Isolation, characterization, and cellular insertion of the flagella from two strains of the archaebacterium Methanospirillum hungatei.

In high (45 mM)-phosphate medium, Methanospirillum hungatei strains GP1 and JF1 grew as very long, nonmotile chains of cells that did not possess flagella. However, growth in lower (3 or 30 mM)-phosphate medium resulted in the production of mostly single cells and short chains that were motile by means of two polar tufts of flagella, which transected the multilayered terminal plug of the cell. Electron microscopy of negatively stained whole mounts revealed a flagellar filament diameter of approximately 10 nm. Flagellar filaments were isolated from either culture fluid or concentrated cell suspensions that were subjected to shearing. Flagellar filaments were sensitive to treatment with both Triton X-100 and Triton X-114 at concentrations as low as 0.1% (vol/vol). The filaments of both strains were composed of two flagellins of Mr 24,000 and 25,000. However, variations in trace element composition of the medium resulted in the production of a third flagellin in strain JF1. This additional flagellin appeared as a ladderlike smear on sodium dodecyl sulfate-polyacylamide gels with a center of intensity of Mr 35,000 and cross-reacted with antisera produced from filaments containing only the Mr-24,000 and -25,000 flagellins. On sodium dodecyl sulfate-polyacrylamide gels, all flagellins stained by the thymol-sulfuric acid and Alcian blue methods, suggesting that they were glycosylated. This was further supported by chemical deglycosylation of the strain JF1 flagellins, which resulted in a reduction in their apparent molecular weight on sodium dodecyl sulfate-polyacylamide gels. Heterologous reactions to sera raised against the flagella from each strain were limited to the Mr-24,000 flagellins.     

Formation of a flagella-like but straight polymer of Salmonella flagellin

Salmonella flagellin (monomer) polymerizes into flagellar filaments with the addition of (NH₄)₂SO₄ (Ada et al., 1963; Wakabayashi et al., 1969). When, however, this process was allowed to take place in the presence of a high concentration of NaCl (about 1.5 m), the product consisted of flagella-like but straight filaments. This phenomenon was common to four kinds of flagellins derived from strains SJ670, SJ25, SJ30 and SJ814. When the straight filament, suspended in 0.15 m-NaCl, was heated, it depolymerized to the monomer, which could in turn be polymerized into flagellar filaments by the addition of short fragments of flagella at room temperature. Nevertheless, attempts at direct transformation between the two types of filaments were unsuccessful. In 0.15 m-NaCl, straight filaments prepared from the four kinds of flagellins had markedly different heat stabilities, which were much lower than that of any kind of flagella. When monomeric flagellin dissolved in 3.5 m-NaCl was seeded with short fragments of straight filaments, the monomer polymerized onto the ends of the short fragments, which consequently grew into long straight filaments. In this type of experiment, monomers and seeds derived from the four strains were able to interact in any combination, suggesting that straight filaments consisting of the four kinds of flagellins have the same substructures. Whether the concentration of added NaCl was 0.15 m or 3.5 m, fragments of flagella (or straight filaments) were unable to act as seeds for the formation of straight filaments (or flagellar filaments). From this and other experimental results, it was concluded that in the two filamentous structures, flagellin molecules may be packed in different ways.     

Reconstitution of <Emphasis Type="Italic">Salmonella</Emphasis> Flagella attached to Cell Bodies

THE reconstitution in vitro of flagellar filaments from their component flagellin monomers in Salmonella has shown that the filaments have structural polarity and grow at an end distal to the cell body¹; flagella in vivo also grow from their tips^2,3. This suggests that even when flagella are attached to living cells, filaments may be reconstituted from exogenous flagellin monomers at the tips in appropriate conditions. In spite of some negative results⁴, we have been encouraged^5–10 to re-examine the question.     

The Synthesis In Vitro of Flagellin by a Cell-Free Extract from Bacillus Pumilus

Abstract

Bacterial flagella are constructed mainly, or perhaps exclusively, of protein subunits, the flagellins. Demonstration is given in this report for the in vitro incorporation of radioactive amino acids into flagellin, it also appears that part of such incorporation reflects de novo synthesis of flagellin moleculus. Cell-free extracts were prepared from flagellated cells of Bacillus pumilus, by digestion of the cell wall with lysozyme, lysis in the Standard buffer of NIRENBERG and MATTHAEI (1961), treatment with deoxyribonuclease and centrifugation at 15.000×g. The reaction mixtures contained the cell-free extract, one or more [¹⁴C]-amino acids and the usual components required for cell-free protein synthesis. After incubation at 37° carrier flagellin was added and the pH of the reaction mixture adjusted to 2. Flagellin, which is soluble at this pH, was purified by disc electrophoresis or by reconstitution of flagellar filament at pH 5.4 followed by electrophoresis on a column of ethanolized cellulose. When an amino acid absent from B. pumilus flagellin (such as tyrosine) was used, the amount of radioactivity incorporated into the flagellin fraction was negligible as compared to that incorporated when radioactive leucine, arginine and lysine were used. The identity of the purified radioactive protein was established more conclusively by tryptic digestion and chromatographic separation of the resulting peptides. The ninhydrin positive peaks were shown to be coincident with the radioactive peaks. The radioactive peaks disappeared when a cell-free extract from non-flagellated mutant cells was used. The incorporation of radioactive methionine in the N-terminal position of the molecule indicated that at least some of the molecules had been synthesized de novo.     

Growth-saturation in vitro of Salmonella flagella

At physiological ionic strength and pH, short fragments of Salmonella flagella (seeds) grow longer in the presence of monomeric flagellin and there exists a one-to-one correspondence between the seeds and fully grown filaments (Asakura et al., 1964). In this study it was shown that when monomer and seed derived from a preparation of flagella (strain SJ25) were mixed in a protein ratio r larger than 20, the filaments stopped growing or became inactive for a long period of time, and the average length of inactive filaments was independent of the value of r. The phenomenon was called growth-saturation. The antibody-labelling technique (Asakura et al., 1968) made it possible to show that, though active filaments having equal lengths grew at various rates ranging between 0 and 0.16 μm/min, the average value of growth rate depended little on length. On the other hand, it was found that the proportion of inactive filament in the total filament increased rapidly as the value of r was increased continuously from 0 to 10. The dependence of the proportion of inactive filament on r suggested that filaments became inactive with a probability independent of their length. The rate of inactivation (or the probability with which a filament becomes inactive during growth by a unit length) had various values when different preparations of flagella were used as starting materials. The distribution of length for an assembly of inactive filaments was determined by low-magnification electron microscopy. The result could be approximated by an exponential distribution: the number-average length was 4.54 μm and the rate of inactivation was 0.224 μm^?1.     

Flagellar characteristics ofRhizobium species

Summary The flagellation and growth characteristics of 82 strains ofRhizobium were studied. The strains were originally isolated from the root nodules of 19 genera and 35 species of leguminous plants. Two morphological types of bacteria were found which differed mainly in the nature of their flagellation. The one type shows a most unusual and unique flagellation with single subpolar flagella of wavelength averaging from 1.9 to 2.2 microns. The other type shows peritrichous flagellation with usually one and, less often, several flagella per flagellated individual. The flagellar wavelength of the latter type averaged from 1.3 to 1.6 microns. Most strains of both types were rather poorly flagellated. An almost perfect correlation was found between the type of flagellation and the growth rate in peptone-mannitol medium. The subpolar types grew relatively slowly and the peritrichous types relatively rapidly. Some strains of the subpolar type showed flagellar variants with multiple flagella of very short wavelength in addition to the normal subpolar flagellum. A few individuals showed the short wavelength flagella only.     

Feeding of a Freshwater Flagellate,Bodo saltans,on Diverse Bacteria1

ABSTRACT Bodo saltans was isolated from a chalk stream and fed with pure cultures of seven bacteria obtained from the same river. The flagellates were allowed to migrate into suspensions of either of two bacterial species in a T-maze at 20–22°C. There was a significant difference (P < 0.01) between the numbers of flagellates which migrated into suspensions of different bacteria, which were subsequently arranged in an order of “attractiveness” to the flagellate. Bodo saltans grew successfully in monoxenic suspensions of all seven bacterial strains, but more rapid growth occurred with non-flagellated than with flagellated bacteria; this may be because while feeding, B. saltans tends to associate with surfaces where non-flagellated bacteria may also congregate. The efficiency with which B. saltans is able to utilize different bacteria may be influenced by the motility or secretory activities of the bacteria. There was no incontrovertible evidence that B. saltans responds to specific bacterial attractants.     

Aspects of nitrogen fixation in Chlorobium

Four strains of the green sulfur bacterium Chlorobium were studied in respect to nitrogen nutrition and nitrogen fixation. All strains grew on ammonia, N₂, or glutamine as sole nitrogen sources; certain strains also grew on other amino acids. Acetylene-reducing activity was detectable in all strains grown on N₂ or on amino acids (except for glutamine). In N₂ grown Chlorobium thiosulfatophilum strain 8327 1 mM ammonia served to switch-off nitrogenase activity, but the effect of ammonia was much less dramatic in glutamate or limiting ammonia grown cells. The glutamine synthetase inhibitor methionine sulfoximine inhibited ammonia switch-off in all but one strain. Cell extracts of glutamate grown strain 8327 reduced acetylene and required Mg²⁺ and dithionite, but not Mn²⁺, for activity. Partially purified preparations of Rhodospirillum rubrum nitrogenase reductase (iron protein) activating enzyme slightly stimulated acetylene reduction in extracts of strain 8327, but no evidence for an indigenous Chlorobium activating enzyme was obtained. The results suggest that certain Chlorobium strains are fairly versatile in their nitrogen nutrition and that at least in vivo, nitrogenase activity in green bacteria is controlled by ammonia in a fashion similar to that described in nonsulfur purple bacteria and in Chromatium.Non-common abbreviations MSX Methionine sulfoximine - MOPS 3-(N-morpholino) propane sulfonic acid This paper is dedicated to Professor Norbert Pfennig on the occasion of his 60th birthday     

Evidence of reversed electron transport in syntrophic butyrate or benzoate oxidation by Syntrophomonas wolfei and Syntrophus buswellii

Syntrophomonas wolfei and Syntrophus buswellii were grown with butyrate or benzoate in a defined binary coculture with Methanospirillum hungatei. Both strains also grew independent of the partner bacteria with crotonate as substrate. Localization of enzymes involved in butyrate oxidation by S. wolfei revealed that ATP synthase, hydrogenase, and butyryl-CoA dehydrogenase were at least partially membrane-associated whereas 3-hydroxybutyryl-CoA dehydrogenase and crotonase were entirely cytoplasmic. Inhibition experiments with copper chloride indicated that hydrogenase faced the outer surface of the cytoplasmic membrane. Suspensions of butyrate-or benzoate-grown cells of either strain accumulated hydrogen during oxidation of butyrate or benzoate to a low concentration that was thermodynamically in equilibrium with calculated reaction energetics. The protonophore carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and the proton-translocating ATPase inhibitor N,Ndicyclohexylcarbodiimide (DCCD) both specifically inhibited hydrogen formation from butyrate or benzoate at low concentrations, whereas hydrogen formation from crotonate was not affected. A menaquinone was extracted from cells of S. wolfei and S. buswellii grown syntrophically in a binary methanogenic culture. The results indicate that a proton-potential-driven process is involved in hydrogen release from butyrate or benzoate oxidation.Abbreviations BES Bromoethanesulfonate - CCCP Carbonyl cyanide-m-chlorophenyl-hydrazone - DCCD N,Ndicyclohexylcarbodiimide - DCPIP Dichlorophenol indophenol - PMS Phenazine methosulfate     

Classification of secondary alcohol-utilizing methanogens including a new thermophilic isolate

A thermophilic coccoid methanogenic bacterium, strain TCI, that grew optimally around 55° C was isolated with 2-propanol as hydrogen donor for methanogenesis from CO₂. H₂, formate or 2-butanol were used in addition. Each secondary alcohol was oxidized to its ketone. Growth occurred in defined freshwater as well as salt (2% NaCl, w/v) medium. Acetate was required as carbon source, and 4-aminobenzoate and biotin as growth factors. A need for molybdate or alternatively tungstate was shown.Strain TCI was further characterized together with two formerly isolated mesophilic secondary alcohol-utilizing methanogens, the coccoid strain CV and the spirilloid strain SK. The guanine plus cytosine content of the DNA of the three strains was 55,47, and 39 mol%, respectively. Determination of the molecular weights of the methylreductase subunits and sequencing of ribosomal 16S RNA of strains TCI and CV revealed close relationships to the genus Methanogenium. The new isolate TCI is classified as a strain of the existing species, Methanogenium thermophilum (thermophilicum). For strain CV, that uses ethanol or 1-propanol in addition, a classification as new species, Methanogenium organophilum, is proposed. Strain SK is affiliated with the existing species, Methanospirillum hungatei. The ability to use secondary alcohols was also tested with described species of methanogens. Growth with secondary alcohols was observed with Methanogenium marisnigri, Methanospirillum hungatei strain GP1 and Methanobacterium bryantii, but not with Methanospirillum strains JF1 and M1h, Methanosarcina barkeri, Methanococcus species or thermophilic strains or species other than the new isolate TCI.     

Heterotrophic growth of Thiobacillus A2 on sugars and organic acids

Thiobacillus A2 grew on a number of organic acids, pentoses, hexoses and -linked disaccharides, but not on -linked disaccharides or galactosides. Growth was slow on glucose, although fast-growing strains were selectively isolated. Additive growth rates occurred on glucose and galactose; growth on glucose with fructose, pyruvate or gluconate was biphasic rather than diauxic; fructose was used preferentially over glucose; slow growth on glucose was accelerated by some disaccharides; growth on acetate, fumarate or succinate with glucose gave diauxic growth with preferential use of the acid and repression of glucose incorporation. Acetate and succinate tended to be used preferentially even with cultures grown on them in mixture with fructose or sucrose.     

Na<Superscript>+</Superscript> and flagella-dependent swimming of alkaliphilic <Emphasis Type="Italic">Bacillus pseudofirmus</Emphasis> OF4: a basis for poor motility at low pH and enhancement in viscous media in an “up-motile” variant

Flagella-based motility of extremely alkaliphilic Bacillus species is completely dependent upon Na⁺. Little motility is observed at pH values < ∼8.0. Here we examine the number of flagella/cell as a function of growth pH in the facultative alkaliphile Bacillus pseudofirmus OF4 and a derivative selected for increased motility on soft agar plates. Flagella were produced by both strains during growth in a pH range from 7.5 to 10.3. The number of flagella/cell and flagellin levels of cells were not strongly dependent on growth pH over this range in either strain although both of these parameters were higher in the up-motile strain. Assays of the swimming speed indicated no motility at pH < 8 with 10 mM Na⁺, but significant motility at pH 7 at much higher Na⁺ concentrations. At pH 8–10, the swimming speed increased with the increase of Na⁺ concentration up to 230 mM, with fastest swimming at pH 10. Motility of the up-motile strain was greatly increased relative to wild-type on soft agar at alkaline pH but not in liquid except when polyvinylpyrrolidone was added to increase viscosity. The up-motile phenotype, with increased flagella/cell may support bundle formation that particularly enhances motility under a subset of conditions with specific challenges.     

Nitrogen source effects on the growth and toxicity of two strains of the ciguatera-causing dinoflagellate Gambierdiscus toxicus

Two Caribbean strains (1651 and 1655) of the ciguatera-causing dinoflagellate Gambierdiscus toxicus were grown in xenic, batch culture under defined, measured nutrient conditions with nitrate, ammonium, urea, a mix of free amino acids (FAA), or putrescine as the nitrogen source. Cultures were maintained at 27 °C, salinity 35, 110 μmol m⁻² s⁻¹ (12 h:12 h light:dark cycle) on L2 medium at an initial nitrogen concentration of 50 μM N. Toxicity was determined using a ouabain/veratridine-dependent cytotoxicity assay (N2A assay) standardized to a ciguatoxin standard. Nitrate, ammonium, FAA, and putrescine supported growth, but urea did not. The appearance of ammonium in the organic nitrogen cultures indicated that G. toxicus and/or associated bacteria remineralized the available organic nitrogen. Both strains were exposed to nitrogen-limiting conditions as evidenced by chlorophyll a content per cell, nitrogen content, and nitrogen (N) to phosphorus (P) (N:P) ratio significantly declining once nitrogen was no longer available in the medium and cells entered stationary phase. Strain 1651 grew significantly faster than strain 1655 when nitrate, FAA, and putrescine was the nitrogen source, but not ammonium. Nitrogen source had no effect on growth rate (0.14 d⁻¹) in strain 1651. The growth rate of strain 1655 (0.10–0.13 d⁻¹) was significantly faster on ammonium than the other nitrogen sources. Strain 1655 was significantly more toxic (10-fold) than strain 1651 except when growing on ammonium at exponential phase. Toxicity ranged from 1.3 to 8.7 fg C-CTX1-Eq cell⁻¹ in strain 1651 and from 30.7 to 54.3 fg C-CTX1-Eq cell⁻¹ in strain 1655. Nitrogen source had no significant affect on toxicity. Toxicity was greater in stationary versus exponential phase cells for strain 1651 when grown on nitrate and strain 1655 regardless of nitrogen source. The difference in toxicity between growth phases may result from an increase in ciguatoxin and/or maitotoxin. Our results suggest that some strains of G. toxicus when associated with bacteria are able to take advantage of organic as well as inorganic nitrogen sources on short time scales to support future growth. The uncoupling of total nitrogen and phosphorus pools from conditions in the water column suggest that instantaneous growth rates can be supported by nutrients acquired hours to days earlier.     

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook‐associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non‐motile mutant cells that are unable to assemble flagellar filaments and pentagon‐shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella‐deficient mutants (i.e., in the hook, rod, or MS‐ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.     

The combined effect of high hydrostatic pressure,heat and bacteriocins on inactivation of foodborne pathogens in milk and orange juice

The objective of this study was to combine pressure (345 MPa) with heat (50 C), and bacteriocins (5000 AU/ml sample) for a short time (5 min) for the inactivation of relatively pressure-resistant strains of four foodborne pathogens: Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella in pasteurized milk and orange juice. Without bacteriocin addition, 5.5 log-cycle reduction was obtained for S. aureus 485 in milk whereas more than 8 log-cycle reduction was achieved for all the other strains studied. After storage of samples for 24 h at 4 C, S. aureus 765 also gave positive results on selective media, where no growth was observed for all the other micro-organisms assayed. Incubation of the same pressurized samples at 37 C for 48 h showed growth of L. monocytogenes strains in addition to S. aureus strains, where still no growth was observed for E. coli O157:H7 and Salmonella strains in their respective selective media. For orange juice samples, more than 8 log-cycle reduction was achieved for all the bacterial species studied. No growth was seen for these species on their respective selective media agar plates after storage at 4 C for 24 h and at 37 C for 48 h. When a bacteriocin-based biopreservative (BP₁) was combined with pressurization, more than 8 log-cycle reduction in cell population of the resistant strains of S. aureus and L. monocytogenes were achieved in milk after pressurization. Milk samples were stored at 25 C up to 30 days to test the effect of treatment and samples showed no growth whereas all the controls were positive.     

Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by the ifm locus

Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures. No information is available regarding the regulation of flagellar formation in B. subtilis in response to contact with a solid surface. One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates. This study describes the swarming ability of a B. subtilis hypermotile mutant harboring a mutation in the ifm locus that has long been known to affect the degree of flagellation and motility in liquid media. On solid media, the mutant produces elongated and hyperflagellated cells displaying a 10-fold increase in extracellular flagellin. In contrast to the mutant, the parental strain, as well as other laboratory strains carrying a wild-type ifm locus, fails to activate a swarm response. Furthermore, it stops to produce flagella when transferred from liquid to solid medium. Evidence is provided that the absence of flagella is due to the lack of flagellin gene expression. However, restoration of flagellin synthesis in cells overexpressing sigma(D) or carrying a deletion of flgM does not recover the ability to assemble flagella. Thus, the ifm gene plays a determinantal role in the ability of B. subtilis to contact with solid surfaces.     

Mixed-acid fermentation and polysaccharide production by Lactobacillus helveticus in milk cultures

Lactobacillus helveticus grown in milk with pH control at 6.2 had a slower growth rate (=0.27 h^–1) and produced less exopolysaccharide (49 mg l^–1) but increased lactic acid production (425 mM) compared to cultures without pH control (=0.5 h^–1, 380 mg exopolysaccharide l^–1, and 210 mM lactate), respectively. Both cultures displayed a mixed-acid fermentation with formation of acetate, which is linked not only to citrate metabolism, but also to alternative pathways from pyruvate.     

Fermentation of glutamate and other compounds by Acidaminobacter hydrogenoformans gen. nov. sp. nov., an obligate anaerobe isolated from black mud. Studies with pure cultures and mixed cultures with sulfate-reducing and methanogenic bacteria

From mud from the Ems-Dollard estuary (The Netherlands) an L-glutamate-fermenting bacterium was isolated. The isolated strain glu 65 is Gram-negative, rodshaped, obligately anaerobic, non-sporeforming and does not contain cytochromes. The G+C content of its DNA is 48 mol percent.Pure cultures of strain glu 65 grew slowly on glutamate (_max 0.06 h^-1) and formed acetate, CO₂, formate and hydrogen, and minor amounts of propionate. A more rapid fermentation of glutamate was achieved in mixed cultures with sulfate-reducing bacteria (Desulfovibrio HL21 or Desulfobulbus propionicus) or methanogens (Methanospirillum hungatei or Methanobrevibacter arboriphilicus AZ). In mixed culture with Desulfovibrio HL21 a _max of 0.10 h^-1 was observed. With Desulfovibrio or the methanogens propionate was a major product (up to 0.47 mol per mol glutamate) in addition to acetate.Extracts of glutamate-grown cells possessed high activities of 3-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate, and very high activities of NAD⁺-dependent glutamate dehydrogenase, an enzyme most likely involved in the pathway to propionate.The following other substrates allowed reasonable to good growth in pure culture: histidine, -ketoglutarate, serine, cysteine, glycine, adenine, pyruvate, oxaloacetate and citrate. Utilization in mixed cultures was demonstrated for: glutamine, arginine, ornithine, threonine, lysine, alanine, valine, leucine and isoleucine (with Desulfovibrio HL21) and malate (with Methanospirillum).The shift in the fermentation of glutamate and the syntrophic utilization of the above substrates are explained in terms of interspecies hydrogen transfer.Strain glu 65 is described as the type strain of Acidaminobacter hydrogenoformans gen. nov. sp. nov.