Pseudomonas aeruginosa is the leading pathogen of chronic cystic fibrosis (CF) lung infection. Life-long persistence in the inflamed and ever fluctuating CF lungs results in the selection of a variety of changes in P. aeruginosa physiology. Accumulating evidence suggests that especially metabolic changes support the survival and growth of P. aeruginosa within the hypoxic and nutritious CF mucus. To investigate if metabolic adaptations we described for hypermutable P. aeruginosa from late CF lung disease (Hoboth et al., 2009. J. Infect. Dis., pp. 118-130) may represent specific changes in response to the selective conditions within the oxygen-restricted CF mucus, we determined the expression of a set of genes during aerobic and hypoxic growth in LB and the artificial sputum medium ASM. We further focused on the regulation of the two isocitrate dehydrogenases Icd and Idh. Interestingly, both isoenzymes may replace each other under aerobic and hypoxic conditions. The NADPH- and RpoS-dependent Icd seems to be the leading isoenzyme under prolonged oxygen limitation and stationary growth phase. LacZ reporter analysis revealed that oxygen-restriction increased the expression levels of azu, cbb3-1, cbb3-2, ccpR, icd, idh and oprF gene, whereas himD and nuoA are increasingly expressed only during hypoxic growth in ASM. Overexpression of the anaerobic regulator Anr improved the expression of azu, ccpR, cbb3-2 and icd. In summary, expression of azu, cbb3-1, cbb3-2, ccpR, icd, idh, oprF, himD, and nuoA appeared to be beneficial for the growth of P. aeruginosa under hypoxic conditions indicating these genes may represent marker genes for the metabolic adaptation to the CF lung environment.
Pseudomnas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene gcd, limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation.
Pseudomonas aeruginosa is a major nosocomial bacterial pathogen causing complicated catheter-associated urinary tract infections (CAUTIs). Here, we present the 6.9-Mb draft genome sequence of P. aeruginosa MH38 isolated from an acute nosocomial CAUTI. It exhibits resistance to several antibiotics but revealed low-level production of virulence factors.
Pseudomonas aeruginosa is a notable nosocomial pathogen causing severe chronic infections. Here we present the draft genome sequence of P. aeruginosa MH27, isolated from a patient with a chronic hospital-acquired catheter-associated urinary tract infection. The 7.1-Mb genome sequence organized in 24 scaffolds contributes to the understanding of biofilm formation and antibiotic resistance.
As is true for many other antibiotic-resistant Gram-negative pathogens, members of the Burkholderia cepacia complex (BCC) are currently being assessed for their susceptibility to phage therapy as an antimicrobial treatment. The objective of this study was to perform genomic and limited functional characterization of the novel BCC phage JG068 (vB_BceP_JG068).
In contrast to the rapidly increasing knowledge on genome content and diversity of bacterial viruses, insights in intracellular phage development and its impact on bacterial physiology are very limited. We present a multifaceted study combining quantitative PCR (qPCR), microarray, RNA-seq, and two-dimensional gel electrophoresis (2D-GE), to obtain a global overview of alterations in DNA, RNA, and protein content in Pseudomonas aeruginosa PAO1 cells upon infection with the strictly lytic phage LUZ19. Viral genome replication occurs in the second half of the phage infection cycle and coincides with degradation of the bacterial genome. At the RNA level, there is a sharp increase in viral mRNAs from 23 to 60% of all transcripts after 5 and 15 min of infection, respectively. Although microarray analysis revealed a complex pattern of bacterial up- and downregulated genes, the accumulation of viral mRNA clearly coincides with a general breakdown of abundant bacterial transcripts. Two-dimensional gel electrophoretic analyses shows no bacterial protein degradation during phage infection, and seven stress-related bacterial proteins appear. Moreover, the two most abundantly expressed early and late-early phage proteins, LUZ19 gene product 13 (Gp13) and Gp21, completely inhibit P. aeruginosa growth when expressed from a single-copy plasmid. Since Gp13 encodes a predicted GNAT acetyltransferase, this observation points at a crucial but yet unexplored level of posttranslational viral control during infection.
Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections.
Pseudomonas aeruginosa is an opportunistic human pathogen, which can cause severe urinary tract infections (UTIs). Because of the high intrinsic antibiotic resistance of P. aeruginosa and its ability to develop new resistances during antibiotic treatment, these infections are difficult to eradicate. The antibiotic susceptibility of 32 P. aeruginosa isolates from acute and chronic UTIs were analysed under standardized conditions showing 19% multi-drug resistant strains. Furthermore, the antibiotic tolerance of two P. aeruginosa strains to ciprofloxacin and tobramycin was analysed under urinary tract-relevant conditions which considered nutrient composition, biofilm growth, growth phase, and oxygen concentration. These conditions significantly enhance the antibiotic tolerance of P. aeruginosa up to 6000-fold indicating an adaptation of the bacterium to the specific conditions present in the urinary tract. This reversible phenomenon is possibly due to the increased formation of persister cells and is based on iron limitation in artificial urine. The results suggest that the general high antibiotic resistance of P. aeruginosa urinary tract isolates together with the increasing tolerance of P. aeruginosa grown under urinary tract conditions decrease the efficiency of antibiotic treatment of UTIs.
Phages could be an important alternative to antibiotics, especially for treatment of multiresistant bacteria as e.g. Pseudomonas aeruginosa. For an effective use of bacteriophages as antimicrobial agents, it is important to understand phage biology but also genes of the bacterial host essential for phage infection.
During chronic infection of the cystic fibrosis (CF) lung, Pseudomonas aeruginosa grows and persists in a microaerobic to anaerobic environment. P. aeruginosa is well adapted to thrive under such conditions and contains multiple enzyme systems for energy generation under oxygen-restricted or even anaerobic conditions. Recent data confirm a heterogeneous environment in the CF lung and indicate that P. aeruginosa induces enzyme systems for microaerobic growth but also denitrification and fermentative pathways. Moreover, stress response systems as universal stress proteins enhance survival under anaerobic energy starvation conditions. Growth in these oxygen-limited environments induces a drastic physiological change in P. aeruginosa, like increased alginate production and alterations in the outer membrane, which contribute to an increased antibiotic tolerance.
Pseudomonas aeruginosa is one of the most frequent agents of urinary tract infections especially in patients with indwelling urethral catheters. A total of 30 P. aeruginosa isolates from urinary tract infections was investigated for their genotypic and phenotypic characteristics. Single Nucleotide Polymorphism chip typing experiments in combination with bioinformatical cluster analyses allowed genotypic grouping of the isolates. Some similarities to strains from lung infections but also to environmental strains were observed. Finally, several urinary tract-specific groups were identified indicating a strong heterogeneity of the urethral isolates. Pyoverdin, protease, and phospholipase A production in combination with quorum sensing activity and biofilm formation were common phenotypic characteristics of these strains. In contrast, swarming phenotypes, the production of pyocyanin, and the extracellular enzymes phospholipase C and elastase were rarely observed. Interestingly, strains isolated from catheter-associated infections showed significantly enhanced biofilm formation, decreased motility, and a slightly increased expression of virulence factors in relation to isolates from acute urinary tract infections.
The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and biofilm formation during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network in response to oxygen tension and NO. Little is known about all members of the Anr and Dnr regulons and the mediated immediate response to oxygen depletion. Comprehensive transcriptome and bioinformatics analyses in combination with a limited proteome analyses were used for the investigation of the P. aeruginosa response to an immediate oxygen depletion and for definition of the corresponding Anr and Dnr regulons. We observed at first the activation of fermentative pathways for immediate energy generation followed by induction of alternative respiratory chains. A solid position weight matrix model was deduced from the experimentally identified Anr boxes and used for identification of 170 putative Anr boxes in potential P. aeruginosa promoter regions. The combination with the experimental data unambiguously identified 130 new members for the Anr and Dnr regulons. The basis for the understanding of two regulons of P. aeruginosa central to biofilm formation and infection is now defined.
Pseudomonas aeruginosa causes lung infections in patients suffering from the genetic disorder Cystic Fibrosis (CF). Once a chronic lung infection is established, P. aeruginosa cannot be eradicated by antibiotic treatment. Phage therapy is an alternative to treat these chronic P. aeruginosa infections. However, little is known about the factors which influence phage infection of P. aeruginosa under infection conditions and suitable broad host range phages.
Pseudomonas aeruginosa produces and secretes several lipolytic enzymes, among them the lipases LipA and LipC. LipA is encoded within the lipA/lipH operon, together with its cognate foldase LipH, which was also found to be required for the functional expression of LipC. At present, the physiological function of LipC is unknown. We have cloned a synthetic operon consisting of the lipC structural gene and the foldase gene lipH obtained from the lipA/lipH operon and have constructed, in parallel, a lipC-deficient P. aeruginosa mutant. Inactivation of the lipC gene significantly impaired type IV pilus-dependent twitching and swarming motility, but also the flagella-mediated swimming motility of P. aeruginosa. Moreover, for the lipC mutant, we observed a significant decrease in the amount of extracellular rhamnolipids. Also, the P. aeruginosa lipC mutant showed a significantly altered biofilm architecture. Proteome analysis revealed the accumulation of the response regulator protein PhoP in the lipC mutant.
Pseudomonas aeruginosa is a versatile opportunistic human pathogen that is able to colonize a broad spectrum of different aquatic and soil habitats. In the environment and during pathogenesis, P. aeruginosa encounters oxygen-limited and anaerobic environments. Particularly during chronic infection of the cystic fibrosis lung, oxygen-limiting conditions seem to contribute to persistent infection. Oxygen limitation increases antibiotic tolerance, robust biofilms and alginate biosynthesis, which contribute to the persistence of this opportunistic pathogen. Despite the importance of anaerobic metabolism during persistent infection of P. aeruginosa, we are just beginning to understand the underlying regulatory network and the molecular basis of how anaerobic metabolism contributes to a persistent infection. A deeper understanding of the anaerobic physiology of P. aeruginosa will allow the identification of new antibiotic targets and new therapeutic strategies.
The Roseobacter clade represents one of the most abundant, metabolically versatile and ecologically important bacterial groups found in marine habitats. A detailed molecular investigation of the regulatory and metabolic networks of these organisms is currently limited for many strains by missing suitable genetic tools.
We present a new method for diparental mating with the outstanding advantage that counterselection of the Escherichia coli donor strain is not required. This improved method uses a new donor strain, E. coli ST18, a hemA deletion mutant defective in tetrapyrrole biosynthesis. The hemA mutation can be complemented by addition of 5-aminolevulinic acid. Therefore, counterselection is carried out only using standard media and growth conditions optimal for the recipient strain. Consequently, recipient strains are isolated in a significantly shorter period.
Pseudomonas putida KT2440 is frequently used in biotechnical research and applications due to its metabolic versatility and organic solvent resistance. A major drawback for a broad application is the inability of the bacterium to survive and grow under anoxic conditions, which prohibits the production of oxygen-sensitive proteins and metabolites. To develop a P. putida strain, which is able to survive under anoxic conditions, the enzymatic systems of anaerobic nitrate and nitrite respiration were introduced into KT2440. For this purpose, two cosmids encoding all structural, maturation and regulatory genes for P. aeruginosa nitrate reductase (pNAR) and nitrite- and nitric oxide reductase (pNIR-NOR) were stably maintained in P. putida KT2440. Transcriptome analyses revealed expression of the encoded nar, nir and nor operons and accessory genes under anoxic conditions. The produced enzyme systems efficiently reduced nitrate or nitrite, respectively, sustaining anaerobic life of recombinant KT2440. Interestingly, anaerobic life of P. putida induced genes involved in arginine-fermentation and genes encoding a putative copper stress resistance operon.
Pseudomonas aeruginosa attracts research attention as a common opportunistic nosocomial pathogen causing severe health problems in humans. Nevertheless, its primary habitat is the natural environment. Here, we relate the genetic diversity of 381 environmental isolates from rivers in northern Germany to ecological factors such as river system, season of sampling and different levels of water quality. From representatives of 99 environmental clones, also in comparison with 91 clinical isolates, we determined motility phenotypes, virulence factors, biofilm formation, serotype and the resistance to seven environmental P.aeruginosa phages. The integration of genetic, ecological and phenotypic data showed (i) the presence of several extended clonal complexes (ecc) which are non-uniformly distributed across different water qualities, and (ii) a correlation of the hosts serotype composition with susceptibility towards distinct groups of environmental phages. For at least one ecc (eccB), we assumed the ecophysiological differences on environmental water adaptation and phage resistance to be so distinct as to reinforce an environmentally driven cladogenic split from the remainder of P.aeruginosa. In summary, we conclude that the majority of the microevolutionary population dynamics of P.aeruginosa were shaped by the natural environment and not by the clinical habitat.
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