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Archives of Microbiology (v.179, #5)
Secretins of Pseudomonas aeruginosa: large holes in the outer membrane by Wilbert Bitter (pp. 307-314).
Pseudomonas aeruginosa produces a large number of exoproteins, ranging from the ADP-ribosyltransferases exotoxin A and ExoS to degradative enzymes, such as elastase and chitinase. As it is a gram-negative bacterium, P. aeruginosa must be able to transport these exoproteins across both membranes of the cell envelope. In addition, also proteins that are part of cellular appendages, such as type IV pili and flagella, have to cross the cell envelope. Whereas the majority of the proteins transported across the inner membrane are dependent on the Sec channel, the systems for translocation across the outer membrane seem to be more diverse. Gram-negative bacteria have invented a number of different strategies during the course of evolution to achieve this goal. Although these transport machineries seem to be radically different, many of them actually depend on a member of the secretin protein family for their function. Recent results show that secretins form a large complex in the outer membrane, which constitutes the actual translocation channel. Understanding the working mechanism of this protein translocation channel could open up new strategies to target molecular machineries at the heart of many important virulence factors.
Keywords: Secretin Outer membrane Exoprotein Pseudomonas aeruginosa Pili
Methanol utilization by a novel thermophilic homoacetogenic bacterium, Moorella mulderi sp. nov., isolated from a bioreactor by Melike Balk; Jan Weijma; Michael W. Friedrich; Alfons J. M. Stams (pp. 315-320).
A thermophilic, anaerobic, spore-forming bacterium (strain TMS) was isolated from a thermophilic bioreactor operated at 65 °C with methanol as the energy source. Cells were gram-positive straight rods, 0.4–0.6 µm×2–8 μm, growing as single cells or in pairs. The temperature range for growth was 40–70 °C with an optimum at 65 °C. Growth was observed from pH 5.5 to 8.5, and the optimum pH was around 7. The salinity range for growth was 0–45 g NaCl l−1 with an optimum at 10 g l−1. The isolate was able to grow on methanol, H2-CO2 (80/20%, v/v), formate, lactate, pyruvate, glucose, fructose, cellobiose and pectin. The bacterium reduced thiosulfate to sulfide. The G+C content of the DNA was 53 mol%. Comparison of 16S rRNA genes revealed that strain TMS is related to Moorella glycerini (96%, sequence similarity), Moorella thermoacetica (92%) and Moorella thermoautotrophica (92%). On the basis of physiological and phylogenetic differences, strain TMS is proposed as a new species within the genus Moorella, Moorella mulderi sp. nov. (=DSM 14980, =ATCC BAA-608).
Keywords: Moorella mulderi sp. nov. Thermophile Syntrophy Methanol oxidation Sporulation
Fermentation of glycolate by a pure culture of a strictly anaerobic gram-positive bacterium belonging to the family Lachnospiraceae by Peter H. Janssen; Philip Hugenholtz (pp. 321-328).
The component bacteria of a three-membered mixed culture able to ferment glycolate to acetate, propionate and CO2 were isolated in pure culture. All three strains were strict anaerobes that, on the basis of comparative 16S rRNA gene sequence analysis, belonged to the order Clostridiales in the phylum Firmicutes (low G+C gram-positive bacteria). Two of the strains were not involved in glycolate metabolism. The third, the glycolate-fermenting strain 19gly4 (DSM 11261), was related to members of the family Lachnospiraceae. The cells of strain 19gly4 were oval- to lemon-shaped, 0.85 µm long and 0.65 µm in diameter, occurring singly, in pairs, or in chains of up to 30 cells. Strain 19gly4 fermented glycolate or fumarate to acetate, succinate, and CO2. Hydrogen was not formed, and strain 19gly4 was able to grow on glycolate in pure culture without any syntrophic hydrogen transfer and without the use of an external electron acceptor. There was no evidence for homoacetogenic metabolism. This bacterium therefore differs in metabolism from previously reported glycolate-utilising anaerobes.
Keywords: Anaerobe Fermentation Glycolate
Isolation of mutants of Vibrio anguillarum defective in haeme utilisation and cloning of huvA, a gene coding for an outer membrane protein involved in the use of haeme as iron source by Ramón Mazoy; Carlos R. Osorio; Alicia E. Toranzo; Manuel L. Lemos (pp. 329-338).
The isolation of Vibrio anguillarum mutants lacking the ability to use haemin and haemoglobin as the only iron sources, as well as the identification of a gene involved in haeme utilisation are described. One of the isolated mutants defective in haeme utilisation lacked an iron-regulated outer membrane protein of 79-kDa. Although growth on haeme as iron source was completely abolished, the haemin and haemoglobin binding activities remained intact in the mutant, suggesting that the absent protein is not the only one involved in haeme binding. The wild-type phenotype in this mutant was restored by transformation with a cosmid clone (pML1) containing a 21-kb DNA fragment isolated from a gene library derived from the parental strain of V. anguillarum . Sequence analysis of pML1 subclones led to the finding of an ORF, huvA, that codes for a 79-kDa protein (HuvA) and whose sequence shows high identity with haeme receptors from Vibrio choleare (HutA) and Vibrio vulnificus (HupA). The sequence of huvA from the V. anguillarum haeme-utilisation mutant revealed a single mutation, leading to the synthesis of a truncated HuvA protein of 70 kDa. The parental strain and the cosmid-complemented mutant showed a higher degree of virulence for fish than the mutant strain in experimental infections in which fish were previously overloaded with haemin. This finding suggests that haeme uptake plays an important role in V. anguillarum multiplication in fish tissues when free haeme is available.
Keywords: Haeme utilisation Iron uptake Vibrio anguillarum
Exploring the Penicillium marneffei genome by Kwok-yung Yuen; Géraldine Pascal; Samson S. Y. Wong; Philippe Glaser; Patrick C. Y. Woo; Frank Kunst; James J. Cai; Elim Y. L. Cheung; Claudine Médigue; Antoine Danchin (pp. 339-353).
Penicillium marneffei is a dimorphic fungus that intracellularly infects the reticuloendothelial system of humans and bamboo rats. Endemic in Southeast Asia, it infects 10% of AIDS patients in this region. The absence of a sexual stage and the highly infectious nature of the mould-phase conidia have impaired studies on thermal dimorphic switching and host-microbe interactions. Genomic analysis, therefore, could provide crucial information. Pulsed-field gel electrophoresis of genomic DNA of P. marneffei revealed three or more chromosomes (5.0, 4.0, and 2.2 Mb). Telomeric fingerprinting revealed 6–12 bands, suggesting that there were chromosomes of similar sizes. The genome size of P. marneffei was hence about 17.8–26.2 Mb. G+C content of the genome is 48.8 mol%. Random exploration of the genome of P. marneffei yielded 2303 random sequence tags (RSTs), corresponding to 9% of the genome, with 11.7, 6.3, and 17.4% of the RSTs having sequence similarity to yeast-specific sequences, non-yeast fungus sequences, and both (common sequences), respectively. Analysis of the RSTs revealed genes for information transfer (ribosomal protein genes, tRNA synthetase subunits, translation initiation, and elongation factors), metabolism, and compartmentalization, including several multi-drug-resistance protein genes and homologues of fluconazole-resistance gene. Furthermore, the presence of genes encoding pheromone homologues and ankyrin repeat-containing proteins of other fungi and algae strongly suggests the presence of a sexual stage that presumably exists in the environment.
Keywords: Penicillium marneffei Genome Genomic analysis
Characterization of unusual hydroxy- and ketocarotenoids in Rubrivivax gelatinosus: involvement of enzyme CrtF or CrtA by Violaine Pinta; Soufian Ouchane; Martine Picaud; Shinichi Takaichi; Chantal Astier; Françoise Reiss-Husson (pp. 354-362).
Carotenoids are widely spread terpenoids found in photosynthetic organisms and a number of non-photosynthetic fungi and bacteria. The photosynthetic non-sulfur purple bacterium Rubrivivax gelatinosus produces carotenoids by both the spheroidene and the normal spirilloxanthin pathways. The characteristics of two carotenogenesis enzymes, spheroidene monooxygenase CrtA and O-methyltransferase CrtF, were investigated. Disruption of the corresponding genes by insertional mutagenesis affected carotenoid species in both pathways, and the genetic evidence indicated that both genes are involved in the two pathways. In these mutants, several unusual hydroxy- and ketocarotenoids were identified by spectroscopic and chemical methods. Moreover, the carotenoid analyses demonstrated that a large number of different carotenoid intermediates are accepted as substrates by the CrtA enzyme. The combined manipulation of crtF and crtA allowed new carotenoids to be produced and broadened the diversity of structurally different carotenoids synthesized by Rvi. gelatinosus. Methylated carotenoids, such as spheroidene and spirilloxanthin, are known to function as accessory pigments in the light-harvesting and reaction-center complexes of purple bacteria; the demethylated carotenoids described here were able to fulfill the same functions in the mutants.
Keywords: Ketocarotenoids Purple bacteria Rubrivivax Carotenoid genes Carotenoid biosynthesis crtA crtF
Activity of wild-type and hybrid Bacillus thuringiensis δ-endotoxins against Agrotis ipsilon by Ruud A. de Maagd; Mieke Weemen-Hendriks; Jos W. Molthoff; Samir Naimov (pp. 363-367).
Twelve Cry1 and two Cry9 δ-endotoxins from Bacillus thuringiensis were tested for their activity against black cutworm (Agrotis ipsilon). A. ipsilon was not susceptible to many toxins, but three toxins had significant activity. Cry9Ca was the most toxic, followed by Cry1Aa and Cry1Fb. Hybrids between these three active proteins were made by in vivo recombination and analyzed for activity against A. ipsilon. Analysis of hybrids between Cry1Aa and Cry1Fb indicated that domain I of Cry1Aa protein was involved in its higher activity.
Keywords: Bacillus thuringiensis δ-Endotoxin Hybrid protein Agrotis ipsilon
Rhodobacter sphaeroides has a family II pyrophosphatase: comparison with other species of photosynthetic bacteria by Heliodoro Celis; Bernardo Franco; Silvia Escobedo; Irma Romero (pp. 368-376).
The cytoplasmic pyrophosphatase from Rhodobacter sphaeroides was purified and characterized. The enzyme is a homodimer of 64 kDa. The N-terminus was sequenced and used to obtain the complete pyrophosphatase sequence from the preliminary genome sequence of Rba. sphaeroides, showing extensive sequence similarity to family II or class C pyrophosphatases. The enzyme hydrolyzes only Mg-PPi and Mn-PPi with a K m of 0.35 mM for both substrates. It is not activated by free Mg 2+, in contrast to the cytoplasmic pyrophosphatase from Rhodospirillum rubrum, and it is not inhibited by NaF, methylendiphosphate, or imidodiphosphate. This work shows that Rba. sphaeroides and Rhodobacter capsulatus cytoplasmic pyrophosphatases belong to family II, in contrast to Rsp. rubrum, Rhodopseudomonas palustris, Rhodopseudomonas gelatinosa, and Rhodomicrobium vannielii cytoplasmic pyrophosphatases which should be classified as members of family I. This is the first report of family II cytoplasmic pyrophosphatases in photosynthetic bacteria and in a gram-negative organism.
Keywords: Photosynthetic bacteria Rhodobacter sphaeroides Rhodospirillum rubrum Rhodobacter capsulatus Family II inorganic pyrophosphatase
Rhodospirillum rubrum has a family I pyrophosphatase: purification, cloning, and sequencing by Irma Romero; Rodolfo García-Contreras; Heliodoro Celis (pp. 377-380).
The cytoplasmic pyrophosphatase of the photosynthetic bacterium Rhodospirillum rubrum was purified to electrophoretic homogeneity. The enzyme is a homohexamer of 20-kDa monomers. The gene was cloned and sequenced. Alignment of the deduced 179-amino-acid protein with known bacterial pyrophosphatases revealed conservation of all residues in the active site. Attempts to obtain an insertion mutant of the cytoplasmic pyrophosphatase gene did not yield any cell completely devoid of cytoplasmic pyrophosphatase activity. The mutants obtained showed 50% of the enzymatic activity and grew in twice the generation time of wild-type cells. This suggests that the membrane-bound pyrophosphatase of Rsp. rubrum is not sufficient for a normal growth rate, whereas the cytoplasmic enzyme is essential for growth. The characteristics of the gene and the encoded protein fit those of prokaryotic family I pyrophosphatases.
Keywords: Rhodospirillum rubrum Family I pyrophosphatases Photosynthetic bacteria Inorganic pyrophosphatase gene
Characterisation of the Escherichia coli mfd promoter by Louise K. Stanley; Nigel J. Savery (pp. 381-385).
The bacterial mfd gene encodes a transcription-repair coupling factor that mediates the preferential repair of DNA damage in the template strand of active transcriptional units. In this report, the transcription start site for the Escherichia coli mfd gene was determined in vivo and in vitro, and the DNA determinants for mfd transcription by deletion and site-directed mutagenesis were defined. A canonical σ70-dependent promoter, mfd P1, was responsible for the majority of mfd transcription, and a core region consisting of residues −42 to +5 was sufficient for full activity in rich and minimal media.
Keywords: Escherichia coli Transcription initiation Promoter Transcription-coupled repair Mfd TRCF