Georg Acker

Physiological ecology of Clostridium glycolicum RD-1, an aerotolerant acetogen isolated from sea grass roots

ABSTRACT An anaerobic, H2-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii. Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H2-CO2, formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O2 in the headspace of static, horizontally incubated culture tubes; the concentration of O2 decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O2. In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H2 became significant end products when RD-1 was grown on glucose in the presence of O2. Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H2 were produced. When the concentration of O2 in the headspace exceeded 1% (vol/vol), supplemental H2 was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that ofClostridium glycolicum DSM 1288T, an organism characterized as a fermentative anaerobe. Comparative experiments with C. glycolicum DSM 1288T demonstrated that it had negligible H2- and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and C.glycolicum DSM 1288T. A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that ofC. glycolicum DSM 1288Tconfirmed that RD-1 was a strain of C.glycolicum. These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O2, (iii) oxic conditions favor the production of ethanol, lactate, and H2by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O2 might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O2.

Clostridium scatologenes strain SL1 isolated as an acetogenic bacterium from acidic sediments

A strictly anaerobic, H2-utilizing bacterium, strain SL1, was isolated from the sediment of an acidic coal mine pond. Cells of strain SL1 were sporulating, motile, long rods with a multilayer cell wall. Growth was observed at 5-35 degrees C and pH 3.9-7.0. Acetate was the sole end product of H2 utilization and was produced in stoichiometries indicative of an acetyl-CoA-pathway-dependent metabolism. Growth and substrate utilization also occurred with CO/CO2, vanillate, syringate, ferulate, ethanol, propanol, 1-butanol, glycerine, cellobiose, glucose, fructose, mannose, xylose, formate, lactate, pyruvate and gluconate. With most substrates, acetate was the main or sole product formed. Growth in the presence of H2/CO2 or CO/CO2 was difficult to maintain in laboratory cultures. Methoxyl, carboxyl and acrylate groups of various aromatic compounds were O-demethylated, decarboxylated and reduced, respectively. Small amounts of butyrate were produced during the fermentation of sugars. The acrylate group of ferulate was reduced. Nitrate, sulfate, thiosulfate, dimethylsulfoxide and Fe(III) were not utilized as electron acceptors. Analysis of the 16S rRNA gene sequence of strain SL1 demonstrated that it is closely related to Clostridium scatologenes (99.6% sequence similarity), an organism characterized as a fermentative anaerobe but not previously shown to be capable of acetogenic growth. Comparative experiments with C. scatologenes DSM 757T demonstrated that it utilized H2/CO2 (negligible growth), CO/CO2 (negligible growth), formate, ethanol and aromatic compounds according to stoichiometries indicative of the acetyl-CoA pathway. CO dehydrogenase, formate dehydrogenase and hydrogenase activities were present in both strain SL1 and C. scatologenes DSM 757T. These results indicate that (i) sediments of acidic coal mine ponds harbour acetogens and (ii) C. scatologenes is an acetogen that tends to lose its capacity to grow acetogenically under H2/CO2 or CO/CO2 after prolonged laboratory cultivation.

Comparison of two laboratory methods for the determination of serum resistance in Borrelia burgdorferi isolates.

A growth inhibition assay (GIA) and an immunofluorescence test detecting deposited complement components C6 and C9 were compared for their ability to classify Borrelia isolates with respect to their resistance to non-immune human serum (NHS). In both assays a total of 34 Borrelia isolates of all three human pathogenic genospecies were tested. Interestingly, 95% of the serum-sensitive or intermediate serum-sensitive isolates belonged to the genospecies B. burgdorferi s. s. and B. garinii, whereas most B. afzelii isolates (83%) proved serum-resistant. Consequently, a strong correlation between the assignment of the isolates to the different genospecies and their degree of serum sensitivity was seen. These findings were supported strongly by the quantitative analysis of the deposited complement components and the location of the terminal complement complex on the bacterial surface as detected by means of immunoelectron microscopy. The GIA displayed an obvious lack of sensitivity to slow growing isolates, whereas the IFA allowed classification of all Borrelia isolates. Discrimination between serum-sensitive and serum-resistant isolates in the IFA was the most specific provided that the detection of C6 and C9 was incorporated into the final classification of isolates. Accordingly, both assays, turned out to be effective and reliable tools for the investigation of borrelial serum sensitivity. The IFA, however, is regarded as superior to the GIA owing to the obvious ease of performance and its rapid capability for the classification of even very slow growing isolates.

Clostridium akagii sp. nov. and Clostridium acidisoli sp. nov. : acid-tolerant, N2-fixing clostridia isolated from acidic forest soil and litter

Two anaerobic acid-tolerant bacteria, CK58T and CK74T, were isolated from acidic beech litter and acidic peat-bog soil, respectively. Both bacteria were spore-forming, motile rods with peritrichous flagella. The capacity to sporulate decreased with prolonged cultivation. Cells of CK58T formed chains or aggregates and were linked by a connecting filament that consisted of a core and a surrounding sheath. Cellobiose, glucose, xylose, arabinose, maltose, mannose and salicin supported growth of CK58T. These substrates, as well as mannitol, lactose, sucrose, glycerol, melezitose, raffinose and rhamnose, supported growth of CK74T. Sorbitol, trehalose, H2/CO2, CO/CO2, vanillate, Casamino acids, peptone, and various purines and pyrimidines did not support the growth of either organism. Growth of CK58T and CK74T on glucose yielded butyrate, lactate, acetate, formate, H2 and CO2 as end products. Growth of CK58T and CK74T was observed at pH 3.7-7.1 and 3.6-6.9, respectively. CK58T and CK74T grew in nitrogen-free medium at pH 3.7 under an N2 atmosphere and reduced acetylene at rates approximating 1 nmol min-1 (mg protein)-1. CK58T and CK74T did not contain carbon monoxide dehydrogenase or cytochromes, produce methane, or dissimilate nitrate or sulfate. Thus, CK58T and CK74T were characterized as nonacetogenic, N2-fixing, fermentative chemo-organotrophs. The G + C contents of CK58T and CK74T were 31.4 and 30.7 mol%, respectively. CK58T and CK74T were phylogenetically most closely related to Clostridium pasteurianum. The 16S rRNA gene sequence similarity values of CK58T and CK74T to C. pasteurianum and each other did not exceed 96.5%, and it is proposed that strains CK58T and CK74T be named Clostridium akagii CK58T (DSM 12554T) and Clostridium acidisoli CK74T (DSM 12555T), respectively. These results suggest that previously uncharacterized clostridial species reside and might fix N2 in the annoxic microzones of acidic forest soil and litter.

Differences of two Borrelia burgdorferi strains in complement activation and serum resistance

Complement activation and serum resistance of the Borrelia burgdorferi strains B31 (American strain) and PKo (European strain) were compared. In 25% (v/v) normal human serum (NHS) free of B. burgdorferi-specific antibodies the cells of the PKo strain were high activators of complement as indicated by rapid and strong C9 consumption, by deposition of up to 336763 C9 molecules per cell and by the formation of the terminal complement complex on the cell surface. By comparison, complement activation by the B31 strain was low with 5.4-fold less C9 deposited per cell. The addition of B. burgdorferi-specific antibodies to NHS either as purified IgG or heat-inactivated patient sera, had no influence on the results with both strains. After an incubation period of 2h at 37 degrees C in 25% (v/v) NHS most cells of the PKo strain had lost their viability as indicated by cell immobilization and failure to multiply in subcultures. In addition, extensive cell fragmentation and bleb formation were observed in the electron microscope. In contrast, the B31 strain remained alive and morphologically intact after the same incubation with NHS. We conclude from our results that complement activation and serum resistance are properties which differ considerably between isolated strains of B. burgdorferi.

In vitro activities of fluoroquinolones against the spirochete Borrelia burgdorferi.

ABSTRACT Little is known to date about the in vitro activity of fluoroquinolones against Borrelia species. Our study aimed at determining the in vitro activities of 15 quinolones against nine isolates of the Borrelia burgdorferi sensu lato complex in addition to one Borrelia valaisiana and oneBorrelia bissettii tick isolate. For the determination of MICs, a standardized colorimetric microdilution method was applied. Determination of minimal borreliacidal concentrations providing 100% killing of the final inoculum (MBCs) after 72 h and time-kill experiments were performed by conventional culture in Barbour-Stoenner-Kelly medium in combination with dark-field microscopy. The rank order of potency on a microgram-per-milliliter basis for the substances with in vitro activity against B. burgdorferi was gemifloxacin (MIC at which 90% of the isolates tested are inhibited [MIC90], 0.12 μg/ml) > sitafloxacin (MIC90, 0.5 μg/ml), grepafloxacin (MIC90, 0.5 μg/ml) > gatifloxacin (MIC90, 1 μg/ml), sparfloxacin (MIC90, 1 μg/ml), trovafloxacin (MIC90, 1 μg/ml) > moxifloxacin (MIC90, 2 μg/ml), ciprofloxacin (MIC90, 2 μg/ml) > levofloxacin (MIC90, 4 μg/ml) > ofloxacin (MIC90, 8 μg/ml), norfloxacin (MIC90, 8 μg/ml) > fleroxacin (MIC90, >16 μg/ml), and pefloxacin (MIC90, 32 μg/ml) > nalidixic acid (MIC90, 256 μg/ml). After 72 h of exposure, gemifloxacin was borreliacidal (100% killing) against the isolates investigated at a median MBC of 4 μg/ml. In the other compounds tested, median MBCs were higher (≥8 μg/ml). Results of electron microscopy and time-kill studies clearly support an in vitro activity of some fluoroquinolones against borreliae. Our study demonstrates for the first time the enhanced in vitro effectiveness of some of the recently introduced 4-quinolones against B. burgdorferi.

Comparison of In Vitro Activities of Ketolides, Macrolides, and an Azalide against the Spirochete Borrelia burgdorferi

ABSTRACT Two ketolides, three macrolides, and one azalide were tested in vitro against 17 isolates of the B. burgdorferi s.l. complex. As measured in micrograms per milliliter, activity was highest for cethromycin (MIC at which 90% of the tested isolates were inhibited [MIC90], 0.0019 μg/ml) and telithromycin (MIC90, 0.0078 μg/ml). Electron-microscope analysis and time-kill studies also supported enhanced effectiveness of both ketolides.

Clostridium uliginosum sp. nov., a novel acid- tolerant, anaerobic bacterium with connecting filaments

An anaerobic, acid-tolerant bacterium, CK55T, was isolated from an acidic forest bog. Cells of CK55T stained Gram-negative but did not have an outer membrane. Cells were spore-forming, motile rods with peritrichous flagella, formed chains or aggregates and were linked by connecting filaments that were composed of a core and outer sheath. Cellobiose, glucose, xylose, mannose, mannitol, sucrose and peptone supported growth. Arabinose, lactose, raffinose, H2/CO2, CO/CO2, vanillate, Casamino acids and various purines and pyrimidines did not support growth. Growth on carbohydrates yielded acetate, butyrate, lactate, formate and H2 as end-products. Growth was observed at pH 4.0-9.0, with an optimum at pH 6.5, and at 10-30 degrees C, with an optimum at 20-25 degrees C. At 20 degrees C, doubling times were 4 and 6 h at pH 6.5 and 4.0, respectively. Hydrogenase activity in cell-free extracts was 12 U (mg protein)-1. CK55T did not: (i) contain detectable levels of CO, formate, lactate dehydrogenases or cytochromes; (ii) carry out dissimilatory reduction of nitrate or sulfate; or (iii) produce methane. Thus, CK55T was characterized as a non-acetogenic, fermentative chemo-organotroph. The G+C content of CK55T was 28.0 mol%. CK55T was phylogenetically most closely related to Clostridium botulinum (types B, E and F), Clostridium acetobutylicum and other saccharolytic clostridia; the 16S rRNA gene sequence similarity values to the nearest relatives of CK55T were approximately 97%. Based on morphological, physiological and phylogenetic properties of CK55T, it is proposed that CK55T be termed Clostridium uliginosum sp. nov. (= DSM 12992T = ATCC BAA-53T).

7 Immunoelectron Microscopy of Surface Antigens (Polysaccharides) of Gram-negative Bacteria using Pre- and Post-embedding Techniques*

Publisher Summary The subcellular localization of the surface antigens presents considerable preparative difficulties because most of them are components of highly hydrated cell structures which collapse during conventional dehydration procedures. Therefore it is advisable to localize such antigens by combining the results of different methods before firm conclusions are drawn on the bacterial surface architecture of living cells. The neglected whole-mount procedure, a very simple and rapid technique, will continue to be an important tool for examining immunologically accessible surface antigens, especially when used in combination with the conventional thin-section technique. In combination with the Lowicryl K4M thin-section technique described, the whole-mount procedure carried out with cells fixed in different concentrations of glutaraldehyde solutions can be useful in determining which fixation conditions lead to an optimal compromise between labeling signal and ultrastructural preservation. The localization of ECA on the outer membrane surface in both frozen-thawed and Lowicryl K4M resin sections is in good agreement with several observations on different smooth and/or encapsulated strains and mutants.

Differences in gene expression between natural and artificially induced leaf senescence in barley

Senescence is the last step of leaf development in the life span of an annual plant. Senescence can be induced prematurely by treating leaf tissues with jasmonic acid methyl ester (methyl jasmonate, MeJA). During both senescence programmes, drastic changes occur at the biochemical, cellular and ultra-structural levels that were compared here for primary leaves of barley (Hordeum vulgare L.). Our findings indicate that both types of senescence are similar with respect to the morphological changes including the loss of chlorophyll, disintegration of thylakoids, and formation of plastoglobules. However, the time elapsed for reaching senescence completion was different and ranged from 7 to 8 days for artificially senescing, MeJA-treated plants to 7-8 weeks for naturally senescing plants. Pulse-labelling studies along with RNA and protein gel blot analyses showed differential changes in the expression of both plastid and nuclear genes coding for photosynthetic proteins. Several unique messenger products accumulated in naturally and artificially senescing, MeJA-treated leaves. Detailed expression and crosslinking studies revealed that pheophorbide a oxygenase (PAO), a previously implicated key enzyme of chlorophyll breakdown, is most likely not rate-limiting for chlorophyll destruction under both senescence conditions. Metabolite profiling identified differential changes in the composition of carotenoid derivatives and prenyl-lipids to occur in naturally senescing and artificially senescing plants that underscored the differences between both senescence programmes.