also observed indicating that mutation supply is not limiting, which was contrasted by an almost complete lack of noncoding and synonymous mutations. Taken together, these results suggest that the majority of the P. aeruginosa genome is constrained by negative selection, with strong positive selection acting on an accessory subset of genes that facilitate adaptation to the biofilm lifecycle. Long-term bacterial evolution is known to proceed via few, nonsynonymous, positively selected mutations, and here we show that similar dynamics govern shortterm, within-population bacterial diversification.dispersal | prophage | haplotypes D iversifying selection within bacterial populations underpins a range of ecological and clinical phenomena, for example niche adaptation (1-3) and antibiotic resistance (4). During diversifying selection, a single population explores multiple fitness peaks, resulting in subpopulations with different adaptive mutations. This kind of within-population genetic diversity can provide the raw material on which long-term evolution acts. However, diversifying selection of bacterial populations is still poorly understood, because previous experimental evolution and epidemiological studies typically have focused on fixed substitutions at the end point of evolution (5), ignoring short-term or withinpopulation effects.Laboratory-grown Pseudomonas aeruginosa biofilms provide an ideal model for investigating within-population diversification. The bacterium P. aeruginosa is a widespread, Gram-negative generalist and can have either a planktonic, motile lifestyle or exist as a biofilm (i.e., a surface-attached cells embedded within an extracellular polymeric matrix). P. aeruginosa has been the focus of extensive research, because of both its status as a model organism and its ability to form opportunistic, chronic, often lethal biof...
Persistent lung infection by Pseudomonas aeruginosa is typically associated with the development of biofilms, the appearance of morphotypic variants and reduction in the expression of acute virulence factors. We have characterised and compared functional traits [carbon substrate utilisation, attachment and biofilm formation, protease and elastase activity, quorum‐sensing (QS)] of the biofilm dispersal populations of a representative P. aeruginosa isolate from a chronically infected cystic fibrosis individual and P. aeruginosa strain PAO1. The dispersal variants of the clinical strain exhibited significantly greater heterogeneity in all of the phenotypes tested. All morphotypic variants from the dispersal population of the clinical strain showed a significant increase in QS signal and elastase production compared to the parental strain. In contrast, isolates from planktonic cultures were phenotypically identical to the inoculum strain, suggesting that the appearance of these variants was biofilm specific. The clinical strain was shown to have a 3.4‐fold higher mutation frequency than PAO1 which corroborated with the increased diversity of dispersal isolates. These data suggest that the development of a chronic infection phenotype can be reversed to recover acute infection isolates and that growth within a biofilm facilitates diversification of P. aeruginosa which is important for ecological adaptation.
By screening a collection of Salmonella mutants deleted for genes encoding small proteins of ≤60 amino acids, we identified three paralogous small genes (ymdF, STM14_1829, and yciG) required for wild-type flagellum-dependent swimming and swarming motility. The ymdF, STM14_1829, and yciG genes encode small proteins of 55, 60, and 60 amino acid residues, respectively. A bioinformatics analysis predicted that these small proteins are intrinsically disordered proteins, and circular dichroism analysis of purified recombinant proteins confirmed that all three proteins are unstructured in solution. A mutant deleted for STM14_1829 showed the most severe motility defect, indicating that among the three paralogs, STM14_1829 is a key protein required for wild-type motility. We determined that relative to the wild type, the expression of the flagellin protein FliC is lower in the ΔSTM14_1829 mutant due to the downregulation of the flhDC operon encoding the FlhDC master regulator. By comparing the gene expression profiles between the wild-type and ΔSTM14_1829 strains via RNA sequencing, we found that the gene encoding the response regulator PhoP is upregulated in the ΔSTM14_1829 mutant, suggesting the indirect repression of the flhDC operon by the activated PhoP. Homologs of STM14_1829 are conserved in a wide range of bacteria, including Escherichia coli and Pseudomonas aeruginosa. We showed that the inactivation of STM14_1829 homologs in E. coli and P. aeruginosa also alters motility, suggesting that this family of small intrinsically disordered proteins may play a role in the cellular pathway(s) that affects motility. IMPORTANCE This study reports the identification of a novel family of small intrinsically disordered proteins that are conserved in a wide range of flagellated and nonflagellated bacteria. Although this study identifies the role of these small proteins in the scope of flagellum-dependent motility in Salmonella, they likely play larger roles in a more conserved cellular pathway(s) that indirectly affects flagellum expression in the case of motile bacteria. Small intrinsically disordered proteins have not been well characterized in prokaryotes, and the results of our study provide a basis for their detailed functional characterization.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.