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Archives of Microbiology (v.185, #5)

Metabolism of lactose by Clostridium thermolacticum growing in continuous culture by Christophe Collet; Laurence Girbal; Paul Péringer; Jean-Paul Schwitzguébel; Philippe Soucaille (pp. 331-339).

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l⁻¹) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h⁻¹. Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h⁻¹. The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD⁺, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H₂ removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H₂ scavenging and acetate producing microorganisms.

Keywords: Clostridium thermolacticum ; Lactose metabolism; Acetate production; Continuous culture; Enzyme activities; Intracellular cofactors

DNA replication defect in the Escherichia coli cgtA(ts) mutant arising from reduced DnaA levels by Aleksandra E. Sikora; Ryszard Zielke; Alicja Węgrzyn; Grzegorz Węgrzyn (pp. 340-347).

In Escherichia coli and other bacteria, the ribosome-associated CgtA GTP-binding protein plays a critical role in many basic cellular processes, including the control of DNA replication and/or segregation. However, the mechanism of this control is largely unknown. Here we report that ectopic expression of the dnaA gene partially restored both early growth in liquid medium and DNA synthesis defects of the cgtA(ts) mutant. Amounts of DnaA protein in the cgtA(ts) mutant incubated at elevated (42°C) temperature were significantly lower relative to wild-type bacteria. Both level of dnaA mRNA and transcriptional activity of the dnaA promoter-lacZ fusion were decreased in the CgtA-deficient cells. The effects of ectopic expression of dnaA were specific as analogous expression of another gene coding for a replication regulator, seqA, had no significant changes in growth and DNA synthesis in the cgtA mutant. Thus, it appears that the DNA replication defect in this mutant is a consequence of reduced DnaA levels.

Keywords: Obg proteins; CgtA protein; GTP-binding proteins; DNA replication; DnaA initiator protein; SeqA protein

Nitric oxide biosynthesis by Leishmania amazonensis promastigotes containing a high percentage of metacyclic forms by Marcelo Genestra; Wilson J. S. Souza; Damiana Guedes-Silva; Gérzia M. C. Machado; Léa Cysne-Finkelstein; Rômulo José Soares Bezerra; Fabiane Monteiro; Leonor L. Leon (pp. 348-354).

Due to the diversity of its physiological and pathophysiological functions and general ubiquity, the study of nitric oxide (NO) has become of great interest. In this work, it was demonstrated that Leishmania amazonensis promastigotes produces NO, a free radical synthesized from l-arginine by nitric oxide synthase (NOS). A soluble NOS was purified from L. amazonensis promastigotes by affinity chromatography (2′, 5′-ADP-agarose) and on SDS-PAGE the enzyme migrates as a single protein band of 116.2 (±6) kDa. Furthermore, the presence of a constitutive NOS was detected through indirect immunofluorescence using anti-cNOS and in NADPH consumption assays. The present work show that NO production, detected as nitrite in culture supernatant, is prominent in promastigotes preparations with high number of metacyclic forms, suggesting an association with the differentiation and the infectivity of the parasite.

Keywords: Leishmania amazonensis ; Nitric oxide; Nitric oxide synthase; Promastigotes; Metacyclogenesis

Characterization of an ecto-ATPase activity in Fonsecaea pedrosoi by Ítalo Collopy-Junior; Lucimar F. Kneipp; Fernanda C. da Silva; Marcio L. Rodrigues; Celuta S. Alviano; José Roberto Meyer-Fernandes (pp. 355-362).

In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5′-triphosphate (ATP) at a rate of 84.6 ± 11.3 nmol Pi h⁻¹ mg⁻¹ mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 ± 27.6 nmol Pi h⁻¹ mg⁻¹) in the presence of 5 mM MgCl₂, with values of V _max and apparent K _m for Mg-ATP²⁻corresponding to 541.9 ± 48.6 nmol Pi h⁻¹ mg⁻¹ cellular dry weight and 1.9 ± 0.2 mM, respectively. The Mg²⁺-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A₁(V-ATPases)_,and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg²⁺-stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4′-diisothiocyanostylbene-2,2′-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P₂purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.

Keywords: Fonsecaea pedrosoi ; Chromoblastomycosis; Ecto-ATPase activity; E-Type ATPase; Ecto-nucleotidase

Chlorobium chlorochromatii sp. nov., a symbiotic green sulfur bacterium isolated from the phototrophic consortium “Chlorochromatium aggregatum” by Kajetan Vogl; Jens Glaeser; Kristina R. Pfannes; Gerhard Wanner; Jörg Overmann (pp. 363-372).

A symbiotic green sulfur bacterium, strain CaD, was isolated from an enrichment culture of the phototrophic consortium “Chlorochromatium aggregatum”. The capability of the epibiont to grow in pure culture indicates that it is not obligately symbiotic. Cells are Gram-negative, nonmotile, rod-shaped and contain chlorosomes. Strain CaD is obligately anaerobic and photolithoautotrophic, using sulfide as electron donor. Acetate and peptone are photoassimilated in the presence of sulfide and hydrogencarbonate. Photosynthetic pigments contain bacteriochlorophylls a and c, and γ-carotene and OH-γ-carotene glucoside laurate as the dominant carotenoids. In cells from pure cultures, chlorosomes are equally distributed along the inner face of the cytoplasmic membrane. In contrast, the distribution of the chlorosomes in symbiotic epibiont cells is uneven, with chlorosomes being entirely absent at the site of attachment to the central bacterium. The symbiotic epibiont cells display a conspicuous additional layered structure at the attachment site. The G + C content of genomic DNA of strain CaD is 46.7 mol%. On the basis of 16S rRNA sequence comparison, the strain is distantly related to Chlorobium species within the green sulfur bacteria phylum (≤94.6% sequence homology). The novel isolate is therefore described as a novel species within the genus Chlorobium, Chlorobium chlorochromatii.

Keywords: Chlorobiaceae; Chlorobium ; Symbiosis; Phototrophic consortia; Carotenoids; “Chlorochromatium aggregatum”

Indole-3-acetic acid improves Escherichia coli’s defences to stress by C. Bianco; E. Imperlini; R. Calogero; B. Senatore; A. Amoresano; A. Carpentieri; P. Pucci; R. Defez (pp. 373-382).

Indole-3-acetic acid (IAA) is a ubiquitous molecule playing regulatory roles in many living organisms. To elucidate the physiological changes induced by IAA treatment, we used Escherichia coli K-12 as a model system. By microarray analysis we found that 16 genes showed an altered expression level in IAA-treated cells. One-third of these genes encode cell envelope components, or proteins involved in bacterial adaptation to unfavourable environmental conditions. We thus investigated the effect of IAA treatment on some of the structural components of the envelope that may be involved in cellular response to stresses. This showed that IAA-treated cells had increased the production of trehalose, lipopolysaccharide (LPS), exopolysaccharide (EPS) and biofilm. We demonstrated further that IAA triggers an increased tolerance to several stress conditions (heat and cold shock, UV-irradiation, osmotic and acid shock and oxidative stress) and different toxic compounds (antibiotics, detergents and dyes) and this correlates with higher levels of the heat shock protein DnaK. We suggest that IAA triggers an increased level of alert and protection against external adverse conditions by coordinately enhancing different cellular defence systems.

Keywords: Trehalose; DnaK; LPS; EPS; Biofilm; Stress response

Francisella sp. (Family Francisellaceae) causing mortality in Norwegian cod (Gadus morhua) farming by Are Nylund; Karl F. Ottem; Kuninori Watanabe; Egil Karlsbakk; Bjørn Krossøy (pp. 383-392).

In 2004, a new disease was detected in cod (Gadus morhua) in western Norway. Affected cod had white granulomas in the visceral organs and skin. A species of Francisella was isolated on blood agar plates from moribund cod. The bacterium could be grown at temperatures ranging from 6 to 22°C, but did not grow at 37°C. Challenge experiments showed that Francisella sp. was the cause for the new disease. The 16S rDNA gene sequence from Francisella sp. showed 99.17% similarity to F. philomiragia, and the 16S–23S ribosomal RNA intergenic spacer (249 nt), shows a similarity with that from Francisella isolated from tilapia and F. tularensis of 96.8 and 35.9%, respectively. The 23S sequence is more similar to F. tularensis, 97.7% (2,862 nt), compared to the tilapia isolate 96.8% (2,131 nt). The partial putative outer membrane protein (FopA) sequence (781 nt) from Francisella sp. shows a similarity with that from F. tularensis and F. philomiragia of 77.3 and 98.2%, respectively. Based on sequence data, culturing temperatures and pathogenicity for cod, it is suggested that this Francisella sp. from cod could be a new species of Francisella, Family Francisellaceae.

Keywords: Cod; Gadus morhua ; Francisella ; rDNA; FopA; Phylogeny

The complete mitochondrial genome of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae: gene order and trn gene clusters reveal a common evolutionary course for all Sordariomycetes, while intergenic regions show variation by Dimitri V. Ghikas; Vassili N. Kouvelis; Milton A. Typas (pp. 393-401).

The mitochondrial genome (mtDNA) of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, with a total size of 24,673 bp, was one of the smallest known mtDNAs of Pezizomycotina. It contained the 14 typical genes coding for proteins related to oxidative phosphorylation, the two rRNA genes, a single intron that harbored an intronic ORF coding for a putative ribosomal protein (rps) within the large rRNA gene (rnl), and a set of 24 tRNA genes which recognized codons for all amino acids, except proline and valine. Gene order comparison with all known mtDNAs of Sordariomycetes illustrated a highly conserved genome organization for all the protein- and rRNA-coding genes, as well as three clusters of tRNA genes. By considering all mitochondrial essential protein-coding genes as one unit a phylogenetic study of these small genomes strongly supported the common evolutionary course of Sordariomycetes (100% bootstrap support) and highlighted the advantages of analyzing small genomes (mtDNA) over single genes. In addition, comparative analysis of three intergenic regions demonstrated sequence variability that can be exploited for intra- and inter-specific identification of Metarhizium.

Keywords: Mitochondrial genome; Gene order; trn gene cluster; Metarhizium anisopliae ; Phylogenetic relationships; Intergenic regions; Identification

Inducible transcription of genes involved in taurine uptake and dissimilation by Silicibacter pomeroyi DSS-3^T by Andzelika K. Gorzynska; Karin Denger; Alasdair M. Cook; Theo H. M. Smits (pp. 402-406).

A largely untested hypothesis for the bacterial dissimilation of taurine was explored in Silicibacter pomeroyi DSS-3, whose genome has been sequenced. Substrate-specific transcription of candidate genes encoding taurine uptake and dissimilation (tauABC, tpa, ald, xsc, pta) was found, which corresponded to the induction of Tpa, Ald, Xsc and Pta, that was observed.

Keywords: Reverse transcriptase PCR; Taurine dissimilatory pathway; Enzymes of taurine degradation

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Archives of Microbiology (v.185, #5)

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Archives of Microbiology (v.185, #5)