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Archives of Microbiology (v.168, #2)

Recent advances in genetic analyses of hyperthermophilic Archaea and Bacteria by K. M. Noll; Madeline Vargas (pp. 73-80).

Hyperthermophilic Archaea and Bacteria are an extraordinarily important class of organisms for which genetic tools remain to be developed. Unique technological obstacles to this goal are posed by the thermophilic and, in some cases, strictly anaerobic nature of these organisms. However, recent advances in the cultivation of hyperthermophiles, in the discovery of genetic elements for vector development, and in the construction of genetic markers point toward the achievement of this goal in the near future. Transformation protocols have already been reported for Sulfolobus and Pyrococcus, and plasmid-mediated conjugation was recently found in Sulfolobus. Plasmids are available for Sulfolobus, Pyrococcus, and the bacterial hyperthermophile Thermotoga, and these provide the bases for vector construction in these hosts. A Desulfurococcus mobile intron may provide a novel means to introduce genes into a variety of archaeal hosts. With full genome sequences of several hyperthermophiles available soon, genetic tools will allow full exploitation of this information to study these organisms in depth and to utilize their unique properties in biotechnological applications.

Keywords: Key wordsArchaea; Hyperthermophile; Thermophile; Sulfolobus; Pyrococcus; Thermotoga; Methanococcus; jannaschii; Vectors; Genetic markers; Thermostable; proteins

Biochemical and genetic characterization of the acetaldehyde dehydrogenase complex from Acetobacter europaeus by Claudia Thurner; Cinzia Vela; Linda Thöny-Meyer; L. Meile; M. Teuber (pp. 81-91).

The aldehyde dehydrogenase complex, which catalyzes the oxidation of acetaldehyde to acetic acid, was purified to apparent homogeneity from the membrane fraction of the industrial vinegar-producing strain Acetobacter europaeus. The determined K _m for acetaldehyde was 2.1 mM. SDS-PAGE of the enzyme complex showed the presence of three different subunits with molecular masses of 79, 46, and 17 kDa, respectively. The two larger subunits contained heme. The difference spectrum indicated a cytochrome c, a heme B, and a [2Fe–2S] cluster. The nucleotide sequence of several cloned fragments of a 6-kb chromosomal DNA segment from A. europaeus was determined. It contains three consecutive open reading frames that correspond to proteins with calculated molecular masses of 84.1, 49.0, and 16.7 kDa; these were assigned to the purified proteins and named aldH, aldF, and aldG, respectively. The N-terminal sequence of the 79-kDa subunit was detected within the predicted amino acid sequence of AldH, which indicated the presence of a leader peptide. Cotranscription of the three genes was shown by Northern hybridization. Sequence analysis and experimental evidence allowed the assignment of the following cofactors to the respective subunits of the aldehyde dehydrogenase complex: heme C to AldF, [2Fe–2S] cluster to AldG, and heme B and a molybdopterin cofactor to AldH. Part of an open reading frame, gdhA, was detected upstream of the operon that showed high similarities to the C-terminal part of several pyrroloquinoline-chinone-dependent glucose dehydrogenases.

Keywords: Key wordsAcetobacter europaeus; Aldehyde; dehydrogenase complex; Aldehyde dehydrogenase-encoding genes; Promoter mapping

Comparative genomic analysis of isolates belonging to the six species of the genus Thermus using pulsed-field gel electrophoresis and ribotyping by Leonilde M. Moreira; Milton S. Da Costa; I. Sá-Correia (pp. 92-101).

Fifty isolates belonging to the six validly described species of the genus Thermus (T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus, T. brockianus, and T. oshimai) isolated from hot springs of different geographical areas were compared using macrorestriction analysis of genomic DNA and ribotyping. With the exception of presumed clones, the macrorestriction patterns of isolates obtained with EcoRI or NdeI were distinct. However, isolates belonging to the same species exhibited similar profiles particularly when they were isolated from the same hot spring. The estimated genomic size of strains of the Thermus spp. varied between approximately 1.8 and 2.5 Mbp. Ribotyping with BamHI and HindIII produced 30 and 35 distinct ribotypes, respectively. In spite of the variability of the hybridization patterns produced, the ribotypes obtained for isolates belonging to the same species also shared, in general, several fragments of identical size, and these fragments were similar when isolates originated from the same spring.

Keywords: Key wordsThermus; Restriction fragment length; polymorphism; Pulsed-field gel electrophoresis; Ribotyping; Genome size

Identification of stsC, the gene encoding the l-glutamine:scyllo-inosose aminotransferase from streptomycin-producing Streptomycetes by Joachim Ahlert; Jürgen Distler; Kambiz Mansouri; W. Piepersberg (pp. 102-113).

Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins – together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster – show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the l-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.

Keywords: Key words Streptomycin biosynthesis; Aminocyclitol; Aminotransferase; Streptomyces griseus

Thermoanaerobacter mathranii sp. nov., an ethanol-producing, extremely thermophilic anaerobic bacterium from a hot spring in Iceland by Lise Larsen; Peter Nielsen; B. K. Ahring (pp. 114-119).

The extremely thermophilic ethanol-producing strain A3 was isolated from a hot spring in Iceland. The cells were rod-shaped, motile, and had terminal spores; cells from the mid-to-late exponential growth phase stained gram-variable but had a gram-positive cell wall structure when viewed by transmission electron microscopy. Strain A3 used a number of carbohydrates as carbon sources, including xylan, but did not utilize microcrystalline cellulose. Fermentation end products were ethanol, acetate, lactate, CO₂, and H₂. The temperature optimum for growth was between 70 and 75° C, and growth occurred in the range of 50–75° C. The pH range for growth was 4.7–8.8, with an optimum at pH 7.0. Strain A3 was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, and vancomycin at 100 mg/l but was not sensitive to chloramphenicol and neomycin at 10 mg/l, which indicates that strain A3 belongs to the eubacteria. Addition of 50.66 kPa H₂ or 2% NaCl did not affect growth. The isolate grew in the presence of exogenously added 4% (w/v) ethanol. The G+C ratio was 37 mol%. 16S rDNA studies revealed that strain A3 belongs to the genus Thermoanaerobacter. Genotypic and phenotypic differences between strain A3 and other related species indicate that strain A3 can be assigned to a new species, and the name Thermoanaerobacter mathranii is proposed.

Keywords: Key words Anaerobe; Thermophile; Xylose; Hemicellulose; Ethanol; Hot spring; Iceland; Thermoanaerobacter mathranii

Carbohydrate metabolism in Thermoproteus tenax: in vivo utilization of the non-phosphorylative Entner-Doudoroff pathway and characterization of its first enzyme, glucose dehydrogenase by B. Siebers; Volker F. Wendisch; Reinhard Hensel (pp. 120-127).

Thermoproteus tenax is a hyperthermophilic, facultative heterotrophic archaeum. In this organism the utilization of the two catabolic pathways, a variant of the Embden-Meyerhof-Parnas (EMP) pathway and the modified (nonphosphorylative) Entner-Doudoroff (ED) pathway, was investigated and the first enzyme of the ED pathway, glucose dehydrogenase, was characterized. The distribution of the ¹³C label in alanine synthesized by cells grown with [1-¹³C]glucose indicated that in vivo the EMP pathway and the modified ED pathway operate parallel, with glucose metabolization via the EMP pathway being prominent. To initiate studies on the regulatory mechanisms governing carbon flux via these pathways, the first enzyme of the ED pathway, glucose dehydrogenase, was purified to homogeneity and its phenotypic properties were characterized. The pyridine-nucleotide-dependent enzyme used both NAD⁺ and NADP⁺ as cosubstrates, showing a 100-fold higher affinity for NADP⁺. Besides glucose, xylose was used as substrate, but with significantly lower affinity. These data suggest that the physiological function of the enzyme is the oxidation of glucose by NADP⁺. A striking feature was the influence of NADP⁺ and NAD⁺ on the quaternary structure and activity state of the enzyme. Without cosubstrate, the enzyme was highly aggregated (mol. mass > 600 kDa) but inactive, whereas in the presence of the cosubstrate the aggregates dissociated into enzymatically active, homomeric dimers with a mol. mass of 84 kDa (mol. mass of subunits: 41 kDa). The N-terminal amino acid sequence showed striking similarity to the respective partial sequences of alcohol dehydrogenases and sorbitol dehydrogenases, but no resemblance to the known pyridine-nucleotide-dependent archaeal and bacterial glucose dehydrogenases.

Keywords: Key words Archaeum; Carbohydrate metabolism; Embden-Meyerhof-Parnas pathway; Modified; (nonphosphorylative) Entner-Doudoroff pathway; Glucose dehydrogenase; Dual cosubstrate specificity

Outer membrane proteins of Methylococcus capsulatus (Bath) by A. Fjellbirkeland; Hans Kleivdal; Carsten Joergensen; Helle Thestrup; Harald B. Jensen (pp. 128-135).

Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed by electron microscopy and by the content of β-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes, this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl.

Keywords: Key wordsMethylococcus capsulatus; Outer membrane proteins; Porin

Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC by Franz Kaufmann; Gert Wohlfarth; G. Diekert (pp. 136-142).

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H₂ plus hydrogenase purified from strain MC.

Keywords: Key words Corrinoid protein; Ether cleavage; Hydrogenase; Methyltransferase; O-demethylase; Strain MC; Tetrahydrofolate

Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes by Heidi Peter; Andrea Bader; Andreas Burkovski; Camille Lambert; R. Krämer (pp. 143-151).

Corynebacterium glutamicum accumulates the compatible solutes proline, glycine betaine, and ectoine under conditions of high osmolality. Uptake of proline is mediated by both a high-affinity and a low-affinity secondary transport system. The low-affinity uptake system also accepts glycine betaine and ectoine as substrates. In the present study, the gene encoding the high-affinity proline uptake system PutP was isolated by heterologous complementation of Escherichia coli mutant strain WG389, which lacks the transport systems BetT, PutP, ProP, and ProU and is unable to synthesize proline and glycine betaine. This gene (putP) encodes a protein of 524 amino acids that shares identity with the proline transport systems PutP of E. coli, Staphylococcus aureus, Salmonella typhimurium, Haemophilus influenzae, and Klebsiella pneumoniae. Functional studies of PutP synthesized in E. coli mutant strain MKH13, which also lacks the transport systems for compatible solutes and is unable to synthesize glycine betaine, revealed that this carrier system is not regulated by the external osmolality on the level of activity. K _m values of 7.6 mM for proline and 1.3 mM for sodium as cotransported ion were determined. Deletion of the putP gene allowed the functional characterization of another proline uptake system with low affinity.

Keywords: Key wordsCorynebacterium glutamicum; PutP; Proline transport; Compatible solutes; Osmoregulation

Cell elongation and septation are two mutually exclusive processes in Escherichia coli by P. Canepari; Caterina Signoretto; Marzia Boaretti; Maria Del Mar Lleò (pp. 152-159).

Bacterial rod morphogenesis was studied in synchronously growing cells of Escherichia coli C600 during the reshaping process that follows the removal of mecillinam, a β-lactam antibiotic that specifically inhibits lateral wall formation of gram-negative rods and causes transition to coccal shape. Removal of mecillinam after 30 min of action did not affect the timing of subsequent cell division, but removal after 50 min delayed resumption of cell division for approximately one generation time. In order to study the interplay between lateral wall elongation and septum formation in determining and maintaining the bacterial rod shape, we evaluated the effect of re-adding mecillinam or of adding aztreonam (a specific inhibitor of septum formation) at various stages of the reshaping process. We conclude that mecillinam was active only during the reshaping process, while aztreonam was active only later when the cells were close to dividing again. These results provide further evidence for our previous proposal according to which elongation and septation are two alternating and competing events of the cell cycle and are linked to each other to force bacterial rods to grow to a given length.

Keywords: Key words Bacterial shape; Peptidoglycan; β-Lactam; antibiotics; Escherichia coli

Sensitivity to pH, product inhibition, and inhibition by NAD+ of 1,3-propanediol dehydrogenase purified from Enterobacter agglomerans CNCM 1210 by Fabien Barbirato; Anne Larguier; Thierry Conte; Suzette Astruc; A. Bories (pp. 160-163).

Because of its key role in the metabolism of glycerol during fermentation, 1,3-propanediol dehydrogenase (EC 1.1.1.202) of Enterobacter agglomerans CNCM 1210 was purified to homogeneity and studied with respect to its sensitivity to pH and to nucleotide and 1,3-propanediol concentrations. Enzyme activity was optimal at pH 7.8. The enzyme was competitively inhibited by NAD⁺ (K_i of 0.29 mM), and 1,3-propanediol exerted a strong inhibitory effect according to a mixed-type inhibition with a K_i of 13.7 mM and an a-factor of 9.0. It is proposed that these dehydrogenase properties be extended to the dehydrogenases of Citrobacter freundii and Klebsiella pneumoniae, which exhibited numerous similar physical properties.

Keywords: Key wordsEnterobacter agglomerans; 1; 3-Propanediol dehydrogenase; Protein purification; Kinetic study; pH; Competitive inhibition; Mixed-type; inhibition

The biotransformation of t-butylacetonitrile and its boron-containing analogue trimethylamine-cyanoborane by Brevibacterium R312 by Anthony J. Millais; Duncan Casson; John Lovelady; Andrea M. Nicholson; Christopher J. Knowles (pp. 164-168).

The ability of the nitrile hydratase/amidase system from Brevibacterium R312 to biotransform tert-butylacetonitrile was studied with a view to their utilisation in the production of novel amino acids from isostructural compounds. Brevibacterium R312 was able to transform nitriles with this structure; however, the wide spectrum amidase from this organism was unable to biotransform the corresponding amide to the carboxylic acid.

Keywords: Key wordsBrevibacterium R312; Amine-cyanoborane; Amine-carboxyborane; t-Butylacetonitrile; t-Butylacetamide; Biotransformation

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Archives of Microbiology (v.168, #2)