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Archives of Microbiology (v.176, #1-2)
No Title by Jan R. van der Ploeg; Eric Eichhorn; Thomas Leisinger (pp. 1-8).
In the absence of sulfate and cysteine, Escherichia coli can use aliphatic sulfonates as a source of sulfur for growth. Starvation for sulfate leads to the expression of the tauABCD and ssuEADCB genes. Each of these gene clusters encodes an ABC-type transport system required for uptake of aliphatic sulfonates and a desulfonation enzyme. The TauD protein is an α-ketoglutarate-dependent dioxygenase that preferentially liberates sulfite from taurine (2-aminoethanesulfonic acid). SsuD is a monooxygenase that catalyzes the oxygenolytic desulfonation of a range of aliphatic sulfonates other than taurine. Its cosubstrate is FMNH2, which is provided by SsuE, an NAD(P)H-dependent FMN reductase. In contrast to many other bacteria, E. coli is unable to grow with arylsulfonates or with sulfate esters as sulfur source. The tau and ssu systems thus provide all genes for the utilization of known organosulfur sources by this organism, except the as yet unidentified gene(s) that enable some E. coli strains to grow with methanesulfonate or cysteate as a sulfur source. Expression of the tau and ssu genes requires the LysR-type transcriptional regulatory proteins CysB and Cbl. Synthesis of Cbl itself is under control of the CysB protein, and the CysB protein may therefore be regarded as the master regulator for sulfur assimilation in E. coli, while the Cbl protein functions as an accessory element specific for utilization of sulfur from organosulfur sources.
Keywords: Sulfonate Sulfate starvation Cysteine biosynthesis Escherichia coli Taurine Oxygenase ABC transporter Dioxygenase CysB Cbl
No Title by Hong Li; Debra M. Sherman; Shilai Bao; Louis A. Sherman (pp. 9-18).
The temporal and spatial accumulation of cyanophycin was studied in two unicellular strains of cyanobacteria, the diazotrophic Cyanothece sp. strain ATCC 51142 and the non-diazotrophic Synechocystis sp. strain PCC 6803. Biochemistry and electron microscopy were used to monitor the dynamics of cyanophycin accumulation under nitrogen-sufficient and nitrogen-deficient conditions. In Cyanothece sp. ATCC 51142 grown under 12 h light/12 h dark nitrogen-fixing conditions, cyanophycin was temporally regulated relative to nitrogenase activity and accumulated in granules after nitrogenase activity commenced. Cyanophycin granules reached a maximum after the peak of nitrogenase activity and eventually were utilized completely. Knock-out mutants were constructed in Synechocystis sp. PCC 6803 cphA and cphB genes to analyze the function of these genes and cyanophycin accumulation under nitrogen-deficient growth conditions. The mutants grew under such conditions, but needed to degrade phycobilisomes as a nitrogen reserve. Granules could be seen in some wild-type cells after treatment with chloramphenicol, but were never found in Δ cphA and Δ cphB mutants. These results led to the conclusion that cyanophycin is temporally and spatially regulated in nitrogen-fixing strains such as Cyanothece sp. ATCC 51142 and represents a key nitrogen reserve in these organisms. However, cyanophycin appeared to play a less important role in the non-diazotrophic unicellular strains and phycobilisomes appeared to be the main nitrogen reserve.
Keywords: Cyanobacteria Nitrogen fixation Cyanophycin Nitrogenase Synechocystis sp. strain PCC 6803 Cyanothece sp. strain ATCC 51142 Nitrogen metabolism Phycobilisomes
No Title by Lina De Smet; Vesna Kostanjevecki; Yves Guisez; Jozef Van Beeumen (pp. 19-28).
Flavocytochrome c-sulfide dehydrogenase (FCSD), an enzyme that catalyzes the reversible conversion of sulfide to elemental sulfur in vitro, is common to bacteria that utilize reduced sulfur compounds as electron donors in the process of carbon dioxide fixation. FCSD is a heterodimer containing two different cofactors, a flavin (FAD) and one or two heme c groups, located on the separate protein subunits. Efforts to produce the holoproteins of the soluble Allochromatium vinosum FCSD and the membrane-bound Ectothiorhodospira vacuolata protein in Escherichia coli using several expression systems were unsuccessful. Although all systems used were able to export the recombinant FCSDs to the periplasm, the proteins did not incorporate heme. In order to develop a new expression system involving photosynthetic hosts (Rhodobacter capsulatus, Rhodobacter sphaeroides and Ect. vacuolata), plasmid mobilisation from E. coli donors was studied. In the search for efficient promoters for such hosts, a system was developed combining the broad-host-range plasmid pGV910 and the promoter of the A. vinosum RuBisCo gene, rbcA. Conjugation was used to enable transfer from the expression plasmid of E. coli into Rba. capsulatus, Rba. sphaeroides strains and into Ect. vacuolata. Both Rhodobacter hosts were able to transcribe the genes coding for FCSD from the rbcA promoter and to produce detectable amounts of recombinant FCSD holoprotein. Western blots showed that the best production was obtained from cells grown photosynthetically on malate or acetate with sulfide. This system may prove to be of general use for the production of recombinant c-type cytochromes in homologous or related host systems.
Keywords: Flavocytochrome c Phototrophic sulfur bacteria Sulfide oxidation rbcA promoter Cytochrome c expression
No Title by Caroline M. Plugge; Johanna M. van Leeuwen; Tineke Hummelen; Melike Balk; Alfons J. Stams (pp. 29-36).
The glutamate catabolism of three thermophilic syntrophic anaerobes was compared based on the combined use of [13C] glutamate NMR measurements and enzyme activity determinations. In some cases the uptake of intermediates from different pathways was studied. The three organisms, Caloramator coolhaasii, Thermanaerovibrio acidaminovorans and strain TGO, had a different stoichiometry of glutamate conversion and were dependent on the presence of a hydrogen scavenger (Methanobacterium thermoautotrophicum Z245) to a different degree for their growth. C. coolhaasii formed acetate, CO2, NH4 + and H2 from glutamate. Acetate was found to be formed through the β-methylaspartate pathway in pure culture as well as in coculture. T. acidaminovorans converted glutamate to acetate, propionate, CO2, NH4 + and H2. Most likely, this organism uses the β-methylaspartate pathway for acetate formation. Propionate formation occurred through a direct oxidation of glutamate via succinyl-CoA and methylmalonyl-CoA. The metabolism of T. acidaminovorans shifted in favour of propionate formation when grown in coculture with the methanogen, but this did not lead to the use of a different glutamate degradation pathway. Strain TGO, an obligate syntrophic glutamate-degrading organism, formed propionate, traces of succinate, CO2, NH4 + and H2. Glutamate was converted to propionate oxidatively via the intermediates succinyl-CoA and methylmalonyl-CoA. A minor part of the succinyl-CoA was converted to succinate and excreted.
Keywords: Anaerobic metabolism Glutamate [13C] Nuclear magnetic resonance Syntrophic interactions Interspecies hydrogen transfer
No Title by Thomas B. Bair; Dale W. Isabelle; Lacy Daniels (pp. 37-43).
The structure of coenzyme F420 in Mycobacterium smegmatis was examined using proton NMR, amino acid analysis, and HPLC. The two major F420 structures were shown to be composed of a chromophore identical to that of F420 from Methanobacterium thermoautotrophicum, with a side chain of a ribityl residue, a lactyl residue and five or six glutamate groups (F420–5 and F420–6). Peptidase treatment studies suggested that L-glutamate groups are linked by γ-glutamyl bonds in the side chain. HPLC analysis indicated that Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium fortuitum have F420–5 and F420–6 as the predominant structures, whereas Mycobacterium avium contains F420–5, F420–6 and F420–7 in significant amounts. 7,8-Didemethyl 8-hydroxy 5-deazariboflavin (FO), an intermediate in F420 biosynthesis, accounted for about 1–7% of the total deazaflavin in cells. Peptidase treatment of F420 created F420 derivatives that may be useful for the assay of enzymes involved in F420 biosynthesis.
Keywords: Coenzyme F420 FO Mycobacterium Polyglutamate 5-deazaflavin Folate
No Title by Andreas Nocker; Nila-Pia Krstulovic; Xavier Perret; Franz Narberhaus (pp. 44-51).
Expression of at least ten genes in Bradyrhizobium japonicum, seven of which code for small heat shock proteins (sHsps), is under the control of ROSE (repression of heat shock gene expression). This negatively cis-acting DNA element confers temperature control to a σ70-type promoter. Here, we show that ROSE elements are not restricted to B. japonicum but are also present in Bradyrhizobium sp. (Parasponia), Rhizobium sp. strain NGR234 and Mesorhizobium loti. An overall alignment of all ROSE sequences reveals a highly conserved and probably functionally important region towards the 3′-end of the element. Moreover, we provide genetic evidence for the previously proposed presence of multiple sHsps in these organisms. Primer-extension data of five newly identified ROSE-associated operons show that transcription is repressed at low temperatures and induced after a temperature upshift. Translational ROSE-hsp′-′lacZ fusions of Bradyrhizobium sp. (Parasponia) and Rhizobium sp. strain NGR234 integrated into the chromosome of B. japonicum were heat-responsive. The functionality of these heterologous ROSE elements hints at a common regulatory principle conserved in various rhizobia.
Keywords: Bradyrhizobium japonicum Heat shock response Small heat-shock protein Regulation Rhizobia
No Title by Pascale Mosoni; Brigitte Gaillard-Martinie (pp. 52-61).
A spontaneous adhesion-defective mutant (mutant D5) of Ruminococcus albus strain 20 was isolated and compared to the parent to investigate the impact of the mutation on cellulolysis and to identify the adhesion mechanism of R. albus. The comparison of kinetics of cellulose degradation by strain 20 and mutant D5 showed that the mutation delayed and reduced bacterial growth on cellulose and cellulose degradation. These results were partly explained by a twofold lower cellulase activity in the mutant than in the parent. The glycocalyx of strain 20, observed by transmission electron microscopy, was large and homogenous, and linked cells to cellulose. The mutant glycocalyx was aggregated at its periphery and cells attached loosely to cellulose. A glycoprotein of 25 kDa (GP25), present in the membrane fraction and the extracellular medium of strain 20, was not detected in the same fractions of mutant D5. Though glycoprotein GP25 did not bind to cellulose, it may be involved in adhesion as an intermediate component. Different cell-surface features of mutant D5 (cellulases, glycoprotein GP25, glycocalyx) were thus affected, any or all of which may be involved in its adhesion-defective phenotype. These results suggest that adhesion and cellulolysis are linked and that adhesion is a multifactorial phenomenon that involves at least the extracellular glycocalyx.
Keywords: Ruminococcus albus Adhesion-defective mutant Glycocalyx Cellulase activity Cellulose-binding proteins Cellulosome-like structure
No Title by Grant Buchanan; Jochen Kuper; Ralf R. Mendel; Günter Schwarz; Tracy Palmer (pp. 62-68).
The mob genes of several bacteria have been implicated in the conversion of molybdopterin to molybdopterin guanine dinucleotide. The mob locus of Rhodobacter sphaeroides WS8 comprises three genes, mobABC. Chromosomal in-frame deletions in each of the mob genes have been constructed. The mobA mutant strain has inactive DMSO reductase and periplasmic nitrate reductase activities (both molybdopterin guanine dinucleotide-requiring enzymes), but the activity of xanthine dehydrogenase, a molybdopterin enzyme, is unaffected. The inability of a mobA mutant to synthesise molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms following iodine oxidation. Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumulate wild-type levels of molybdopterin and molybdopterin guanine dinucleotide, indicating they are not compromised in molybdenum cofactor synthesis. In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor insertion is not necessarily a pre-requisite for export.
Keywords: Rhodobacter sphaeroides Dimethylsulfoxide reductase Molybdenum cofactor Molybdopterin guanine dinucleotide Twin arginine translocation Protein FA MobA
No Title by Ho-Yong Sohn; Hiroshi Kuriyama (pp. 69-78).
We previously demonstrated that periodic H2S production during aerobic continuous culture of Saccharomyces cerevisiae resulted in ultradian respiratory oscillation, and that H2S production was dependent on the activity of sulfate uptake and the level of sulfite. To investigate the mechanism of regulation of the sulfate assimilation pathway and of respiratory oscillation, several amino acids were pulse-injected into cultures during respiratory oscillation. Injection of sulfur amino acids or their derivatives perturbed respiratory oscillation, with changes in the H2S production profile. Four major regulators of H2S production in the sulfate assimilation pathway and respiratory oscillation were identified: (1) O-acetylhomoserine, not O-acetylserine, as a sulfide acceptor, (2) homoserine/threonine as a regulator of O-acetylhomoserine supply, (3) methionine/S-adenosyl methionine as a negative regulator of sulfate assimilation, and (4) cysteine (or its derivatives) as an essential regulator. The results obtained after the addition of DL-propargylglycine (5 µM and 100 µM) and cystathionine (50 µM) suggested that the intracellular cysteine level and cystathionine γ-lyase, rather than methionine/S-adenosylmethionine, play an essential role in the regulation of sulfate assimilation and respiratory oscillation. Based on these results and those of our previous reports, we propose that periodic depletion of cysteine (or its derivatives), which is involved in the detoxification of toxic materials originating from respiration, causes periodic H2S production.
Keywords: Cysteine Hydrogen sulfide Sulfate assimilation pathway Saccharomyces cerevisiae Ultradian respiratory oscillation
No Title by Athina Amanatidou; Marjon H. Bennik; Leon G. Gorris; Eddy J. Smid (pp. 79-88).
In this study, the responses of two Lactobacillus sake strains to elevated oxygen concentrations at 8 °C were investigated. L. sake DSM 6333 (L. sake sens), unlike L. sake NCFB 2813 (L. sake ins), showed a low growth rate in the presence of 90% O2 and a rapid loss in viability shortly after entry into stationary phase. The steady-state cytosolic superoxide radical (O2 –) concentration in L. sake sens was 0.134 µM and in the oxygen-insensitive mutant LSUV4 it was 0.013 µM. The nine- to ten-fold decrease in the rate of O2 – elimination in L. sake sens indicates the significance of the O2 – -scavenging system in protecting against elevated O2. The superoxide dismutase (SOD) activity was 10- to 20-fold higher in L. sake ins than in L. sake sens, depending on the growth phase. An oxygen-insensitive mutant of L. sake sens, designated as strain LSUV4, had a ten-fold higher SOD activity than the wild-type strain, which likely restored its oxygen tolerance. Damage to proteins in L. sake sens was evidenced by the increased protein carbonyl content and reduced activities of the [Fe-S]-cluster-containing enzymes fumarase and fumarate reductase. This study forms a physiological basis for understanding the significance of elevated oxygen stress as an additional method for inhibition of microbial growth in relation to food preservation.
Keywords: Lactobacillus sake High oxygen concentration Superoxide radical NADH oxidase Superoxide dismutase Protein carbonyl content Oxidative stress
No Title by Karin Denger; Alasdair M. Cook (pp. 89-95).
Aerobic enrichment cultures (11) yielded three cultures able to utilise ethane-1,2-disulfonate as sole source of carbon and energy in salts medium. Two pure cultures were obtained and we worked with strain EDS1, which was assigned to the genus Ralstonia on the basis of its 16S rDNA sequence and simple taxonomic tests. Strain EDS1 utilised at least seven alkane(di)sulfonates, ethane-1,2-disulfonate, taurine, isethionate, sulfoacetate, sulfoacetaldehyde and propane-1,3-disulfonate, as well as methanesulfonate and formate. Growth with ethanedisulfonate was concomitant with substrate disappearance and the formation of 2 mol sulfate per mol substrate. The growth yield, 7 g protein (mol C)–1, indicated quantitative utilisation of the substrate. Ethanedisulfonate-dependent oxygen uptake of whole cells during growth rose to a maximum before the end of growth and then sank rapidly; this was interpreted as evidence for an inducible desulfonative oxygenase that was not active in cell extracts. Inducible sulfoacetaldehyde sulfo-lyase was detected at high activity. Inducible degradation of taurine or isethionate or sulfoacetate via sulfoacetaldehyde sulfo-lyase is interpreted from the data.
Keywords: Desulfonation Disulfonates Oxygenase Ralstonia Sulfoacetaldehyde sulfo-lyase
No Title by Harma T. Brondijk; Wil N. Konings; Bert Poolman (pp. 96-105).
Solute transport in Saccharomyces cerevisiae can be regulated through mechanisms such as trans-inhibition and/or catabolite inactivation by nitrogen or carbon sources. Studies in hybrid membranes of S. cerevisiae suggested that the maltose transport system Mal61p is fully reversible and capable of catalyzing both influx and efflux transport. This conclusion has now been confirmed by studies in a S. cerevisiae strain lacking the maltase enzyme. Whole cells of this strain, wherein the orientation of the maltose transporter is fully preserved, catalyze fully reversible maltose transport. Catabolite inactivation of the maltose transporter Mal61p was studied in the presence and absence of maltose metabolism and by the use of different glucose analogues. Catabolite inactivation of Mal61p could be triggered by maltose, provided the sugar was metabolized, and the rate of inactivation correlated with the rate of maltose influx. We also show that 2-deoxyglucose, unlike 6-deoxyglucose, can trigger catabolite inactivation of the maltose transporter. This suggests a role for early glycolytic intermediates in catabolite inactivation of the Mal61 protein. However, there was no correlation between intracellular glucose-6-phosphate or ATP levels and the rate of catabolite inactivation of Mal61p. On the basis of their identification in cell extracts, we speculate that (dideoxy)-trehalose and/or (deoxy)-trehalose-6-phosphate trigger catabolite inactivation of the maltose transporter.
Keywords: Membrane transport Post-translational regulation Trans-inhibition Yeast Catabolite inactivation
No Title by Ramón J. De Lucas; Ana I. Domínguez; Yolanda Higuero; Óscar Martínez; Beatriz Romero; Alfonso Mendoza; Francisco J. Garcia-Bustos; Fernando Laborda (pp. 106-113).
The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5′-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5′ and 3′ flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC + transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.
Keywords: Aspergillus fumigatus sC gene Transformation system Homologous transformation 2-way selection ATP sulfurylase Sulfate adenyltransferase Selenate resistance Pathogenic fungus Aspergillosis
No Title by Luis A. Sayavedra-Soto; Chelsea M. Byrd; Daniel J. Arp (pp. 114-120).
The induction of the enzyme activities involved in butane metabolism in Pseudomonas butanovora was characterized. P. butanovora was grown on butane or its metabolites, both singly and in mixtures with other growth substrates. Cells grown in each of the butane metabolites readily consumed the growth substrate and downstream metabolites, but consumed the upstream butane metabolites more slowly. Upstream activities in the butane metabolism could be induced by downstream metabolites, but to much lower levels than with the primary substrate. The induction of butane oxidation was not repressed when P. butanovora was grown or incubated in a mixture of butane and 1-butanol, butyraldehyde or butyrate. However, no induction of butane consumption was observed in a mixture of butane and lactate, which is indicative of catabolite repression. In lactate-grown cells that were rid of the growth substrate and incubated with butane and acetylene (to inactivate newly formed butane monooxygenase), the consumption of butane, 1-butanol and butyraldehyde consumption was not induced. The overall results suggest an independent regulatory mechanism for each of the enzyme activities in butane metabolism. In addition, a low, constitutive butane oxidation was observed in cells grown on substrates other than butane metabolites.
Keywords: Alkane metabolism Butane oxidation pathway Pseudomonas butanovora Catabolite repression
No Title by Shu-Fen Weng; Pan-Ming Tai; Cheng-Hwa Yang; Cheng-Der Wu; Wan-Ju Tsai; Juey-Wen Lin; Yi-Hsiung Tseng (pp. 121-128).
Sequencing of a 6.4-kb DNA fragment, cloned from the plant pathogenic bacterium Xanthomonas campestris pv. campestris 17 revealed five ORFs whose deduced amino acid sequences show strong similarities to the bacterial HrcA, GrpE, DnaK, DnaJ, and PdxK. The four heat shock genes are organized in the order hrcA-grpE-dnaK-dnaJ, a genome organization found in many gram-positive bacteria, but only in one gram-negative species (Xylella fastidiosa). These observations suggest that the HrcA-CIRCE system, comprising at least four genes arranged in this order, already existed for the regulation of stress responses before bacteria diverged into gram-negative and gram-positive groups. Primer-extension results suggested the presence of promoters at the regions upstream of grpE and dnaK. In the presence of stress, heat or ethanol (4%), the X. campestris pv. campestris 17 grpE and dnaK promoters were induced two- to three-fold over controls. Since the grpE and dnaK promoters possess E. coli σ32 promoter-like sequences, they are functional in E. coli, although at levels much lower than in X. campestris pv. campestris 17. Furthermore, expression of the X. campestris pv. campestris 17 dnaK promoter in E. coli was elevated by the cloned X. campestris σ32 gene, indicating that the cognate σ32 works more efficiently for the X. campestris promoters.
Keywords: Stress-responsive genes Promoter σ32 HrcA Evolution
No Title by Olaf Kniemeyer; Johann Heider (pp. 129-135).
The initial steps in the anaerobic oxidation of the aromatic hydrocarbon ethylbenzene by denitrifying bacteria are two sequential dehydrogenation reactions of ethylbenzene to (S)-1-phenylethanol and further to acetophenone. The enzyme catalysing the second oxidation step, (S)-1-phenylethanol dehydrogenase, was analysed in the denitrifying bacterium Azoarcus sp. strain EbN1. An NAD+-dependent 1-phenylethanol dehydrogenase for each of the enantiomers of 1-phenylethanol was identified in this bacterium; the two enzymes were induced under different growth conditions. (S)-1-phenylethanol dehydrogenase from ethylbenzene-grown cells was purified and biochemically characterised. The enzyme is a typical secondary alcohol dehydrogenase and consists of two subunits of 25.5 kDa. The enantioselective enzyme catalyses the oxidation of (S)-1-phenylethanol or the reduction of acetophenone and is inhibited by high concentrations of (R)-1-phenylethanol. The enzyme exhibits low apparent K m values for (S)-1-phenylethanol and acetophenone and is rather substrate-specific, using only a few chemically similar secondary alcohols, such as 1-phenylpropanol and isopropanol.
Keywords: Anaerobic catabolism Ethylbenzene 1-Phenylethanol Acetophenone Short-chain alcohol dehydrogenase
No Title by Socorro Mesa; Michael Göttfert; Eulogio J. Bedmar (pp. 136-142).
Cleavage of genomic DNA from Bradyrhizobium japonicum strain 3I1b110 by the restriction enzymes PmeI, PacI, and SwaI has been used together with pulsed-field gel electrophoresis and Southern hybridization to locate the nirK, norCBQD, and nosRZDFYLX denitrification genes on the chromosomal map of B. japonicum strain 110spc4. Mutant strains GRK13, GRC131, and GRZ25 were obtained by insertion of plasmid pUC4-KIXX-aphII-PSP, which carries recognition sites for the enzymes PacI, PmeI and SwaI, into the B. japonicum 3I1b110 nirK, norC and nosZ genes, respectively. Restriction of strain 3I1b110 genomic DNA with PacI, PmeI and SwaI yielded three, five and nine fragments, respectively. Pulsed-field gel electrophoresis of restricted mutant DNAs resulted in an altered fragment pattern that allowed determination of the position of the selected genes. Complementary mapping data were obtained by hybridization using digoxigenin-labeled B. japonicum 3I1b110 nirK, norBQD and nosZD as gene probes. The nirK, norCBQD and nosRZDFYLX genes were located close to the groEL 2 , cycH and cycVWX genes, respectively, on the strain 110spc4 genetic map. In contrast to other denitrifiers, B. japonicum 3I1b110 denitrification genes were dispersed over the entire chromosome.
Keywords: Hybridization nir nor nos genes Pulsed-field gel electrophoresis Genetic map Bradyhizobium japonicum
No Title by Zofia Tynecka; Izabela Korona-Głowniak; Renata Łoś (pp. 143-150).
2-[14C]oxoglutarate uptake in resting cells of Staphylococcus aureus 17810S occurs via two kinetically different systems: (1) a secondary, electrogenic 2-oxoglutarate:H+ symporter (K m=0.105 mM), energized by an electrochemical proton potential (ΔµH+) that is generated by the oxidation of endogenous amino acids and sensitive to ionophores, and (2) a ΔµH+-independent facilitated diffusion system (K m=1.31 mM). The 2-oxoglutarate transport system of S. aureus 17810S can be classified as a new member of the MHS (metabolite:H+ symporter) family. This transporter takes up various dicarboxylic acids in the order of affinity: succinate = malate > fumarate > 2-oxoglutarate > glutamate. Energy conservation with 2-oxoglutarate was studied in starved cells of strain 17810S. Initial transport of 2-oxoglutarate in these cells is energized by ΔµH+ generated via hydrolysis of residual ATP. Subsequent oxidation of the accumulated 2-oxoglutarate generates ΔµH+ for further, autoenergized transport of this 2-oxoacid and also for ΔµH+-linked resynthesis of ATP. In the cadmium-sensitive S. aureus 17810S, Cd2+ accumulation strongly inhibits energy conservation with 2-oxoglutarate at the level of ΔµH+ generation, without direct blocking of the 2-oxoglutarate transport system or ATP synthase complex. In the cadmium-resistant S. aureus 17810R, Cd2+ does not affect energy conservation due to its extrusion by the Cd2+ efflux system (Cd2+-ATPase of P-type), which prevents Cd2+ accumulation.
Keywords: 2-Oxoglutarate Transport Energetics Kinetics Specificity Starvation Autoenergization Energy conservation Cadmium Staphylococcus aureus
No Title by Miguel Alaminos; Juan L. Ramos (pp. 151-154).
Biosynthesis of methionine from homoserine in Pseudomonas putida takes place in three steps. The first step is the acylation of homoserine to yield an acyl-L-homoserine. This reaction is catalyzed by the products of the metXW genes and is equivalent to the first step in enterobacteria, gram-positive bacteria and fungi, except that in these microorganisms the reaction is catalyzed by a single polypeptide (the product of the metA gene in Escherichia coli and the met5 gene product in Neurospora crassa). In Pseudomonas putida, as in gram-positive bacteria and certain fungi, the second and third steps are a direct sulfhydrylation that converts the O-acyl-L-homoserine into homocysteine and further methylation to yield methionine. The latter reaction can be mediated by either of the two methionine synthetases present in the cells.
Keywords: Auxotrophic mutants Methionine biosynthesis Peudomonas putida met genes
No Title by Alexander Tkachenko; Larisa Nesterova; Michael Pshenichnov (pp. 155-157).
Putrescine up-regulated, in a concentration-dependent manner, the expression levels of the oxyR and katG genes of Escherichia coli cells exposed to hydrogen peroxide. Its stimulatory effect was more pronounced under conditions of strong oxidative stress. 1,4-Diamino-2-butanone, a specific inhibitor of putrescine synthesis, also inhibited oxyR expression under oxidative stress. When added to inhibited cells, putrescine relieved this inhibitory effect. Addition of putrescine to E. coli cultures exposed to oxidative stress led to increased cell survival.
Keywords: Polyamines Putrescine Oxidative stress oxyR katG Expression level Cell survival