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Archives of Microbiology (v.180, #3)
New ubiquitous translocators: amino acid export by Corynebacterium glutamicum and Escherichia coli by Lothar Eggeling; Hermann Sahm (pp. 155-160).
Molecular access to amino acid excretion by Corynebacterium glutamicum and Escherichia coli led to the identification of structurally novel carriers and novel carrier functions. The exporters LysE, RhtB, ThrE and BrnFE each represent the protoype of new transporter families, which are in part distributed throughout all of the kingdoms of life. LysE of C. glutamicum catalytes the export of basic amino acids. The expression of the carrier gene is regulated by the cell-internal concentration of basic amino acids. This serves, for example, to maintain homoeostasis if an excess of l-lysine or l-arginine inside the cell should arise during growth on complex media. RhtB is one of five paralogous systems in E. coli, of which at least two are relevant for l-threonine production. A third system is relevant for l-cysteine production. It is speculated that the physiological function of these paralogues is related to quorum sensing. ThrE of C. glutamicum exports l-threonine and l-serine. However, a ThrE domain with a putative hydrolytic function points to an as yet unknown role of this exporter. BrnFE in C. glutamicum is a two-component permease exporting branched-chained amino acids from the cell, and an orthologue in B. subtilis exports 4-azaleucine.
Keywords: Carrier; Exporter; Efflux; Topology; Metabolite control; Amino acids; l-Cysteine; l-Threonine; l-Lysine; Biotechnology
Genetic analysis of bacteriophage λN-dependent antitermination suggests a possible role for the RNA polymerase α subunit in facilitating specific functions of NusA and NusE by Agnieszka Szalewska-Pałasz; Barbara Strzelczyk; Anna Herman-Antosiewicz; Grzegorz Węgrzyn; Mark S. Thomas (pp. 161-168).
A role for the Escherichia coli RNA polymerase α subunit in transcription antitermination dependent on bacteriophage λ N protein has been previously inferred from the isolation of rpoA mutants that alter the efficiency of this process. This report describes studies on the efficiency of N-dependent transcription antitermination in a strain containing the rpoA341 mutation, which interferes with this process. The effect of mutations in genes coding for different Nus factors and/or plasmids overexpressing nus genes on bacteriophage λ development in an E. coli rpoA341 host was examined. In addition, the effect of overproduction of the N protein in these genetic backgrounds was assessed. Analogous bacterial strains were employed to measure the efficiency of the antitermination process using the lacZ reporter gene under control of the λ p R promoter, and containing the phage nutR region and the t R1 terminator between the promoter and lacZ. The experimental results suggest interactions between components of the N-antitermination complex, which have been established biochemically, as well as additional functional relationships within the complex. Furthermore, the results indicate that amino acid substitution in the α subunit C-terminal domain encoded by the rpoA341 mutation may specifically disrupt the function of the NusA and NusE proteins. During this analysis, it was also found that the E. coli nusA1 mutant exhibits a conditional lethal phenotype.
Keywords: Bacteriophage λ; Transcription antitermination; Nus factors; λ N protein; RNA polymerase α subunit
The putrescine analogue 1-aminooxy-3-aminopropane perturbs polyamine metabolism in the phytopathogenic fungus Sclerotinia sclerotiorum by Andrés Gárriz; María C. Dalmasso; Fernando L. Pieckenstain; Oscar A. Ruiz (pp. 169-175).
The effects of the putrescine analogue 1-aminooxy-3-aminopropane on fungal polyamine metabolism were evaluated using Sclerotinia sclerotiorum as an experimental model. The compound inhibited ornithine decarboxylase, spermidine synthase, and S -adenosyl-methionine decarboxylase in mycelial extracts. Addition of 1-aminooxy-3-aminopropane at 1 mM to the culture medium did not reduce mycelial growth and caused a 29% decrease in free spermidine and a two-fold increase in free spermine. When added 4.5 h before the determination of ornithine decarboxylase, 1-aminooxy-3-aminopropane reduced in vivo activity of this enzyme by 40–50%. When added 48 h before the determination, 1-aminooxy-3-aminopropane at 0.01 and 0.1 mM caused a slight increase of in vivo ornithine decarboxylase activity, while it had no effect at 1 mM. Comparison of the action of 1-aminooxy-3-aminopropane with that of other inhibitors of polyamine biosynthesis suggested that its effects on in vivo ornithine decarboxylase activity resulted from a balance between direct inhibition of enzyme activity and indirect stimulation of enzyme synthesis and/or activity mediated by the decrease in spermidine levels, which in turn was due to inhibition of spermidine synthase and S -adenosyl-methionine decarboxylase. The potential of 1-aminooxy-3-aminopropane as a tool for studies on fungal polyamine metabolism and for the control of plant diseases of fungal origin is discussed.
Keywords: S-Adenosyl-methionine decarboxylase; 1-Aminooxy-3-aminopropane; Ornithine decarboxylase; Polyamines; Sclerotinia sclerotiorum ; Spermidine synthase
Repressed multidrug resistance genes in Streptomyces lividans by Li-Fong Lee; Yi-Jiun Huang; Carton W. Chen (pp. 176-184).
Multidrug resistance (MDR) systems are ubiquitously present in prokaryotes and eukaryotes and defend both types of organisms against toxic compounds in the environment. Four families of MDR systems have been described, each family removing a broad spectrum of compounds by a specific membrane-bound active efflux pump. In the present study, at least four MDR systems were identified genetically in the soil bacterium Streptomyces lividans. The resistance genes of three of these systems were cloned and sequenced. Two of them are accompanied by a repressor gene. These MDR gene sequences are found in most other Streptomyces species investigated. Unlike the constitutively expressed MDR genes in Escherichia coli and other gram-negative bacteria, all of the Streptomyces genes were repressed under laboratory conditions, and resistance arose by mutations in the repressor genes.
Keywords: Multidrug resistance; Streptomyces ; Ethidium bromide; Gene expression; Efflux
Molecular cloning, expression and characterization of three distinctive genes encoding methionine aminopeptidases in cyanobacterium Synechocystis sp. strain PCC6803 by Anelia Atanassova; Mamoru Sugita; Masahiro Sugiura; Tamara Pajpanova; Ivan Ivanov (pp. 185-193).
Methionine aminopeptidase, known to be encoded by single genes in prokaryotes, is a cobalt-dependent enzyme that catalyzes the removal of N-terminal methionine residues from nascent polypeptides. Three ORFs encoding putative methionine aminopeptidases from the genome of cyanobacterium Synechocystis sp. strain PCC6803, designated as slr0786 (map-1), slr0918 (map-2) and sll0555 (map-3) were cloned and expressed in Escherichia coli. The purified recombinant proteins encoded by map-1 and map-3 had much higher methionine aminopeptidase activity than the recombinant protein encoded by map-2. Comparative analysis revealed that the three recombinant enzymes differed in their substrate specificity, divalent ion requirement, pH, and temperature optima. The broad activities of the iso-enzymes are discussed in light of the structural similarities with other peptidase families and their levels of specificity in the cell. Potential application of cyanobacterial MetAPs in the production of recombinant proteins used in medicine is proposed. This is the first report of a prokaryote harboring multiple methionine aminopeptidases.
Keywords: Cobalt-dependent enzyme; Cyanobacteria; Escherichia coli ; Iso-enzymes; Methionine aminopeptidase; Recombinant proteins
Physiological role of the F420-non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis by Alexander Stojanowic; Gerd J. Mander; Evert C. Duin; Reiner Hedderich (pp. 194-203).
F420-non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis is a [NiFe] hydrogenase composed of the three subunits MvhA, MvhG, and MvhD. Subunits MvhA and MvhG form the basic hydrogenase module conserved in all [NiFe] hydrogenases, whereas the 17-kDa MvhD subunit is unique to Mvh. The function of this extra subunit is completely unknown. In this work, the physiological function of this hydrogenase, and in particular the role of the MvhD subunit, is addressed. In cells of Mt. marburgensis from Ni2+-limited chemostat cultures the amount of Mvh decreased about 70-fold. However, the amounts of mvh transcripts did not decrease in these cells as shown by competitive RT-PCR, arguing against a regulation at the level of transcription. In cells grown in the presence of non-limiting amounts of Ni2+, Mvh was found in two chromatographically distinct forms—a free form and in a complex with heterodisulfide reductase. In cells from Ni2+-limited chemostat cultures, Mvh was only found in a complex with heterodisulfide reductase. The EPR spectrum of the purified enzyme reduced with sodium dithionite was dominated by a signal with g zyx =2.006, 1.936 and 1.912. The signal could be observed at temperatures up to 80 K without broadening, indicative of a [2Fe–2S] cluster. Subunit MvhD contains five cysteine residues that are conserved in MvhD homologues of other organisms. Four of these conserved cysteine residues can be assumed to coordinate the [2Fe–2S] cluster that was detected by EPR spectroscopy. The MvhG subunit contains 12 cysteine residues, which are known to ligate three [4Fe–4S] clusters. Data base searches revealed that in some organisms, including the Methanosarcina species and Archaeoglobus fulgidus, a homologue of mvhD is fused to the 3′ end of an hdrA homologue, which encodes a subunit of heterodisulfide reductase. These data allow the conclusion that the only function of Mvh is to provide reducing equivalents for heterodisulfide reductase and that the MvhD subunit is an electron transfer protein that forms the contact site to heterodisulfide reductase.
Keywords: [NiFe] hydrogenases; Methanothermobacter marburgensis ; Heterodisulfide reductase; Methanogenic archaea
Characterization of the Penicillium chrysogenum antifungal protein PAF by Lydia Kaiserer; Christoph Oberparleiter; Renate Weiler-Görz; Wolfgang Burgstaller; Eva Leiter; Florentine Marx (pp. 204-210).
The filamentous fungus Penicillium chrysogenum abundantly secretes the small, highly basic and cysteine-rich protein PAF (Penicillium antifungal protein). In this study, the antifungal activity of PAF is described. PAF inhibited the growth of a variety of filamentous fungi, including opportunistic human pathogenic and phytopathogenic fungi, whereas bacterial and yeast cells were unaffected. PAF reduced the conidial germination and hyphal extension rates in a dose-dependent manner and induced severe changes in cell morphology that resulted in crippled and distorted hyphae and atypical branching. Growth-affected hyphae suffered from oxidative stress, plasma membrane leakage, and metabolic inactivity, which points to an induction of multifactorial effects in sensitive fungi. In contrast to other known antifungal proteins, the effects of PAF were only partially antagonized by cations.
Keywords: Antifungal activity; Penicillium chrysogenum ; Morphology; Membrane leakage; Oxidative stress
In situ growth of the novel SM1 euryarchaeon from a string-of-pearls-like microbial community in its cold biotope, its physical separation and insights into its structure and physiology by Christine Moissl; Christian Rudolph; Reinhard Rachel; Marcus Koch; Robert Huber (pp. 211-217).
Recently, a unique archaeal/bacterial community that grows in a macroscopically visible string-of-pearls-like structure in cold (~10 °C), sulfurous marsh water was discovered. Here, a new technique is described that allows the fast and reliable growth of these string-of-pearls-like microbial communities in larger quantities on polyethylene nets in nature. The microbial net population, estimated to consist of about 10,000 single pearls, can be harvested once a week and the archaeal cells selectively separated by density gradient centrifugation. As in native pearls, the archaeal cell fraction obtained consisted of a single type of coccoid cells only, 0.6 µm in diameter. This novel type of euryarchaea has been tentatively named SM1 euryarchaeon. Electron microscopy and immuno-fluorescence in situ hybridization (immuno-FISH) revealed that about 100 pili-like fibers, up to 3 µm in length, emanate radially from the surface of each cell. The SM1 euryarchaeal cells exhibited a viability of about 90%. The optimal conditions for viability were temperatures between −2 °C and 20 °C, pH 5–9, and low salt conditions; cell viability was independent of oxygen partial pressures. The cultures stained gram-positive, the cell wall was sensitive to SDS, EDTA and Proteinase K treatment. The cells did not exhibit the typical fluorescence for methanogens and did not contain coenzyme F420. The G+C-content was 34.5 mol%.
Keywords: Archaea; Microbial community; Sulfurous spring; String-of-pearls; SM1 euryarchaeon; Psychrophilic; Genome; Immuno-FISH
Type III secretion of EspB in enterohemorrhagic Escherichia coli O157:H7 by Hao-Jie Chiu; Wen-Shiun Lin; Wan-Jr Syu (pp. 218-226).
EspB of enterohemorrhagic Escherichia coli O157:H7 is one of the type III proteins, categorized as translocators, that are secreted in abundance. To define the secretion determinants, different fragments of EspB were fused in recombinant proteins and the proteins secreted into media analyzed by Western blot. The results indicated that the C-terminal 30 residues of EspB were dispensable for secretion whereas the N-terminal first 117 residues played a major role. However, this N-terminal segment alone was not sufficient to confer the secretion. To acquire basic activity, the EspB fusion protein had to contain the N-terminal segment and another segment consisting of either residues 118–190 or residues 191–282. It is possible that the N-terminal region may act as the primary component of the secretion signal while other determinants help to maintain a conformation of EspB favorable for secretion. However, alternative mechanisms cannot be completely excluded. Not withstanding this, the signal for the type III secretion of EspB is apparently distinct from those previously described for the secretion of effector proteins such as Yops in Yersinia.
Keywords: EHEC; EspB; Escherichia coli ; Type III secretion system
Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi by Kozo Hirokawa; Keiko Gomi; Mikio Bakke; Naoki Kajiyama (pp. 227-231).
Our fungal culture collection was screened for fructosyl peptide oxidase, an enzyme that could be used for the determination of glycated hemoglobin in diabetic subjects with hyperglycemia. Fructosyl peptide oxidases were found in strains of eight genera: Achaetomiella, Achaetomium, Chaetomium, Coniochaeta, Eupenicillium, Gelasinospora, Microascus and Thielavia. By their substrate specificity toward N α -fructosyl valyl-histidine (α-keto-amine) and N ε -fructosyl lysine (ε-keto-amine), fructosyl peptide oxidases could be categorized into two groups: (1) enzymes that oxidize both α-keto-amine and ε-keto-amine, and (2) enzymes that preferably oxidize α-keto-amine. A fructosyl peptide oxidase from Achaetomiella virescens ATCC 32393, active toward both N α -fructosyl valyl-histidine and N ε -fructosyl lysine, was purified to homogeneity and characterized. The enzyme was monomeric (M r=50,000), was most active at 40 °C and pH 8.0, and had a covalently bound flavin as a prosthetic group. Apparent K m values for N α -fructosyl valyl-histidine and N ε -fructosyl lysine were 2.30 and 1.69 mM, respectively. N α -fructosyl valyl-histidine was consumed and the same molar amount of valyl-histidine was produced by the fructosyl peptide oxidase reaction. This enzyme could be useful for the measurement of hemoglobin A1C, the N-terminal valine residue of the β-subunit of which is glycated.
Keywords: Fructosyl peptide oxidase; Fructosyl amino acid oxidase; Hemoglobin A1C ; Achaetomiella virescens ATCC 32393; Glycated protein