Abstract
A protein glycosylation system related to that for protein mannosylation in yeast is present in many actinomycetes. This system involves polyprenyl phosphate mannose synthase (Ppm), protein ...mannosyl transferase (Pmt), and lipoprotein N-acyl transferase (Lnt). In this study, we obtained a series of mutants in the ppm (sco1423), lnt1 (sco1014), and pmt (sco3154) genes of Streptomyces coelicolor, which encode Ppm, Lnt1, and Pmt, to analyze their requirement for glycosylation of the heterologously expressed Apa glycoprotein of Mycobacterium tuberculosis. The results show that both Ppm and Pmt were required for Apa glycosylation, but that Lnt1 was dispensable for both Apa and the bacteriophage φC31 receptor glycosylation. A bacterial two-hybrid assay revealed that contrary to M. tuberculosis, Lnt1 of S. coelicolor does not interact with Ppm. The D2 catalytic domain of M. tuberculosisPpm was sufficient for complementation of an S. coelicolor double mutant lacking Lnt1 and Ppm, both for Apa glycosylation and for glycosylation of φC31 receptor. On the other hand, M. tuberculosisPmt was not active in S. coelicolor, even when correctly localized to the cytoplasmic membrane, showing fundamental differences in the requirements for Pmt activity in these two species.
Polyprenyl phosphate mannose synthase (Ppm) and protein mannosyl transferase (Pmt) are required for protein mannosylation of the Mycobacterium tuberculosis Apa protein in Streptomyces coelicolor, but lipoprotein N-acyl transferase (Lnt) is dispensable.
ABSTRACT
Pseudomonas aeruginosa is an opportunistic human pathogen that is able to produce several virulence factors such as pyocyanin, rhamnolipids and elastase. In the clinical reference strain ...PAO1, synthesis of these virulence factors is regulated transcriptionally by quorum sensing (QS) and post-transcriptionally by the Rsm system. Herein, we investigated the role of these systems in the control of the pyocyanin, rhamnolipids and elastase production in the marine strain ID4365. We found that this strain carries a nonsense mutation in lasR that makes it a natural mutant in the Las QS system. However, its QS response is still functional with the Rhl system activating virulence factors synthesis. We found that the Rsm system affects virulence factors production, since overexpression of RsmA reduces pyocyanin production whereas RsmY overexpression increases its synthesis. Unexpectedly, and in contrast to the type strain PAO1, inactivation of rsmA increases pyocyanin but reduces elastase and rhamnolipids production by a reduction of RhlR levels. Thus, QS and Rsm systems are involved in regulating virulence factors production, but this regulation is different to the PAO1 strain even though their genomes are highly conserved. It is likely that these differences are related to the different ecological niches in which these strains lived.
Quorum sensing systems and Rsm system in Pseudomonas aeruginosa ID4365.
ABSTRACT
Pseudomonas aeruginosa is one of the main models to study social behaviors in bacteria since it synthesizes several exoproducts, including exoproteases and siderophores and release them to ...the environment. Exoproteases and siderophores are public goods that can be utilized by the individuals that produce them but also by non-producers, that are considered social cheaters. Molecularly exoprotease cheaters are mutants in regulatory genes such as lasR, and are commonly isolated from chronic infections and selected in the laboratory upon serial cultivation in media with protein as a sole carbon source. Despite that the production of exoproteases is exploitable, cooperators have also ways to restrict the growth and selection of social cheaters, for instance by producing toxic metabolites like pyocyanin. In this work, using bacterial competitions, serial cultivation and growth assays, we demonstrated that rhamnolipids which production is regulated by quorum sensing, selectively affect the growth of lasR mutants and are able to restrict social cheating, hence contributing to the maintenance of cooperation in Pseudomonas aeruginosa populations.
Rhamnolipids are amphiphilic molecules produced by Pseudomonas aeruginosa; here it is demonstrated that they selectively inhibit the growth of lasR mutants, decreasing the exploitation of exoproteases produced by the wild-type strain.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
is a wide-spread γ-proteobacterium that produces the biosurfactant rhamnolipid that has a great commercial value due to excellent properties of low toxicity and high biodegradability. However, this ...bacterium is an opportunist pathogen that constitutes an important health hazard due to its production of virulence-associated traits and its high antibiotic resistance. Thus, it is highly desirable to have a non-virulent
strain for rhamnolipid production. It has been reported that strain ATCC 9027 is avirulent in mouse models of infection, and it is still able to produce rhamnolipid. Thus, it has been proposed to be suitable for it industrial production, since it encodes a defective LasR quorum sensing (QS) transcriptional regulator that is the head of this regulatory network. However, the restoration of virulence factor production by overexpression of
(the gene encoding a QS-transcriptional regulator which is under the transcriptional control of LasR) is not sufficient to restore its virulence in mice. It is desirable to obtain a deeper understanding of ATCC 9027 attenuated-virulence phenotype and to assess the safety of this strain to be used at an industrial scale. In this work we determined whether increasing the expression of the pore-forming toxin encoded by the
operon in strain ATCC 9027 had an impact on its virulence using
and mouse models of infections. We increased the expression of the
operon by overexpressing from a plasmid its transcriptional activator Vfr or of the Vfr ligand cyclic AMP produced by CyaB. We found that in
ATCC 9027/pUCP24-
and ATCC 9027/pUCP24-
gained a virulent phenotype, but these strains remained avirulent in murine models of
infection. These results reinforce the possibility of using ATCC 9027 for industrial biosurfactants production.
Objective
To construct
Pseudomonas aeruginosa
PA14 derivatives that overproduce rhamnolipids (RL) by blocking the synthesis of the carbon-storage polymer polyhydroxyalkanoates (PHA) and by ...overexpressing the
rhlAB
-
R
operon that encodes for enzymes of RL synthesis and the RhlR transcriptional regulator.
Results
In contrast to previous results showing that overexpression of
rhlAB
-
R
genes in two
P. aeruginosa
strains (PAO1 and ATCC 9027) is sufficient to overproduce RL, we show that a PA14 derivative overexpressing the
rhlAB
-
R
operon did not increase the synthesis of these biosurfactants. In addition, PA14 mutants deficient in PHA production did not overproduce RL either. However, if the
rhlAB
-
R
genes were expressed in a mutant that is completely impaired in PHA synthesis, a significant increase in RL production was observed (59%). These results show that RL production in PA14 is limited both by the availability of fatty acid precursors and by the levels of the RhlA and RhlB enzymes that are involved in the synthesis of mono-RL.
Conclusions
The limitation of RL production by
P. aeruginosa
PA14 is multifactorial and diverse from the results obtained with other strains. Thus, the factors that limit RL production are particular to each
P. aeruginosa
strain, so strain-specific strategies should be developed to increase their production.
is a metabolically versatile bacterium and also an important opportunistic pathogen. It has a remarkable genomic structure since the genetic information encoding its pathogenicity-related traits ...belongs to its core-genome while both environmental and clinical isolates are part of the same population with a highly conserved genomic sequence. Unexpectedly, considering the high level of sequence identity and homologue gene number shared between different
isolates, the presence of specific essential genes of the two type strains PAO1 and PA14 has been reported to be highly variable. Here we report the detailed bioinformatics analysis of the essential genes of
PAO1 and PA14 that have been previously experimentally identified and show that the reported gene variability was owed to sequencing and annotation inconsistencies, but that in fact they are highly conserved. This bioinformatics analysis led us to the definition of 348
.
general essential genes. In addition we show that 342 of these 348 essential genes are conserved in
a nitrogen-fixing, cyst-forming, soil bacterium. These results support the hypothesis of
having a polyphyletic origin with a Pseudomonads genomic backbone, and are a challenge to the accepted theory of bacterial evolution.
Graphical abstract
A
Pseudomonas aeruginosa
MAZ105 strain belonging to phylogroup 3 has a defective Pqs QS system due to a polar mutation in
pqsA
but can produce pyocyanin because it can express PqsE ...in low-phosphate medium.
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