scholarly journals Impacts of Ser/Thr Protein Kinase Stk1 on the Proteome, Twitching Motility, and Competitive Advantage in Pseudomonas aeruginosa

2021 ◽  
Vol 12 ◽  
Author(s):  
Xuan Zhu ◽  
Chao Feng ◽  
Lantian Zhou ◽  
Zhenzhen Li ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protein Kinase ◽  
Physiological Role ◽  
Protein Domain ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Related Proteins

Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium in the environment and a leading cause of nosocomial infections worldwide. Therefore, it is listed by the WHO as a human pathogen that urgently needs the development of new antibacterial drugs. Recent findings have demonstrated that eukaryote-type Ser/Thr protein kinases play a vital role in regulating various bacterial physiological processes by catalyzing protein phosphorylation. Stk1 has proven to be a Ser/Thr protein kinase in P. aeruginosa. However, the regulatory roles of Stk1 have not yet been revealed. Thus, we constructed a stk1 knockout mutant (∆stk1) from the P. aeruginosa PAO1 strain and employed a Tandem Mass Tag (TMT) labeling-based quantitative proteomic strategy to characterize proteome-wide changes in response to the stk1 knockout. In total, 620 differentially expressed proteins, among which 288 proteins were upregulated and 332 proteins were downregulated, were identified in ∆stk1 compared with P. aeruginosa PAO1. A detailed bioinformatics analysis of these differentially expressed proteins was performed, including GO annotation, protein domain profile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, subcellular localization and enrichment analysis. Notably, the downregulation of type IV pilus-related proteins and upregulation of T6SS-H1-related proteins were found in the ∆stk1 strain, and the results were corroborated by quantitative PCR at the mRNA level. Further experiments confirmed that the loss of stk1 weakens bacterial twitching motility and promotes a growth competition advantage, which are, respectively, mediated by type IV pilus-related proteins and T6SS-H1-related proteins. These findings contribute to a better understanding of the physiological role of Stk1, and proteomic data will help further investigations of the roles and mechanisms of Stk1 in P. aeruginosa, although the detailed regulation and mechanism of Stk1 still need to be revealed.

scholarly journals CryoEM map of Pseudomonas aeruginosa PilQ enables structural characterization of TsaP

2020 ◽  
Author(s):  
Matthew McCallum ◽  
Stephanie Tammam ◽  
John L. Rubinstein ◽  
Lori L. Burrows ◽  
P. Lynne Howell
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Virulence ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Natural Competence ◽  
Over Expression ◽  
Type Iv ◽  
Signal Cascade ◽  
Surface Sensing

ABSTRACTThe type IV pilus machinery is a multi-protein complex that polymerizes and depolymerizes a pilus fibre used for attachment, twitching motility, phage adsorption, natural competence, protein secretion, and surface-sensing. An outer membrane secretin pore is required for passage of the pilus fibre out of the cell. Herein, the structure of the tetradecameric secretin, PilQ, from the Pseudomonas aeruginosa type IVa pilus system was determined to 4.3 Å and 4.4 Å resolution in the presence and absence of C7 symmetric spokes, respectively. The heptameric spokes were found to be the two tandem C-terminal domains of TsaP. TsaP forms a belt around PilQ and while the protein is not essential for twitching motility, over-expression of TsaP triggers a signal cascade upstream of PilY1 leading to cyclic di-GMP up-regulation. These results resolve the identity of the spokes identified with Proteobacterial PilQ homologs and may reveal a new component of the surface-sensing cyclic di-GMP signal cascade.IMPACT STATEMENTThe type IV pilus is critical for bacterial virulence. The co-structure of the pilus secretin PilQ and TsaP is determined. Characterization of TsaP implicates it in surface-sensing signal transduction.

scholarly journals Pseudomonas aeruginosa AlgR Regulates Type IV Pilus Biosynthesis by Activating Transcription of the fimU-pilVWXY1Y2E Operon

2008 ◽  
Vol 190 (6) ◽  
pp. 2023-2030 ◽  
Author(s):  
Belen Belete ◽  
Haiping Lu ◽  
Daniel J. Wozniak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Response Regulator ◽  
Transcriptional Profiling ◽  
Pcr Analysis ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Mobility Shift ◽  
Type Iv ◽  
Type Iv Pilin ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The response regulator AlgR is required for Pseudomonas aeruginosa type IV pilus-dependent twitching motility, a flagellum-independent mode of solid surface translocation. Prior work showed that AlgR is phosphorylated at aspartate 54, and cells expressing an AlgR variant that cannot undergo phosphorylation (AlgRD54N) lack twitching motility. However, the mechanism by which AlgR controls twitching motility is not completely understood. We hypothesized that AlgR functioned by activating genes within the prepilin fimU-pilVWXY1Y2E cluster that are necessary for type IV pilin biogenesis. Reverse transcriptase PCR analysis showed that the fimU-pilVWXY1Y2E genes are cotranscribed in an operon, which is under the control of AlgR. This supports prior transcriptional profiling studies of wild-type strains and algR mutants. Moreover, expression of the fimU-pilVWXY1Y2E operon was reduced in strains expressing AlgRD54N. DNase footprinting and electrophoretic mobility shift assays demonstrate that AlgR but not AlgRD54N bound with high affinity to two sites upstream of the fimU-pilVWXY1Y2E operon. Altogether, our findings indicate that AlgR is essential for proper pilin localization and that phosphorylation of AlgR results in direct activation of the fimU-pilVWXY1Y2E operon, which is required for the assembly and export of a functional type IV pilus.

scholarly journals Disparate Subcellular Localization Patterns of Pseudomonas aeruginosa Type IV Pilus ATPases Involved in Twitching Motility

2005 ◽  
Vol 187 (3) ◽  
pp. 829-839 ◽  
Author(s):  
Poney Chiang ◽  
Marc Habash ◽  
Lori L. Burrows
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Distribution Patterns ◽  
Opportunistic Pathogen ◽  
Twitching Motility ◽  
Polar Localization ◽  
Type Iv ◽  
And Function

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.

scholarly journals Single-Residue Changes in the C-Terminal Disulfide-Bonded Loop of the Pseudomonas aeruginosa Type IV Pilin Influence Pilus Assembly and Twitching Motility

2009 ◽  
Vol 191 (21) ◽  
pp. 6513-6524 ◽  
Author(s):  
Hanjeong Harvey ◽  
Marc Habash ◽  
Francisca Aidoo ◽  
Lori L. Burrows
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antigenic Variation ◽  
Intact Protein ◽  
Twitching Motility ◽  
Beta Turn ◽  
Type Iv ◽  
Pilin Subunit ◽  
Type Iv Pilin ◽  
Intersubunit Interactions ◽  
Pilus Assembly

ABSTRACT PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.

scholarly journals The role of core and accessory type IV pilus genes in natural transformation and twitching motility in the bacterium Acinetobacter baylyi

PLoS ONE ◽  
2017 ◽  
Vol 12 (8) ◽  
pp. e0182139 ◽  
Author(s):  
Colleen G. Leong ◽  
Rebecca A. Bloomfield ◽  
Caroline A. Boyd ◽  
Amber J. Dornbusch ◽  
Leah Lieber ◽  
...  
Keyword(s):  
Natural Transformation ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Acinetobacter Baylyi ◽  
Type Iv

scholarly journals Mucin inhibitsPseudomonas aeruginosabiofilm formation by significantly enhancing twitching motility

2014 ◽  
Vol 60 (3) ◽  
pp. 155-166 ◽  
Author(s):  
Cecily L. Haley ◽  
Cassandra Kruczek ◽  
Uzma Qaisar ◽  
Jane A. Colmer-Hamood ◽  
Abdul N. Hamood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iv Pili ◽  
Dependent Manner ◽  
Twitching Motility ◽  
Strain Pao1 ◽  
Concentration Dependent Manner ◽  
Dependent Effect ◽  
Type Iv ◽  
Biofilm Development

In Pseudomonas aeruginosa, type IV pili (TFP)-dependent twitching motility is required for development of surface-attached biofilm (SABF), yet excessive twitching motility is detrimental once SABF is established. In this study, we show that mucin significantly enhanced twitching motility and decreased SABF formation in strain PAO1 and other P. aeruginosa strains in a concentration-dependent manner. Mucin also disrupted partially established SABF. Our analyses revealed that mucin increased the amount of surface pilin and enhanced transcription of the pilin structural gene pilA. Mucin failed to enhance twitching motility in P. aeruginosa mutants defective in genes within the pilin biogenesis operons pilGHI/pilJK-chpA-E. Furthermore, mucin did not enhance twitching motility nor reduce biofilm development by chelating iron. We also examined the role of the virulence factor regulator Vfr in the effect of mucin. In the presence or absence of mucin, PAOΔvfr produced a significantly reduced SABF. However, mucin partially complemented the twitching motility defect of PAOΔvfr. These results suggest that mucin interferes with SABF formation at specific concentrations by enhancing TFP synthesis and twitching motility, that this effect, which is iron-independent, requires functional Vfr, and only part of the Vfr-dependent effect of mucin on SABF development occurs through twitching motility.

Two direct gene targets contribute to Clp-dependent regulation of type IV pilus-mediated twitching motility in Lysobacter enzymogenes OH11

2018 ◽  
Vol 102 (17) ◽  
pp. 7509-7519 ◽  
Author(s):  
Jiaojiao Chen ◽  
Danyu Shen ◽  
Benard Omondi Odhiambo ◽  
Dan Xu ◽  
Sen Han ◽  
...  
Keyword(s):  
Twitching Motility ◽  
Type Iv Pilus ◽  
Lysobacter Enzymogenes ◽  
Type Iv ◽  
Direct Gene

scholarly journals Pseudomonas aeruginosa Twitching Motility-Mediated Chemotaxis towards Phospholipids and Fatty Acids: Specificity and Metabolic Requirements

2008 ◽  
Vol 190 (11) ◽  
pp. 4038-4049 ◽  
Author(s):  
Rhea M. Miller ◽  
Andrew P. Tomaras ◽  
Adam P. Barker ◽  
Dennis R. Voelker ◽  
Edward D. Chan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alternative Energy ◽  
Isocitrate Lyase ◽  
Malate Synthase ◽  
Glyoxylate Shunt ◽  
Twitching Motility ◽  
Long Chain Fatty Acid ◽  
Type Iv ◽  
Energy Taxis ◽  
Lipid Metabolism Genes

ABSTRACT Pseudomonas aeruginosa demonstrates type IV pilus-mediated directional twitching motility up a gradient of phosphatidylethanolamine (PE). Only one of four extracellular phospholipases C of P. aeruginosa (i.e., PlcB), while not required for twitching motility per se, is required for twitching-mediated migration up a gradient of PE or phosphatidylcholine. Whether other lipid metabolism genes are associated with this behavior was assessed by analysis of transcription during twitching up a PE gradient in comparison to transcription during twitching in the absence of any externally applied phospholipid. Data support the hypothesis that PE is further degraded and that the long-chain fatty acid (LCFA) moieties of PE are completely metabolized via β-oxidation and the glyoxylate shunt. It was discovered that P. aeruginosa exhibits twitching-mediated chemotaxis toward unsaturated LCFAs (e.g., oleic acid), but not saturated LCFAs (e.g., stearic acid) of corresponding lengths. Analysis of mutants that are deficient in glyoxylate shunt enzymes, specifically isocitrate lyase (ΔaceA) and malate synthase (ΔaceB), suggested that the complete metabolism of LCFAs through this pathway was required for the migration of P. aeruginosa up a gradient of PE or unsaturated LCFAs. At this point, our data suggested that this process should be classified as energy taxis. However, further evaluation of the ability of the ΔaceA and ΔaceB mutants to migrate up a gradient of PE or unsaturated LCFAs in the presence of an alternative energy source clearly indicated that metabolism of LCFAs for energy is not required for chemotaxis toward these compounds.

scholarly journals Differential Regulation of Twitching Motility and Elastase Production by Vfr in Pseudomonas aeruginosa

2002 ◽  
Vol 184 (13) ◽  
pp. 3605-3613 ◽  
Author(s):  
Scott A. Beatson ◽  
Cynthia B. Whitchurch ◽  
Jennifer L. Sargent ◽  
Roger C. Levesque ◽  
John S. Mattick
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Binding Pocket ◽  
Receptor Protein ◽  
Twitching Motility ◽  
Wild Type ◽  
Type Iv ◽  
Allelic Deletion ◽  
Pyocyanin Production ◽  
Null Mutants

ABSTRACT Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasI::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr. This allele (VfrΔEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrΔEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrΔEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

scholarly journals Phosphorylation of the Pseudomonas aeruginosa Response Regulator AlgR Is Essential for Type IV Fimbria-Mediated Twitching Motility

2002 ◽  
Vol 184 (16) ◽  
pp. 4544-4554 ◽  
Author(s):  
Cynthia B. Whitchurch ◽  
Tatiana E. Erova ◽  
Jacqui A. Emery ◽  
Jennifer L. Sargent ◽  
Jonathan M. Harris ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Twitching Motility ◽  
Type Iv ◽  
Molecular Events ◽  
And Function ◽  
Alginate Biosynthesis

ABSTRACT The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P. aeruginosa.

Sign in / Sign up

Export Citation Format

Share Document