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Archives of Microbiology (v.175, #2)
No Title by Jan Roelof van der Meer; Roald Ravatn; Vladimir Sentchilo (pp. 79-85).
Genes for metabolic pathways in bacteria that degrade aromatic or aliphatic pollutants have mostly been confined to either plasmid DNAs or to the chromosome. For a few pathways, including classical pathways for chlorocatechol and biphenyl degradation, recent evidence has been obtained for location of the pathway genes on mobile DNA elements which employ phage-like integrases. This enables the DNA elements to integrate into specific sites on the chromosome and yet to excise and transfer to other host bacteria. This mini-review gives an overview of those elements and their relationship to an increasing number of phage-like elements associated with bacterial virulence.
Keywords: clc Element Pseudomonas sp. strain B13 Chlorocatechol degradation Biphenyl degradation Gene island Phage-like integrase
No Title by Alicja Węgrzyn; Grzegorz Węgrzyn (pp. 86-93).
Early models of the regulation of initiation of DNA replication by protein complexes predicted that binding of a replication initiator protein to a replicator region is required for initiation of each DNA replication round, since after the initiation event the replication initiator should dissociate from DNA. It was, therefore, assumed that binding of the replication initiator is a signal for triggering DNA replication. However, more recent investigations have revealed that in many replicons this is not the case. Studies on the regulation of the replication of plasmids derived from bacteriophage λ demonstrated that, once assembled, the replication complex can be inherited by one of the two daughter plasmid copies after each replication round and may function in subsequent replication rounds. Since this DNA-bound protein complex bears information about specific initiation of DNA replication, this phenomenon has been called "protein inheritance." A similar phenomenon has recently been reported for oriJ-based plasmids. Moreover, the current model of the initiation of DNA replication in the yeast Saccharomyces cerevisiae proposes that the origin recognition complex (ORC) remains bound to one copy of the ori sequence (the ARS region) after initiation of DNA replication. Thus, it seems plausible that protein inheritance is not unique for λ plasmids, but may be a common phenomenon in the control of DNA replication, at least in microbes.
Keywords: Regulation of DNA replication Replication complex Transcriptional activation of origin Protein inheritance
No Title by Dimitry Y. Sorokin; J. Gijs Kuenen; Mike S.M. Jetten (pp. 94-101).
Thioalkalivibrio denitrificans is the first example of an alkaliphilic, obligately autotrophic, sulfur-oxidizing bacterium able to grow anaerobically by denitrification. It was isolated from a Kenyan soda lake with thiosulfate as electron donor and N2O as electron acceptor at pH 10. The bacterium can use nitrite and N2O, but not nitrate, as electron acceptors during anaerobic growth on reduced sulfur compounds. Nitrate is only utilized as nitrogen source. In batch culture at pH 10, rapid growth was observed on N2O as electron acceptor and thiosulfate as electron donor. Growth on nitrite was only possible after prolonged adaptation of the culture to increasing nitrite concentrations. In aerobic thiosulfate-limited chemostats, Thioalkalivibrio denitrificans strain ALJD was able to grow between pH values of 7.5 and 10.5 with an optimum at pH 9.0. Growth of the organism in continuous culture on N2O was more stable and faster than in aerobic cultures. The pH limit for growth on N2O was 10.6. In nitrite-limited chemostat culture, growth was possible on thiosulfate at pH 10. Despite the observed inhibition of N2O reduction by sulfide, the bacterium was able to grow in sulfide-limited continuous culture with N2O as electron acceptor at pH 10. The highest anaerobic growth rate with N2O in continuous culture at pH 10 was observed with polysulfide (S8 2–) as electron donor. Polysulfide was also the best substrate for oxygen-respiring cells. Washed cells at pH 10 oxidized polysulfide to sulfate via elemental sulfur in the presence of N2O or O2. In the absence of the electron acceptors, elemental sulfur was slowly reduced which resulted in regeneration of polysulfide. Cells of strain ALJD grown under anoxic conditions contained a soluble cd 1-like cytochrome and a cytochrome-aa 3-like component in the membranes.
Keywords: Thioalkalivibrio denitrificans Alkaliphilic Denitrification Nitrous oxide (N2O) Nitrite
No Title by Ulrike Kappler; Cornelius G. Friedrich; Hans G. Trüper; Christiane Dahl (pp. 102-111).
The pathway of thiosulfate oxidation in the facultatively chemolithotrophic, sulfur-oxidizing bacterium Starkeya novella (formerly Thiobacillus novellus) has not been established beyond doubt. Recently, isolation of the sorAB genes, which encode a soluble sulfite:cytochrome c oxidoreductase, has been reported, indicating that a thiosulfate-oxidizing pathway not involving a multienzyme complex may exist in this organism. Here we report the cloning and sequencing of the soxBCD genes from S. novella, which are closely related to the corresponding genes encoding the thiosulfate-oxidizing multienzyme complex from Paracoccus pantotrophus. These findings suggest two distinct pathways for thiosulfate oxidation in S. novella. The expression of sorAB and soxC in cells grown on thiosulfate- and/or glucose-containing media was studied by Western blot analysis. The results showed that the SorAB protein is synthesized in the presence of thiosulfate irrespective of the presence of glucose. In contrast, the SoxC protein is subject to repression by glucose; the repression, however, appears to be dependent on the relative amounts of glucose and thiosulfate present. The regulatory effects observed for the expression of sorAB are likely to be mediated by an extracytoplasmic function sigma factor encoded by the sigE gene identified upstream of sorAB.
Keywords: Thiosulfate oxidation pathway Thiobacillus novellus Starkeya novella Sulfite Sulfite oxidation Multienzyme complex Paracoccus pantotrophus Sigma factor ECF Thiosulfate
No Title by Markus Hartmann; Gabriele Heinrich; Gerhard H. Braus (pp. 112-121).
Two novel genes, aroF and aroG, from the filamentous fungus Aspergillus nidulans were isolated and the regulative fine-tuning between the encoded, differentially regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases was analyzed. A wide range of DAHP synthase isoenzymes of various organisms are known, but only a few have been characterized further. DAHP synthases (EC 4.1.2.15) catalyze the first committed step of the shikimate pathway, which is a putative target for anti-weed drugs. The reaction is the condensation of erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to yield DAHP. The two purified DAHP synthases showed different affinities for the substrates: 175 µM for PEP and 341 µM for E4P for the aroFp isoenzyme and weaker affinities of 239 µM (PEP) and 475 µM (E4P) for the aroGp isoenzyme. The enzymes are differentially regulated by tyrosine (aroFp) and phenylalanine (aroGp). The calculated k cat values are 7.0 s–1 for the tyrosine-inhibitable (aroFp) and 5.5 s–1 for the phenylalanine inhibitable (aroGp) enzyme. Tyrosine is a competitive inhibitor of the aroFp DAHP synthase in its reaction with PEP. Phenylalanine is a competitive inhibitor of the isoenzyme aroGp in its reaction with E4P. Both enzymes are inhibited by the chelating agent EDTA, which indicates a metal ion as cofactor.
Keywords: Aspergillus nidulans Protein purification Enzyme kinetics Regulation 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase
No Title by Gerhard Weidner; Stefan Steidl; Axel A. Brakhage (pp. 122-132).
In β-lactam-antibiotic-producing fungi, such as Aspergillus (Emericella) nidulans, L-α-aminoadipic acid is the branching point of the lysine and penicillin biosynthesis pathways. To obtain a deeper insight into the regulation of lysine biosynthesis genes, the regulation of the A. nidulans lysF gene, which encodes homoaconitase, was studied. Band-shift assays indicated that the A. nidulans multimeric CCAAT-binding complex AnCF binds to two of four CCAAT motifs present in the lysF promoter region. AnCF consists at least of three different subunits, designated HapB, HapC, and HapE. In both a ΔhapB and a ΔhapC strain, the expression of a translational lysF-lacZ gene fusion integrated in single copy at the chromosomal argB gene locus was two to three-fold higher than in a wild-type strain. These data show that AnCF negatively regulates lysF expression. The results of Northern blot analysis and lysF-lacZ expression analysis did not indicate a lysine-dependent repression of lysF expression. Furthermore, mutational analysis of the lysF promoter region revealed that two GATA sites matching the GATA consensus sequence HGATAR positively affected lysF-lacZ expression. Results of Northern blot analysis also excluded that the global nitrogen regulator AreA is the responsible trans-acting GATA-binding factor.
Keywords: Aspergillus nidulans Homoaconitase Lysine biosynthesis lysF CCAAT GATA
No Title by Antje Breitenstein; Aimo Saano; Mirja Salkinoja-Salonen; Jan R. Andreesen; Ute Lechner (pp. 133-142).
An anaerobic, 2,4,6-trichlorophenol ortho-dehalogenating mixed culture was enriched from sediment of the river Saale (Germany). Two isolated dechlorinating colonies (MK1 and MK2) consisted of rods of different lengths and thicknesses, indicating heterogeneity. Following subcultivation with thiosulfate as alternative electron acceptor and cocultivation with Clostridium celerecrescens T, the 2,4,6-trichlorophenol-dehalogenating bacterium Desulfitobacterium frappieri strain TCP-A was isolated and characterized regarding its taxonomic properties and the spectrum of chlorophenols that it dehalogenated. Four other bacterial strains were coenriched and identified as organisms with closest phylogenetic relatedness to the Clostridium type strains C. indolis, C. glycolicum, C. hydroxybenzoicum and C. sporosphaeroides (16S rDNA sequence identities of 99.5, 99.2, 94.4, and 93.5%, respectively). Amplified ribosomal DNA restriction analysis of the original dehalogenating cultures MK1 and MK2 (when not exposed to thiosulfate) confirmed the microbial heterogeneity and revealed the presence of two additional species related to the type strains of C. celerecrescens and Clostridium propionicum. Only one copy of the 16S rRNA genes of Desulfitobacterium frappieri in each of the clone libraries of MK1 and MK2 (containing 136 and 56 clones, respectively) was found by dot-blot hybridization, suggesting a relatively low number of the dehalogenating bacterium within the enrichment culture.
Keywords: Desulfitobacterium frappieri Clostridium hydroxybenzoicum Clostridium celerecrescens Reductive dechlorination 2,4,6-Trichlorophenol
No Title by Monika Janczarek; Anna Skorupska (pp. 143-151).
The pssB gene of Rhizobium leguminosarum bv. trifolii encodes a protein of 284 amino acids with sequence similarity to eukaryotic inositol monophosphatases. The gene was cloned and overexpressed in Escherichia coli. The purified gene product of pssB showed inositol monophosphatase activity with a K m of 0.23 mM, and a V max of 3.27 µmol Pi min–1 (mg protein)–1. Its substrate specificity, Mg+2 requirement, Li+ inhibition, and subunit association (dimerization) were studied and compared to those of other inositol monophosphatases. Western immunoblotting with anti-PssB antibodies showed the presence of PssB in R. leguminosarum bv. trifolii strain TA1 and lack of this protein in the pssB mutant strain Rt12A. The presence of PssB protein in R. leguminosarum bv. trifolii TA1 was correlated with phosphatase activity with myo-inositol 1-phosphate as a substrate. Evidence for a regulatory function of PssB protein in exopolysaccharide (EPS) synthesis is presented. The mutation in pssB caused EPS overproduction, and introduction of pssB into the wild-type TA1 strain reduced EPS synthesis. The changes in the level of EPS production were correlated with a non-nitrogen-fixing phenotype of rhizobia.
Keywords: Rhizobium leguminosarum bv. trifolii pssB gene Inositol monophosphatase EPS production
No Title by Robert J.H. Okker; Herman P. Spaink; Ben J.J. Lugtenberg; Helmi R.M. Schlaman (pp. 152-160).
The highly conserved nod box sequence in the promoters of the inducible nodulation genes of rhizobia is required for transcription activation together with NodD, a LysR-type transcriptional regulator, and a flavonoid as a coinducer. DNA fragments containing nod box sequences form two binding complexes when crude preparations of Rhizobium leguminosarum bv. viciae are used: a NodD-dependent and an additional, NodD-independent complex. The role of individual nucleotides in the conserved nod box sequence in complex formation and in nodulation gene expression was investigated by introducing 13 individual base-pair substitutions in the nodF nod box of R. leguminosarum bv. viciae and studying their effect on promoter activity and protein–DNA complex formation. Two mutants showed decreased NodD binding and decreased promoter activity. Five mutants showed a NodD-dependent complex as with the wild-type nodF nod box, whereas their promoter activity was severely reduced after induction. This result is in agreement with earlier observations that NodD DNA binding also occurs in the absence of inducer. Four mutants were impaired in the formation of the NodD-independent retardation complex. Three of them showed no alterations in promoter activity, meaning that no specific role for the protein forming the NodD-independent complex could be established. The two mutants in the highly conserved LysR motif of the nod box were unable to direct coinducer-dependent promoter activity but, unexpectedly, their retardation patterns were not altered. The remaining two mutants showed constitutive promoter activity. The results are discussed in terms of the relevance of conserved nucleotides and motifs identified in the nod box.
Keywords: Rhizobium nod Box nodD Nodulation Transcriptional regulation