Abstract
Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four transmembrane regions and a cytoplasmic domain ...of approximately 100 residues, which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The 3D structure of the stator shows that the cytosolic interface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66 and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to the inner membrane play important roles in various processes, such as the hoop-like function in securing the stability of the stator complex and the ion conduction pathway, which may lead to the elucidation of the ion permeation and assembly mechanism of the stator.
Graphical Abstract
Graphical Abstract
The bacterium
is capable of two kinds of flagellum-mediated motility: swimming, which occurs in liquid, and swarming, which occurs on a surface. Swarming is distinct from swimming in that it requires ...secretion of a surfactant, an increase in flagellar density, and perhaps additional factors. Here we report a new gene,
, located within the 32 gene
operon dedicated to flagellar biosynthesis and chemotaxis, which when mutated abolished swarming motility. SwrD was not required for surfactant production, flagellar gene expression, or an increase in flagellar number. Instead, SwrD was required to increase flagellar power. Mutation of
reduced swimming speed and torque of tethered flagella, and all
-related phenotypes were restored when the stator subunits MotA and MotB were overexpressed either by spontaneous suppressor mutations or by artificial induction. We conclude that swarming motility requires flagellar power in excess of that which is needed to swim.
Bacteria swim in liquid and swarm over surfaces by rotating flagella, but the difference between swimming and swarming is poorly understood. Here we report that SwrD of
is necessary for swarming because it increases flagellar torque and cells mutated for
swim with reduced speed. How flagellar motors generate power is primarily studied in
, and SwrD likely increases power in other organisms, like the
,
,
, and the
.
Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive ...force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA (similar to MotA in Escherichia coli) and PomB (similar to MotB in E. coli), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the
PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after cross-linking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner.
The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by cross-linking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.
The Peptidoglycan (PG) cell wall of the Lyme disease (LD) spirochete, Borrelia burgdorferi (Bb), contributes to structural and morphological integrity of Bb; is a persistent antigen in LD patients; ...and has a unique pentapeptide with L-Ornithine as the third amino acid that cross-links its glycan polymers. A borrelial homolog (BB_0167) interacted specifically with borrelilal PG via its peptidoglycan interacting motif (MHELSEKRARAIGNYL); was localized to the protoplasmic cylinder of Bb; and was designated as Borrelia peptidoglycan interacting Protein (BpiP). A bpiP mutant displayed no defect under in vitro growth conditions with similar levels of several virulence-related proteins. However, the burden of bpiP mutant in C3H/HeN mice at day 14, 28 and 62 post-infection was significantly lower compared to control strains. No viable bpiP mutant was re-isolated from any tissues at day 62 post-infection although bpiP mutant was able to colonize immunodeficient SCID at day 28 post-infection. Acquisition or transmission of bpiP mutant by Ixodes scapularis larvae or nymphs respectively, from and to mice, was significantly lower compared to control strains. Further analysis of bpiP mutant revealed increased sensitivity to vancomycin, osmotic stress, lysosomal extracts, human antimicrobial peptide cathelicidin-LL37, complement-dependent killing in the presence of day 14 post-infection mouse serum and increased internalization of CFSC-labeled bpiP mutant by macrophages and dendritic cells compared to control strains. These studies demonstrate the importance of accessory protein/s involved in sustaining integrity of PG and cell envelope during different phases of Bb infection.
The basic functions of a propionate-oxidizing bacterium Pelotomaculum thermopropionicum flagellum, such as motility and chemotaxis, have not been studied. To investigate its motility, we compared ...with that of Syntrophobacter fumaroxidans, an aflagellar propionate-oxidizing bacterium, in soft agar medium. P. thermopropionicum cells spread, while S. fumaroxidans cells moved downward slightly, indicating flagellum-dependent motility in P. thermopropionicum SI. The motility of P. thermopropionicum was inhibited by the addition of carbonyl cyanide m-chlorophenyl hydrazone, a proton uncoupler, which is consistent with the fact that stator protein, MotB of P. thermopropionicum, shared sequence homology with proton-type stators. In addition, 5-N-ethyl-N-isopropyl amiloride, an Na
+
channel blocker, showed no inhibitory effect on the motility. Furthermore, motAB of P. thermopropionicum complemented the defective swimming ability of Escherichia coli ∆motAB. These results suggest that the motility of P. thermopropionicum SI depends on the proton-type flagellar motor.
Cells of Pelotomaculum thermopropionicum SI spread in a soft agar medium by flagellum, that was inhibited by CCCP. The stator proteins, MotAB, also function in Escherichia coli as proton type.
The stator units of the flagellum supply power to the flagellar motor via ion transport across the cytoplasmic membrane and generate torque on the rotor for rotation. Flagellar motors across ...bacterial species have evolved adaptations that impact and enhance stator function to meet the demands of each species, including producing stator units using different fuel types or various stator units for different motility modalities.
produces one of the most complex and powerful flagellar motors by positioning 17 stator units at a greater radial distance than in most other bacteria to increase power and torque for high velocity of motility. We report another evolutionary adaptation impacting flagellar stators by identifying FlgX as a chaperone for
stator units to ensure sufficient power and torque for flagellar rotation and motility. We discovered that FlgX maintains MotA and MotB stator protein integrity likely through a direct interaction with MotA that prevents their degradation. Suppressor analysis suggested that the physiology of
drives the requirement for FlgX to protect stator units from proteolysis by the FtsH protease complex.
Δ
was strongly attenuated for colonization of the natural avian host, but colonization capacity was greatly restored by a single mutation in MotA. These findings suggest that the likely sole function of FlgX is to preserve stator unit integrity for the motility required for host interactions. Our findings demonstrate another evolved adaptation in motile bacteria to ensure the equipment of the flagellar motor with sufficient power to generate torque for motility.
The bacterial flagellum is a reversible rotating motor powered by ion transport through stator units, which also exert torque on the rotor component to turn the flagellum for motility. Species-specific adaptations to flagellar motors impact stator function to meet the demands of each species to sufficiently power flagellar rotation. We identified another evolutionary adaptation by discovering that FlgX of
preserves the integrity of stator units by functioning as a chaperone to protect stator proteins from degradation by the FtsH protease complex due to the physiology of the bacterium. FlgX is required to maintain a level of stator units sufficient to power the naturally high-torque flagellar motor of
for motility in intestinal mucosal layers to colonize hosts. Our work continues to identify an increasing number of adaptations to flagellar motors across bacterial species that provide the mechanics necessary for producing an effective rotating nanomachine for motility.
The bacteriophage T4 early gene product MotB binds tightly but nonspecifically to DNA, copurifies with the host Nucleoid Associated Protein (NAP) H-NS in the presence of DNA and improves T4 fitness. ...However, the T4 transcriptome is not significantly affected by a
knockdown. Here we have investigated the phylogeny of MotB and its predicted domains, how MotB and H-NS together interact with DNA, and how heterologous overexpression of
impacts host gene expression. We find that
is highly conserved among
. Although the MotB sequence has no homology to proteins of known function, predicted structure homology searches suggest that MotB is composed of an N-terminal Kyprides-Onzonis-Woese (KOW) motif and a C-terminal DNA-binding domain of oligonucleotide/oligosaccharide (OB)-fold; either of which could provide MotB's ability to bind DNA. DNase I footprinting demonstrates that MotB dramatically alters the interaction of H-NS with DNA in vitro. RNA-seq analyses indicate that expression of plasmid-borne
up-regulates 75 host genes; no host genes are down-regulated. Approximately 1/3 of the up-regulated genes have previously been shown to be part of the H-NS regulon. Our results indicate that MotB provides a conserved function for
and suggest a model in which MotB functions to alter the host transcriptome, possibly by changing the association of H-NS with the host DNA, which then leads to conditions that are more favorable for infection.
The presence of
was demonstrated in body lice, however, little is known about the mechanism of natural lice infection. In 2013 and 2014, cross-sectional one-day studies were therefore performed ...within two Marseille homeless shelters to assess the presence of
DNA on human skin, blood and in body lice collected from the same homeless individuals. All 332 participants completed questionnaires, were examined for dermatologic signs, and provided four skin samples (hair, neck, armpits, and pelvic belt), blood samples and body lice (if any). We developed a new real-time PCR tool targeting the
gene for the detection of
for all collected samples. Blood culture was also performed. Body lice were found in 24/325 (7.4%) of subjects. We showed a prevalence of
DNA skin-carriage in 33/305 (10.8%) of subjects. No difference was found in
DNA prevalence according to body sites. A strong association between body lice infestation (OR = 3.07,
= 0.029) and
DNA skin-carriage was noted. In lice,
DNA was detected in 59/219 arthropods (26.9%). All blood cultures and real-time PCR on blood samples were negative for
. Lice probably get infected with
while biting through the colonized skin and likely transmit the bacteria in their feces. We found no evidence that lice facilitate the invasion of
into the blood stream. Further investigations are needed to compare phenotypic and genotypic features of
isolates from human skin and lice from the same individuals.
The lytic bacteriophage T4 employs multiple phage-encoded early proteins to takeover the
host. However, the functions of many of these proteins are not known. In this study, we have characterized the ...T4 early gene
, located in a dispensable region of the T4 genome. We show that heterologous production of MotB is highly toxic to
, resulting in cell death or growth arrest depending on the strain and that the presence of
increases T4 burst size 2-fold. Previous work suggested that
affects middle gene expression, but our transcriptome analyses of T4
vs. T4 wt infections reveal that only a few late genes are mildly impaired at 5 min post-infection, and expression of early and middle genes is unaffected. We find that MotB is a DNA-binding protein that binds both unmodified host and T4 modified (glucosylated, hydroxymethylated-5 cytosine, (GHme-C) DNA with no detectable sequence specificity. Interestingly, MotB copurifies with the host histone-like proteins, H-NS and StpA, either directly or through cobinding to DNA. We show that H-NS also binds modified T4 DNA and speculate that MotB may alter how H-NS interacts with T4 DNA, host DNA, or both, thereby improving the growth of the phage.
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