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The strategy of a novel way to enhance bacterial motility

ABSTRACT: Bacterial motility shows a strong evolvable feature depending on the environment. Hyper-motile E. coli could be isolated by evolving non-motile E. coli due to the mutations that enhanced transcriptional expression of the master regulator of the flagellum biosynthesis, FlhDC. These hyper-motile isolates showed reduced growth fitness but with the molecular mechanisms unrevealed. Here we obtained a novel type of hyper-motile isolates by evolving a weakly-motile E. coli K12 strain on the soft agar plates. These isolates carried high accumulated FlhDC proteins and they shared one single point mutation of ClpXV78F. The V78F affected the ATP binding to ClpX via steric repulsive effect and the mutated ClpXP protease lost most of its ability to degraded FlhDC and some other of its known targets. The signal tag of FlhDC for ClpXP recognition was also characterized. Intriguingly, in the hyper-motile strains, the highly enhanced expression of the motility genes was accompanied by the reduced expression of stress resistance genes relating to the reduced fitness of these isolates. Hence, ClpX appeared to be a novel and hot locus during the evolution of bacterial motility and the molecular mechanism of the trade-off between motility and growth was proposed for the first time.

ORGANISM(S): Escherichia coli

PROVIDER: GSE165151 | GEO | 2021/11/15

REPOSITORIES: GEO

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The strategy of a novel way to enhance bacterial motility

Project description:Bacterial motility shows a strong evolvable feature depending on the environment. Hyper-motile E. coli could be isolated by evolving non-motile E. coli due to the mutations that enhanced transcriptional expression of the master regulator of the flagellum biosynthesis, FlhDC. These hyper-motile isolates showed reduced growth fitness but with the molecular mechanisms unrevealed. Here we obtained a novel type of hyper-motile isolates by evolving a weakly-motile E. coli K12 strain on the soft agar plates. These isolates carried high accumulated FlhDC proteins and they shared one single point mutation of ClpXV78F. The V78F affected the ATP binding to ClpX via steric repulsive effect and the mutated ClpXP protease lost most of its ability to degraded FlhDC and some other of its known targets. The signal tag of FlhDC for ClpXP recognition was also characterized. Intriguingly, in the hyper-motile strains, the highly enhanced expression of the motility genes was accompanied by the reduced expression of stress resistance genes relating to the reduced fitness of these isolates. Hence, ClpX appeared to be a novel and hot locus during the evolution of bacterial motility and the molecular mechanism of the trade-off between motility and growth was proposed for the first time.

2021-11-25 | PXD023645 | Pride

A comparison between two Escherichia coli K-12 MG1655 substrains possessing different swimming motility

Project description:Substrains in Escherichia coli K-12 MG1655 can possess various swimming motility, which is mostly resulted from different expression levels of flhDC. Here, we studied the swimming motility of two MG1655 substrains, CY562 and CY570. Our results showed that CY562 had no insertion at the promoter region of flhDC and possessed no swimming motility. In contrast, CY570 had an IS-element insertion at the promoter region of flhDC and showed a hyper-motile phenotype. Transcriptomic data suggest that expression of flhDC and the other known flagella genes was much lower in CY562 than that in CY570. Moreover, CY562 possessed higher expression levels for genes involved in stress response, especially acid-stress response, than CY570. Consistently, CY562 showed a higher survival rate under acid stress than CY570. Our data indicate that there are mechanisms conversely regulating motility and stress response in E. coli.

2021-11-15 | GSE165438 | GEO

Rapid accumulation of motility-activating mutations in resting liquid culture of Escherichia coli

Project description:Expression and use of motility genes is a potentially beneficial but costly process in bacteria. Interestingly, many isolate strains of Escherichia coli possess motility genes but have lost the ability to activate them in conditions in which motile cells are advantageous, raising the question of how they respond to these situations. Through transcriptome profiling of strains in the E. coli single-gene knockout Keio collection, we noticed drastic upregulation of motility genes in many of the deletion strains as compared to its typically non-motile parent strain (BW25113). This switch to a motile phenotype is not a direct consequence of the genes deleted, but is instead due to a variety of secondary mutations that increase the synthesis of the major motility regulator, FlhDC. We found that a phenotypic switch to motility at a population level can be induced in non-motile E. coli strains by incubation in non-shaking liquid medium overnight but not in shaking media. Individual isolates after the overnight incubation acquired distinct mutations upstream of the flhDC operon, including different insertion sequence (IS) elements and, to a lesser extent, point mutations. The rapid sweep in the non-shaking population shows that non-motile strains without existing regulatory mechanisms can quickly adapt to a motile lifestyle by quickly acquired genetic changes.

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Project description:These series of experiments compares the expression profile of the motility variants of E.coli MG1655 ( Motile and NonMotile isolates) to an isogenic E.coli MG1655 strain in which the IS5 upstream of flhDC has been deleted. The expression profiles of genes in the E.coli MG1655 motile isolate and E.coli MG1655 Non_Motile isolate also compared.

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Comparison of IS5 Deletion mutant to E.coli Motile and NonMotile Isolates

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Motility-activating mutations upstream of flhDC reduce acid shock survival of Escherichia coli

Project description:Many neutralophilic bacterial species try to evade acid stress with an escape strategy, which is reflected in the increased expression of genes coding for flagellar components. Extremely acid-tolerant bacteria, such as Escherichia coli, survive the strong acid stress, e.g. in the stomach of vertebrates. Recently, we were able to show that the induction of motility genes in E. coli is strictly dependent on the degree of acid stress, i.e. they are induced under mild acid stress but not under severe acid stress. However, it was not known to what extent fine-tuned expression of motility genes is related to fitness and the ability to survive periods of acid shock. In this study, we demonstrate that the expression of FlhDC, the master regulator of flagellation, is inversely correlated with acid shock survival of E. coli. We encountered this phenomenon when analyzing mutants from the Keio collection in which the expression of flhDC was altered by an IS element. These results suggest a fitness trade-off between acid tolerance and motility.

2024-04-14 | GSE260455 | GEO

Comparison of the transcription profile of B. subtilis clpX strain with B. subtilis clpX spx strain, reporting on Spx activity

Project description:In B. subtilis the unusual transcription factor Spx controls the thiol stress response regulon (Nakano et al 2003 PNAS 100:13603; Rochat et a. 2012 NAR 40:9751). Under normal growth conditions Spx is constantly degraded by the AAA+ protease complex ClpXP (Nakano et al 2001 MolMi 42:383). Comparing a B. subtilis clpX strain, where Spx levels are high, with a B. subtilis clpX spx double mutant strain which lacks Spx, will report on Spx dependent transcriptional control. We prepared total RNA of exponentially grown B. subtilis clpX and of B. subtilis clpX spx cells. Using a merged dye swap experiments of red or green labelled cDNA of the above described RNA preparations-

2014-09-01 | E-GEOD-50102 | biostudies-arrayexpress

Comparison of the transcription profile of B. subtilis clpX strain with B. subtilis clpX spx strain, reporting on Spx activity

2014-09-01 | GSE50102 | GEO

Chemical cross-linking enables drafting ClpXP proximity maps and taking snapshots of in vivo interaction networks

Project description:Protein-protein interactions within complexes and networks are often dynamic and their elucidation remains a challenging task. Here, we show on the example of the proteolytic ClpXP complex the power of combined chemical cross-linking and mass-spectrometry to capture transient binding interactions within ClpP and ClpX as well as across the enigmatic ClpX hexamer – ClpP heptamer interface. Our data suggests that a few hot spot lysine residues located in signature loops in ClpX mediate the ClpX-ClpP interaction. This study further confirms that Listeria monocytogenes ClpX solely interacts with the heterooligomeric ClpP1/2 complex via the ClpP2 apical site. Moreover, the cellular interaction network of human and bacterial proteases was elucidated via in situ chemical cross-linking followed by an antibody-based pull-down against ClpP from genetically unmodified cells. A subsequent gel-free, quantitative mass spectrometric analysis demonstrated an up to 3-fold higher coverage compared to conventional co-immunoprecipitation without cross-linker revealing unprecedented insight into intracellular ClpXP networks.

2018-11-21 | PXD009224 | Pride

flagellar regulon studies

Project description:This study examined the genes under the control of FlhDC and sigmaF in E. coli. Keywords: wild-type, deletion and overexepression strains Under defined steady-state growth condition, we used two different genetic approaches to alter the modulator concentration in cells; (1) moderately expressing FlhDC or sigmaF from anhydrotetracycline (aTc) inducible and Tet repressor-controlled PLtet promoter in a plasmid-borne flhDC or fliA gene; (2) disrupting the expression of FlhDC or sigmaF in flhDC or fliA deletion mutant strains. Samples were taken from culture with wild-type or deletion strains at mid log phase (OD=0.2) or from overexpression strains at mid log phase (OD=0.2) before or 5 minutes after moderate induction. Samples were then RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChip E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).

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