MPIase is a glycolipid involved in protein insertion into and preprotein translocation across the cytoplasmic membranes of E. coli. MPIase is upregulated in the cold conditions to overcome the ...cold-sensitive protein export. CdsA, a CDP-diacylglycerol synthase, catalyzes the first reaction in MPIase biosynthesis. An open reading frame for a peptide of 50 amino acids is encoded immediately after ispU, a neighboring upstream gene of cdsA, and overlaps cdsA to a large extent. Mutational analysis revealed that the expression of this peptide is essential for upregulation of MPIase in the cold. Consistently, expression of this peptide in trans resulted in cold upregulation of MPIase. We therefore named this peptide MucA after its function (MPIase upregulation in the cold). When the partially purified MucA was added to the reaction of the intermediate in MPIase biosynthesis, a significant increase in the product formation was observed, supporting the function of MucA. The possible role of MucA in MPIase biosynthesis is discussed.
•Glycolipid MPIase, a membrane protein integrase, is upregulated in the cold•MucA, a small peptide, stimulates the CdsA activity to upregulate glycolipid MPIase•MucA is important for cell growth in the cold•The gene for MucA largely overlaps the coding sequence of CdsA (>90 %)
is an opportunistic pathogen that causes chronic airway infection in bronchiectasis patients and is closely associated with poor prognosis. Strains isolated from chronically infected patients ...typically have a mucoid phenotype due to the overproduction of alginate. In this study, we isolate a
.
strain from the sputum of a patient with bronchiectasis and find that a truncated mutation occurred in
, which is named
.
causes the strain to transform into a mucoid phenotype, downregulates the expression of T3SS and inflammasome ligands such as
and allows it to avoid inflammasome activation. The truncated mutation of the MucA protein may help
.
escape clearance by the immune system, enabling long-term colonization.
is an important pathogen of the immunocompromised, causing both acute and chronic infections. In cystic fibrosis (CF) patients,
causes chronic disease. The impressive sensory network of
allows the ...bacterium to sense and respond to a variety of stimuli found in diverse environments. Transcriptional regulators, including alternative sigma factors and response regulators, integrate signals changing gene expression, allowing
to cause infection. The two-component transcriptional regulator AlgR is important in
pathogenesis in both acute and chronic infections. In chronic infections, AlgR and the alternative sigma factor AlgU activate the genes responsible for alginate production. Previous work demonstrated that AlgU controls
expression. RsmA is a posttranscriptional regulator that is antagonized by two small RNAs, RsmY and RsmZ. In this work, we demonstrate that AlgR directly activates
expression from the same promoter as AlgU. In addition, phosphorylation was not necessary for AlgR activation of
using
and
mutant strains. AlgU and AlgR appear to affect the antagonizing small RNAs
and
indirectly. RsmA was active in a
mutant strain using leader fusions of two RsmA targets,
and
AlgU and AlgR were necessary for posttranscriptional regulation of
and
Altogether, our work demonstrates that the alginate regulators AlgU and AlgR are important in the control of the RsmA posttranscriptional regulatory system. These findings suggest that RsmA plays an unknown role in mucoid strains due to AlgU and AlgR activities.
infections are difficult to treat and frequently cause significant mortality in CF patients. Understanding the mechanisms of persistence is important. Our work has demonstrated that the alginate regulatory system also significantly impacts the posttranscriptional regulator system RsmA/Y/Z. We demonstrate that AlgR directly activates
expression, and this impacts the RsmA regulon. This leads to the possibility that the RsmA/Y/Z system plays a role in helping
persist during chronic infection. In addition, this furthers our understanding of the reach of the alginate regulators AlgU and AlgR.
The prevalence of clinical multidrug-resistant (MDR) Pseudomonas aeruginosa has been increasing rapidly worldwide over the years and responsible for a wide range of acute and chronic infections with ...high mortalities. Although hundreds of complete genomes of clinical P. aeruginosa isolates have been sequenced, only a few complete genomes of mucoid strains are available, limiting a comprehensive understanding of this important group of opportunistic pathogens. Herein, the complete genome of a clinically isolated mucoid strain P. aeruginosa JNQH-PA57 was sequenced and assembled using Illumina and Oxford nanopore sequencing technologies. Genomic features, phylogenetic relationships, and comparative genomics of this pathogen were comprehensively analyzed using various bioinformatics tools. A series of phenotypic and molecular-genetic tests were conducted to investigate the mechanisms of carbapenem resistance in this strain.
Several genomic features of MDR P. aeruginosa JNQH-PA57 were identified based on the whole-genome sequencing. We found that the accessory genome of JNQH-PA57 including several prophages, genomic islands, as well as a PAPI-1 family integrative and conjugative element (ICE), mainly contributed to the larger genome of this strain (6,747,067 bp) compared to other popular P. aeruginosa strains (with an average genome size of 6,445,223 bp) listed in Pseudomonas Genome Database. Colony morphology analysis and biofilm crystal staining assay respectively demonstrated an enhanced alginate production and a thicker biofilm formation capability of JNQH-PA57. A deleted mutation at nt 424 presented in mucA gene, resulted in the upregulated expression of a sigma-factor AlgU and a GDP mannose dehydrogenase AlgD, which might explain the mucoid phenotype of this strain. As for the carbapenem resistance mechanisms, our results revealed that the interplay between impaired OprD porin, chromosomal β-lactamase OXA-488 expression, MexAB-OprM and MexXY-OprM efflux pumps overexpression, synergistically with the alginates-overproducing protective biofilm, conferred the high carbapenem resistance to P. aeruginosa JNQH-PA57.
Based on the genome analysis, we could demonstrate that the upregulated expression of algU and algD, which due to the truncation variant of MucA, might account for the mucoid phenotype of JNQH-PA57. Moreover, the resistance to carbapenem in P. aeruginosa JNQH-PA57 is multifactorial. The dataset presented in this study provided an essential genetic basis for the comprehensive cognition of the physiology, pathogenicity, and carbapenem resistance mechanisms of this clinical mucoid strain.
Abstract Pseudomonas aeruginosa is isolated in sputum cultures from cystic fibrosis (CF) patients and adults with bronchiectasis (BS) and chronic obstructive pulmonary disease, but it is not well ...known if the characteristics of colonization in these latter patients are similar to those with CF. We examined 125 P. aeruginosa isolates obtained from 31 patients suffering from these diseases by pulsed field gel electrophoresis and genotyping of mucA and fpvA genes. The pattern of colonization, with dominance of a clonal strain and incidence of mucoid phenotypes, was similar in every group of patients; however, in some CF and BS patients, we detected the replacement or coexistence of 2 main clones. The main differences were found in the nucleotide position of less common mucA mutations, other than mucA22 , and in the predominance of the different types of the pyoverdine receptor. Our results support a similar colonization pattern by P. aeruginosa in the different obstructive pulmonary diseases.
The bacterium Pseudomonas fluorescens switches to an alginate-producing phenotype when the pleiotropic anti-sigma factor MucA is inactivated. The inactivation is accompanied by an increased biomass ...yield on carbon sources when grown under nitrogen-limited chemostat conditions. A previous metabolome study showed significant changes in the intracellular metabolite concentrations, especially of the nucleotides, in mucA deletion mutants compared to the wild-type. In this study, the P. fluorescens SBW25 wild-type and an alginate non-producing mucA- ΔalgC double-knockout mutant are investigated through model-based (13)C-metabolic flux analysis ((13)C-MFA) to explore the physiological consequences of MucA inactivation at the metabolic flux level. Intracellular metabolite extracts from three carbon labelling experiments using fructose as the sole carbon source are analysed for (13)C-label incorporation in primary metabolites by gas and liquid chromatography tandem mass spectrometry.
From mass isotopomer distribution datasets, absolute intracellular metabolic reaction rates for the wild type and the mutant are determined, revealing extensive reorganisation of carbon flux through central metabolic pathways in response to MucA inactivation. The carbon flux through the Entner-Doudoroff pathway was reduced in the mucA- ΔalgC mutant, while flux through the pentose phosphate pathway was increased. Our findings also indicated flexibility of the anaplerotic reactions through down-regulation of the pyruvate shunt in the mucA- ΔalgC mutant and up-regulation of the glyoxylate shunt.
Absolute metabolic fluxes and metabolite levels give detailed, integrated insight into the physiology of this industrially, medically and agriculturally important bacterial species and suggest that the most efficient way of using a mucA- mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation.
Pulmonary infections involving Pseudomonas aeruginosa (PA) are a complication faced by cystic fibrosis (CF) patients. The bacteria can turn into a mucoid called mucA22 by mutation of the mucA gene, ...which decreases the effectiveness of antibiotics. Acidified nitrite (NaNO2) was shown to be an antibacterial agent against mucA22. Previously, it was shown that the mucA22 galU double mutant had increased susceptibility to acidified nitrite treatment. The complements of this mutant bacteria were investigated to assure that there were no downstream effects after the genetic modification of the bacteria. This experiment used PA01, mucA22, mucA22 galU, and their complements in conditions mimicking those of CF patients. The hypothesis was that the genetic modification galU of PA and mucA22 should be the only mutation that affects the level of protection against acidified nitrite toxicity and not any other genes downstream of these bacteria. Bacterial cultures, incubated for 24 hours, were used to perform ten-fold serial dilutions. They were spotted onto lysogeny broth agar pH 6.5 containing nitrate as a control or nitrate plus nitrite as an experimental plate. Plates were incubated in either anaerobic (15 mM nitrite) or aerobic (30 mM nitrite) conditions. Results were recorded and then reported in CFUs/mL. The results through p-value were statistically significant and demonstrated that the galU complements were complementable. Therefore, galU is one of the genes that is involved in the protection of PA from nitrite treatment. This could lead to a potential therapeutic in the future to help ease the problems of CF patients.
Metabolic profiling of
Pseudomonas fluorescens
SBW25 and various mutants derived thereof was performed to explore how the bacterium adapt to changes in carbon source and upon induction of alginate ...synthesis. The experiments were performed at steady-state conditions in nitrogen-limited chemostats using either fructose or glycerol as carbon source. Carbon source consumption was up-regulated in the alginate producing mutant with inactivated anti-sigma factor MucA. The
mucA
- mutants (also non-alginate producing
mucA
- control strains) had a higher dry weight yield on carbon source implying a change in carbon and energy metabolism due to the inactivation of the anti-sigma factor MucA. Both LC–MS/MS and GC–MS methods were used for quantitative metabolic profiling, and major reorganization of primary metabolite pools in both an alginate producing and a carbon source dependent manner was observed. Generally, larger changes were observed among the phosphorylated glycolytic metabolites, the pentose phosphate pathway metabolites and the nucleotide pool than among amino acids and citric acid cycle compounds. The most significant observation at the metabolite level was the significantly reduced energy charge of the
mucA
- mutants (both alginate producing and non-producing control strains) compared to the wild type strain. This reduction was caused more by a strong increase in the AMP pool than changes in the ATP and ADP pools. The alginate-producing
mucA
- mutant had a slightly increased GTP pool, while the GDP and GMP pools were strongly increased compared to non-producing
mucA
- strains and to the wild type. Thus, whilst changes in the adenosine phosphate nucleotide pool are attributed to the
mucA
inactivation, adjustments in the guanosine phosphate nucleotide pool are consequences of the GTP-dependent alginate production induced by the
mucA
inactivation. This metabolic profiling study provides new insight into carbon and energy metabolism of the alginate producer
P. fluorescens
.
Cystic fibrosis (CF) is caused by a defect in the CF transmembrane regulator that leads to depletion and dehydration of the airway surface liquid (ASL) of the lung epithelium, providing an ...environment that can be infected by bacteria leading to increased morbidity and mortality. Pseudomonas aeruginosa chronically infects more than 80% of CF patients and one hallmark of infection is the emergence of a mucoid phenotype associated with a worsening prognosis and more rapid decline in lung function. Hypertonic saline (HS) is a clinically proven treatment that improves mucociliary clearance through partial rehydration of the ASL of the lung. Strikingly, while HS therapy does not alter the prevalence of P. aeruginosa in the CF lung it does decrease the frequency of episodes of acute, severe illness known as infective exacerbations among CF patients. In this article, we propose a hypothesis whereby the positive clinical effects of HS treatment are explained by the osmotic sensitivity of the mucoid sub-population of P. aeruginosa in the CF lung leading to selection against this group in favor of the osmotically resistant non-mucoid variants.
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