scholarly journals Roles of Charged Residues in the C-Terminal Region of PomA, a Stator Component of the Na+-Driven Flagellar Motor

2008 ◽  
Vol 190 (10) ◽  
pp. 3565-3571 ◽  
Author(s):  
Madoka Obara ◽  
Toshiharu Yakushi ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Electrostatic Interactions ◽  
Intramolecular Interactions ◽  
The Other ◽  
Flagellar Motor ◽  
Wild Type ◽  
Mutant Proteins ◽  
E Coli ◽  
Flagellar Motors ◽  
Charged Residues ◽  
Suppressor Analysis

ABSTRACT Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in Escherichia coli. Mutational studies have indicated that the Na+-driven motor of Vibrio alginolyticus may incorporate interactions similar to those of the E. coli motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na+-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na+-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of V. alginolyticus or with the Na+-driven chimeric motor of E. coli. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type V. alginolyticus. The R207E mutant conferred motility with the motor of V. alginolyticus but not with the chimeric motor of E. coli. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in V. alginolyticus but was greatly reduced in E. coli. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex.

scholarly journals Roles of Charged Residues of Rotor and Stator in Flagellar Rotation: Comparative Study using H+-Driven and Na+-Driven Motors in Escherichia coli

2006 ◽  
Vol 188 (4) ◽  
pp. 1466-1472 ◽  
Author(s):  
Toshiharu Yakushi ◽  
Junghoon Yang ◽  
Hajime Fukuoka ◽  
Michio Homma ◽  
David F. Blair
Keyword(s):  
Escherichia Coli ◽  
Comparative Study ◽  
Electrostatic Interactions ◽  
General Feature ◽  
Flagellar Motor ◽  
Motor Patterns ◽  
E Coli ◽  
Flagellar Motors ◽  
Charged Residues ◽  
Flagellar Rotation

ABSTRACT In Escherichia coli, rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na+-driven polar flagellum of Vibrio alginolyticus, but mutational studies in V. alginolyticus suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E. coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V. alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in E. coli. The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus may be enhanced by the proteins MotX and MotY.

scholarly journals Biphasic chemotaxis ofEscherichia colito the microbiota metabolite indole

2020 ◽  
Vol 117 (11) ◽  
pp. 6114-6120 ◽  
Author(s):  
Jingyun Yang ◽  
Ravi Chawla ◽  
Kathy Y. Rhee ◽  
Rachit Gupta ◽  
Michael D. Manson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Microbial Communities ◽  
Bacterial Chemotaxis ◽  
Dynamic Responses ◽  
Time Dependent ◽  
Flagellar Motor ◽  
Wild Type ◽  
Gi Tract ◽  
Flagellar Motors

Bacterial chemotaxis to prominent microbiota metabolites such as indole is important in the formation of microbial communities in the gastrointestinal (GI) tract. However, the basis of chemotaxis to indole is poorly understood. Here, we exposedEscherichia colito a range of indole concentrations and measured the dynamic responses of individual flagellar motors to determine the chemotaxis response. Below 1 mM indole, a repellent-only response was observed. At 1 mM indole and higher, a time-dependent inversion from a repellent to an attractant response was observed. The repellent and attractant responses were mediated by the Tsr and Tar chemoreceptors, respectively. Also, the flagellar motor itself mediated a repellent response independent of the receptors. Chemotaxis assays revealed that receptor-mediated adaptation to indole caused a bipartite response—wild-type cells were attracted to regions of high indole concentration if they had previously adapted to indole but were otherwise repelled. We propose that indole spatially segregates cells based on their state of adaptation to repel invaders while recruiting beneficial resident bacteria to growing microbial communities within the GI tract.

The Bβ-sheet in the PAI-1 Molecule Plays an Important Role for Its Stability

2000 ◽  
Vol 83 (06) ◽  
pp. 896-901 ◽  
Author(s):  
Guang-Chao Sui ◽  
Björn Wiman
Keyword(s):  
Amino Acids ◽  
Half Life ◽  
Ph Dependence ◽  
The Other ◽  
Wild Type ◽  
E Coli ◽  
Reactive Center ◽  
The Stability ◽  
Β Sheet ◽  
Pai 1

SummaryWe have investigated the B β-sheet in PAI-1 regarding its role for the stability of the molecule. The residues from His219 to Tyr241 (except for Gly230 and Pro240), covering the s2B and s3B strands, and in addition His185 and His190 were substituted by amino acids with opposite properties. The 23 generated single-site changed mutants and also wild type PAI-1 (wtPAI-1) were expressed in E. coli. Subsequently they were purified by heparin-Sepharose and anhydrotrypsin agarose affinity chromatographies. The stability of the purified PAI-1 variants was analyzed at 37° C and at different pHs (5.5, 6.5 or 7.5). At pH 7.5 and 37° C, single substitutions of the residues in the central portions of both strands 2 and 3 in the B β-sheet (Ile223 to Leu226 on s2B and Met235 to Ile237 on s3B), caused a significant decrease in stability, yielding half-lives of about 10–25% as compared to wtPAI-1. On the other hand, mutations at both sides of the central portion of the B β-sheet (Tyr221, Asp222, Tyr228 and Thr232) frequently resulted in an increased PAI-1 stability (up to 7-fold). While wtPAI-1 exhibited prolonged half-lives at pH 6.5 and 5.5, the PAI-1 variant Y228S was more stable at neutral pH (half-life of 9.6 h at pH 7.5) as compared to its half-life at pH 5.5 (1.1 h). One of the 4 modified histidine residues (His229) resulted in a variant with a clearly affected stability as a function of pH, suggesting that it may, at least in part, be of importance for the pH dependence of the PAI-1 stability. Thus, our data demonstrate that the B β-sheet is of great importance for the stability of the molecule. Modifications in this part causes decreased or increased stability in a certain pattern, suggesting effects on the insertion rate of the reactive center loop into the A β-sheet of the molecule.

scholarly journals GFP Fusion to the N-Terminus of MotB Affects the Proton Channel Activity of the Bacterial Flagellar Motor in Salmonella

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1255
Author(s):  
Yusuke V. Morimoto ◽  
Keiichi Namba ◽  
Tohru Minamino
Keyword(s):  
Electrostatic Interactions ◽  
Proton Translocation ◽  
Proton Channel ◽  
Flagellar Motor ◽  
Wild Type ◽  
Over Expression ◽  
N Terminus ◽  
Flow Through ◽  
Proton Leakage ◽  
Bacterial Flagellar Motor

The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-motBfliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-motBfliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA–FliG interaction responsible for torque generation.

scholarly journals Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

2007 ◽  
Vol 75 (7) ◽  
pp. 3315-3324 ◽  
Author(s):  
Eric J. Gauger ◽  
Mary P. Leatham ◽  
Regino Mercado-Lubo ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Region ◽  
Flagellar Motor ◽  
Wild Type ◽  
E Coli ◽  
Mouse Intestine ◽  
Flagellum Biosynthesis ◽  
Colonizing Ability

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

scholarly journals Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

2003 ◽  
Vol 47 (3) ◽  
pp. 1037-1046 ◽  
Author(s):  
Christian H. Gross ◽  
Jonathan D. Parsons ◽  
Trudy H. Grossman ◽  
Paul S. Charifson ◽  
Steven Bellon ◽  
...  
Keyword(s):  
Amino Acid ◽  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Mutant Proteins ◽  
E Coli ◽  
Novobiocin Resistance

ABSTRACT DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

scholarly journals BACTERIOPHAGE T4 ENDONUCLEASES II AND IV, OPPOSITELY AFFECTED BY dCMP HYDROXYMETHYLASE ACTIVITY, HAVE DIFFERENT ROLES IN THE DEGRADATION AND IN THE RNA POLYMERASE-DEPENDENT REPLICATION OF T4 CYTOSINE-CONTAINING DNA

Genetics ◽  
1986 ◽  
Vol 114 (3) ◽  
pp. 669-685
Author(s):  
Karin Carlson ◽  
Aud Ȗvervatin
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
Bacteriophage T4 ◽  
The Other ◽  
Wild Type ◽  
E Coli ◽  
Phage Dna ◽  
Dna Template ◽  
Do So

ABSTRACT Bacteriophage T4 mutants defective in gene 56 (dCTPase) synthesize DNA where cytosine (Cyt) partially or completely replaces hydroxymethylcytosine (HmCyt). This Cyt-DNA is degraded in vivo by T4 endonucleases II and IV, and by the exonuclease coded or controlled by genes 46 and 47.—Our results demonstrate that T4 endonuclease II is the principal enzyme initiating degradation of T4 Cyt-DNA. The activity of endonuclease IV, but not that of endonuclease II, was stimulated in the presence of a wild-type dCMP hydroxymethylase, also when no HmCyt was incorporated into phage DNA, suggesting the possibility of direct endonuclease IV-dCMP hydroxymethylase interactions. Endonuclease II activity, on the other hand, was almost completely inhibited in the presence of very small amounts of HmCyt (3-9% of total Cyt + HmCyt) in the DNA. Possible mechanisms for this inhibition are discussed.—The E. coli RNA polymerase modified by the products of T4 genes 33 and 55 was capable of initiating DNA synthesis on a Cyt-DNA template, although it probably cannot do so on an HmCyt template. In the presence of an active endonuclease IV, Cyt-DNA synthesis was arrested 10-30 min after infection, probably due to damage to the template. Cyt-DNA synthesis dependent on the unmodified (33  -  55  -) RNA polymerase was less sensitive to endonuclease IV action.

scholarly journals Reengineering of a Corynebacterium glutamicuml-Arginine and l-Citrulline Producer

2009 ◽  
Vol 75 (6) ◽  
pp. 1635-1641 ◽  
Author(s):  
Masato Ikeda ◽  
Satoshi Mitsuhashi ◽  
Kenji Tanaka ◽  
Mikiro Hayashi
Keyword(s):  
Point Mutations ◽  
Amino Acid Biosynthesis ◽  
The Other ◽  
No Production ◽  
Wild Type ◽  
Acid Biosynthesis ◽  
E Coli ◽  
Lysine Producer ◽  
Efficient Producer ◽  
Type Strains

ABSTRACT Toward the creation of a robust and efficient producer of l-arginine and l-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92 up, and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (ΔargR), the production was further increased. The best mutation set, ΔargR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical l-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefold-increased production without retardation, revealing that the limitation in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance, thus allowing significantly higher productivity of arginine/citrulline even at the suboptimal 38°C.

scholarly journals Investigation of the in vitro and in vivo activity of the type IV pilus extension ATPase BfpD

2020 ◽  
Author(s):  
Jinlei Zhao ◽  
Shahista Nisa ◽  
Michael S. Donnenberg
Keyword(s):  
Atpase Activity ◽  
Loss Of Function ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Mutant Proteins ◽  
E Coli ◽  
Type Iv ◽  
Kinetics Of

AbstractType IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

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