Search

Establishment of an axis of cell polarity and differentiation of the cell poles are fundamental aspects of cellular development in many organisms. We compared the effects of two bacterial cytoskeletal-like systems, the MreB and MinCDE systems, on these processes in Escherichia coli. We report that the Min proteins are capable of establishing an axis of oscillation that is the initial step in establishment of polarity in spherical cells, in a process that is independent of the MreB cytoskeleton. In contrast, the MreB system is required for establishment of the rod shape of the cell and for polar targeting of other polar constituents, such as the Shigella virulence factor IcsA and the aspartate chemoreceptor Tar, in a process that is independent of the Min system. Thus, the two bacterial cytoskeletal-like systems act independently on different aspects of cell polarization. [ABSTRACT FROM AUTHOR]

Measurement of the correlation between sensor-protein expression, motility and environmental change is important for understanding the adaptation process of cells during their change of generation. We have developed a novel assay exploiting the on-chip cultivation system, which enabled us to observe the change of the localization of expressed sensor-protein and the motility for generations. Localization of the aspartate sensitive sensor protein at two poles in Escherichia coli decreased quickly after the aspartate was added into the cultivation medium. However, it took more than three generations for recovering the localization after the removal of aspartate from the medium. Moreover, the tumbling frequency was strongly related to the localization of the sensor protein in a cell. The results indicate that the change of the spatial localization of sensor protein, which was inherited for more than three generations, may contribute to cells, motility as the inheritable information. [ABSTRACT FROM AUTHOR]

We have shown that a hybrid motor consisting of proton-type Rhodobacter sphaeroides MotA and sodium-type Vibrio alginolyticus PomB, MotX and MotY, can work as a sodium-driven motor in Vibrio cells. In this study, we tried to substitute the B subunits, which contain a putative ion-binding site in tile transmembrane region. Rhodobacter sphaeroides MotB did not work with either MotA or PomA in Vibrio cells. Therefore, we constructed chimeric proteins (MomB), which had N-terminal MotB and C4erminal PomB. MomB proteins, with the entire transmembrane region derived from the H+-type MotB, gave rise to an Na+ motor with MotA. The other two MomB proteins, in which tile junction sites were within tile transmembrane region, also formed Na+ motors with PomA, but were changed for Na+ or Li+ specificity. These results show that the channel part consisting of the transmembrane regions from the A and B subunits can interchange Na+- and H+-type subunits and this can affect the ion specificity. This is tile first report to have changed the specificity of tile coupling ions in a bacterial flagellar motor. [ABSTRACT FROM AUTHOR]

The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, respectively), have been detected in polar flagella. PF47 and PF45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF60. We found that PF60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindHI restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF47 flagellin. [ABSTRACT FROM AUTHOR]

Polar flagellum-defective mutants (Pof− Laf−) have been isolated from a lateral flagella-defective mutant (Pof+ Laf−). Among these Pof− Laf− mutants, polar-filamentless mutants, which have the hook structure but not the filament, were identified by electron microscopy. Their hooks were covered with a sheath structure which is contiguous to the outer membrane. The filament proteins, flagellins, were shed into the culture medium of these mutants. These flagellins could be sedimented by high-speed centrifugation even after heat or low pH treatment whereas the depolymerized flagellin of the Pof+ strain was degraded by these treatments. After Triton X-100 treatment, most flagellin of the filamentless mutants could no longer be sedimented, and was degraded. We observed vesicle-like structures on the tips of the hooks and in the flagellin fraction sedimented by high speed centrifugation. These results suggest that flagellin of the filamentless mutants is not assembled into the tip of the hook, but is excreted together with a membrane structure which is probably the sheath of polar flagella. [ABSTRACT FROM AUTHOR]

The bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of H+ or Na+ ions to drive flagellar rotation. In proton-driven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectivity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in ΔpomAB Vibrio alginolyticus. Most hypermotile segregants isolated from this weakly motile strain contain mutations in motB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7E) functioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7E) supported sodium-driven motility in motA or motB E. coli cells, and the swimming speed was even higher than with the original stator of E. coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7E) supported sodium-driven motility in a MotX/Y-dependent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor. [Copyright &y& Elsevier]

The article provides information on voltage-gated Na+ channel NavBP when it localizes with methyl accepting chemotaxis protein at cell poles of alkaliphilic Bacillus pseudofirmus OF4. It notes that Nav , found in alkaliphilic Bacillus pseudofirmus OF4, is a member of the bacterial voltage-gated Na+ channel superfamily. NavBP is needed by alkaliphile during the normal chemotaxis responses and for optimal pH homeostasis during a shift to alkaline conditions of suboptimally low Na+ concentrations. Interaction of NavBP with one or more other proteins in vivo, specially methyl-accepting chemotaxis protein (MCP) is involved in the activation of the channel under the pH conditions existing in extremophile.

The chemoreceptor Tcp of Salmonella enterica serovar Typhimurium can sense citrate and a metal-citrate complex as distinct attractants. In this study, we tried to investigate the molecular mechanism of this discrimination. That citrate binds directly to Tcp was verified by the site-specific thiol modification assays using membrane fractions prepared from Escherichia coli cells expressing the mutant Tcp receptors in which single Cys residues were introduced at positions in the putative ligand-binding pocket. To determine the region responsible for the ligand discrimination, we screened for mutations defective in taxis to magnesium in the presence of citrate. All of the isolated mutants from random mutagenesis with hydroxylamine were defective in both citrate and metal-citrate sensing, and the mutated residues are located in or near the α1-α2 and α3-α4 loops within the periplasmic domain. Further analyses with site-directed replacements around these regions demonstrated that the residue Asn67, which is presumed to lie at the subunit interface of the Tcp homodimer, plays a critical role in the recognition of the metal-citrate complex but not that of citrate. Various amino acids at this position differentially affect the citrate and metal-citrate sensing abilities. Thus, for the first time, the abilities to sense the two attractants were genetically dissected. Based on the results obtained in this study, we propose models in which the discrimination of the metal-citrate complex from citrate involves cooperative interaction at Asn67 and allosteric switching. [ABSTRACT FROM AUTHOR]

Escherichia coli chemoreceptors can sense changes in temperature for thermotaxis. Here we found that the aerotaxis transducer Aer, a homolog of chemoreceptors lacking a periplasmic domain, mediates thermoresponses. We propose that thermosensing by the chemoreceptors is a general attribute of their highly conserved cytoplasmic domain (or their less conserved transmembrane domain). [ABSTRACT FROM AUTHOR]

Uropathogenic Escherichia coli (UPEC) is a major causative agent of urinary tract infections. The bacteria internalize into the uroepithelial cells, where aggregate and form microcolonies. UPEC fimbriae and flagella are important for the formation of microcolonies in uroepithelial cells. PapB/FocB family proteins are small DNA-binding transcriptional regulators consisting of approximately 100 amino acids that have been reported to regulate the expression of various fimbriae, including P, F1C, and type 1 fimbriae, and adhesins. In this study, we show that TosR, a member of the PapB/FocB family is the activator of flagellar expression. The tosR mutant had similar expression levels of type 1, P and F1C fimbriae as the parent strain, but flagellar production was markedly lower than in the parent strain. Flagellin is a major component of flagella. The gene encoding flagellin, fliC , is transcriptionally activated by the sigma factor FliA. The fliA expression is induced by the flagellar master regulator FlhDC. The flhD and flhC genes form an operon. The promoter activity of fliC, fliA and flhD in the tosR mutant was significantly lower than in the parent strain. The purified recombinant TosR does not bind to fliC and fliA but to the upstream region of the flhD gene. TosR is known to bind to an AT-rich DNA sequence consisting of 29 nucleotides. The characteristic AT-rich sequence exists 550-578 bases upstream of the flhD gene. The DNA fragment lacking this sequence did not bind TosR. Furthermore, loss of the tosR gene reduced motility and the aggregation ability of UPEC in urothelial cells. These results indicate that TosR is a transcriptional activator that increases expression of the flhDC operon genes, contributing to flagellar expression and optimal virulence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hirakawa, Shimokawa, Noguchi, Tago, Matsuda, Takita, Suzue, Tajima, Kawagishi and Tomita.)

Chemical sensing is vital to the survival of all organisms. Bacterial chemotaxis is conducted by multiple receptors that sense chemicals to regulate a single signalling system controlling the transition between the direction (clockwise vs. counterclockwise) of flagellar rotation. Such an integrated system seems better suited to judge chemicals as either favourable or unfavourable, but not for identification purposes though differences in their affinities to the receptors may cause difference in response strength. Here, an experimental setup was developed to monitor behaviours of multiple cells stimulated simultaneously as well as a statistical framework based on Bayesian inferences. Although responses of individual cells varied substantially, ensemble averaging of the time courses seemed characteristic to attractant species, indicating we can extract information of input chemical species from responses of the bacterium. Furthermore, two similar, but distinct, beverages elicited attractant responses of cells with profiles distinguishable with the Bayesian procedure. These results provide a basis for novel bio-inspired sensors that could be used with other cell types to sense wider ranges of chemicals., (© 2022. The Author(s).)

The increasing occurrence of multidrug-resistant Mycobacterium tuberculosis ( Mtb ) is a serious threat to global public health. Among the many mechanisms of drug resistance, only resistance-nodulation-division (RND)-type multidrug efflux systems can simultaneously render bacteria tolerant to numerous toxic compounds, including antibiotics. The elevated expression of RND-type xenobiotic efflux transporter complexes, which consist of an inner membrane transporter, membrane fusion protein, and outer membrane channel, plays a major role in multidrug resistance. Among the 14 mycobacterial membrane protein large (MmpL) proteins identified as inner membrane transporters of Mtb , MmpL5 is known to participate in the acquisition of resistance to bedaquiline and clofazimine. MmpL5 exports these drugs by forming a complex with the membrane fusion protein mycobacterial membrane protein small 5 (MmpS5). However, the role of MmpS5 in the efflux of antituberculous drugs by MmpL5 remains unclear. In this study, we focused on the in vivo dynamics of MmpL5 using green fluorescent protein (GFP). Single-molecule observations of MmpL5 showed substantial lateral displacements of MmpL5-GFP without the expression of MmpS5. Nondiffusing MmpL5-GFP foci typically showed three-step photobleaching, suggesting that MmpL5 formed a homotrimeric functional complex on the inner membrane in the presence of MmpS5. These results suggest that the expression of MmpS5 facilitates the assembly of monomeric MmpL5 into a homotrimer that is anchored to the inner membrane to transport various antimycobacterial drugs. IMPORTANCE It has been reported that mycobacterial membrane protein large 5 (MmpL5), a resistance-nodulation-division (RND)-type inner membrane transporter in Mycobacterium tuberculosis ( Mtb ), is involved in the transport of antimycobacterial drugs. However, the functional roles of the membrane fusion protein mycobacterial membrane protein small 5 (MmpS5), organized as an operon with MmpL5, are unclear. Via the single-molecule imaging of MmpL5, we uncovered the maintenance of the functional trimeric complex structure of MmpL5 in the presence of MmpS5. These findings demonstrate that the assembly mechanisms of mycobacterial RND efflux systems are the dynamically regulated process through interactions among the components. This represents the first report of the single-molecule observation of Mtb efflux transporters, which may enhance our understanding of innate antibiotic resistance., (Copyright © 2021 Yamamoto et al.)

Environmental sensing is crucial for bacterial survival and pathogenicity. Bacteria sense environmental chemicals using chemoreceptor proteins, such as Methyl-accepting Chemotaxis Proteins (MCPs). Vibrio cholerae, the etiological agent of cholera, has at least 44 chemoreceptor proteins homologous to MCP-Like Proteins (MLPs). Mlp24 and Mlp37 are dCACHE type chemoreceptors that senses various amino acids. Mlp24 is important for cholera toxin production, whereas Mlp37 is related to biofilm formation. The periplasmic ligand binding regions of Mlp24 and Mlp37 (Mlp24p and Mlp37p, respectively) share similar amino acid sequences, tertiary and quaternary structures, and a common mechanism for the ligand amino acid backbone recognition. However, Mlp37p recognizes various l-amino acids and taurine with similar affinity whereas Mlp24p shows different binding affinities for various l-amino acids and does not bind taurine. Here we solved the crystal structure of Mlp37p in complex with l-arginine and compared it with previously determined structures of Mlp37p, Mlp24p and their ligand complexes. We found that Mlp37p changes the conformation of the loop that forms the upper wall of the ligand binding pocket according to size and shape of the ligand, and thereby show similar affinity for various ligands., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

High pressure below 100 MPa interferes inter-molecular interactions without causing pressure denaturation of proteins. In Escherichia coli, the binding of the chemotaxis signaling protein CheY to the flagellar motor protein FliM induces reversal of the motor rotation. Using molecular dynamics (MD) simulations and parallel cascade selection MD (PaCS-MD), we show that high pressure increases the water density in the first hydration shell of CheY and considerably induces water penetration into the CheY-FliM interface. PaCS-MD enabled us to observe pressure-induced dissociation of the CheY-FliM complex at atomic resolution. Pressure dependence of binding free energy indicates that the increase of pressure from 0.1 to 100 MPa significantly weakens the binding. Using high-pressure microscopy, we observed that high hydrostatic pressure fixes the motor rotation to the counter-clockwise direction. In conclusion, the application of pressure enhances hydration of the proteins and weakens the binding of CheY to FliM, preventing reversal of the flagellar motor.

Bacteria sense environmental chemicals using chemosensor proteins, most of which are present in the cytoplasmic membrane. Canonical chemoreceptors bind their specific ligands in their periplasmic domain, and the ligand binding creates a molecular stimulus that is transmitted into the cytoplasm, leading to various cellular responses, such as chemotaxis and specific gene expression. Vibrio cholerae , the causative agent of cholera, contains about 44 putative sensor proteins, which are homologous to methyl-accepting chemotaxis proteins involved in chemotaxis. Two of them, Mlp24 and Mlp37, have been identified as chemoreceptors that mediate chemotactic responses to various amino acids. Although most of the residues of Mlp37 involved in ligand binding are conserved in Mlp24, these chemoreceptors bind the same ligands with different affinities. Moreover, they have distinct cellular roles. Here we determined a series of ligand complex structures of the periplasmic domains of Mlp24 (Mlp24p). The structures revealed that Ca 2+ binds to the loop that forms the upper wall of the ligand-binding pocket. Ca 2+ does not bind to the corresponding loop of Mlp37, implying that the structural difference of the loop may cause the ligand affinity difference. Isothermal titration calorimetry (ITC) measurements indicated that Ca 2+ changes the ligand binding affinity of Mlp24p. Furthermore, Ca 2+ affected chemotactic behaviors to various amino acids mediated by Mlp24. Thus, Ca 2+ is suggested to serve as a cosignal for the primary signal mediated by Mlp24p, and V. cholerae fine-tunes its chemotactic behavior depending on the Ca 2+ concentration by modulating the ligand sensitivity of Mlp24. IMPORTANCE Mlp24 and Mlp37 are homologous chemoreceptors of Vibrio cholerae that bind various amino acids. Although most of the residues involved in ligand interaction are conserved, these chemoreceptors show different affinities for the same ligand and play different cellular roles. A series of ligand complex structures of the periplasmic region of Mlp24 (Mlp24p) and following ITC analysis revealed that Ca 2+ binds to the loop of Mlp24p and modulates the ligand binding affinity of Mlp24p. Moreover, Ca 2+ changes the chemotactic behaviors mediated by Mlp24. We propose that Ca 2+ acts as a cosignal that modulates the affinity of Mlp24 for the primary signal, thereby changing the chemotactic behavior of V. cholerae ., (Copyright © 2019 American Society for Microbiology.)

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Vibrio cholerae, the causative agent of cholera, swims in aqueous environments with a single polar flagellum. In a spatial gradient of a chemical, the bacterium can migrate in "favorable" directions, a property that is termed chemotaxis. The chemotaxis of V. cholerae is not only critical for survival in various environments and but also is implicated in pathogenicity. In this chapter, we describe how to characterize the chemotactic behaviors of V. cholerae: these methods include swarm assay, temporal stimulation assay, capillary assay, and receptor methylation assay.

Selection-based recombineering is a flexible and proven technology to precisely modify bacterial genomes at single base resolution. It consists of two steps of homologous recombination followed by selection/counter-selection. However, the shortage of efficient counter-selectable markers limits the throughput of this method. Additionally, the emergence of 'selection escapees' can affect recombinant pools generated through this method, and they must be manually removed at each step of selection-based recombineering. Here, we report a series of efforts to improve the throughput and robustness of selection-based recombineering and to achieve seamless and automatable genome engineering. Using the nucleoside kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the non-natural nucleoside dP, a highly efficient, rapid, and liquid-based counter-selection system was established. By duplicating hsvtk gene, combined with careful control of the population size for the subsequent round, we effectively eliminated selection escapes, enabling seamless and multiple insertions/replacement of gene-size fragments in the chromosome. Four rounds of recombineering could thus be completed in 10 days, requiring only liquid handling and without any need for colony isolation or genotype confirmation. The simplicity and robustness of our method make it broadly accessible for multi-locus chromosomal modifications.

Vibrio cholerae has three sets of chemotaxis-related signaling proteins, of which only System II has been shown to be involved in chemotaxis. Here, we examined localization of green fluorescent protein (GFP)-fused components of System I. The histidine kinase (CheA1) and the adaptor (CheW0) of System I localized to polar and lateral membrane regions with standing incubation (microaerobic conditions), but their localization was lost after shaking (aerobic conditions). A transmembrane receptor of System I also showed polar and lateral localization with standing incubation. By contrast, GFP-fused components of System II localized constitutively to the flagellated pole. Nitrogen gas, sodium azide or carbonylcyanide m-chlorophenylhydrazone induced localization of CheA1-GFP even with shaking incubation, suggesting that the localization is controlled in response to changes in energy metabolism. Fluorescently labeled tetracysteine-tagged CheA1 also showed azide-induced localization, arguing against artifactual effects of GFP fusions. These results suggest that System I components are assembled into the supramolecular signaling complex in response to reduced cellular energy states, raising the possibility that the System I complex plays a role in sensing and signaling under microaerobic environments, such as in the host intestine., (© 2014 John Wiley & Sons Ltd.)

Burkholderia sp. NK8 can utilize 3-chlorobenzoate (3CB) as a sole source of carbon because it has a megaplasmid (pNK8) that carries the gene cluster (tfdT-CDEF) encoding chlorocatechol-degrading enzymes. The expression of tfdT-CDEF is induced by 3CB. In this study, we found that NK8 cells were attracted to 3CB and its degradation products, 3- and 4-chlorocatechol, and β-ketoadipate. Capillary assays revealed that a pNK8-eliminated strain (NK82) was defective in chemotaxis toward β-ketoadipate. The introduction of a plasmid carrying a putative outer membrane porin gene, which we name ompNK8, into strain NK82 restored chemotaxis toward β-ketoadipate. RT-PCR analyses demonstrated that the transcription of the ompNK8 gene was enhanced in the presence of 3CB.