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Archives of Microbiology (v.172, #6)

Microbial metabolism of methanesulfonic acid by D. P. Kelly; J. C. Murrell (pp. 341-348).

Methanesulfonic acid is a very stable strong acid and a key intermediate in the biogeochemical cycling of sulfur. It is formed in megatonne quantities in the atmosphere from the chemical oxidation of atmospheric dimethyl sulfide (most of which is of biogenic origin) and deposited on the Earth in rain and snow, and by dry deposition. Methanesulfonate is used by diverse aerobic bacteria as a source of sulfur for growth, but is not known to be used by anaerobes either as a sulfur source, a fermentation substrate, an electron acceptor, or as a methanogenic substrate. Some specialized methylotrophs (including Methylosulfonomonas, Marinosulfonomonas, and strains of ¶Hyphomicrobium and Methylobacterium) can use it as a carbon and energy substrate to support growth. Methanesulfonate oxidation is initiated by cleavage catalysed by methanesulfonate monooxygenase, the properties and molecular biology of which are discussed.

Keywords: Key words Methanesulfonate; Oxidation; Reduction; Oxygenase; Energy; Methylotrophy

Carboxylase genes of Sulfolobus metallicus by N. P. Burton; T. D. Williams; P. R. Norris (pp. 349-353).

Carbon dioxide limitation of Sulfolobus metallicus resulted in increased cellular concentrations of polypeptides that were predicted to be biotin carboxylase and biotin carboxyl-carrier-protein components of a protein complex. These polypeptides were coeluted from a native polyacrylamide gel and were estimated at 19 and 59 kDa after separation by denaturing gel electrophoresis. Their encoding genes were identified, sequenced and shown to code for polypeptides of 18,580 and 58,235 Da with similarities to biotin carboxyl carrier proteins and biotin carboxylases, respectively. The genes overlapped at the second of two stop codons that terminated the carboxylase gene. A third gene occurred on the opposite strand, 293 bp upstream of the biotin carboxylase gene. Its deduced amino acid sequence was similar to those of carboxyl transferase subunits of carboxylase enzymes, in particular to those of the propionyl-CoA carboxylases. It is proposed that the three described genes could encode the key enzyme complex responsible for carbon dioxide fixation during autotrophic growth of the thermoacidophilic archaea.

Keywords: Key wordsSulfolobus metallicus; Autotrophy; Carboxylases

Identification and molecular characterization of the eugenol hydroxylase genes (ehyA/ehyB) of Pseudomonas sp. strain HR199 by H. Priefert; J. Overhage; A. Steinbüchel (pp. 354-363).

The gene loci ehyA and ehyB, which are involved in the bioconversion of eugenol to coniferyl alcohol by Pseudomonas sp. strain HR199 (DSM 7063), were identified as the structural genes of a eugenol hydroxylase that represents an enzyme of the flavocytochrome c class. These genes were localized downstream of the eugenol catabolism genes vanA and vanB, encoding vanillate-O-demethylase, on an EcoRI fragment (E230) that has recently been cloned from a Pseudomonas sp. strain HR199 genomic library. The gene encoding the cytochrome c subunit (ehyA) was identified on a subfragment (K18) of E230 by complementation of a nitrosoguanidine-induced, eugenol-negative mutant of strain HR199. The nucleotide sequences of fragment K18 and adjacent regions were determined, revealing open reading frames of 354 and 1,554 bp that represent ehyA and ehyB, respectively. These genes are most probably organized in one operon together with a third open reading frame (ORF2) of 687 bp that was located between ehyA and ehyB. The deduced amino acid sequences of ehyA and ehyB exhibited up to 29 and 55% amino acid identity to the corresponding subunits of p-cresol methylhydroxylase from Pseudomonas putida. Moreover, the amino-terminal sequences of the α- and β-subunits reported recently for a eugenol dehydrogenase of Pseudomonas fluorescens E118 corresponded well with appropriate regions of ehyA and ehyB. The sequence of ORF2 and the deduced amino acid sequence exhibited no significant similarities to any DNA or amino acid sequence from the databases. The eugenol hydroxylase genes were amplified by PCR, cloned in pBluescript SK^–, and functionally expressed in Escherichia coli. Transfer of a DNA fragment comprising ehyA and ehyB to various strains of Pseudomonas species that were unable to utilize eugenol as a carbon source conferred to these bacteria the ability to grow on this substrate.

Keywords: Key wordsPseudomonas; Eugenol hydroxylase; Flavocytochrome c; Formaldehyde dehydrogenase; γ-Glutamylcysteine synthetase; Vanillin production; ehyA; ehyB; fdh; gcs

Identification of three genes expressed primarily during development in Physarum polycephalum by J. Bailey; L. J. Cook; R. Kilmer-Barber; E. Swanson; L. Solnica-Krezel; K. Lohman; W. F. Dove; J. Dee; R. W. Anderson (pp. 364-376).

During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA. In order to identify genes involved in regulating plasmodium formation, we constructed a subtracted cDNA library from cells undergoing development. Three genes that have their highest levels of expression during plasmodium development were identified: redA, redB (regulated in development) and mynD (myosin). Both redA and redB are single-copy genes and are not members of gene families. Although redA has no significant sequence similarities to known genes, redB has sequence similarity to invertebrate sarcoplasmic calcium-binding proteins. The mynD gene is closely related to type II myosin heavy-chain genes from many organisms and is one of a family of type II myosin genes in P. polycephalum. Our results indicate that many more red genes remain to be identified, some of which may play key roles in controlling plasmodium formation.

Keywords: Key words Slime moulds; Physarum polycephalum; Plasmodium development; Differential gene expression; Myosin; Calcium-binding protein

Identification of salt-regulated genes in the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 by subtractive RNA hybridization by J. Vinnemeier; M. Hagemann (pp. 377-386).

To identify genes transcribed preferentially under salt stress, a subtractive RNA hybridization procedure was applied to the cyanobacterium Synechocystis sp. PCC 6803. The screening of a genomic library led to the identification of several RNA species that were more abundant in salt-stressed cells than in control cells. Salt-dependent transcription of the identified genes was verified in Northern blot experiments. In addition to the previously characterized genes cpn60 (encoding GroEL; a molecular chaperone) and isiA (encoding a chlorophyll-binding protein), genes encoding a protein of unknown function (slr0082) and a putative RNA helicase (slr0083) were identified as salt-regulated genes in Synechocystis. Genes slr0082 and slr0083, located at sites adjacent to each other on the Synechocystis chromosome, were transcribed from separate promoters and showed the most significant induction 1–3 h after salt shock. The salt-regulated promoters of these two genes were mapped. Genes cpn60, slr0082, and slr0083 were also found to be induced by a cold shock. The possible role of the identified gene products for salt adaptation of Synechocystis is discussed.

Keywords: Key words Chaperone; Cyanobacteria; groEL; isiA; RNA helicase; Salt stress; Subtractive RNA ¶hybridization; Synechocystis

Desulfonation of propanesulfonic acid by Comamonas acidovorans strain P53: evidence for an alkanesulfonate sulfonatase and an atypical sulfite dehydrogenase by W. Reichenbecher; D. P. Kelly; J. C. Murrell (pp. 387-392).

Evidence is presented for the presence in propanesulfonate-grown Comamonas acidovorans strain P53 of a cytoplasmically located sulfonatase that does not sediment at 100,000 × g. This enzyme catalysed the sulfonate-dependent oxidation of NADH or NADPH, indicating a monooxygenase that effects the addition of molecular oxygen to C₃-C₆ 1-alkanesulfonates. Enzyme activity was proportional to protein concentration only above approximately 2 mg cytoplasmic fraction protein ml^–1, suggesting that the sulfonatase is a multicomponent enzyme, possibly comparable with methanesulfonate monooxygenase. Enzyme activity was strongly inhibited by divalent metal-chelating agents, but was insensitive to cyanide and azide. Sulfite released from sulfonates by Comamonas acidovorans was oxidized by an unusual sulfite dehydrogenase. This was purified approximately 230-fold and was shown to have a molecular mass of 74.4 kDa, comprising two or more subunits. The enzyme activity was specific in vitro for ferricyanide as an electron acceptor and, unlike other bacterial sulfite dehydrogenases, did not contain native cytochrome c or reduce added cytochrome c. It was a basic protein, insensitive to chloride and sulfate, and exhibited a K _m for sulfite of approximately 45 μM.

Keywords: Key wordsComamonas acidovorans; Alkanesulfonates; Sulfonatase; Monooxygenase; Sulfite oxidase; Sulfite dehydrogenase

Degradation of chlorinated and brominated hydrocarbons by Methylomicrobium album BG8 by J. -I. Han; S. Lontoh; J. D. Semrau (pp. 393-400).

The degradation kinetics of ten halogenated hydrocarbons by Methylomicrobium album BG8 expressing particulate methane monooxygenase (pMMO) and the inhibitory effects of these compounds on microbial growth and whole-cell pMMO activity were measured. When M. album BG8 was grown with methane, growth was completely inhibited by dichloromethane (DCM), bromoform (BF), chloroform (CF), vinyl chloride (VC), 1,1-dichloroethylene (1,1-DCE), and cis-dichloroethylene (cis-DCE). Trichloroethylene (TCE) partially inhibited growth on methane, while dibromomethane (DBM), trans-dichloroethylene (trans-DCE), and 1,1,1-trichloroethane (1,1,1-TCA) had no effect. If the cells were grown with methanol, DCM, BF, CF, and 1,1-DCE completely inhibited growth, while VC, trans-DCE, TCE, and 1,1,1-TCA partially inhibited growth. Both DBM and cis-DCE had no effect on growth with methanol. Whole-cell pMMO activity was also affected by these compounds, with all but 1,1,1-TCA, DCM, and DBM reducing activity by more than 25%. DCM, DBM, VC, trans-DCE, cis-DCE, 1,1-DCE, and TCE were degraded and followed Michaelis-Menten kinetics. CF, BF, and 1,1,1-TCA were not measurably degraded. These results suggested that the products of DCM, TCE, VC, and 1,1-DCE inactivated multiple enzymatic processes, while trans-DCE oxidation products were also toxic but to a lesser extent. cis-DCE toxicity, however, appeared to be localized to pMMO. Finally, DBM and 1,1,1-TCA were not inhibitory, and CF and BF were themselves toxic to M. album BG8. Based on these results, the compounds could be separated into four general categories, namely (1) biodegradable with minimal inactivation, (2) biodegradable with substantial inactivation, (3) not biodegradable with minimal inactivation, and (4) not biodegradable but substantial inactivation of cell activity.

Keywords: Key words Methanotrophs; Bioremediation; Halogenated hydrocarbons; Kinetics

Phosphofructokinase activities within the order Spirochaetales and the characterisation of the pyrophosphate-dependent phosphofructokinase from Spirochaeta thermophila by R. S. Ronimus; H. W. Morgan; Y.-H. R.. Ding (pp. 401-406).

The subtype of phosphofructokinase activity, either ATP-, ADP- or pyrophosphate-dependent, present in members of three genera from the Spirochaetales was investigated. The individual species/strains examined included Spirochaeta alkalica, S. asiatica, S. halophila, S. isovalerica, S. litoralis, S. zuelzerae, S. thermophila, two thermophilic spirochetes, Treponema bryantii, T. denticola, ¶T. pectinovorum, Leptospira biflexa and L. interrogans. All of the Spirochaeta strains, regardless of their phenotype, possessed primarily a pyrophosphate-dependent phosphofructokinase. In contrast, T. bryantii, T. denticola and L. biflexa had predominantly an ATP-dependent activity, whereas no activity was detected in T. pectinovorum or ¶L. interrogans. The results suggest that pyrophosphate-dependent phosphofructokinase activity may be a reliable phenotypic marker for the genus Spirochaeta and that there are potentially interesting differences in how the catabolism of saccharides is controlled among members of genera within the Spirochaetales. The pyrophosphate-dependent phosphofructokinase from S. thermophila strain RI 19.B1 was purified (303-fold) to homogeneity and biochemically characterised. The S. thermophila enzyme displayed hyperbolic kinetics with respect to both the forward and reverse cosubstrates and was not significantly affected by traditional activators or inhibitors of phosphofructokinase. The biochemical characterisation represents the first spirochete phosphofructokinase to be described.

Keywords: Key words Spirochetes; Phosphofructokinase; Treponema; Leptospira; Spirochaeta; Glycolysis; ¶Pyrophosphate; Thermophile

Differential enumeration and in situ localization of microorganisms in the hindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes by M. Berchtold; A. Chatzinotas; W. Schönhuber; A. Brune; R. Amann; D. Hahn; H. König (pp. 407-416).

We examined the abundance and spatial distribution of major phylogenetic groups of the domain Bacteria in hindguts of the Australian lower termite Mastotermes darwiniensis by using in situ hybridization with group-specific, fluorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 ± 7.2% and 52.3 ± 8.2% of the DAPI-stained cells in different hindgut fractions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flagellates of the thin-walled anterior region (P3a) and the thick wall of the posterior region (P3b/P4) of the hindgut, as shown by DAPI staining. At most, half of the EUB338-detected cells hybridized with one of the other probes that targeted a smaller assemblage within the bacterial domain. In most fractions, cells were found in varying numbers with probe ALF1b, which targeted members of the α-Proteobacteria, whereas substantial amounts of sulfate-reducing bacteria, gram-positive bacteria with a high DNA G+C content and members of the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum could be detected only in the wall fraction of P3b/P4. This clearly indicates that the hindgut microhabitats differ in the composition of their microbial community. In situ hybridization of cryosections through the hindgut showed only low numbers of bacteria attached to the P3a wall. In contrast, the wall of P3b was densely colonized by rod- and coccus-shaped bacteria, which could be assigned to the Cytophaga-Flavobacterium cluster of the CFB phylum and to the group of gram-positive bacteria with a high DNA G+C content, respectively. Oxygen concentration profiles determined with microelectrodes revealed steep oxygen gradients both in P3a and P3b. Oxygen was consumed within 100 μm below the gut surface, and anoxic conditions prevailed in the central portions of both gut regions, indicating that oxygen consumption in the hindgut does not depend on the presence of a biofilm on the hindgut wall.

Keywords: Key words Freeze sectioning; Fluorescent ¶oligonucleotide probes; In situ hybridization; Intestinal microorganisms; Mastotermes darwiniensis; Oxygen microsensors; rRNA; Termites

Methyl ketone formation during degradation of phenoxybutyric acid by Penicillium canescens SBUG-M 1139 by J. Lottmann; E. Hammer; F. Schauer (pp. 417-420).

Penicillium canescens SBUG-M 1139 was shown to be able to grow using phenoxybutyric acid as the sole carbon source. The rapid conversion of the phenoxyalkanoic acid resulted in the formation of phenol, which was metabolized completely. These reactions were accompanied by an accumulation of the methyl ketone phenoxypropan-2-one. Furthermore, during the metabolism of phenoxybutyric acid, 4-phenoxy-2,3-dehydrobutyric acid, 4-phenoxy-3-hydroxybutyric acid, phenoxyacetic acid, and phenoxypropan-2-ol accumulated in minor amounts. Clearly, fungi can metabolize phenoxyalkanoic acids to produce methyl ketones in a manner analogous to that used for the conversion of short- or medium-chain fatty acids by fungi.

Keywords: Key wordsPenicillium canescens; Moulds; Methyl ¶ketones; Phenoxybutyric acid

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Archives of Microbiology (v.172, #6)