Check out our New Publishers' Select for Free Articles

Archives of Microbiology (v.169, #2)

Identification of the Bradyrhizobium japonicum degP gene as part of an operon containing small heat-shock protein genes by F. Narberhaus; Wolfgang Weiglhofer; Hans-Martin Fischer; Hauke Hennecke (pp. 89-97).

A degP (htrA)-like gene of Bradyrhizobium japonicum was identified immediately downstream of two genes (hspB and hspC) coding for small heat-shock proteins. All three genes are oriented in the same direction and are separated by only 85 and 72 bp, and a heat-inducible transcript covering hspB, hspC, and degP was detected by RT-PCR. These results show that the genes are organized in an operon. Two mutants, a degP insertion mutant and a ΔhspBCdegP mutant, were constructed by marker replacement mutagenesis. Immunoblot analysis performed with a serum raised against the amino-terminal end of IbpA, an HspB homolog of Escherichia coli, identified three heat-inducible protein bands in B. japonicum extract, one of which was missing in the deletion mutant. None of the mutants showed an obvious defect during growth at different temperatures, after heat-shock treatment, or in the presence of solvents. Moreover, they were not affected in root-nodule symbiosis, indicating that the small heat-shock proteins HspB and HspC and the DegP homolog of B. japonicum are not required under a wide range of growth conditions.

Keywords: Key words Bradyrhizobium japonicum; Heat shock; DegP; HtrA; Periplasmic serine protease; Small; heat-shock protein; Mutants

Complex I of Rhodobacter capsulatus and its role in reverted electron transport by Stefan Michael Herter; Christiane Maria Kortlüke; G. Drews (pp. 98-105).

The activities of NAD⁺-photoreduction and NADH/decyl-ubiquinone reductase in membrane preparations of Rhodobacter capsulatus changed to the same extent under different conditions. These results indicated that NADH:ubiquinone oxidoreductase (complex I) catalyzes the electron transport in the downhill direction (respiratory chain) and in the uphill direction (reverted electron flow). This conclusion was confirmed by the characterization of a complex-I-deficient mutant of R. capsulatus. The mutant was not able to reduce NAD⁺ in the light. Since this mutant was not able to grow photoautotrophically, we concluded that complex I is the enzyme that catalyzes the reverted electron flow to NAD⁺ to provide reduction equivalents for CO₂ fixation. Complex I is not essential for the reverted electron flow to nitrogenase since the mutant grew under nitrogen-fixing conditions. As shown by immunological means, NuoE, a subunit of complex I from R. capsulatus having an extended C-terminus, was modified depending on the nitrogen source present in the growth medium. When the organism used N₂ instead of NH₄ ⁺, a smaller NuoE polypeptide was synthesized. The complex-I-deficient mutant was not able to modify NuoE. The function of the modification is discussed.

Keywords: Key words Rhodobacter capsulatus; Complex I; Reverted electron flow; NAD+-photoreduction; CO2 fixation; N2 fixation; NuoE; NuoF

Amygdalin degradation by Mucor circinelloides and Penicillium aurantiogriseum: mechanisms of hydrolysis by Leon Brimer; Anna Rita Cicalini; F. Federici; Maurizio Petruccioli (pp. 106-112).

Mucor circinelloides LU M40 and Penicillium aurantiogriseum P 35 produce extracellular β-glycosidases that are active on the cyanogenic glycoside amygdalin. From the culture broths of M. circinelloides, only one β-glycosidase could be identified, while two different enzymes – both having amygdalase activity – were found in culture broths of P. aurantiogriseum. The study of the mechanism of hydrolysis of the β-bis-glycoside amygdalin with purified enzymes from the two organisms indicated a possible sequential (two-step) reaction. In all cases, the first step of hydrolysis from amygdalin to prunasin was very rapid, while the second step from prunasin to cyanohydrin was much slower. No cyanohydrin lyase activity was found in the culture broths of either fungus.

Keywords: Key words Penicillium aurantiogriseum; Mucor; circinelloides; Fungal amygdalases; Hydrolysis; mechanism

Effects of the insertion of a nonapeptide from murine IL-1β on the immunogenicity of carrier proteins delivered by live attenuated Salmonella by Inês Chen; Mariagrazia Pizza; R. Rappuoli; S. M. C. Newton (pp. 113-119).

A nonapeptide from IL-1β has been reported to be an immunostimulant and adjuvant. To investigate the possibility of enhancing the immunogenicity of recombinant antigens delivered by live-attenuated Salmonella strains, we inserted an oligonucleotide coding for the nonapeptide from murine IL-1β into the genes of three model proteins: LamB, MalE, and flagellin. The hybrid proteins were expressed and delivered in vivo by Salmonella aroA strains, and serum antibody responses were analyzed. The results showed that the nonapeptide induced an increase in the immune response against Salmonella-delivered flagellin, measured on day 28 post-immunization. However, the adjuvant effect was lost by day 42. In no case was an adjuvant effect detected for Salmonella-delivered LamB or MalE. Thus, by comparing the immune responses raised by purified MalE with and without the peptide, we investigated whether the insertion of the peptide affected the immunogenicity of the protein itself. Also in this case, a modest adjuvant effect was shown only after primary immunization and when very low doses of antigen were used. In conclusion, the immunomodulatory properties of the IL-1β peptide can also be detected when it is delivered in vivo by Salmonella; however, the effect is modest and antigen-dependent.

Keywords: Key words IL-1β nonapeptide; Salmonella; Adjuvant

The ecological niche of the consortium “Pelochromatium roseum” by J. Overmann; Christian Tuschak; Henrik Sass; J. Fröstl (pp. 120-128).

A dense accumulation of the phototrophic consortium “Pelochromatium roseum” in a small, eutrophic, freshwater lake (Dagowsee, Brandenburg, Germany) was investigated. Within the chemocline, the number of epibionts of the consortia represented up to 19% of the total number of bacteria. Per “P. roseum” a mean value of 20 epibionts was determined. Similar to other aquatic habitats, consortia in the Dagowsee were found only at low light intensities (< 7 μmol quanta m^–2 s^–1) and low sulfide concentrations (0–100 μM). In dialysis cultures of “P. roseum”, bacterial cells remained in a stable association only when incubated at light intensities between 5 and 10 μmol quanta m^–2 s^–1. Intact consortia from natural samples had a buoyant density of 1046.8 kg m^–3, which was much higher than that of ambient chemocline water (995.8 kg m^–3). Under environmental conditions and without motility, this density difference would result in rapid sedimentation of consortia toward the lake bottom. Our results indicate that (1) consortia are adapted to a very narrow regime of light intensities and sulfide concentrations, (2) motility and tactic responses must be of ecological significance for the colonization of the free water column of lakes, and (3) phototrophic growth of consortia can be explained only by a cycling of sulfur species in the chemocline, possibly within the consortia themselves.

Keywords: Key words Phototrophic consortia; Green sulfur; bacteria; Chlorobiaceae; “Chlorochromatium”; “Pelochromatium”; Syntrophy; Sulfur cycle

Physiology and tactic response of the phototrophic consortium “Chlorochromatium aggregatum” by Jürgen M. Fröstl; J. Overmann (pp. 129-135).

The phototrophic consortium “Chlorochromatium aggregatum” was enriched from sediment samples of a eutrophic freshwater lake and was maintained at high numbers in anoxic sulfide-reduced medium. Growth of intact consortia was observed only in the light and in the presence of 2-oxoglutarate as an organic carbon source. Consortia of “C. aggregatum” reached maximum growth rates at light intensities ≥ 5 μmol quanta m^–2 s^–1. Of ten compounds tested, sulfide, thiosulfate, 2-oxoglutarate, and citrate served as a chemoattractant for “C. aggregatum”. When incubated in the presence of sulfide and in the light, epibionts reduced the fluorochrome 5-cyano-2,3-di-4-tolyl-tetrazolium chloride (CTC). Reduction of CTC was not observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or in the dark, indicating that sulfide serves as an electron donor for the phototrophic epibiont. Motile consortia accumulated scotophobically in microcuvettes at a wavelength of 740 nm. Since this wavelength corresponds to the position of the absorption maximum of bacteriochlorophylls c or d, the photosynthetic pigments are most likely the photoreceptors of the scotophobic response. It is concluded that, within the consortia, a rapid interspecies signal transfer occurs between the nonmotile, green-colored epibiont and the motile, colorless central bacterium.

Keywords: Key words Phototrophic consortia; “Chlorochromatium”; Syntrophy; Phototaxis; Chemotaxis

Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes by S. Spring; Ulysses Lins; R. Amann; Karl-Heinz Schleifer; Luis C. S. Ferreira; Darci M. S. Esquivel; Marcos Farina (pp. 136-147).

Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the α-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.

Keywords: Key words Magnetic bacteria; Biomineralization; Magnetite; 16S rRNA; In situ hybridization; Ultrastructure; Electron microscopy

A novel pink-pigmented facultative methylotroph, Methylobacterium thiocyanatum sp. nov., capable of growth on thiocyanate or cyanate as sole nitrogen sources by A. P. Wood; Donovan P. Kelly; I. R. McDonald; Sarah L. Jordan; Thomas D. Morgan; Sajdah Khan; J. Colin Murrell; Elena Borodina (pp. 148-158).

The isolation and properties of a novel species of pink-pigmented methylotroph, Methylobacterium thiocyanatum, are described. This organism satisfied all the morphological, biochemical, and growth-substrate criteria to be placed in the genus Methylobacterium. Sequencing of the gene encoding its 16S rRNA confirmed its position in this genus, with its closest phylogenetic relatives being M. rhodesianum, M. zatmanii and M. extorquens, from which it differed in its ability to grow on several diagnostic substrates. Methanol-grown organisms contained high activities of hydroxypyruvate reductase [3 μmol NADH oxidized min^–1 (mg crude extract protein)^–1], showing that the serine pathway was used for methylotrophic growth. M. thiocyanatum was able to use thiocyanate or cyanate as the sole source of nitrogen for growth, and thiocyanate as the sole source of sulfur in the absence of other sulfur compounds. It tolerated high concentrations (at least 50 mM) of thiocyanate or cyanate when these were supplied as nitrogen sources. Growing cultures degraded thiocyanate to produce thiosulfate as a major sulfur end product, apparently with the intermediate formation of volatile sulfur compounds (probably hydrogen sulfide and carbonyl sulfide). Enzymatic hydrolysis of thiocyanate by cell-free extracts was not demonstrated. Cyanate was metabolized by means of a cyanase enzyme that was expressed at approximately sevenfold greater activity during growth on thiocyanate [V _max 634 ± 24 nmol NH₃ formed min^–1 (mg protein)^–1] than on cyanate [89 ± 9 nmol NH₃ min^–1 (mg protein)^–1]. Kinetic study of the cyanase in cell-free extracts showed the enzyme (1) to exhibit high affinity for cyanate (K _m 0.07 mM), (2) to require bicarbonate for activity, (3) to be subject to substrate inhibition by cyanate and competitive inhibition by thiocyanate (K _i 0.65 mM), (4) to be unaffected by 1 mM ammonium chloride, (5) to be strongly inhibited by selenocyanate, and (6) to be slightly inhibited by 5 mM thiosulfate, but unaffected by 0.25 mM sulfide or 1 mM thiosulfate. Polypeptides that might be a cyanase subunit (mol.wt. 17.9 kDa), a cyanate (and/or thiocyanate) permease (mol.wt. 25.1 and 27.2 kDa), and a putative thiocyanate hydrolase (mol.wt. 39.3 kDa) were identified by SDS-PAGE. Correlation of the growth rate of cultures with thiocyanate concentration (both stimulatory and inhibitory) and the kinetics of cyanase activity might indicate that growth on thiocyanate involved the intermediate formation of cyanate, hence requiring cyanase activity. The very high activity of cyanase observed during growth on thiocyanate could be in compensation for the inhibitory effect of thiocyanate on cyanase. Alternatively, thiocyanate may be a nonsubstrate inducer of cyanase, while thiocyanate degradation itself proceeds by a carbonyl sulfide pathway not involving cyanate. A formal description of the new species (DSM 11490) is given.

Keywords: Key words Methylobacterium thiocyanatum; Thiocyanate metabolism; Cyanase; 16S rRNA sequence; SDS-PAGE; Pink-pigmented facultative methylotroph

Anaerobic and aerobic oxidation of ferrous iron at neutral pH by chemoheterotrophic nitrate-reducing bacteria by M. Benz; Andreas Brune; Bernhard Schink (pp. 159-165).

Nine out of ten anaerobic enrichment cultures inoculated with sediment samples from various freshwater, brackish-water, and marine sediments exhibited ferrous iron oxidation in mineral media with nitrate and an organic cosubstrate at pH 7.2 and 30° C. Anaerobic nitrate-dependent ferrous iron oxidation was a biological process. One strain isolated from brackish-water sediment (strain HidR2, a motile, nonsporeforming, gram-negative rod) was chosen for further investigation of ferrous iron oxidation in the presence of acetate as cosubstrate. Strain HidR2 oxidized between 0.7 and 4.9 mM ferrous iron aerobically and anaerobically at pH 7.2 and 30° C in the presence of small amounts of acetate (between 0.2 and 1.1 mM). The strain gained energy for growth from anaerobic ferrous iron oxidation with nitrate, and the ratio of iron oxidized to acetate provided was constant at limiting acetate supply. The ability to oxidize ferrous iron anaerobically with nitrate at approximately pH 7 appears to be a widespread capacity among mesophilic denitrifying bacteria. Since nitrate-dependent iron oxidation closes the iron cycle within the anoxic zone of sediments and aerobic iron oxidation enhances the reoxidation of ferrous to ferric iron in the oxic zone, both processes increase the importance of iron as a transient electron carrier in the turnover of organic matter in natural sediments.

Keywords: Key words Iron oxidation; Ferrous iron; Ferric iron; Nitrate reduction; Sediments

Purification and chacterization of acid-type phosphatases from a heavy-metal-accumulating Citrobacter sp. by Byeong C. Jeong; Philip S. Poole; Anthony C. Willis; L. E. Macaskie (pp. 166-173).

An acid phosphatase from a heavy-metal-accumulating strain of a Citrobacter sp. was resolved into two forms on the basis of their nonbinding (phosphatase I) or binding (phosphatase II) behaviour on the cation-exchange resin SP-Sephadex C50. Both holoenzymes had a molecular mass of 103–108 kDa as determined by Superose Q-6 column chromatography in the presence of 150 mM KCl and a subunit molecular mass of 27 kDa as determined by SDS-PAGE; the enzyme was tetrameric. Both enzymes had a pI ≈ 9.0 and were immunologically cross-reactive. There were minor differences in amino acid composition and in peptide maps following tryptic digest. The pH optimum for phosphatases I and II was 5.5 and 6.25, respectively; phosphatase II alone retained activity at pH values up to 9.0. Phosphatase I was more resistant to mechanical shear, γ-irradiation, high temperature, and toxins (F^– and formaldehyde). Glycerol increased the thermostability of both enzymes, particularly the more thermosensitive phosphatase II. Phosphatase II had a lower K _m and a lower V _max for glycerol 2-phosphate hydrolysis. The production of enzyme isoforms is a phenomenon similar to that described previously for the alkaline phosphatase of Escherichia coli, where the isoforms relate to precursive and final processed forms of the enzyme. Acid phosphatase is physiologically distinct, with a role that is still obscure but that may relate to cellular stress responses.

Keywords: Key words Phosphatases; Citrobacter; Acid; phosphatase; Phosphatase isoforms

In vitro protein phosphorylation associated with cellular differentiation of Streptomyces griseus by Susumu Okamoto; Masayoshi Itoh; K. Ochi (pp. 174-177).

In vitro phosphorylation reactions with crude cellular extracts revealed that phosphorylation of a 17-kDa protein is associated with the onset of aerial mycelium formation in solid culture (but not submerged spore formation in liquid culture) of Streptomyces griseus. The possible importance of the 17-kDa protein phosphorylation in cellular differentiation was further indicated by inducing aerial mycelium formation in the presence of decoyinine and in studies using certain developmental mutants (relC, afsA, and M-1). It is proposed that the 17-kDa protein may play a role in cellular differentiation of S. griseus via its phosphorylation.

Keywords: Key words Protein phosphorylation; Cellular; differentiation; Streptomyces griseus

Sections

Personal tools

Archives of Microbiology (v.169, #2)

Sections

Personal tools

Local resources

Archives of Microbiology (v.169, #2)