Showing papers in "Archives of Microbiology in 2003"
TL;DR: This survey shows that laccase-like enzymes occur in many gram-negative and gram-positive bacteria and databank searches and experimental data now provide evidence for their distribution in prokaryotes.
Abstract: Laccases are copper-containing proteins that require O(2) to oxidize phenols, polyphenols, aromatic amines, and different non-phenolic substrates by one-electron transfer, resulting in the formation of reactive radicals. Although their specific physiological functions are not completely understood, there are several indications that laccases are involved in the morphogenesis of microorganisms (e.g., fungal spore development, melanization) and in the formation and/or degradation of complex organic substances such as lignin or humic matter. Owing to their high relative non-specific oxidation capacity, laccases are useful biocatalysts for diverse biotechnological applications. To date, laccases have been found only in eukaryotes (fungi, plants); however, databank searches and experimental data now provide evidence for their distribution in prokaryotes. This survey shows that laccase-like enzymes occur in many gram-negative and gram-positive bacteria. Corresponding genes have been found in prokaryotes that are thought to have branched off early during evolution, e.g., the extremely thermophilic Aquifex aeolicus and the archaeon Pyrobaculum aerophilum. Phylogenetically, the enzymes are members of the multi-copper protein family that have developed from small-sized prokaryotic azurins to eukaryotic plasma proteins.
443 citations
TL;DR: A novel feather-degrading microorganism was isolated from poultry waste, producing a high keratinolytic activity when cultured on broth containing native feather, and complete feather degradation was achieved during cultivation.
Abstract: A novel feather-degrading microorganism was isolated from poultry waste, producing a high keratinolytic activity when cultured on broth containing native feather. Complete feather degradation was achieved during cultivation. The bacterium presents potential use for biotechnological processes involving keratin hydrolysis. Chryseobacterium sp. strain kr6 was identified based on morphological and biochemical tests and 16S rRNA sequencing. The bacterium presented optimum growth at pH 8.0 and 30 degrees C; under these conditions, maximum feather-degrading activity was also achieved. Maximum keratinase production was reached at 25 degrees C, while concentration of soluble protein was similar at both 25 and 30 degrees C. Reduction of disulfide bridges was also observed, increasing with cultivation time. The keratinase of strain kr6 was active on azokeratin and azocasein as substrates, and presented optimum pH and temperature of 7.5 and 55 degrees C, respectively. The keratinase activity was inhibited by 1,10-phenanthroline, EDTA, Hg(2+), and Cu(2+) and stimulated by Ca(2+).
280 citations
TL;DR: PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix and allowed the genus of the toxin producer to be identified.
Abstract: Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.
263 citations
TL;DR: In addition to the risk for infection, the allergenic and toxigenic properties, as well as the inflammatory effects are discussed in this review as possible health impacts of bioaerosols.
Abstract: Airborne fungal contaminants are increasingly gaining importance in view of health hazards caused by the spores themselves or by microbial metabolites. In addition to the risk for infection, the allergenic and toxigenic properties, as well as the inflammatory effects are discussed in this review as possible health impacts of bioaerosols. A major problem is the lack of threshold values for pathogenic and non-pathogenic fungi, both in the workplace and in outdoor air. While the relevance of mycotoxins has been intensely studied in connection with contamination of food and feed, the possible respiratory uptake of mycotoxins from the air has so far not been sufficiently taken into account. Toxic secondary metabolites are expected to be present in airborne spores, and may thus occur in airborne dust and bioaerosols. Potential health risks cannot be estimated reliably unless exposure to mycotoxins is determined qualitatively and quantitatively. Microbial volatile organic compounds (MVOC) have been suggested to affect human health, causing lethargy, headache, and irritation of the eyes and mucous membranes of the nose and throat. The production of MVOC by fungi has been discussed in connection with domestic indoor microbial pollution, but the relevance of fungal metabolites in working environments remains insufficiently studied.
234 citations
TL;DR: It is concluded that Crenarchaeota exhibit a mosaic of three or possibly four autotrophic pathways, supporting their role in CO2 fixation and the distribution of the pathways so far correlates with the 16S-rRNA-based taxa of the Cren Archaeota.
Abstract: Representative autotrophic and thermophilic archaeal species of different families of Crenarchaeota were examined for key enzymes of the known autotrophic CO(2) fixation pathways. Pyrobaculum islandicum ( Thermoproteaceae) contained key enzymes of the reductive citric acid cycle. This finding is consistent with the operation of this pathway in the related Thermoproteus neutrophilus. Pyrodictium abyssi and Pyrodictium occultum ( Pyrodictiaceae) contained ribulose 1,5-bisphosphate carboxylase, which was active in boiling water. Yet, phosphoribulokinase activity was not detectable. Operation of the Calvin cycle remains to be demonstrated. Ignicoccus islandicus and Ignicoccus pacificus ( Desulfurococcaceae) contained pyruvate oxidoreductase as potential carboxylating enzyme, but apparently lacked key enzymes of known pathways; their mode of autotrophic CO(2) fixation is at issue. Metallosphaera sedula, Acidianus ambivalens and Sulfolobus sp. strain VE6 ( Sulfolobaceae) contained key enzymes of a 3-hydroxypropionate cycle. This finding is in line with the demonstration of acetyl-coenzyme A (CoA) and propionyl-CoA carboxylase activities in the related Acidianus brierleyi and Sulfolobus metallicus. Enzymes of central carbon metabolism in Metallosphaera sedula were studied in more detail. Enzyme activities of the 3-hydroxypropionate cycle were strongly up-regulated during autotrophic growth, supporting their role in CO(2) fixation. However, formation of acetyl-CoA from succinyl-CoA could not be demonstrated, suggesting a modified pathway of acetyl-CoA regeneration. We conclude that Crenarchaeota exhibit a mosaic of three or possibly four autotrophic pathways. The distribution of the pathways so far correlates with the 16S-rRNA-based taxa of the Crenarchaeota.
177 citations
TL;DR: Seven strains of marine aerobic anoxygenic phototrophs belonging to the genus Erythrobacter were isolated and required an organic carbon substrate for growth; however, they are able to supplement a significant fraction of their metabolic requirements with photosynthetically derived energy.
Abstract: Seven strains of marine aerobic anoxygenic phototrophs belonging to the genus Erythrobacter were isolated. The strains were characterized regarding their physiological and biochemical properties, 16S rDNA and pufM gene sequences, morphological features, substrate preference, as well as pigment and lipid composition. All strains had functional type-2 reaction centers containing bacteriochlorophyll, served by small, light-harvesting complex 1, and were photosynthetically competent. In addition, large pools of carotenoids were found, but only some of the accessory pigments transfer energy to the reaction centers. All of the isolates were facultative photoheterotrophs. They required an organic carbon substrate for growth; however, they are able to supplement a significant fraction of their metabolic requirements with photosynthetically derived energy.
158 citations
TL;DR: These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.
Abstract: The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min−1mg protein-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.
151 citations
TL;DR: The survival of Kluyveromyces thermotolerans and Torulaspora delbrueckii in mixed cultures with Saccharomyces cerevisiae was examined at low oxygen availability in a defined grape juice medium and the early deaths seemed to be mediated by a cell-to-cell contact mechanism at high cell densities of S. Cerevisiae.
Abstract: The survival of Kluyveromyces thermotolerans and Torulaspora delbrueckii in mixed cultures with Saccharomyces cerevisiae was examined at low oxygen availability in a defined grape juice medium. In these fermentations, K. thermotolerans and T. delbrueckii died off earlier than S. cerevisiae, and K. thermotolerans and T. delbrueckii exhibited parabolic death kinetics. Furthermore, the early deaths seemed to be non-apoptotic in nature. In order to understand the mechanism causing the early deaths, various single- and mixed-culture fermentations were carried out. The early deaths could not be explained by nutrient depletion or the presence of toxic compounds. Rather, they seemed to be mediated by a cell-to-cell contact mechanism at high cell densities of S. cerevisiae, and to a lesser ability of K. thermotolerans and T. delbrueckii to compete for space, as compared to S. cerevisiae. These results contribute to an increased understanding of why K. thermotolerans and T. delbrueckii die off before S. cerevisiae in wine fermentations.
135 citations
TL;DR: A genetic system for Magnetospirillumgryphiswaldense includes a plating technique that allows the screening of magnetic vs non-magnetic colonies, and a protocol for the transfer of foreign DNA by electroporation and high-frequency conjugation.
Abstract: Genetic analysis of bacterial magnetosome biomineralization has been hindered by the lack of an appropriate methodology for cultivation and genetic manipulation of most magnetotactic bacteria. In this report, a genetic system for Magnetospirillum gryphiswaldense is described. The system includes a plating technique that allows the screening of magnetic vs non-magnetic colonies, and a protocol for the transfer of foreign DNA by electroporation and high-frequency conjugation. Various broad-host-range vectors of the IncQ, IncP, and pBBR1 groups were found to be capable of replication in M. gryphiswaldense. Several antibiotic resistance markers that can be expressed in M. gryphiswaldense were identified. Tn5 transposons delivered on a suicide plasmid showed transpositional insertion into random chromosomal sites.
130 citations
TL;DR: PAF inhibited the growth of a variety of filamentous fungi, including opportunistic human pathogenic and phytopathogenic fungi, whereas bacterial and yeast cells were unaffected, and points to an induction of multifactorial effects in sensitive fungi.
Abstract: 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.
117 citations
TL;DR: Moderately thermophilic acidophilic bacteria were isolated from geothermal (30–83 °C) acidic (pH 2.7–3.7) sites in Yellowstone National Park, and eight of the bacteria were able to catalyze the dissimilatory oxidation of ferrous iron, and eleven could reduce ferric iron to ferrousIron in anaerobic cultures.
Abstract: Moderately thermophilic acidophilic bacteria were isolated from geothermal (30–83 °C) acidic (pH 2.7–3.7) sites in Yellowstone National Park. The temperature maxima and pH minima of the isolates ranged from 50 to 65 °C, and pH 1.0–1.9. Eight of the bacteria were able to catalyze the dissimilatory oxidation of ferrous iron, and eleven could reduce ferric iron to ferrous iron in anaerobic cultures. Several of the isolates could also oxidize tetrathionate. Six of the iron-oxidizing isolates, and one obligate heterotroph, were low G+C gram-positive bacteria (Firmicutes). The former included three Sulfobacillus-like isolates (two closely related to a previously isolated Yellowstone strain, and the third to a mesophilic bacterium isolated from Montserrat), while the other three appeared to belong to a different genus. The other two iron-oxidizers were an Actinobacterium (related to Acidimicrobium ferrooxidans) and a Methylobacterium-like isolate (a genus within the α-Proteobacteria that has not previously been found to contain either iron-oxidizers or acidophiles). The other three (heterotrophic) isolates were also α-Proteobacteria and appeared be a novel thermophilic Acidisphaera sp. An ARDREA protocol was developed to discriminate between the iron-oxidizing isolates. Digestion of amplified rRNA genes with two restriction enzymes (SnaBI and BsaAI) separated these bacteria into five distinct groups; this result was confirmed by analysis of sequenced rRNA genes.
TL;DR: 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.
Abstract: 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.
TL;DR: This is the first report describing reductive dechlorination of hexachlorobenzene and pentachlorabenzene via dehalorespiration by a pure bacterial culture and indicates different reductiveDehalogenases might be induced by the different electron acceptors.
Abstract: The chlororespiring anaerobe Dehalococcoides sp. strain CBDB1 used hexachlorobenzene and pentachlorobenzene as electron acceptors in an energy-conserving process with hydrogen as electron donor. Previous attempts to grow Dehalococcoides sp. strain CBDB1 with hexachlorobenzene or pentachlorobenzene as electron acceptors failed if these compounds were provided as solutions in hexadecane. However, Dehalococcoides sp. strain CBDB1 was able to grow with hexachlorobenzene or pentachlorobenzene when added in crystalline form directly to cultures. Growth of Dehalococcoides sp. strain CBDB1 by dehalorespiration resulted in a growth yield ( Y) of 2.1+/-0.24 g protein/mol Cl(-) released with hexachlorobenzene as electron acceptor; with pentachlorobenzene, the growth yield was 2.9+/-0.15 g/mol Cl(-). Hexachlorobenzene was reductively dechlorinated to pentachlorobenzene, which was converted to a mixture of 1,2,3,5- and 1,2,4,5-tetrachlorobenzene. Formation of 1,2,3,4-tetrachlorobenzene was not detected. The final end-products of hexachlorobenzene and pentachlorobenzene dechlorination were 1,3,5-trichlorobenzene, 1,3- and 1,4-dichlorobenzene, which were formed in a ratio of about 3:2:5. As reported previously, Dehalococcoides sp. strain CBDB1 converted 1,2,3,5-tetrachlorobenzene exclusively to 1,3,5-trichlorobenzene, and 1,2,4,5-tetrachlorobenzene exclusively to 1,2,4-trichlorobenzene. The organism therefore catalyzes two different pathways to dechlorinate highly chlorinated benzenes. In the route leading to 1,3,5-trichlorobenzene, only doubly flanked chlorine substituents were removed, while in the route leading to 1,3-and 1,4-dichlorobenzene via 1,2,4-trichlorobenzene singly flanked chlorine substituents were also removed. Reductive dehalogenase activity measurements using whole cells pregrown with different chlorobenzene congeners as electron acceptors indicated that different reductive dehalogenases might be induced by the different electron acceptors. To our knowledge, this is the first report describing reductive dechlorination of hexachlorobenzene and pentachlorobenzene via dehalorespiration by a pure bacterial culture.
TL;DR: This first global analysis of the early transcriptional response of exponentially growing Pseudomonas aeruginosa to iron permitted identification of 107 Pa genes whose mRNA levels were not previously known to be affected by iron.
Abstract: To successfully infect humans, Pseudomonas aeruginosa (Pa) must overcome the low iron availability in host tissues. A transcriptome comparison was carried out between iron-starved cells of Pa treated with iron and untreated controls. The present study is the first global analysis of the early transcriptional response of exponentially growing Pa to iron. Approximately 1.3% of the Pa genes displayed > or = 5.0-fold changes in mRNA levels in iron-treated cells. Treatment affected the mRNA levels of many genes required for iron acquisition as well as several genes with relevance to virulence previously known to be regulated by iron. More importantly, the analysis permitted identification of 107 Pa genes whose mRNA levels were not previously known to be affected by iron. These genes are good candidates for mutagenesis studies aimed at identifying novel functions relevant to iron metabolism in Pa. Some of these genes encode predicted siderophore receptors, iron transport systems, TonB-dependent receptors, regulatory proteins, and proteins relevant to virulence. Notably, 49 genes encode hypothetical or conserved hypothetical proteins of unknown function, suggesting that they are involved directly or indirectly in iron metabolism or metabolic adaptation to different iron-availability conditions.
TL;DR: The PduP protein is a polyhedral-body-associated CoA-acylating aldehyde dehydrogenase important for 1,2-propanediol degradation by S. enterica, consistent with the proposal that these structures function to minimize propionaldehyde toxicity during the growth of S. entersica.
Abstract: Salmonella enterica forms polyhedral bodies involved in coenzyme-B12-dependent 1,2-propanediol degradation. Prior studies showed that these bodies consist of a proteinaceous shell partly composed of the PduA protein, coenzyme-B12-dependent diol dehydratase, and additional unidentified proteins. In this report, we show that the PduP protein is a polyhedral-body-associated CoA-acylating aldehyde dehydrogenase important for 1,2-propanediol degradation by S. enterica. A PCR-based method was used to construct a precise nonpolar deletion of the gene pduP. The resulting pduP deletion strain grew poorly on 1,2-propanediol minimal medium and expressed 105-fold less propionaldehyde dehydrogenase activity (0.011 micromol min(-1) mg(-1)) than did wild-type S. enterica grown under similar conditions (1.15 micromol min(-1) mg(-1)). An Escherichia coli strain was constructed for high-level production of His8-PduP, which was purified by nickel-affinity chromatography and shown to have 15.2 micromol min(-1) mg(-1) propionaldehyde dehydrogenase activity. Analysis of assay mixtures by reverse-phase HPLC and mass spectrometry established that propionyl-CoA was the product of the PduP reaction. For subcellular localization, purified His8-PduP was used as antigen for the preparation of polyclonal antiserum. The antiserum obtained was shown to have high specificity for the PduP protein and was used in immunogold electron microscopy studies, which indicated that PduP was associated with the polyhedral bodies involved in 1,2-propanediol degradation. Further evidence for the localization of the PduP enzyme was obtained by showing that propionaldehyde dehydrogenase activity co-purified with the polyhedral bodies. The fact that both Ado-B12-dependent diol dehydratase and propionaldehyde dehydrogenase are associated with the polyhedral bodies is consistent with the proposal that these structures function to minimize propionaldehyde toxicity during the growth of S. enterica on 1,2-propanediol.
TL;DR: The overall results suggest that isiA/isiAB as well as idiB transcription in response to oxidative stress might be controlled by a transcriptional repressor possibly of the PerR-type, which explains the observed cross-talk between IdiB- and Fur-mediated transcriptional regulation of gene expression.
Abstract: In the mesophilic cyanobacterium Synechococcus elongatus PCC 7942, iron starvation induces the expression of a number of proteins, including IdiA and IsiA. Whereas IdiA protects photosystem (PS) II under mild iron limitation against oxidative stress in a yet unknown way, prolonged iron starvation leads to the formation of the PS I–IsiA supercomplex. Transcription of idiA is positively regulated by IdiB under iron starvation, and Fur represses transcription of isiAB under iron-sufficient growth conditions. In this report, data are presented suggesting a strong interrelationship between iron homeostasis and oxidative stress in S. elongatus PCC 7942, and showing that transcription of major iron-regulated genes, such as isiA, isiAB, idiA, idiB, mapA, and irpA, is induced by oxidative stress within a few minutes by treatment of cells with hydrogen peroxide or methylviologen. The overall results suggest that isiA/isiAB as well as idiB transcription in response to oxidative stress might be controlled by a transcriptional repressor possibly of the PerR-type. This fact also explains the observed cross-talk between IdiB- and Fur-mediated transcriptional regulation of gene expression and for the role of H2O2 as a superior trigger coordinating expression of iron-regulated genes under iron starvation and oxidative stress. Measuring 77 K chlorophyll a fluorescence, it is shown that hydrogen peroxide treatment causes a transient short-term modification of PS II and PS I most likely leading to increased cyclic electron transport around PS I. In this context, the intriguing observation was made that idiB is transcribed as part of an operon together with a gene encoding a potential [2Fe–2S]-protein. This protein has similarity to [Fe–S]-proteins involved in the electron transport activity of the NDH I complex in eubacteria. Since the NDH I complex is involved in cyclic electron transport activity around PS I in cyanobacteria and both adaptation to iron starvation and adaptation to oxidative stress lead to an enhanced cyclic electron transport activity around PS I, this potential [Fe–S]-protein might participate in the overall adaptational response to iron starvation and/or oxidative stress in Synechococcus.
TL;DR: A comparative discussion of the underlying mechanisms of DNA transfer in mesophilic and extremely thermophilic bacteria is placed on, highlighting conserved and distinctive features of these transformation machineries.
Abstract: Horizontal gene flow is a driving force for bacterial adaptation. Among the three distinct mechanisms of gene transfer in bacteria, conjugation, transduction, and transformation, the latter, which includes competence induction, DNA binding, and DNA uptake, is perhaps the most versatile mechanism and allows the incorporation of free DNA from diverse bacterial species. Here we review DNA transport machineries mediating uptake of naked DNA in gram-positive and gram-negative bacteria. Different putative models of transformation machineries comprising components similar to proteins of type IV pili are presented. Emphasis is placed on a comparative discussion of the underlying mechanisms of DNA transfer in mesophilic and extremely thermophilic bacteria, highlighting conserved and distinctive features of these transformation machineries.
TL;DR: Fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum, which is shown to be tetrameric and inhibited by the monovalent cation Li+ and AMP and phosphoenolpyruvate.
Abstract: The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K
m value of about 14 µM and a V
max of about 5.4 µmol min−1 mg−1 and k
cat of about 3.2 s−1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg2+ or Mn2+ and was inhibited by the monovalent cation Li+ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTΔfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.
TL;DR: For the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either β- or ε-end groups and the results indicate that both enzymes might have originated from duplication of a single gene.
Abstract: Chlorophyll-b-possessing cyanobacteria of the genus Prochlorococcus share the presence of high amounts of α- and β-carotenoids with green algae and higher plants. The branch point in carotenoid biosynthesis is the cyclization of lycopene, for which in higher plants two distinct enzymes are required, e- and β-lycopene cyclase. All cyanobacteria studied so far possess a single β-cyclase. Here, two different Prochlorococcus sp. MED4 genes were functionally identified by heterologous gene complementation in Escherichia coli to encode lycopene cyclases. Whereas one is both functionally and in sequence highly similar to the β-cyclase of Synechococcus sp. strain PCC 7942 and other cyanobacteria, the other showed several intriguing features. It acts as a bifunctional enzyme catalyzing the formation of e- as well as of β-ionone end groups. Expression of this cyclase in E. coli resulted in the simultaneous accumulation of α- β-, δ-, and e-carotene. Such an activity is in contrast to all lycopene e-cyclases known so far, including those of the higher plants. Thus, for the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either β- or e-end groups. These two genes are suggested to be designated as crtL-b and crtL-e. The results indicate that both enzymes might have originated from duplication of a single gene. Consequently, we suggest that multiple gene duplications followed by functional diversification resulted several times, and in independent lineages, in the appearance of enzymes for the biosynthesis of cyclic carotenoids.
TL;DR: A gene cluster involved in the utilization of tetrahydrofuran by Pseudonocardia sp.
Abstract: A gene cluster involved in the utilization of tetrahydrofuran by Pseudonocardia sp. strain K1 was cloned and sequenced. Analysis of a 9.2-kb DNA fragment revealed eight ORFs. The genes designated as thmADBC encode the components of a putative monooxygenase exhibiting a high similarity to different binuclear-iron-containing multicomponent monooxygenases. thmA encodes the derived 545-amino-acid oxygenase α-subunit, thmD the 360-amino-acid reductase component, thmB the 346-amino-acid oxygenase β-subunit, and thmC the 117-amino-acid coupling protein. Upstream of the thm genes, an additional ORF (sad) was identified coding for a protein with high similarity to various aldehyde dehydrogenases. A succinate semialdehyde dehydrogenase activity was specifically expressed in tetrahydrofuran-grown cells. N-terminal sequence analysis of the purified protein revealed that it is encoded by sad. Northern blot analysis indicated that transcription of the thm genes and sad was specifically induced during growth on tetrahydrofuran. Mono-, di- and polycistronic transcripts of these genes were detected. Primer-extension analysis identified transcriptional start sites 37, 61, and 41 bp upstream of the translation start of sad, thmA, and thmB, respectively. Additional ORFs were identified upstream (orfY) and downstream (orfZ and aldH) of the thm genes. Furthermore, the data indicated that the analyzed gene cluster was present as a single copy and located on a plasmid.
TL;DR: The hypothesis that the microflora of the gastrointestinal tract of an individual influences the particular isoflavone metabolites produced following consumption is corroborated.
Abstract: Isoflavonoids found in legumes, such as soybeans, are converted by intestinal bacteria to metabolites that might have increased or decreased estrogenic activity. Variation in the effects of dietary isoflavonoids among individuals has been attributed to differences in their metabolism by intestinal bacteria. To investigate this variation, the metabolism of the isoflavonoid daidzein by bacteria from ten fecal samples, provided at different times by six individuals on soy-containing diets, was compared. After anaerobic incubation of bacteria with daidzein for 2 weeks, four samples had metabolized daidzein and six samples had not. Three of the positive samples were from individuals whose microflora had not metabolized daidzein in previous samples. Dihydrodaidzein was observed in one sample, dihydrodaidzein and equol in another sample, and equol and O-desmethylangolensin in two other samples. These results corroborate the hypothesis that the microflora of the gastrointestinal tract of an individual influences the particular isoflavone metabolites produced following consumption.
TL;DR: The physiological function of F420-non-reducing hydrogenase (Mvh) is addressed and it is concluded that the only function of Mvh is to provide reducing equivalents for heterodisulfide reductase and that the M vhD subunit is an electron transfer protein that forms the contact site to heterod isulfide reducesase.
Abstract: 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.
TL;DR: The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectromaetry (ESI MS/MS).
Abstract: The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.
TL;DR: Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, andHymA and HymB, identified after determination of their N-terminal sequences, which might represent the most simple type of a formate hydrogen lyase.
Abstract: Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.
TL;DR: The presence of the putative esterase domain makes it highly possible that cop1, cmt1, and cmt2 encode enzymes synthesizing TDCM from trehalose monocorynomycolate.
Abstract: By data mining in the sequence of the Corynebacterium glutamicum ATCC 13032 genome, six putative mycolyltransferase genes were identified that code for proteins with similarity to the N-terminal domain of the mycolic acid transferase PS1 of the related C glutamicum strain ATCC 17965 The genes identified were designated cop1, cmt1, cmt2, cmt3, cmt4, and cmt5 (cmt from corynebacterium mycolyltransferases) cop1 encodes a protein of 657 amino acids, which is larger than the proteins encoded by the cmt genes with 365, 341, 483, 483, and 411 amino acids Using bioinformatics tools, it was shown that all six gene products are equipped with signal peptides and esterase domains Proteome analyses of the cell envelope of C glutamicum ATCC 13032 resulted in identification of the proteins Cop1, Cmt1, Cmt2, and Cmt4 All six mycolyltransferase genes were used for mutational analysis cmt4 could not be mutated and is considered to be essential cop1 was found to play an additional role in cell shape formation A triple mutant carrying mutations in cop1, cmt1, and cmt2 aggregated when cultivated in MM1 liquid medium This mutant was also no longer able to synthesize trehalose dicorynomycolate (TDCM) Since single and double mutants of the genes cop1, cmt1, and cmt2 could form TDCM, it is concluded that the three genes, cop1, cmt1, and cmt2, are involved in TDCM biosynthesis The presence of the putative esterase domain makes it highly possible that cop1, cmt1, and cmt2 encode enzymes synthesizing TDCM from trehalose monocorynomycolate
TL;DR: A molecular phylogeny of the paralogous cofG and cofH genes is consistent with the genes being vertically inherited within the euryarchaeal, cyanobacterial, and actinomycetal lineages.
Abstract: The hydride carrier coenzyme F420 contains the unusual chromophore 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Microbes that generate F420 produce this FO moiety using a pyrimidine intermediate from riboflavin biosynthesis and the 4-hydroxyphenylpyruvate precursor of tyrosine. The fbiC gene, cloned from Mycobacterium smegmatis, encodes the bifunctional FO synthase. Expression of this protein in Escherichia coli caused the host cells to produce FO during growth, and activated cell-free extracts catalyze FO biosynthesis in vitro. FO synthase in the methanogenic euryarchaeon Methanocaldococcus jannaschii comprises two proteins encoded by cofG (MJ0446) and cofH (MJ1431). Both subunits were required for FO biosynthesis in vivo and in vitro. Cyanobacterial genomes encode homologs of both genes, which are used to produce the coenzyme for FO-dependent DNA photolyases. A molecular phylogeny of the paralogous cofG and cofH genes is consistent with the genes being vertically inherited within the euryarchaeal, cyanobacterial, and actinomycetal lineages. Ancestors of the cyanobacteria and actinomycetes must have acquired the two genes, which subsequently fused in actinomycetes. Both CofG and CofH have putative radical S-adenosylmethionine binding motifs, and pre-incubation with S-adenosylmethionine, Fe2+, sulfide, and dithionite stimulates FO production. Therefore a radical reaction mechanism is proposed for the biosynthesis of FO.
TL;DR: Recent results show that secretins form a large complex in the outer membrane, which constitutes the actual translocation channel, which could open up new strategies to target molecular machineries at the heart of many important virulence factors.
Abstract: Pseudomonas aeruginosa produces a large number of exoproteins, ranging from the ADP-ribosyltransferases exotoxin A and ExoS to degradative enzymes, such as elastase and chitinase. As it is a gram-negative bacterium, P. aeruginosa must be able to transport these exoproteins across both membranes of the cell envelope. In addition, also proteins that are part of cellular appendages, such as type IV pili and flagella, have to cross the cell envelope. Whereas the majority of the proteins transported across the inner membrane are dependent on the Sec channel, the systems for translocation across the outer membrane seem to be more diverse. Gram-negative bacteria have invented a number of different strategies during the course of evolution to achieve this goal. Although these transport machineries seem to be radically different, many of them actually depend on a member of the secretin protein family for their function. Recent results show that secretins form a large complex in the outer membrane, which constitutes the actual translocation channel. Understanding the working mechanism of this protein translocation channel could open up new strategies to target molecular machineries at the heart of many important virulence factors.
TL;DR: An ABC-type transporter in Escherichia coli that transports both l- and d-methionine, but not other natural amino acids, was identified and was designated the methionine uptake transporter (MUT) family, the first functionally characterized member of a novel family of bacterial permeases within the ABC superfamily.
Abstract: An ABC-type transporter in Escherichia coli that transports both L- and D-methionine, but not other natural amino acids, was identified. This system is the first functionally characterized member of a novel family of bacterial permeases within the ABC superfamily. This family was designated the methionine uptake transporter (MUT) family (TC #3.A.1.23). The proteins that comprise the transporters of this family were analyzed phylogenetically, revealing the probable existence of several sequence-divergent primordial paralogues, no more than two of which have been transmitted to any currently sequenced organism. In addition, MetJ, the pleiotropic methionine repressor protein, was shown to negatively control expression of the operon encoding the ABC-type methionine uptake system. The identification of MetJ binding sites (in gram-negative bacteria) or S-boxes (in gram-positive bacteria) in the promoter regions of several MUT transporter-encoding operons suggests that many MUT family members transport organic sulfur compounds.
TL;DR: The glutathione-mediated pathway for the detoxification of endogenously and exogenously derived toxic compounds was investigated using a pigment that accumulates in certain adenine biosynthetic mutants of yeasts to obtain insight regarding new genes involved in the three phases of this detoxification pathway.
Abstract: The glutathione-mediated pathway for the detoxification of endogenously and exogenously derived toxic compounds was investigated using a pigment that accumulates in certain adenine biosynthetic mutants of yeasts. The ade1 / ade2 mutants of Saccharomyces cerevisiae, when grown on adenine-limiting medium, accumulate a characteristic red pigment (ade pigment) in their vacuoles. The precursors of the ade pigments are toxic intermediates that form conjugates with glutathione, followed by their transport inside the vacuole. In this study, this red pigment was used as a phenotypic screen to obtain insight regarding new genes involved in the three phases of this detoxification pathway: the activation phase (phase I), the conjugation phase (phase II), and the efflux phase (phase III). Components of the phase III (efflux) pathway which includes, in addition to the previously characterized Ycf1p and Bpt1p, another member of the 'Ycf1p family', Bat1p, as well as a vacuolar H(+)-ATPase-dependent transport were identified. In the investigation of phase II (conjugation), it was found that glutathione S-transferases, encoded by GTT1 and GTT2,do not appear to play a role in this process. By contrast, two other previously characterized genes, the oxidative stress transcription factor gene, SKN7, and the yeast caesin protein kinase gene, YCK1, of S. cerevisiae do participate in this pathway.
TL;DR: 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.
Abstract: Recently, a unique archaeal/bacterial community that grows in a macroscopically visible string-of-pearls-like structure in cold (~10 degrees 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 micro m 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 micro m 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 degrees C and 20 degrees 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 F(420). The G+C-content was 34.5 mol%.