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Archives of Microbiology (v.179, #3)

Laccases and their occurrence in prokaryotes by Harald Claus (pp. 145-150).

Laccases are copper-containing proteins that require O₂ 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.

Keywords: Laccase Multi-copper protein Prokaryote Phylogeny

Utilization of acidic amino acids and their amides by pseudomonads: role of periplasmic glutaminase-asparaginase by Avinash Sonawane; Ute Klöppner; Christian Derst; Klaus-Heinrich Röhm (pp. 151-159).

The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) support rapid growth of a variety of Pseudomonas strains when provided as the sole source of carbon and nitrogen. All key enzymes of glutamate metabolism were detected in P. fluorescence, with glutaminase and asparaginase showing the highest specific activities. A periplasmic glutaminase/asparaginase activity (PGA) was found in all pseudomonads examined, including a number of root-colonizing biocontrol strains. The enzyme was purified and shown to be identical with the ansB gene product described previously. In addition to PGA, P. fluorescens contains a cytoplasmic asparaginase with marked specificity for Asn. PGA is strongly and specifically induced by its substrates (Asn, Gln) but also by the reaction products (Asp, Glu). In addition, PGA is subject to efficient carbon catabolite repression by glucose and by citrate cycle metabolites. A mutant of P. putida KT2440 with a disrupted ansB gene was unable to utilize Gln, whereas growth of the mutant on other amino acids was normal.

Keywords: Glutamine Asparagine Amino acid metabolism Regulation Pseudomonas Glutaminase/asparaginase

Autotrophic CO₂ fixation pathways in archaea (Crenarchaeota) by Michael Hügler; Harald Huber; Karl Otto Stetter; Georg Fuchs (pp. 160-173).

Representative autotrophic and thermophilic archaeal species of different families of Crenarchaeota were examined for key enzymes of the known autotrophic CO₂ 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 CO₂ 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 CO₂ 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.

Keywords: CO2 fixation Autotrophy Archaea Ribulose 1,5-bisphosphate carboxylase 3-Hydroxypropionate cycle Biotin

A new purple sulfur bacterium isolated from a littoral microbial mat, Thiorhodococcus drewsii sp. nov. by Annette Zaar; Georg Fuchs; Jochen R. Golecki; Jörg Overmann (pp. 174-183).

A new strain of purple sulfur bacterium was isolated from a marine microbial mat sampled in Great Sippewissett Salt Marsh at the Atlantic coast (Woods Hole, Mass., USA). Single cells of strain AZ1 were coccus-shaped, highly motile by means of a single flagellum, and did not contain gas vesicles. Intracellular membranes were of the vesicular type. However, additional concentric membrane structures were present. The photosynthetic pigments were bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series, with rhodopin as the dominant carotenoid. Hydrogen sulfide (up to 11 mM), sulfur, thiosulfate, and molecular hydrogen were used as electron donors during anaerobic phototrophic growth. During growth on sulfide, elemental sulfur globules were transiently stored inside the cells. Strain AZ1 is much more versatile than most other Chromatiaceae with respect to electron donor and organic substrates. In the presence of CO₂, it is capable of assimilating C₁–C₅ fatty acids, alcohols, and intermediates of the tricarboxylic acid cycle. Strain AZ1 could also grow photoorganotrophically with acetate as the sole photosynthetic electron donor. Chemotrophic growth in the dark under microoxic conditions was not detected. Optimum growth occurred at pH 6.5–6.7, 30–35 °C, ≥50 µmol quanta m⁻² s⁻¹, and 2.4–2.6% NaCl. The DNA base composition was 64.5 mol% G+C. Comparative sequence analysis of the 16S rRNA gene confirmed that the isolate is a member of the family Chromatiaceae. Sequence similarity to the most closely related species, Thiorhodococcus minor DSMZ 11518^T, was 97.8%; however, the value for DNA-DNA hybridization between both strains was only 20%. Because of the low genetic similarity and since strain AZ1 physiologically differs considerably from all other members of the Chromatiaceae, including Trc. minor, the new isolate is described as a new species of the genus Thiorhodococcus, Thiorhodococcus drewsii sp. nov.

Keywords: Chromatiaceae Purple sulfur bacteria Thiorhodococcus Microbial mats Bacteriochlorophyll a Spirilloxanthin Rhodopin Sulfur Anoxygenic photosynthesis Fatty acid metabolism

Characterization of the gene encoding glutamate dehydrogenase (gdhA) from the ruminal bacterium Ruminococcus flavefaciens FD-1 by Dionysios A. Antonopoulos; Rustam I. Aminov; Paul A. Duncan; Bryan A. White; Roderick I. Mackie (pp. 184-190).

The gene encoding glutamate dehydrogenase (gdhA) in the ruminal bacterium Ruminococcus flavefaciens FD-1 was cloned. A degenerate primer based on the N-terminal amino acid sequence of the purified protein was used in conjunction with genome walking to obtain the complete ORF of 1,365 bp, capable of encoding a polypeptide of 455 amino acid residues. The translated ORF contained the amino acid motifs characteristic of the subfamily GDH S_50_I small glutamate dehydrogenases, including the catalytic site, and matched the originally deduced N-terminal amino acid sequence. BLAST search yielded high scores with other GdhA sequences from a variety of organisms, the closest match being with the GdhA sequence of Corynebacterium glutamicum (63% amino acid identity). Classification of the GdhA enzyme from R. flavefaciens FD-1 as a GDH S_50_I subfamily member was further supported by phylogenetic analysis. The transcript size determined by Northern blot analysis was in good agreement with the putative regulatory region of the gene and confirmed its monocistronic structure. R. flavefaciens GdhA activity appears to be regulated primarily at the level of transcription. Brief exposure to 20 mM NH₄Cl prior to extraction did not alter the level of activity. Transcriptional regulation, studied with quantitative real-time RT-PCR, demonstrated a three-fold increase of the gdhA transcript concentration in ammonia-limited cells in comparison with an excess of ammonia in the medium. This is in agreement with the enzyme activity data obtained under ammonia- and carbon-limited growth conditions.

Keywords: Ruminococcus Glutamate dehydrogenase Transcriptional regulation

Saccharin as a sole source of carbon and energy for Sphingomonas xenophaga SKN by David Schleheck; Alasdair M. Cook (pp. 191-196).

A bacterium, strain SKN, that was able to utilize saccharin as the sole source of carbon and energy for aerobic growth, was enriched and isolated from communal sewage. The isolate was identified as a strain of Sphingomonas xenophaga. Saccharin was quantitatively converted to cell material, sulfate, ammonium and, presumably, CO₂. The specific rate of saccharin-dependent oxygen uptake during growth reached a maximum before the culture entered the stationary phase and then fell to undetectable levels. Saccharin was degraded only in the presence of molecular oxygen. Catechol was detected as an intermediate during degradation of saccharin in whole cells and catechol 1,2-dioxygenase was expressed inducibly during growth with saccharin. There was an apparent requirement of 2 mol O₂/mol saccharin to remove the substituents on the ring and to cleave the ring. We presume that S. xenophaga SKN synthesizes a multi-component saccharin dioxygenase that simultaneously cleaves off both vicinal substituents from the aromatic ring to yield catechol and the undefined precursor of CO₂ as well as sulfate and ammonium ions.

Keywords: Saccharin degradation Saccharin dioxygenase Sphingomonas xenophaga Catechol 1,2 dioxygenase

A two [4Fe-4S]-cluster-containing ferredoxin as an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans by Wiebke Thamer; Irina Cirpus; Marcus Hans; Antonio J. Pierik; Thorsten Selmer; Eckhard Bill; Dietmar Linder; Wolfgang Buckel (pp. 197-204).

The key step in the fermentation of glutamate by Acidaminococcus fermentans is a reversible syn-elimination of water from (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA catalyzed by 2-hydroxyglutaryl-CoA dehydratase, a two-component enzyme system. The actual dehydration is mediated by component D, which contains 1.0 [4Fe-4S]²⁺ cluster, 1.0 reduced riboflavin-5′-phosphate and about 0.1 molybdenum (VI) per heterodimer. The enzyme has to be activated by the extremely oxygen-sensitive [4Fe-4S]^1+/2+-cluster-containing homodimeric component A, which generates Mo(V) by an ATP/Mg²⁺-induced one-electron transfer. Previous experiments established that the hydroquinone state of a flavodoxin (m=14.6 kDa) isolated from A. fermentans served as one-electron donor of component A, whereby the blue semiquinone is formed. Here we describe the isolation and characterization of an alternative electron donor from the same organism, a two [4Fe-4S]^1+/2+-cluster-containing ferredoxin (m=5.6 kDa) closely related to that from Clostridium acidiurici. The protein was purified to homogeneity and almost completely sequenced; the magnetically interacting [4Fe-4S] clusters were characterized by EPR and Mössbauer spectroscopy. The redox potentials of the ferredoxin were determined as −405 mV and −340 mV. Growth experiments with A. fermentans in the presence of different iron concentrations in the medium (7–45 μM) showed that flavodoxin is the dominant electron donor protein under iron-limiting conditions. Its concentration continuously decreased from 3.5 μmol/g protein at 7 μM Fe to 0.02 μmol/g at 45 μM Fe. In contrast, the concentration of ferredoxin increased stepwise from about 0.2 μmol/g at 7–13 μM Fe to 1.1±0.1 μmol/g at 17–45 μM Fe.

Keywords: Acidaminococcus fermentans Ferredoxin Flavodoxin 2-Hydroxyglutaryl-CoA dehydratase ATP-induced electron transfer [4Fe-4S] cluster Iron limitation

Molecular characterization of HPr and related enzymes, and regulation of HPr phosphorylation in the ruminal bacterium Streptococcus bovis by Narito Asanuma; Tsuneo Hino (pp. 205-213).

Molecular properties of HPr, enzyme I, and HPr kinase in Streptococcus bovis, and the regulation of HPr phosphorylation were examined. The genes encoding HPr (ptsH) and enzyme I (ptsI) were found to be cotranscribed. Two transcriptional start sites were detected in a region upstream of the HPr kinase gene (hprK). HPr kinase had both HPr-phosphorylating and HPr-dephosphorylating activities. The importance of phosphorylation of Ser-46 in HPr was shown by using a mutant HPr in which Ser-46 was replaced by Ala. When S. bovis was grown in glucose-limited medium, the amount of seryl-phosphorylated HPr (HPr-[Ser-P]) decreased drastically as the growth rate decreased. In contrast, the amount of histidyl-phosphorylated HPr (HPr-[His-P]) increased gradually as the growth rate decreased. The amount of HPr kinase did not greatly change with the growth phase, whereas the intracellular P_i concentration increased as the growth rate decreased. HPr-[Ser-P] decreased as the intracellular P_i increased as a consequence of inhibition of HPr kinase activity by P_i and simultaneous enhancement of HPr-[Ser-P] phosphatase activity by P_i. Thus, it is conceivable that the ratio of HPr-[Ser-P] to HPr-[His-P] is regulated by the bifunctional activity of HPr kinase in response to intracellular P_i concentration.

Keywords: Ruminal bacterium Streptococcus bovis HPr Enzyme I HPr kinase

Suppression-subtractive hybridisation reveals variations in gene distribution amongst the Burkholderia cepacia complex, including the presence in some strains of a genomic island containing putative polysaccharide production genes by Yasmin N. Parsons; Rebecca Banasko; Maria G. Detsika; Kwanjit Duangsonk; Lucille Rainbow; C. Anthony Hart; Craig Winstanley (pp. 214-223).

Some strains of the Burkholderia cepacia complex, including the ET12 lineage, have been implicated in epidemic spread amongst cystic fibrosis (CF) patients. Suppression-subtractive hybridisation was used to identify genomic regions within strain J2315 (ET12 lineage; genomovar IIIA) that were absent from a non-transmissible genomovar IIIB strain. Sequence data from 15 subtracted clones were used to interrogate the genome sequence of strain J2315 and identify genomic regions incorporating the subtracted sequences. Many of the genomic regions displayed abnormally low GC content and similarity to sequences implicated in gene transfer. The distribution of three subtracted regions amongst members of the B. cepacia complex varied. A large cluster of genes with strong sequence similarity to capsular production genes from Burkholderia mallei and other bacterial pathogens was identified. This genomic island was detected in some but not all representatives of genomovar IIIA, two out of four genomovar I strains, and one of two strains of Burkholderia multivorans, but was not detected in Burkholderia stabilis, Burkholderia vietnamiensis, genomovar VI or Burkholderia. ambifaria. The polysaccharide production gene cluster of strain J2315 carries an IS407-like sequence within the gene similar to B. mallei wcbO that is lacking in other ET12 isolates. Genes from this cluster are expressed during exponential growth in broth.

Keywords: Burkholderia cepacia Cystic fibrosis Subtraction hybridisation Capsule

Growth of sulfate-reducing bacteria and methanogenic archaea with methylated sulfur compounds: a commentary on the thermodynamic aspects by Johannes C. M. Scholten; J. Colin Murrell; Donovan P. Kelly (pp. 224-224).

2002 Acknowledgement to Reviewers (pp. 225-226).

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Archives of Microbiology (v.179, #3)