The acetic acid bacteria, 1941–1961

Summary Leifson's (1954) differentiation of the acetic acid bacteria into (1)Acetobacter—peritrichously flagellated and acetate oxidising, and (2)Acetomonas—polarly flagellated and acetate non-oxidising, has, by various workers, been (a) fully confirmed, (b) doubted, or only partly accepted, and (c) denied altogether. Also the priority ofAcetomonas overGluconobacter has been questioned, as has also facile mutability inAcetobacter. By a critical comparative review of all the publications concerned, and by the experimental examination of the various cultures claimed to contraveneLeifson's correlation, we have attempted to clarify the confusion, and to give a clear picture of the true position, which is really quite simple. The result of so doing has been to vindicateLeifson's findings in every case, to refute criticism of facile mutability inAcetobacter, and to show whyGluconobacter Asai cannot claim priority overAcetomonas Leifson.     

Flagellation of“Gluconobacter” melanogenus

Summary Electron-micrographs ofGluconobacter melanogenus strain AC 8 (formerlyG. liquefaciens), andG. melanogenus strain U 4, have conclusively confirmed the findings ofShimwell andCarr (1959),Stouthamer (1960), andLeifson (private communication), that these strains are not polarly flagellatedAcetomonas orGluconobacter strains, but peritrichously flagellated acetate-oxidisingAcetobacter ones. When transferred to the latter genus all evidence that these two genera are derived from a “common pool of ancestors”, as claimed byDe Ley (1961), disappears.     

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.     

Polar, peritrichous, and lateral flagella belong to three distinguishable flagellar families

The bacterial flagellum transforms its shape into several distinguishable helical shapes (polymorphs) under various environmental conditions. Polymorphs of each type of flagellum stay on a circle in the pitch-diameter (P versus πD) plot, indicating that they all belong to one family. Previously, we showed that the flagellar family of a marine bacterium Idiomarina loihiensis (Family II) differed from the conventional flagellar family of Salmonella typhimurium (Family I). The pitch and diameter of Family II flagella are half those of Family I flagella. We have suggested that Family I encompasses peritrichous flagella, while Family II forms a polar flagellum. In this study, we have surveyed the polymorphs of flagella from 18 other species and categorized their family types. Previous observations were confirmed; Family I form peritrichous flagella and Family II form polar flagella. Furthermore, we found that lateral flagella had helical parameters much smaller than those of the other two Families and thus belong to a new family (Family III).     

Effect of growth rate on the production of<Emphasis Type="SmallCaps">l</Emphasis>-proline in the fed-batch culture of<Emphasis Type="Italic">Corynebacterium acetoacidophilum</Emphasis>

Corynebacterium acetoacidophilum RYU3161 was cultivated in al-histidine-limited fed-batch culture. To investigate the effect of cell growth on thel-proline production, 5l fed-batch culture was performed using an exponential feeding rate to obtain the specific growth rates (μ) of 0.04, 0.06, 0.08, and 0.1 h⁻¹. The results show that the highest production ofl-proline was obtained at μ=0.04 h⁻¹. The specificl-proline production rate (Q_p) increased proportionally as a function of the specific growth rate, but decreased after it revealed the maximum value at μ=0.08 h⁻¹. Thus, the highest productivity ofl-proline was 1.66 g L⁻¹ h⁻¹ at μ=0.08 h⁻¹. The results show that the production of L-proline inC. acetoacidophilum RYU3161 has mixed growth-associated characteristics.     

Giant flagellar bundles ofVibrio alginolyticus (NCMB 1803)

Summary Following swarming ofVibrio alginolyticus on solid medium a large number of giant flagellar bundles appear behind the growth front. The suggested sequence of events leading to bundle formation is as follows. After inoculation from liquid to solid media the short rods with a single polar sheathed flagellum develop peritrichous nonsheathed flagella and elongate into long filamentous swarmers. After division into short rods, some of the cells become spherical in shape with many peritrichous flagella concentrated at one pole in close association with the sheathed polar flagellum. These tufted spherical bodies form the template upon which masses of loose peritrichous flagella spontaneously aggregate.Flagellar bundles formed when bacteria are grown at pH 8.5 are longer than those formed at pH 7.2 and shorter when grown at pH 6.5. In distilled water the flagellar bundles disintegrate into masses of flagellar fragments.     

The nitrogen requirements ofGluconobacter,Acetobacter andFrateuria

The nitrogen requirements of 96Gluconobacter, 55Acetobacter and 7Frateuria strains were examined. Only someFrateuria strains were able to grow on 0.5% yeast extract broth or 0.5% peptone broth. In the presence ofd-glucose ord-mannitol as a carbon source, ammonium was used as the sole source of nitrogen by all three genera. With ethanol, only a fewAcetobacter strains grew on ammonium as a sole nitrogen source. Singlel-amino acids cannot serve as a sole source of carbon and nitrogen for growth ofGluconobacter, Acetobacter orFrateuria. The singlel-amino acids which were used by most strains as a sole nitrogen source for growth are: asparagine, aspartic acid, glutamine, glutamic acid, proline and alanine. SomeAcetobacter andGluconobacter strains deaminated alanine, asparagine, glutamic acid, threonine, serine and proline. NoFrateuria strain was able to develop on cysteine, glycine, threonine or tryptophan as a sole source of nitrogen for growth. An inhibitory effect of valine may explain the absence of growth on this amino acid. No amino acid is “essential” forGluconobacter, Acetobacter orFrateuria.     

The cell biology of peritrichous flagella in Bacillus subtilis

Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly (< 5 min) but the assembly of flagella capable of supporting motility is rate limited by filament polymerization (> 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid‐like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.     

Heliobacterium sulfidophilum sp. nov. andHeliobacterium undosum sp. nov.: Sulfideoxidizing heliobacteria from thermal sulfidic springs

Two new species of heliobacteria isolated from cyanobacterial mats of two alkaline sulfidic hot springs are formally described. Strains BR4 and BG29 are assigned to anoxygenic phototrophic bacteria of the familyHeliobacteriaceae, since they possess the unique properties of this taxon: strict anaerobiosis, formation of bacteriochlorophyllg, the lack of extensive intracytoplasmic membranes and chlorosomes, an unusual cell wall structure, and phylogenetic relatedness to the low G+C gram-positive eubacteria. Based on the 16S rDNA sequence similarity, strains BR4 and BG29 are assigned to the genusHeliobacterium and described as two new species of this genus:Heliobacterium sulfidophilum sp. nov. andHeliobacterium undosum sp. nov. The G+C content of the DNA is 51.3 mol % inHbt. sulfidophilum and 57.2-57.7 mol % inHbt. undosum. The cells ofHbt. sulfidophilum are rods, and the cells ofHbt. undosum are slightly twisted spirilla or short rods. Both new bacteria are motile by peritrichous flagella.Hbt. sulfidophilum produces endospores. The new bacteria are strict anaerobes growing photoheterotrophically on a limited range of organic compounds. In the dark, they can switch from photosynthesis to the slow fermentation of pyruvate. Biotin is required as a growth factor. Both species are highly tolerant to sulfide (up to 2 mM at pH 7.5) and oxidize it photoheterotrophically to elemental sulfur; photoautotrophic growth was not observed. The temperature optimal for growth ofHbt. sulfidophilum andHbt undosum is 30–35‡C, and the optimal pH is 7–8.     

Flagellar Morphology of Vibrio parahaemolyticus (Fujino et al) Sakazaki,Iwanami and Fukumi 19631

The flagellar morphology of 88 Vibrio parahaemolyticus strains, including a strain descended from Fujino's original strain EB101 (= ATCC17802 = KM1339) was studied. EB101 and 83 other strains (95%) showed mixed polar and peritrichous type of flagellation when grown on modified MOF (MMOF) agar after 16-hr incubation at 20 C. Cultures containing numerous peritrichous cells showed wiggly movements in moist preparations and rapidly spreading growth in semisolid agar plates. Peritrichous flagella were easily removed mechanically from the soma. The mean wavelengths of polar and peritrichous flagella were 2.53 μm (normal type) and 1.72 μm (atypical curly type) respectively. Peritrichous cells on solid media appeared after incubation for 2.5 hr at 37 C and 7 hr at 20 C. Overnight incubation at 37 C and acidity of the medium due to fermentation of carbohydrate markedly ruined peritrichous flagella. Electron micrograph of cells grown on MMOF agar revealed a sheathed polar flagellum and unsheathed peritrichous flagella. A hook structure was demonstrated at the proximal end of the latter. Polar monotrichous cultures in MMOF broth sometimes contained some cells having several or many peritrichous flagella of atypical curly type. Seven strains of Vibrio cholerae were exclusively polar monotrichous on solid and in liquid media. The flagellation of V. parahaemolyticus is concluded as being a mixed polar-peritrichous type. This fact would indicate that V. parahaemolyticus should be excluded from the genus Vibrio, since the genus Vibrio was defined as polar monotrichous.     

Flagella and Pili of Iron-Oxidizing Thiobacilli Isolated from a Uranium Mine in Northern Ontario, Canada

Five strains of Thiobacillus ferrooxidans, which included three recent isolates from a uranium mine, possessed flagella. Three of the strains had several pili per cell. The dimensions, fine structure, and orientation of the flagella were different. Both polar and peritrichous flagella were observed, indicating strain-dependent ultrastructural variation in acidophilic thiobacilli. Neither flagella nor pili were detected in eight other strains of T. ferrooxidans and two strains of Thiobacillus acidophilus by electron microscopy, although all of the cultures contained motile cells.     

Isolation and identification of threeCellulomonas spp. from forest soils

Three stains of cellulose-degrading, aerobic, mesophilic bacteria were isolated from forest soils and, from their cultural, biochemical, and physiological characteristics, they were identified as members of the genusCellulomonas. Unusual biochemical characteristics, e.g. urea hydrolysis, were observed in two isolates. These characteristics have not previously been reported for cellulomonads and may prove to be significant for characterization ofCellulomonas spp. The isolates were able to use urea as a N source in cellulose fermentation. All three strains were motile, with one to four peritrichous flagella observed. Amino acid and polysaccharide composition of the cell walls of the three isolates were identical.     

Effect of NaCl on kinetics ofd-glucosamine uptake in yeasts differing in halotolerance

The initial rate ofd-glucosamine uptake by the non-halotolerant yeastSaccharomyces cerevisiae was approximately halved as the apparent half saturation constant (K_m) and the apparent maximum velocity (V_max) changed from 6.6mm to 16.4mm and from 22 μmol · g⁻¹ · min⁻¹ to 16 μmol · g⁻¹ · min⁻¹, respectively, when the salinity in the medium was increased from zerom to 0.68m NaCl. Corresponding changes in a high affinity transport system in the halotolerant yeastDebaryomyces hansenii were from 1.1mm to 4.6mm and from 3.1 μmol · g⁻¹ · min⁻¹ to 4.5 μmol · g⁻¹ · min⁻¹, implying a practically unchanged transport capacity. In 2.7m NaCl, K_m and V_max in this system were 24.5mm and 1.1 μmol · g⁻¹ · min⁻¹, respectively, representing a marked decrease in transport capability. Nevertheless, the degree of affinity in this extreme salinity must still be regarded as noteworthy. In addition to the high affinity transport system inD. hansenii, a low affinity system, presumably without relevance ind-glucosamine transport, was observed.     

Die Rolle der Fimbrien bei der eigenartigen Sternbildung von Agrobacterium luteum

Zusammenfassung Die bemerkenswerte Sternbildung von Agrobacterium luteum Stamm A61 (Moll et al., 1967; Ahrens, 1968) ist auf polare Fimbrien zurückzuführen, die um die Sternaggregate ausgebreitet sind. Es wird angenommen, daß sich Schwärmzellen an den Fimbrien verfangen und durch Kontraktion rasch zum Stern gezogen werden. Nach elektronenoptischen Präparaten sind die Fimbrien bis zu 10,5 lang und können die beobachtete Sternbildung gut erklären. Agrobacterium luteum Stamm B14 besitzt ebenfalls lange polare Fimbrien. Beide Stämme tragen peritriche Geißeln, deren Struktur sich deutlich von den dünnen und unregelmäßigen Fimbrien unterscheidet.

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The function of fimbriae in the peculiar star formation of Agrobacterium luteum

Summary The remarkable way of star formation in Agrobacterium luteum strain A61 (Moll et al., 1967; Ahrens, 1968) was found to be due to polar fimbriae which are spread around the star-shaped aggregates. It is assumed that swarmers adhere to the fimbriae and, by contraction, are swiftly pulled towards the star. As seen in electron microscopic preparations, the fimbriae are up to 10,5 long and may well explain the star formation observed. Agrobacterium luteum strain B 14 also possesses long polar fimbriae. Both strains have peritrichous flagella, the structure of which being clearly distinct from the delicate and irregular fimbriae.

     

Mechanosensitive Channels in the Cell Body of Chlamydomonas

Mechanosensitive channels appear ubiquitous but they have not been well characterized in cells directly responding to mechanical stimuli. Here, we identified tension-sensitive channel currents on the cell body of Chlamydomonas, a protist that shows a marked behavioral response to mechanical stimulation. When a negative pressure was applied to the cell body with a patch clamp electrode, single-ion-channel currents of 2.4 pA in amplitude were observed. The currents were inhibited by 10 μm gadolinium, a general blocker of mechanosensitive channels. The currents were most likely due to Ca²⁺ influxes because the current was absent in Ca²⁺-free solutions and the reversal potential was 98 mV positive to the resting potential. The distribution of channel-open times conformed to a single exponential component and that of closed times to two exponential components. This mechanosensitive channel was similar to the one found in the flagella in the following respects: both channels were inhibited by Gd³⁺ at 10 μm but not at 1 μm; both passed Ca²⁺ and Ba²⁺; their kinetic parameters for channel opening were similar. These observations raise the possibility that identical mechanosensitive channels may function both in the behavioral control through the mechanoreception by the flagella and in the regulation of cellular physiology in response to mechanical perturbation on the cell body. Received: 13 May 1998/Revised: 2 September 1998     

Inhibition of large-conductance calcium-activated potassium channel by 2-methoxyestradiol in cultured vascular endothelial (HUV-EC-C) cells

2-Methoxyestradiol, an endogenous metabolite of 17β-estradiol, is known to have antitumor and antiangiogenic actions. The effects of 2-methoxyestradiol on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In the whole-cell patch-clamp configuration, 2-methoxyestradiol (0.3–30 μm) reversibly suppressed the amplitude of K⁺ outward currents. The IC ₅₀ value of the 2-methoxyestradiol-induced decrease in outward current was 3 μm. Evans blue (30 μm) or niflumic acid (30 μm), but not diazoxide (30 μm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol (3 μm) to the bath did not modify the single-channel conductance of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 μm) produced a shift in the activation curve of BK_Ca channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BK_Ca channels is primarily mediated by a decrease in the number of long-lived openings. 2-Methoxyestradiol-induced inhibition of the channel activity was potentiated by membrane stretch. In contrast, neither 17β-estradiol (10 μm) nor estriol (10 μm) affected BK_Ca channel activity, whereas 2-hydroxyestradiol (10 μm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 μm) caused membrane depolarization and Evans blue (30 μm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress the activity of BK_Ca channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional activity of endothelial cells. Received: 27 November 2000/Revised: 13 April 2001     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

A Function of Polar Flagellum and Anisotropic Growth in Vibrio alginolyticus Early-Phase Colonies

We continuously observed growth of Vibrio alginolyticus early-phase colonies on agar plates by phase-contrast microscopy. Two mutants defective in motility on solid surfaces were used in this study: one (YM4) can swim in liquid environments using its polar flagellum, and the other (NMB198) cannot swim because it lacks any flagella. We found that isolated colonies of YM4 were generally more circular than those of NMB198. This observation suggests that YM4 cells moved slightly within a colony by the function of their polar flagella. For clustered colonies, where the distance between the colonies was short (<50 μm), the colonies of YM4 grew rapidly along the line between them, but they grew slowly in the lateral directions. Some colonies of NMB198 grew toward neighboring colonies. These observations indicate colony-to-colony interaction.     

Endosymbiotic bacteria associated with the intracellular green algae ofHydra viridis

Gram-negative bacteria 4.5–5.5 μm in length and 1.2 μm in diameter are found in gastrodermal cells of three stains of freshwater green hydras,Hydra viridis (Ohio and Carolina from North America, Jubilee strain from England). They are motile via single polar flagella. They were detected in live animals, Jensen stained material, and electron micrographic sections. Bacteria lose motility quickly upon release from hydra cells. Green hydras harbor strain-specific numbers of chlorellae in these cells. Other tissue types lack algae. The chlorella-hydra symbiosis can be disassociated and the partners grown separately; transfer of photosynthate from algae to hydra occurs. Here we report the presence of endocellular bacterial vesicles specifically associated with cells that contain the symbiotic chlorellae. No cells that contained algae and lacked bacteria were seen. Vesicles, especially conspicuous in sexually mature green hydras, are probably produced upon extrusion from the cell. They contain either algae and bacteria or bacteria alone and are often expelled to the surrounding medium via the coelenteron. Bacteria are absent in nerve cells, interstitial cells, nematocysts, mucous cells, sperm, and probably in most of the other cell types that lack algae. They are present in at least one cell type that lacked algae: the columnar ovarian cells. Bacteria were lost in “bleached” hydras, those induced to lose their algae by high intensity light in a solution of DCMU, a standard inhibitor of photosynthesis. They were absent in a fourth strain of green hydra (Connecticut Valley,H. viridis) and inH. fusca andH. littoralis, two freshwater nonsymbiotic hydras. All of the hydra lacking bacteria contain conspicuous lipid droplets in their cells. The presence of large numbers of bacteria has implications for interpretations of metabolic exchange between host and algal symbionts and for extrapolation of metabolic data from strain to strain ofH. viridis.     

Production and characteristics of the exocellular polysaccharide in mutant strains ofXanthomonas fuscans

Production of the exocellular polysaccharide of the phytopathogenic bacteriumXanthomonas fuscans was investigated with respect to its possible use in utilization of industrial wastes containing lactose. Six stablelac ⁺ mutants were obtained after the treatment withN-methyl-N′-nitroso-N′-nitroguanidine. The mutants were compared with the parent strain. Morphological and cultivation characteristics, as well as production of the exooellular polysaccharide were compared. The production was found to be maximal during the stationary phase of growth in strains cultivated under submerged conditions. Gas chromatography revealed that the polysaccharide of the parent strain is formed by α- and β-D-glucose and α- and β-d-mannose with a small amount ofd-ribose and 6-deoxy-l-mannose. Composition of the polysaccharides produced by the mutant strains (lac ⁺) does not qualitatively differ from that of the parent strain. However, they were found to contain a higher quantity ofd-mannose, which is favourable for their industrial utilization.