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Archives of Microbiology (v.172, #3)

Recent advances in understanding resin acid biodegradation: microbial diversity and metabolism by Vincent J. J. Martin; Zhongtang Yu; W. W. Mohn (pp. 131-138).

Resin acids are tricyclic diterpenoids that are found in the oleoresin of coniferous trees. Resin-acid-degrading microorganisms are ubiquitous in the environment. The bacterial isolates that grow on resin acids as sole organic substrates are physiologically and phylogenetically diverse, and include psychrotolerant, mesophilic, and thermophilic bacteria. Recent studies of the biodegradation of resin acids by these organisms have demonstrated that in gram-negative bacteria, distinct biochemical pathways exist for the degradation of abietane- and pimerane-type resin acids. One of these organisms, Pseudomonas abietaniphila BKME-9, harbors a convergent pathway that channels the nonaromatic abietanes and dehydroabietic acid into 7-oxodehydroabietic acid. This dioxygenolytic pathway is encoded by the recently cloned and sequenced dit gene cluster. The dit cluster encodes the ferredoxin and the α- and β-subunits of a new class of ring-hydroxylating dioxygenases as well as an extradiol ring-cleavage dioxygenase. Although it was previously thought that resin acids are very recalcitrant under anoxic conditions, recent investigations have demonstrated that they are partially metabolized under anoxic conditions by undefined microorganisms. The anaerobic degradation of resin acids principally generates aromatized and decarboxylated products (such as retene) that are thought to persist in the environment.

Keywords: Key words Resin acid; Abietane; Pimerane; Diversity; Biodegradation; Dioxygenase; Diterpene

Isolation and characterization of a Clostridium sp. with cinnamoyl esterase activity and unusual cell envelope ultrastructure by C. S. McSweeney; Amanda Dulieu; Richard I. Webb; Therese Del Dot; Linda L. Blackall (pp. 139-149).

Microorganisms that hydrolyse the ester linkages between phenolic acids and polysaccharides in plant cell walls are potential sources of enzymes for the degradation of lignocellulosic waste. An anaerobic, mesophilic, spore-forming, xylanolytic bacterium with high hydroxy cinnamic acid esterase activity was isolated from the gut of the grass-eating termite Tumilitermes pastinator. The bacterium was motile and rod-shaped, stained gram-positive, had an eight-layered cell envelope, and formed endospores. Phylogenetic analysis based on 16S rRNA indicated that the bacterium is closely related to Clostridium xylanolyticum and is grouped with polysaccharolytic strains of clostridia. A wide range of carbohydrates were fermented, and growth was stimulated by either xylan or cellobiose as substrates. The bacterium hydrolysed and then hydrogenated the hydroxy cinnamic acids (ferulic and p-coumaric acids), which are esterified to arabinoxylan in plant cell walls. Three cytoplasmic enzymes with hydroxy cinnamic acid esterase activity were identified using non-denaturing gel electrophoresis. This bacterium possesses an unusual multilayered cell envelope in which both leaflets of the cytoplasmic membrane, the peptidoglycan layer and the S layer are clearly discernible. The fate of all these components was easily followed throughout the endospore formation process. The peptidoglycan component persisted during the entire morphogenesis. It was seen to enter the septum and to pass with the engulfing membranes to surround the prespore. It eventually expanded to form the cortex, verification for the peptidoglycan origin of the cortex. Sporogenic vesicles, which are derived from the cell wall peptidoglycan, were associated with the engulfment process. Spore coat fragments appeared early, in stage II, though spore coat formation was not complete until after cortex formation.

Keywords: Key wordsClostridium xylanolyticum; Cinnamic acid; Esterase; Lignocellulose; Sporogenesis; Ultrastructure; Cell envelope

Aerobic turnover of dimethyl sulfide by the anoxygenic phototrophic bacterium Thiocapsa roseopersicina by Henk M. Jonkers; Michael Jansen; Marc J. E. C. Van der Maarel; H. Van Gemerden (pp. 150-156).

This is the first report describing the complete oxidation of dimethyl sulfide (DMS) to sulfate by an anoxygenic, phototrophic purple sulfur bacterium. Complete DMS oxidation was observed in cultures of Thiocapsa roseopersicina M11 incubated under oxic/light conditions, resulting in a yield of 30.1 mg protein mmol^–1. No oxidation of DMS occurred under anoxic/light conditions. Chloroform, methyl butyl ether, and 3-amino-1,2,4-triazole, which are specific inhibitors of aerobic DMS oxidation in thiobacilli and hyphomicrobia, did not affect DMS oxidation in strain M11. This could be due to limited transport of the inhibitors through the cell membrane. The growth yield on sulfide as sole electron donor was 22.2 mg protein mmol^–1 under anoxic/light conditions. Since aerobic respiration of sulfide would have resulted in yields lower than 22 mg protein mmol^–1, the higher yield on DMS under oxic/light conditions suggests that the methyl groups of DMS have served as an additional carbon source or as an electron donor in addition to the sulfide moiety. The kinetic parameters V _max and K _m for DMS oxidation under oxic/light conditions were 12.4 ± 1.3 nmol (mg protein)^–1 min^–1 and 2 μM, respectively. T. roseopersicina M11 also produced DMS by cleavage of dimethylsulfoniopropionate (DMSP). Specific DMSP cleavage rates increased with increasing initial substrate concentrations, suggesting that DMSP lyase was only partly induced at lower initial DMSP concentrations. A comparison of T. roseopersicina strains revealed that only strain M11 was able to oxidize DMS and cleave DMSP. Both strain M11 and strain 5811 accumulated DMSP intracellularly during growth, while strain 1711 showed neither of these characteristics. Phylogenetic comparison based on 16S rRNA gene sequence revealed a similarity of 99.0% between strain M11 and strain 5811, and 97.6% between strain M11 and strain 1711. DMS and DMSP utilization thus appear to be strain-specific.

Keywords: Key words Purple sulfur bacterium; Thiocapsa; roseopersicina; Dimethyl sulfide; Dimethylsulfoniopropionate

Sexual development of Aspergillus nidulans in tryptophan auxotrophic strains by Sabine E. Eckert; Bernd Hoffmann; Christoph Wanke; G. H. Braus (pp. 157-166).

The interplay between sexual development and amino acid biosynthesis in the ascomycete Aspergillus nidulans was studied. The growth and differentiation of four tryptophan auxotrophic strains that are unable to regulate their tryptophan biosynthesis, were examined. These strains are sterile on medium containing low tryptophan concentrations. Fruitbody formation was restored by supplementation with high concentrations of tryptophan and was promoted by supplementation with indole or auxin. Tryptophan supplementation resulted in auxin production of A. nidulans. Fertility of ascospores of the tryptophan auxotrophic strains could only be partially re-established. trpC transcript levels and enzyme activities remained stable in cleistothecia and conidiophore extracts as compared to those of mycelium, while levels of gene transcripts involved in glycolysis were lower in fruitbodies and conidiospores. Auxotrophic strains unable to form fruitbodies at intermediate amino acid supplementation levels turned on the cross-pathway regulatory system that is induced by amino acid starvation. We conclude that there is a connection between the genetic network of cross-pathway control and sexual development in A. nidulans.

Keywords: Key wordsAspergillus nidulans; Tryptophan; biosynthesis; Fruitbody formation; Auxin

Heliorestis daurensis, gen. nov. sp. nov., an alkaliphilic rod-to-coiled-shaped phototrophic heliobacterium from a Siberian soda lake by Irina A. Bryantseva; Vladimir M. Gorlenko; Elena I. Kompantseva; Laurie A. Achenbach; M. T. Madigan (pp. 167-174).

A novel alkaliphilic heliobacterium was isolated from microbial mats of a low-salt alkaline Siberian soda lake. Cells of the new organism were tightly coiled when grown in coculture with a rod-shaped bacterium, but grew as short filaments when finally obtained in pure culture. The new phototroph, designated strain BT-H1, produced bacteriochlorophyll g and a neurosporene-like pigment, and lacked internal photosynthetic membranes. Similar to other heliobacteria, strain BT-H1 grew photoheterotrophically on a limited range of organic compounds including acetate and pyruvate. Sulfide was oxidized to elemental sulfur and polysulfides under photoheterotrophic conditions; however, photoautotrophic growth was not observed. Cultures of strain BT-H1 were alkaliphilic, growing optimally at pH 9, and unlike other heliobacteria, they grew optimally at a temperature of 25 °C rather than at 40 °C or above. Analysis of the 16S rRNA gene sequence of the new organism showed that it groups within the heliobacterial clade. However, its branching order was phylogenetically basal to all previously investigated species of heliobacteria. The G+C content of the DNA of strain BT-H1 (44.9 mol%) was also quite distinct from that of other heliobacteria. This unique assemblage of properties implicates strain BT-H1 as a new genus and species of the heliobacteria, Heliorestis daurensis, named for its unusual morphology (“restis” is Latin for “rope”) and for the Daur Steppe in Russia in which these soda lakes are located.

Keywords: Key words Photosynthesis; Anoxygenic phototrophic; bacteria; Heliobacteria; Bacteriochlorophyll g; Alkaliphiles; Soda lakes

Purification of cold-shock-like proteins from Stigmatella aurantiaca– molecular cloning and characterization of the cspA gene by Irmela Stamm; Andreas Leclerque; W. Plaga (pp. 175-181).

Prominent low-molecular-weight proteins were isolated from vegetative cells of the myxobacterium Stigmatella aurantiaca and were found to be members of the cold-shock protein family. A first gene of this family (cspA) was cloned and sequenced. It encodes a protein of 68 amino acid residues that displays up to 71% sequence identity with other bacterial cold-shock(-like) proteins. A cysteine residue within the RNP-2 motif is a peculiarity of Stigmatella CspA. A cspA::(Δtrp-lacZ) fusion gene construct was introduced into Stigmatella by electroporation, a method that has not been used previously for this strain. Analysis of the resultant transformants revealed that cspA transcription occurs at high levels during vegetative growth at 20 and 32 °C, and during fruiting body formation.

Keywords: Key words Cold-shock protein family; Protein; purification; Electroporation; Gene expression; Stigmatella aurantiaca; Myxobacteria

Purification and characterization of 5-oxo-l-prolinase from Paecilomyces varioti F-1, an ATP-dependent hydrolase active with l-2-oxothiazolidine-4-carboxylic acid by K. Mochizuki (pp. 182-185).

An enzyme cleaving l-2-oxothiazolidine-4-carboxylic acid to l-cysteine was purified 75-fold with 8% recovery to near homogeneity from crude extracts of Paecilomyces varioti F-1, which had been isolated as a fungus able to assimilate l-2-oxothiazolidine-4-carboxylic acid. The molecular mass was estimated to be 260 kDa by gel filtration. The purified preparation migrated as a single band of molecular mass 140 kDa upon SDS-PAGE. The maximum activity was observed at a range of pH 7.0–8.0 and at 50 °C. The enzyme activity was completely inhibited by SH-blocking reagents such as AgNO₃, p-chloromercuribenzoic acid, N-ethylmaleimide, and N-bromosuccinimide. The enzyme required ATP, Mg²⁺, and KCl for the cleavage of l-2-oxothiazolidine-4-carboxylic acid. The enzyme also cleaved 5-oxo-l-proline to l-glutamic acid and is considered to be 5-oxo-l-prolinase.

Keywords: Key wordsl-2-Oxothiazolidine-4-carboxylic acid; 5-Oxo-l-proline; 5-Oxo-l-prolinase; Paecilomyces; varioti F-1

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Archives of Microbiology (v.172, #3)