The Pseudomonas aeruginosa substrate-binding protein Ttg2D functions as a general glycerophospholipid transporter across the periplasm.
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ABSTRACT:
SUBMITTER: Yero D
PROVIDER: S-EPMC8035174 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Filamentous phage impact biofilm development, stress tolerance, virulence, biofilm dispersal, and colony variants. Previously, we identified 137 Pseudomonas aeruginosa PA14 mutants with more than threefold enhanced and 88 mutants with more than 10-fold reduced biofilm formation by screening 5850 transposon mutants (PLoS Pathogens5: e1000483, 2009). Here, we characterized the function of one of these 225 mutations, dppA1 (PA14_58350), in regard to biofilm formation. DppA1 is a substrate-binding protein (SBP) involved in peptide utilization via the DppBCDF ABC transporter system. We show that compared to the wild-type strain, inactivating dppA1 led to 68-fold less biofilm formation in a static model and abolished biofilm formation in flow cells. Moreover, the dppA1 mutant had a delay in swarming and produced 20-fold less small-colony variants, and both biofilm formation and swarming were complemented by producing DppA1. A whole-transcriptome analysis showed that only 10 bacteriophage Pf5 genes were significantly induced in the biofilm cells of the dppA1 mutant compared to the wild-type strain, and inactivation of dppA1 resulted in a 600-fold increase in Pf5 excision and a million-fold increase in phage production. As expected, inactivating Pf5 genes PA0720 and PA0723 increased biofilm formation substantially. Inactivation of DppA1 also reduced growth (due to cell lysis). Hence, DppA1 increases biofilm formation by repressing Pf5 prophage.
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Project description:The essential functions of a bacterial pathogen reflect the most basic processes required for its viability and growth, and represent potential therapeutic targets. Most screens for essential genes have assayed a single condition--growth in a rich undefined medium--and thus have not distinguished genes that are generally essential from those that are specific to this particular condition. To help define these classes for Pseudomonas aeruginosa, we identified genes required for growth on six different media, including a medium made from cystic fibrosis patient sputum. The analysis used the Tn-seq circle method to achieve high genome coverage and analyzed more than 1,000,000 unique insertion positions (an average of one insertion every 6.0 bp). We identified 352 general and 199 condition-specific essential genes. A subset of assignments was verified in individual strains with regulated expression alleles. The profile of essential genes revealed that, compared with Escherichia coli, P. aeruginosa is highly vulnerable to mutations disrupting central carbon-energy metabolism and reactive oxygen defenses. These vulnerabilities may arise from the stripped-down architecture of the organism's carbohydrate utilization pathways and its reliance on respiration for energy generation. The essential function profile thus provides fundamental insights into P. aeruginosa physiology as well as identifying candidate targets for new antibacterial agents.
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Project description:In this study, we show that the dppBCDF operon of Pseudomonas aeruginosa PA14 encodes an ABC transporter responsible for the utilization of di/tripeptides. The substrate specificity of ABC transporters is determined by its associated substrate-binding proteins (SBPs). Whereas in E. coli only one protein, DppA, determines the specificity of the transporter, five orthologous SBPs, DppA1-A5 are present in P. aeruginosa. Multiple SBPs might broaden the substrate specificity by increasing the transporter capacity. We utilized the Biolog phenotype MicroArray technology to investigate utilization of di/tripeptides in mutants lacking either the transport machinery or all of the five SBPs. This high-throughput method enabled us to screen hundreds of dipeptides with various side-chains, and subsequently, to determine the substrate profile of the dipeptide permease. The substrate spectrum of the SBPs was elucidated by complementation of a penta mutant, deficient of all five SBPs, with plasmids carrying individual SBPs. It became apparent that some dipeptides were utilized with different affinity for each SBP. We found that DppA2 shows the highest flexibility on substrate recognition and that DppA2 and DppA4 have a higher tendency to utilize tripeptides. DppA5 was not able to complement the penta mutant under our screening conditions. Phaseolotoxin, a toxic tripeptide inhibiting the enzyme ornithine carbamoyltransferase, is also transported into P. aeruginosa via the DppBCDF permease. The SBP DppA1, and with much greater extend DppA3, are responsible for delivering the toxin to the permease. Our results provide a first overview of the substrate pattern of the ABC dipeptide transport machinery in P. aeruginosa.
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Project description:The stringent response (SR) is a highly conserved stress response in bacteria. It is composed of two factors, (i) a nucleotide alarmone, guanosine tetra- and pentaphosphate ((p)ppGpp), and (ii) an RNA polymerase-binding protein, DksA, that regulates various phenotypes, including bacterial virulence. The clinically significant opportunistic bacterial pathogen Pseudomonas aeruginosa possesses two genes, dksA1 and dksA2, that encode DksA proteins. It remains elusive, however, which of these two genes plays a more important role in SR regulation. In this work, we compared genome-wide, RNA-Seq-based transcriptome profiles of ΔdksA1, ΔdksA2, and ΔdksA1ΔdksA2 mutants to globally assess the effects of these gene deletions on transcript levels coupled with phenotypic analyses. The ΔdksA1 mutant exhibited substantial defects in a wide range of phenotypes, including quorum sensing (QS), anaerobiosis, and motility, whereas the ΔdksA2 mutant exhibited no significant phenotypic changes, suggesting that the dksA2 gene may not have an essential function in P. aeruginosa under the conditions used here. Of note, the ΔdksA1 mutants displayed substantially increased transcription of genes involved in polyamine biosynthesis, and we also detected increased polyamine levels in these mutants. Because SAM is a shared precursor for the production of both QS autoinducers and polyamines, these findings suggest that DksA1 deficiency skews the flow of SAM toward polyamine production rather than to QS signaling. Together, our results indicate that DksA1, but not DksA2, controls many important phenotypes in P. aeruginosa We conclude that DksA1 may represent a potential target whose inhibition may help manage recalcitrant P. aeruginosa infections.
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Project description:Locus PA4043 in the genome of Pseudomonas aeruginosa PAO1 has been annotated as coding for a farnesyl pyrophosphate synthase (FPPS). This open reading frame was cloned and expressed recombinantly in Escherichia coli. The dimeric enzyme shows farnesyl pyrophosphate synthase activity and is strongly inhibited by ibandronate and zoledronate, drugs that are presently in clinical use. The structures of the unliganded enzyme and complexes with the substrate geranyl diphosphate (GPP), the inhibitor ibandronate and two compounds obtained from a differential scanning fluorimetry-based screen of a fragment library were determined by X-ray crystallography to resolutions of better than 2.0?Å. The enzyme shows the typical ?-helical fold of farnesyl pyrophosphate synthases. The substrate GPP binds in the S1 substrate site in an open conformation of the enzyme. In the enzyme-ibandronate complex three inhibitor molecules are bound in the active site of the enzyme. One inhibitor molecule occupies the allylic substrate site (S1) of each subunit, as observed in complexes of nitrogen-containing bisphosphonate inhibitors of farnesyl synthases from other species. Two (in subunit A) and one (in subunit B) additional ibandronate molecules are bound in the active site. The structures of the fragment complexes show two molecules bound in a hydrophobic pocket adjacent to the active site. This allosteric pocket, which has previously only been described for FPPS from eukaryotic organisms, is thus also present in enzymes from pathogenic prokaryotes and might be utilized for the design of inhibitors of bacterial FPPS with a different chemical scaffold to the highly charged bisphosphonates, which are less likely to pass bacterial membranes.
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Project description:One of the major proteins secreted by Pseudomonas aeruginosa is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263-270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of Streptomyces olivaceoviridis and CBP21 of Serratia marcescens and to the cellulose-binding protein p40 of Streptomyces halstedii. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of Vibrio harveyi. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of P. aeruginosa, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D).
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Project description:The beta-carbon of the Pseudomonas aeruginosa 1244 pilin C-terminal Ser is a site of glycosylation. The present study was conducted to determine the pilin structures necessary for glycosylation. It was found that although Thr could be tolerated at the pilin C terminus, the blocking of the Ser carboxyl group with the addition of an Ala prevented glycosylation. Pilin from strain PA103 was not glycosylated by P. aeruginosa 1244, even when the C-terminal residue was converted to Ser. Substituting the disulfide loop region of strain PA103 pilin with that of strain 1244 allowed glycosylation to take place. Neither conversion of 1244 pilin disulfide loop Cys residues to Ala nor the deletion of segments of this structure prevented glycosylation. It was noted that the PA103 pilin disulfide loop environment was electronegative, whereas that of strain 1244 pilin had an overall positive charge. Insertion of a positive charge into the PA103 pilin disulfide loop of a mutant containing Ser at the C terminus allowed glycosylation to take place. Extending the "tail" region of the PA103 mutant pilin containing Ser at its terminus resulted in robust glycosylation. These results suggest that the terminal Ser is the major pilin glycosylation recognition feature and that this residue cannot be substituted at its carboxyl group. Although no other specific recognition features are present, the pilin surface must be compatible with the reaction apparatus for glycosylation to occur.
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Project description:The genome of Pseudomonas aeruginosa contains three open reading frames, PA2966, PA1869, and PA3334, which encode putative acyl carrier proteins, AcpP1, AcpP2, and AcpP3, respectively. In this study, we found that, although these apo-ACPs were successfully phosphopantetheinylated by P. aeruginosa phosphopantetheinyl transferase (PcpS) and all holo-forms of these proteins could be acylated by Vibrio harveyi acyl-ACP synthetase (AasS), only AcpP1 could be used as a substrate for the synthesis of fatty acids, catalyzed by P. aeruginosa cell free extracts in vitro, and only acpP1 gene could restore growth in the Escherichia coliacpP mutant strain CY1877. And P. aeruginosaacpP1 could not be deleted, while disruption of acpP2 or acpP3 in the P. aeruginosa genome allowed mutant strains to grow as well as the wild type strain. These findings confirmed that only P. aeruginosa AcpP1 functions in fatty acid biosynthesis, and that acpP2 and acpP3 do not play roles in the fatty acid synthetic pathway. Moreover, disruption of acpP2 and acpP3 did not affect the ability of P. aeruginosa to produce N-acylhomoserine lactones (AHL), but replacement of P. aeruginosaacpP1 with E. coliacpP caused P. aeruginosa to reduce the production of AHL molecules, which indicated that neither P. aeruginosa AcpP2 nor AcpP3 can act as a substrate for synthesis of AHL molecules in vivo. Furthermore, replacement of acpP1 with E. coliacpP reduced the ability of P. aeruginosa to produce some exo-products and abolished swarming motility in P. aeruginosa.
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Project description:Penicillin-binding proteins (PBPs) function as transpeptidases, carboxypeptidases, or endopeptidases during peptidoglycan synthesis in bacteria. As the well-known drug targets for ?-lactam antibiotics, the physiological functions of PBPs and whether they are essential for growth are of significant interest. The pathogen Pseudomonas aeruginosa poses a particular risk to immunocompromised and cystic fibrosis patients, and infections caused by this pathogen are difficult to treat due to antibiotic resistance. To identify potential drug targets among the PBPs in P. aeruginosa, we performed gene knockouts of all the high-molecular-mass (HMM) PBPs and determined the impacts on cell growth and morphology, susceptibility to ?-lactams, peptidoglycan structure, virulence, and pathogenicity. Disruptions of the transpeptidase domains of most HMM PBPs, including double disruptions, had only minimal effects on cell growth. The exception was PBP3, where cell growth occurred only when the protein was conditionally expressed on an integrated plasmid. Conditional deletion of PBP3 also caused a defect in cell division and increased susceptibility to ?-lactams. Knockout of PBP1a led to impaired motility, and this observation, together with its localization at the cell poles, suggests its involvement in flagellar function. Overall, these findings reveal that PBP3 represents the most promising target for drug discovery against P. aeruginosa, whereas other HMM PBPs have less potential.
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