Regulation of Pseudomonas aeruginosa virulence factors by two novel RNA thermometers.

In a number of bacterial pathogens, the production of virulence factors is induced at 37 °C; this effect is often regulated by mRNA structures formed in the 5' untranslated region (UTR) that block translation initiation of genes at environmental temperatures. At 37 °C, the RNA structures become unstable and ribosomes gain access to their binding sites in the mRNAs. Pseudomonas aeruginosa is an important opportunistic pathogen and the expression of many of its virulence-associated traits is regulated by the quorum-sensing (QS) response, but the effect of temperature on virulence-factor expression is not well-understood. The aim of this work is the characterization of the molecular mechanism involved in thermoregulation of QS-dependent virulence-factor production. We demonstrate that traits that are dependent on the QS transcriptional regulator RhlR have a higher expression at 37 °C, correlating with a higher RhlR concentration as measured by Western blot. We also determined, using gene fusions and point mutations, that RhlR thermoregulation is a posttranscriptional effect dependent on an RNA thermometer of the ROSE (Repression Of heat-Shock gene Expression) family. This RNA element regulates the expression of the rhlAB operon, involved in rhamnolipid production, and of the downstream rhlR gene. We also identified a second functional thermometer in the 5' UTR of the lasI gene. We confirmed that these RNA thermometers are the main mechanism of thermoregulation of QS-dependent gene expression in P. aeruginosa using quantitative real-time PCR. This is the first description, to our knowledge, of a ROSE element regulating the expression of virulence traits and of an RNA thermometer controlling multiple genes in an operon through a polar effect.

Pseudomonas aeruginosa ATCC 9027 is a non-virulent strain suitable for mono-rhamnolipids production.

Rhamnolipids produced by Pseudomonas aeruginosa are biosurfactants with a high biotechnological potential, but their extensive commercialization is limited by the potential virulence of P. aeruginosa and by restrictions in producing these surfactants in heterologous hosts. In this work, we report the characterization of P. aeruginosa strain ATCC 9027 in terms of its genome-sequence, virulence, antibiotic resistance, and its ability to produce mono-rhamnolipids when carrying plasmids with different cloned genes from the type strain PAO1. The genes that were expressed from the plasmids are those coding for enzymes involved in the synthesis of this biosurfactant (rhlA and rhlB), as well as the gene that codes for the RhlR transcriptional regulator. We confirm that strain ATCC 9027 forms part of the PA7 clade, but contrary to strain PA7, it is sensitive to antibiotics and is completely avirulent in a mouse model. We also report that strain ATCC 9027 mono-rhamnolipid synthesis is limited by the expression of the rhlAB-R operon. Thus, this strain carrying the rhlAB-R operon produces similar rhamnolipids levels as PAO1 strain. We determined that strain ATCC 9027 with rhlAB-R operon was not virulent to mice. These results show that strain ATCC 9027, expressing PAO1 rhlAB-R operon, has a high biotechnological potential for industrial mono-rhamnolipid production.

Pseudomonas aeruginosa clinical and environmental isolates constitute a single population with high phenotypic diversity.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen with a high incidence of hospital infections that represents a threat to immune compromised patients. Genomic studies have shown that, in contrast to other pathogenic bacteria, clinical and environmental isolates do not show particular genomic differences. In addition, genetic variability of all the P. aeruginosa strains whose genomes have been sequenced is extremely low. This low genomic variability might be explained if clinical strains constitute a subpopulation of this bacterial species present in environments that are close to human populations, which preferentially produce virulence associated traits. RESULTS: In this work, we sequenced the genomes and performed phenotypic descriptions for four non-human P. aeruginosa isolates collected from a plant, the ocean, a water-spring, and from dolphin stomach. We show that the four strains are phenotypically diverse and that this is not reflected in genomic variability, since their genomes are almost identical. Furthermore, we performed a detailed comparative genomic analysis of the four strains studied in this work with the thirteen previously reported P. aeruginosa genomes by means of describing their core and pan-genomes. CONCLUSIONS: Contrary to what has been described for other bacteria we have found that the P. aeruginosa core genome is constituted by a high proportion of genes and that its pan-genome is thus relatively small. Considering the high degree of genomic conservation between isolates of P. aeruginosa from diverse environments, including human tissues, some implications for the treatment of infections are discussed. This work also represents a methodological contribution for the genomic study of P. aeruginosa, since we provide a database of the comparison of all the proteins encoded by the seventeen strains analyzed.

Long-term survival, growth, and reproduction of Acropora palmata sexual recruits outplanted onto Mexican Caribbean reefs.

Acropora palmata is a foundational yet endangered Caribbean reef-building coral species. The lack of recovery after a disease outbreak and low recruitment has led to widespread use of fragmentation to restore populations. Another option is the production of sexual recruits (settlers) via assisted reproduction to improve the genetic diversity of depleted populations; however, the viability of this approach has not been tested over the long term. In 2011 and 2012, A. palmata larvae were cultured, settled, and the sexual recruits raised in an ex-situ nursery. Survival and growth were monitored over time. In 2014, these two F1 cohorts were moved to an in-situ nursery and after one year, a subset (29 colonies) was outplanted onto Cuevones Reef in the Mexican Caribbean. Growth and survival of these colonies were monitored periodically and compared to colonies that remained in the in-situ nursery. In 2019, samples were collected and analyzed for fertility and fecundity. 53% of the colonies were gravid and fecundity was 5.61 ± 1.91 oocytes and 3.04 ± 0.26 spermaries per polyp. A further 14 colonies from these two cohorts were outplanted in 2020 onto Picudas Reef and monitored during the subsequent spawning seasons. Two years after outplanting onto Picudas Reef, all colonies were alive and spawning of three of these colonies was recorded in 2022 in synchrony with the wild population. Gametes were collected from two colonies and crossed, with 15% fertilization success. Spermatozoa from wild colonies were then added and fertilization success increased to 95%. The resultant larvae followed normal development and symbiont uptake was visible within two weeks. The F2 generation was settled, maintained in an ex-situ nursery, and monitored for survival and growth. Both F1 and F2 generations followed a Type III survival curve with high initial mortality while in the ex-situ nursery and low later-stage mortality. The growth rates of these colonies increased three-fold after outplanting when compared to their growth rates in the ex-situ and in-situ nurseries. All colonies survived while in the in-situ nursery and for an additional nine years after outplanting onto Cuevones Reef. Overall, our results show that colonies produced by assisted breeding, once outplanted, may contribute to the genetic diversity and establishment of self-sustaining sexually-reproducing populations, which is an overarching goal of coral restoration programs.

Coral affected by stony coral tissue loss disease can produce viable offspring.

Stony coral tissue loss disease (SCTLD) has caused high mortality of at least 25 coral species across the Caribbean, with Pseudodiploria strigosa being the second most affected species in the Mexican Caribbean. The resulting decreased abundance and colony density reduces the fertilization potential of SCTLD-susceptible species. Therefore, larval-based restoration could be of great benefit, though precautionary concerns about disease transmission may foster reluctance to implement this approach with SCTLD-susceptible species. We evaluated the performance of offspring obtained by crossing gametes of a healthy P. strigosa colony (100% apparently healthy tissue) with that of a colony affected by SCTLD (>50% tissue loss) and compared these with prior crosses between healthy parents. Fertilization and settlement were as high as prior crosses among healthy parents, and post-settlement survivorship over a year in outdoor tanks was 7.8%. After thirteen months, the diseased-parent recruits were outplanted to a degraded reef. Their survivorship was ∼44% and their growth rate was 0.365 mm ± 1.29 SD per month. This study shows that even diseased parent colonies can be effective in assisted sexual reproduction for the restoration of species affected by SCTLD.