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Archives of Microbiology (v.169, #4)

Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue by F. Oxelfelt; Paula Tamagnini; Peter Lindblad (pp. 267-274).

Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N₂-fixing and non-N₂-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme.

Keywords: Key words Cyanobacteria; Uptake hydrogenase; hupSL; Nostoc

Genes coding for respiratory complexes map on all three chromosomes of the Paracoccus denitrificans genome by Christine Winterstein; B. Ludwig (pp. 275-281).

The genome of Paracoccus denitrificans (strain Pd1222) consists of three distinct DNA molecules when separated by standard pulsed-field gel electrophoresis with apparent molecular sizes of approximately 2, 1.1, and 0.64 Mb. When the separated chromosomes are digested by restriction enzymes and sizes of resulting fragments are summed up, the three chromosomes are composed of 1.83, 1.16, and 0.67 Mb. Since their migration behavior relative to size standards is largely independent of electrophoresis conditions, at least the two smaller chromosomes most likely represent linear molecules. The size analysis presented here allows an unequivocal distinction between groups of different strains of P. denitrificans and of Thiosphaera pantotropha, confirming an earlier cytochrome c analysis. When the genome was analyzed with different probes coding for respiratory enzymes, essential genes were found spread over all three chromosomes without any obvious clustering on any of the three forms.

Keywords: Key words Pulsed-field gel electrophoresis; Megaplasmid; Thiosphaera pantotropha; Paracoccus denitrificans; Genome size

High rate of aerobic nitrification and denitrification by Nitrosomonas eutropha grown in a fermentor with complete biomass retention in the presence of gaseous NO2 or NO by D. Zart; Eberhard Bock (pp. 282-286).

A pure culture of the obligately lithoautotrophic ammonia-oxidizer Nitrosomonas eutropha was grown in a laboratory-scale bioreactor with complete biomass retention. The air supply was supplemented with nitrogen dioxide (NO₂; 25 or 50 ppm) or nitric oxide (NO; 25 or 50 ppm). Compared to cultures grown without these nitrogenous oxides, the addition of NO₂ or NO to the culture resulted in a significant increase of the nitrification rate, specific activity of ammonia oxidation, growth rate, and maximum cell densities. In contrast, the growth yield slightly decreased in the presence of NO or NO₂. Maximum cell densities of about 2 × 10¹⁰ cells ml^–1 and a maximum nitrification rate of about 221 mmol NH₄ ⁺ l^–1 day^–1 were obtained after 3 weeks in the presence of 50 ppm NO₂. Furthermore, in the stationary phase about 50% of the nitrite produced was aerobically denitrified to dinitrogen (N₂) and traces of nitrous oxide (N₂O). When cells were supplemented with NO, a high rate of aerobic denitrification occurred only during the first days of the exponential growth phase.

Keywords: Key wordsNitrosomonas eutropha; NO2; NO; Nitrification; Denitrification; Nitrogen loss

Methanobrevibacter filiformis sp. nov., a filamentous methanogen from termite hindguts by Jared R. Leadbetter; Laurel D. Crosby; J. A. Breznak (pp. 287-292).

A morphologically distinct, filamentous methanogen was isolated from hindguts of the subterranean termite, Reticulitermes flavipes (Kollar) (Rhinotermitidae), wherein it was part of the microbiota colonizing the hindgut wall. Individual filaments of strain RFM-3 were 0.23–0.28 μm in diameter and usually > 50 μm in length and aggregated into flocs that were often ≥ 0.1 mm in diameter. Optimal growth of strain RFM-3 was obtained at pH 7.0–7.2 and 30° C with a yeast-extract-supplemented, dithiothreitol-reduced medium in which cells produced stoichiometric amounts of methane from H₂ + CO₂. The morphology and gram-positive staining reaction of strain RFM-3, as well as its resistance to cell lysis by various chemical agents and its restriction to H₂ + CO₂ as an energy source, suggested that it was a member of the Methanobacteriaceae. The nucleotide sequence of the SSU-rRNA-encoding gene of strain RFM-3 confirmed this affiliation and also supported its recognition as a new species of Methanobrevibacter, for which the epithet filiformis is herewith proposed. Although M. filiformis was one of the dominant methanogens in R. flavipes collected from Woods Hole (Mass., USA), cells of similar morphology were not consistently observed in R. flavipes collected from different geographical locations.

Keywords: Key words Archaea; Methanobacteriaceae; Gut; microbiota; Symbiosis; Insect

Characterization of Aquamicrobium defluvii gen. nov. sp. nov., a thiophene-2-carboxylate-metabolizing bacterium from activated sludge by Andreas Bambauer; Fred A. Rainey; Erko Stackebrandt; J. Winter (pp. 293-302).

A gram-negative bacterium was isolated from activated sewage sludge with thiophene-2-carboxylate as the sole source of carbon and with nitrate as an electron acceptor. The isolate, strain NKK, was a motile, oxidase- and catalase-positive, rod-like bacterium with a G+C content of 61.7 mol%. Besides nitrate, oxygen could serve as a terminal electron acceptor. Among many carbon sources tested, only a few sugars, fatty acids, and thiophene-2-carboxylate supported growth. Other heterocyclic compounds were not used. The sulfur atom of thiophene-2-carboxylate was oxidized to thiosulfate when cells were grown aerobically, or to elemental sulfur when cells were grown anaerobically with nitrate. Nitrate was reduced to nitrite. Growth on thiophene-2-carboxylate was dependent on the addition of molybdate to the medium. Tungstate, a specific antagonist of molybdate, inhibited growth on thiophene-2-carboxylate at concentrations > 10^–7 M. Three inducible enzymes involved in the metabolism of thiophene-2-carboxylate were detected: an ATP-, CoA-, thiophene-2-carboxylate- and Mg²⁺-dependent thiophene-2-carboxyl-CoA ligase (AMP-forming), a molybdenum-containing thiophene-2-carboxyl-CoA dehydrogenase, and a thiophene-2-carboxyl-CoA thioesterase. The sequence of the 16S rRNA gene suggested a classification of strain NKK within the α-subgroup of the Proteobacteria as a new genus and species, Aquamicrobium defluvii gen. nov. sp. nov. (DSM 11603), closely related to Mesorhizobium sp. and Phyllobacterium sp., but representing a distinct lineage equal in depth to those of the two mentioned genera. Aquamicrobium defluvii can be distinguished from both genera by a distinct spectrum of substrates, the maximal growth temperature, and a different salt tolerance.

Keywords: Key wordsAquamicrobium defluvii; Thiophene-2-carboxylic acid; Thiophene-2-carboxy-CoA ligase; Thiophene-2-carboxyl-CoA thioesterase; Thiophene-2-carboxyl-CoA dehydrogenase; Thiosulfate; Sulfur; Molybdenum; Nitrate; Nitrite

Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product by Andreas Tauch; Thomas Hermann; Andreas Burkovski; Reinhard Krämer; Alfred Pühler; J. Kalinowski (pp. 303-312).

By complementation analysis of an isoleucine-uptake-deficient Escherichia coli strain, it was shown that a 1.6-kb HindIII-StuI fragment of Corynebacterium glutamicum ATCC 13032, located downstream of the aecD gene, encodes an isoleucine uptake system. Sequence analysis revealed that the complementing fragment carried an open reading frame, termed brnQ, that encodes a protein with sequence similarities to branched-chain amino acid carriers of gram-positive and gram-negative bacteria. The brnQ gene specifies a predominantly hydrophobic protein of 426 amino acid residues with a calculated molecular mass of 44.9 kDa. A topology prediction by neural network computer analysis suggests the existence of 12 hydrophobic segments that most probably form transmembrane α-helices. A C. glutamicum mutant strain harboring a defined deletion of brnQ in the chromosome showed a considerably lower isoleucine uptake rate of 0.04 nmol min^–1 mg (dry mass)^–1 as compared to the wild-type strain rate of 1.2 nmol min^–1 mg (dry mass)^–1. Overexpression of brnQ by means of a tac promotor resulted in an elevated uptake rate for isoleucine of 11.3 nmol min^–1 mg (dry mass)^–1. Evidently, the brnQ gene encodes the only transport system in C. glutamicum directing isoleucine uptake.

Keywords: Key wordsCorynebacterium glutamicum; Amino acid; production; Branched-chain amino acids; Isoleucine; uptake

Dehalobacter restrictus gen. nov. and sp. nov., a strictly anaerobic bacterium that reductively dechlorinates tetra- and trichloroethene in an anaerobic respiration by C. Holliger; Dittmar Hahn; Hermie Harmsen; Wolfgang Ludwig; Wolfram Schumacher; Brian Tindall; Francisco Vazquez; Norbert Weiss; Alexander J. B. Zehnder (pp. 313-321).

The highly enriched anaerobic bacterium that couples the reductive dechlorination of tetrachloroethene to growth, previously referred to as PER-K23, was obtained in pure culture and characterized. The bacterium, which does not form spores, is a small, gram-negative rod with one lateral flagellum. It utilized only H₂ as an electron donor and tetrachloroethene and trichloroethene as electron acceptors in an anaerobic respiration process; it could not grow fermentatively. Acetate served as a carbon source in a defined medium containing iron as the sole trace element, the two vitamins thiamine and cyanocobalamin, and the three amino acids arginine, histidine, and threonine. The cells contained menaquinones and b-type cytochromes. The G+C content of the DNA was 45.3 ± 0.3 mol%. The cell wall consisted of type-A3γ peptidoglycan with ll-diaminopimelic acid and one glycine as an interpeptide bridge. The cells are surrounded by an S-layer; an outer membrane was absent. Comparative sequence analysis of the 16S rRNA sequence showed that PER-K23 is related to gram-positive bacteria with a low G+C content of the DNA. Based on the cytological, physiological, and phylogenetic characterization, it is proposed to affiliate the isolate to a new genus, Dehalobacter, with PER-K23 as the type strain of the new species Dehalobacter restrictus.

Keywords: Key words Anaerobic respiration; Reductive; dechlorination; Tetrachloroethene; Trichloroethene; Hydrogen oxidation; Dehalobacter restrictus

The genes lmbB1 and lmbB2 of Streptomyces lincolnensis encode enzymes involved in the conversion of l-tyrosine to propylproline during the biosynthesis of the antibiotic lincomycin A by Dietmar Neusser; Heike Schmidt; Jaroslav Spizèk; Jitka Novotnà; Ursula Peschke; Stefan Kaschabeck; Pavel Tichy; W. Piepersberg (pp. 322-332).

The genes lmbA,B1,B2 in the lincomycin A production gene cluster of Streptomyces lincolnensis were shown to form a common transcription unit with the promoter located directly upstream of lmbA. The proteins LmbB1 (mol. mass, 18 kDa) and LmbB2 (mol. mass 34 kDa), when over-produced together in Escherichia coli, brought about enzyme activities for the specific conversion of both l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA) to a yellow-colored product. The LmbB1 protein alone catalyzed the conversion of l-DOPA, but not of l-tyrosine. The purified LmbB1 protein showed a K _m for l-DOPA of 258.3 μM. The l-tyrosine converting activity could not been demonstrated in vitro. The preliminary interpretation of these data suggests that the protein LmbB1 is an l-DOPA extradiol-cleaving 2,3-dioxygenase and that the protein LmbB2, either alone or in accord with LmbB1, represents an l-tyrosine 3-hydroxylase. This sequence of putative oxidation reactions on l-tyrosine seems to represent a new pathway different from the ones catalyzed by mammalian l-tyrosine hydroxylases or the wide-spread tyrosinases. The protein LmbA seemed not to be involved in this process. The labile, yellow-colored product from l-DOPA could not be converted to a picolinic acid derivative [3-(2-carboxy-5-pyridyl)alanine] in the presence of ammonia. Therefore, it probably is not a derivative of a cis,cis-3-hydroxymuconic acid semialdehyde; instead, its speculative structure represents a heterocyclic precursor of the propylhygric acid moiety of lincomycin A.

Keywords: Key words Antibiotic biosynthesis; Lincomycin; Streptomyces lincolnensis; Oxidases; Propylhygric acid

Anaerobic degradation of α-resorcylate by Thauera aromatica strain AR-1 proceeds via oxidation and decarboxylation to hydroxyhydroquinone by Corinna Gallus; B. Schink (pp. 333-338).

Anaerobic degradation of α-resorcylate (3,5-dihydroxybenzoate) was studied with the denitrifying strain AR-1, which was assigned to the described species Thauera aromatica. α-Resorcylate degradation does not proceed via the benzoyl-CoA, the resorcinol, or the phloroglucinol pathway. Instead, α-resorcylate is converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by dehydrogenative oxidation and decarboxylation. Nitrate, K₃[Fe(CN)₆], dichlorophenol indophenol, and the NAD⁺ analogue 3-acetylpyridine adeninedinucleotide were suitable electron acceptors for the oxidation reaction; NAD⁺ did not function as an electron acceptor. The oxidation reaction was strongly accelerated by the additional presence of the redox carrier phenazine methosulfate, which could also be used as sole electron acceptor. Oxidation of α-resorcylate with molecular oxygen in cell suspensions or in cell-free extracts of α-resorcylate- and nitrate-grown cells was also detected although this bacterium did not grow with α-resorcylate under an air atmosphere. α-Resorcylate degradation to hydroxyhydroquinone proceeded in two steps. The α-resorcylate-oxidizing enzyme activity was membrane-associated and exhibited maximal activity at pH 8.0. The primary oxidation product was not hydroxyhydroquinone. Rather, formation of hydroxyhydroquinone by decarboxylation of the unknown intermediate in addition required the cytoplasmic fraction and needed lower pH values since hydroxyhydroquinone was not stable at alkaline pH.

Keywords: Key wordsα-resorcylate (3; 5-dihydroxybenzoate); Hydroxyhydroquinone (1; 2; 4-trihydroxybenzene); Aromatic compounds; Anaerobic degradation

Structure, organization, and expression of genes coding for envelope components in the archaeon Methanosarcina mazei S-6 by Linda E. Mayerhofer; E. Conway de Macario; Rong Yao; Alberto J. L. Macario (pp. 339-345).

The antigenic mosaics of archaeal species are complex and lead to the distinction of different immunotypes. We began the dissection of the antigenic mosaic of the methanogen Methanosarcina mazei S-6 by gene cloning and sequencing. The analysis of the sequence, organization, and in vitro (heterologous) and in vivo expression of two three-gene clusters that encode proteins localized to the cell envelope and that are recognized by antibodies for surface structures is presented in this report. The amino acid sequences and compositions share characteristics with S-layer proteins and, most notably, have repeats of conserved sequences and secondary structures. Expressed genes produced proteins with a tendency to oligomerize, and one of these proteins was susceptible to breakdown at regular intervals. Altogether, the data reveal a modular system (clusters of homologous genes, proteins of similar sequences with conserved repeats) seemingly suitable for assembling an enormous variety of final molecular structures by rearranging and combining genes, proteins, and repeats, and thus generate the observed wide spectrum of antigenic diversity.

Keywords: Key words Archaea; Cell envelope genes; Heterologous expression; In vivo expression; Methanosarcina mazei; Cell envelope proteins; Repeats; S-layer; Antigenic diversity

Investigation of the fumarate metabolism of the syntrophic propionate-oxidizing bacterium strain MPOB by Bernardina L. M. Van Kuijk; Elvire Schlösser; Alfons J. M. Stams (pp. 346-352).

The growth of the syntrophic propionate-oxidizing bacterium strain MPOB in pure culture by fumarate disproportionation into carbon dioxide and succinate and by fumarate reduction with propionate, formate or hydrogen as electron donor was studied. The highest growth yield, 12.2 g dry cells/mol fumarate, was observed for growth by fumarate disproportionation. In the presence of hydrogen, formate or propionate, the growth yield was more than twice as low: 4.8, 4.6, and 5.2 g dry cells/mol fumarate, respectively. The location of enzymes that are involved in the electron transport chain during fumarate reduction in strain MPOB was analyzed. Fumarate reductase, succinate dehydrogenase, and ATPase were membrane-bound, while formate dehydrogenase and hydrogenase were loosely attached to the periplasmic side of the membrane. The cells contained cytochrome c, cytochrome b, menaquinone-6 and menaquinone-7 as possible electron carriers. Fumarate reduction with hydrogen in membranes of strain MPOB was inhibited by 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO). This inhibition, together with the activity of fumarate reductase with reduced 2,3-dimethyl-1,4-naphtoquinone (DMNH₂) and the observation that cytochrome b of strain MPOB was oxidized by fumarate, suggested that menequinone and cytochrome b are involved in the electron transport during fumarate reduction in strain MPOB. The growth yields of fumarate reduction with hydrogen or formate as electron donor were similar to the growth yield of Wolinella succinogenes. Therefore, it can be assumed that strain MPOB gains the same amount of ATP from fumarate reduction as W. succinogenes, i.e. 0.7 mol ATP/mol fumarate. This value supports the hypothesis that syntrophic propionate-oxidizing bacteria have to invest two-thirds of an ATP via reversed electron transport in the succinate oxidation step during the oxidation of propionate. The same electron transport chain that is involved in fumarate reduction may operate in the reversed direction to drive the energetically unfavourable oxidation of succinate during syntrophic propionate oxidation since (1) cytochrome b was reduced by succinate and (2) succinate oxidation was similarly inhibited by HOQNO as fumarate reduction.

Keywords: Key words Syntrophy; Fumarate reduction; Propionate; oxidation; Anaerobic growth; Electron transport chain

Net efflux of citrate in Penicillium simplicissimum is mediated by a transport protein by Martin Gallmetzer; Brigitte Müller; W. Burgstaller (pp. 353-359).

Penicillium simplicissimum excreted citrate, isocitrate, and succinate when grown in a strongly buffered medium [1 M Mes (pH 6) or 1 M Hepes (pH 7.3)]. Growth in a weakly buffered medium did not lead to citrate excretion despite a similar intracellular citrate concentration. When nongrowing, citrate-excreting hyphae were aerated in a glucose solution, the following steady-state intracellular concentrations of organic acids were measured: succinate (25 mM); citrate, isocitrate, malate, and fumarate (all less than 5 mM). After 2 h of incubation, the extracellular concentrations of these acids were [μmol (g dry wt.)^–1]: isocitrate [100], citrate [60], succinate [30], and malate, fumarate, and α-ketoglutarate [<5]. The excretion of citrate was due neither to an unspecific change in the permeability of the plasma membrane nor to simple diffusion of undissociated citric acid. The involvement of a transport protein in citrate excretion was indicated because N-ethylmaleimide and sodium azide inhibited citrate excretion strongly despite an unchanged outward-directed citrate gradient. Arguments are given why efflux via a citrate uptake carrier is not considered probable. These results indicate that citrate is excreted by P. simplicissimum via a transport protein that probably specifically mediates the efflux of citrate.

Keywords: Key wordsPenicillium simplicissimum; Efflux of; citrate; Efflux of isocitrate; Basal permeability; Specific excretion system

Regulation of poly(β-hydroxybutyrate) synthesis in Methylobacterium rhodesianum MB 126 growing on methanol or fructose by G. Mothes; Jörg-Uwe Ackermann; Wolfgang Babel (pp. 360-363).

The intracellular concentration of CoA metabolites and nucleotides was determined in batch cultures of Methylobacterium rhodesianum grown on methanol and shifted to growth on fructose. The intracellular concentration of CoA decreased from a high value of 0.6 nmol/mg poly(β-hydroxybutyrate)-free bacterial dry mass during growth on methanol to a low value of 0.03 nmol/mg poly(β-hydroxybutyrate)-free bacterial dry mass after a shift to fructose as a carbon source. The levels of NADH, NADPH, and acetyl-CoA were also lower. Under these conditions, acetyl-CoA was metabolized by both citrate synthase and β-ketothiolase, and poly(β-hydroxybutyrate) synthesis and growth occurred simultaneously during growth on fructose. Moreover, the level of ATP was approximately 50% lower during growth on fructose, supporting the hypothesis of a bottleneck in the energy supply during the growth of M. rhodesianum with fructose.

Keywords: Key words Poly(β-hydroxybutyrate); Methylobacterium rhodesianum; Fructose; Regulation; Intracellular metabolites

Phylogenetic position of an obligately chemoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus, on the basis of 16S rRNA gene sequences and two form I RuBisCO gene sequences by H. Nishihara; Toshiaki Yaguchi; Seon Yong Chung; Ken-ichiro Suzuki; M. Yanagi; K. Yamasato; T. Kodama; Y. Igarashi (pp. 364-368).

Two form ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes from the obligately autotrophic, marine hydrogen oxidizer Hydrogenovibrio marinus were sequenced. The deduced amino acid sequences of both RuBisCOs revealed that they are similar to those of sulfur oxidizers (Thiobacillus) and a purple sulfur bacterium (Chromatium vinosum). According to the 16S rRNA gene sequences, H. marinus is also affiliated with these microorganisms, members of Thiomicrospira being the closest relatives. Sequence similarities of the 16S rRNA genes and of the RuBisCO genes among these γ-Proteobacteria suggest a common autotrophic ancestry. An ancestor of purple sulfur bacteria might be a common root of H. marinus and related sulfur oxidizers.

Keywords: Key wordsHydrogenovibrio marinus; Hydrogen-oxidizing bacterium; Marine bacteria; Obligate chemolithoautotroph; Form I RuBisCO; sequences; 16S rRNA gene sequence; Phylogeny

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Archives of Microbiology (v.169, #4)