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Archives of Microbiology (v.187, #1)
Distinct mechanisms regulate expression of the two major groEL homologues in Rhizobium leguminosarum by Phillip Gould; Maria Maguire; Peter A. Lund (pp. 1-14).
We investigated the regulation of the two of the three groE operons (cpn.1 and cpn.2) of the root-nodulating bacterium R. leguminosarum strain A34. Both are heat inducible, and both have a CIRCE sequence in their upstream regions, suggesting regulation by an HrcA repressor. Mutagenesis of the CIRCE sequence upstream of cpn.1 led to an increase in the levels of cpn.1 mRNA, and knock-out of the hrcA gene increased the level of Cpn60.1 protein (the GroEL homologue encoded by the cpn.1 operon). Inactivation of the hrcA gene also caused increased expression of a 29 kDa protein that was identified as RhiA, a component of a quorum-sensing system. However, neither loss of the upstream CIRCE sequence, nor loss of HrcA function, had any effect on expression from the cpn.2 promoter. Further analysis of the cpn.2 upstream region suggested regulation could be mediated by an RpoH system, and this was confirmed by deleting the rpoH gene from the chromosome, which led to a decreased level of Cpn60.2 expression. Inactivation of RpoH led to a reduction in growth rate which could be partly compensated for by inactivation of HrcA, indicating an overlap in the in vivo function of the proteins regulated by these two systems.
Keywords: Chaperonin; Rhizobium ; HrcA; Heat shock; RpoH
Sporotalea propionica gen. nov. sp. nov., a hydrogen-oxidizing, oxygen-reducing, propionigenic firmicute from the intestinal tract of a soil-feeding termite by Hamadi I. Boga; Rong Ji; Wolfgang Ludwig; Andreas Brune (pp. 15-27).
An unusual propionigenic bacterium was isolated from the intestinal tract of the soil-feeding termite Thoracotermes macrothorax. Strain TmPN3 is a motile, long rod that stains gram-positive, but reacts gram-negative in the KOH test. It forms terminal endospores and ferments lactate, glucose, lactose, fructose, and pyruvate to propionate and acetate via the methyl-malonyl-CoA pathway. Propionate and acetate are formed at a ratio of 2:1, typical of most propionigenic bacteria. Under a H2/CO2 atmosphere, the fermentation product pattern of glucose, fructose, and pyruvate shifts towards propionate formation at the expense of acetate. Cell suspensions reduce oxygen with lactate, glucose, glycerol, or hydrogen as electron donor. In the presence of oxygen, the product pattern of lactate fermentation shifts from propionate to acetate production. 16S rRNA gene sequence analysis showed that strain TmPN3 is a firmicute that clusters among the Acidaminococcaceae, a subgroup of the Clostridiales comprising obligately anaerobic, often endospore-forming bacteria that possess an outer membrane. Based on phenotypic differences and less than 92% sequence similarity to the 16S rRNA gene sequence of its closest relative, the termite hindgut isolate Acetonema longum, strain TmPN3T is proposed as the type species of a new genus, Sporotalea propionica gen. nov. sp. nov. (DSM 13327T, ATCC BAA-626T).
Keywords: Acidaminococcaceae; Propionate formation; Oxygen reduction; Hydrogen oxidation; Lactate fermentation; Termite gut
Peroxidase activity of selenoprotein GrdB of glycine reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum by Tina Gröbe; Michael Reuter; Torsten Gursinsky; Brigitte Söhling; Jan R. Andreesen (pp. 29-43).
The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific “protein B”-complex of glycine reductase. The “protein B”-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)−1 with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)−1 in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep-tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.
Keywords: Selenocysteine; Glycine reductase; Peroxidase activity; Anaerobes; Eubacterium acidaminophilum
Biotransformation of mercury in pH-stat cultures of eukaryotic freshwater algae by David J. A. Kelly; Kenneth Budd; Daniel D. Lefebvre (pp. 45-53).
Eukaryotic algae were studied to determine their ability to biotransform HgII under aerated and pH controlled conditions. All algae converted HgII into β-HgS and Hg0 to varying degrees. When HgII was administered as HgCl2 to the algae, biotransformation by species of Chlorophyceae (Selenastrum minutum and Chlorella fusca var. fusca) was initiated with β-HgS synthesis (K 1/2 of hours) and concomitant Hg° evolution occurred in the first hour. Hg° synthesis was impeded by the formation of β-HgS and this inhibition was released in C. fusca var. fusca when cellular thiols were oxidized by the addition of dimethylfumarate (DMF). The diatom, Navicula pelliculosa (Bacillariophyceae), converted a substantially greater proportion of the applied HgII into Hg0, whereas the thermophilic alga, Galdieria sulphuraria (Cyanidiophyceae), rapidly biotransformed as much as 90% of applied HgII into β-HgS (K 1/2 ≈ 20 min). This thermophile was also able to generate Hg0 even after all exogenously applied HgCl2 had been biotransformed. The results suggest that β-HgS may be the major dietary mercurial for grazers of contaminated eukaryotic algae.
Keywords: Eukaryotic algae; Metals; Mercury sulfide; Metacinnabar
Protein kinase C isoforms from Giardia duodenalis: identification and functional characterization of a β-like molecule during encystment by María Luisa Bazán-Tejeda; Raúl Argüello-García; Rosa María Bermúdez-Cruz; Martha Robles-Flores; Guadalupe Ortega-Pierres (pp. 55-66).
Protein kinase C (PKC) is a family of serine/threonine kinases that regulate many different cellular processes such as cell growth and differentiation in eukaryotic cells. Using specific polyclonal antibodies raised against mammalian PKC isoforms, it was demonstrated here for the first time that Giardia duodenalis expresses several PKC isoforms (beta, delta, epsilon, theta and zeta). All PKC isoforms detected showed changes in their expression pattern during encystment induction. In addition, selective PKC inhibitors blocked the encystment in a dose-dependent manner, suggesting that PKC isozymes may play important roles during this differentiation process. We have characterized here the only conventional-type PKC member found so far in Giardia, which showed an increased expression and changes in its intracellular localization pattern during cyst formation. The purified protein obtained by chromatography on DEAE-cellulose followed by size-exclusion chromatography, displayed in vitro kinase activity using histone HI-IIIS as substrate, which was dependent on cofactors required by conventional PKCs, i.e., phospholipids and calcium. An open reading frame in the Giardia Genome Database that encodes a homolog of PKCβ catalytic domain was identified and cloned. The expressed recombinant protein was also recognized by a mammalian anti-PKCβ antibody and was referred as giardial PKCβ on the basis of all these experimental evidence.
Keywords: Giardia; Protein kinase C; Encystment; Phosphorylation; Translocation; Lower eukaryotes; Protozoan differentiation
Multiple regulators of the Flavohaemoglobin (hmp) gene of Salmonella enterica serovar Typhimurium include RamA, a transcriptional regulator conferring the multidrug resistance phenotype by Elizabeth Hernández-Urzúa; David S. Zamorano-Sánchez; José Ponce-Coria; Enrique Morett; Susan Grogan; Robert K. Poole; Jorge Membrillo-Hernández (pp. 67-77).
Microbial flavohaemoglobins are proteins with homology to haemoglobins from higher organisms, but clearly linked to nitric oxide (NO) metabolism by bacteria and yeast. hmp mutant strains of several bacteria are hypersensitive to NO and related compounds and hmp genes are up-regulated by the presence of NO. The regulatory mechanisms involved in hmp induction by NO and the superoxide-generating agent, methyl viologen (paraquat; PQ), are complex, but progressively being resolved. Here we show for the first time that, in Salmonella enterica serovar Typhimurium, hmp transcription is increased on exposure to PQ and demonstrate that RamA, a homologue of MarA is responsible for most of the hmp paraquat regulation. In addition we demonstrate NO-dependent elevation of Salmonella hmp transcription and Hmp accumulation. In both Escherichia coli and Salmonella modest transcriptional repression of hmp is exerted by the iron responsive transcriptional repressor Fur. Finally, in contrast to previous reports, we show that in E. coli and Salmonella, hmp induction by both paraquat and sodium nitroprusside is further elevated in a fur mutant background, indicating that additional regulators are implicated in this control process.
Keywords: Flavohaemoglobin; Nitric oxide; Paraquat; Fur; Iron regulation; RamA
Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti by Bacem Mnasri; Moncef Mrabet; Gisèle Laguerre; Mohamed Elarbi Aouani; Ridha Mhamdi (pp. 79-85).
Nodulation of common bean was explored in six oases in the south of Tunisia. Nineteen isolates were characterized by PCR–RFLP of 16S rDNA. Three species of rhizobia were identified, Rhizobium etli, Rhizobium gallicum and Sinorhizobium meliloti. The diversity of the symbiotic genes was then assessed by PCR–RFLP of nodC and nifH genes. The majority of the symbiotic genotypes were conserved between oases and other soils of the north of the country. Sinorhizobia isolated from bean were then compared with isolates from Medicago truncatula plants grown in the oases soils. All the nodC types except for nodC type p that was specific to common bean isolates were shared by both hosts. The four isolates with nodC type p induced N2-fixing effective nodules on common bean but did not nodulate M. truncatula and Medicago sativa. The phylogenetic analysis of nifH and nodC genes showed that these isolates carry symbiotic genes different from those previously characterized among Medicago and bean symbionts, but closely related to those of S. fredii Spanish and Tunisian isolates effective in symbiosis with common bean but unable to nodulate soybean. The creation of a novel biovar shared by S. meliloti and S. fredii, bv. mediterranense, was proposed.
Keywords: Biovar mediterranense ; Genetic diversity; Medicago ; Phaseolus vulgaris ; Rhizobia; Salt tolerance