Castellaniella gen. nov., to accommodate the phylogenetic lineage of Alcaligenes defragrans, and proposal of Castellaniella defragrans gen. nov., comb. nov. and Castellaniella denitrificans sp. nov.

Comparative 16S rRNA gene sequence analysis indicates that two distinct sublineages exist within the genus Alcaligenes: the Alcaligenes faecalis lineage, comprising Alcaligenes aquatilis and A. faecalis (with the three subspecies A. faecalis subsp. faecalis, A. faecalis subsp. parafaecalis and A. faecalis subsp. phenolicus), and the Alcaligenes defragrans lineage, comprising A. defragrans. This phylogenetic discrimination is supported by phenotypic and chemotaxonomic differences. It is proposed that the A. defragrans lineage constitutes a distinct genus, for which the name Castellaniella gen. nov. is proposed. The type strain for Castellaniella defragrans gen. nov., comb. nov. is 54PinT (=CCUG 39790T = CIP 105602T = DSM 12141T). Finally, on the basis of data from the literature and new DNA-DNA hybridization and phenotypic data, the novel species Castellaniella denitrificans sp. nov. (type strain NKNTAUT = DSM 11046T = CCUG 39541T) is proposed for two strains previously identified as strains of A. defragrans.

Parvibaculum lavamentivorans converts linear alkylbenzenesulphonate surfactant to sulphophenylcarboxylates, alpha,beta-unsaturated sulphophenylcarboxylates and sulphophenyldicarboxylates, which are degraded in communities.

AIMS: The aims were to test whether Parvibaculum lavamentivoransT degraded commercial linear alkylbenzenesulphonate (LAS) surfactant via omega-oxygenation and beta-oxidation to sulphophenylcarboxylates (SPCs), whether the organism was widespread and reisolable, and whether the degradative community used the 4-sulphocatechol 1,2-dioxygenase to cleave the aromatic ring from LAS. METHODS AND RESULTS: Heterotrophic P. lavamentivoransT converted LAS (side chain length C10-C13) to SPCs (C4-C13), alpha,beta-unsaturated SPCs (C4-C13) and sulphophenyldicarboxylates (SPdCs) (at least C8-C12). Identifications came from high performance liquid chromatography (HPLC) separation, an electrospray interface and mass spectrometry. No evidence for other paths was found. The degradation of LAS in trickling filters inoculated with environmental samples always showed transient SPC intermediates (HPLC) and the presence of the P. lavamentivorans morphotype in the community. One new isolate was obtained. A community able to mineralize LAS contained 4-sulphocatechol-1,2-dioxygenase at high specific activity. CONCLUSIONS: Parvibaculum lavamentivoransT degrades commercial LAS via omega-oxygenation, oxidation and chain shortening through beta-oxidation to yield a wide range of SPCs. The latter are degraded in bacterial communities which contain organisms like P. lavamentivorans, and which utilize sulphocatechol dioxygenase for ring cleavage. SIGNIFICANCE AND IMPACT OF THE STUDY: There is one widespread pathway to degrade LAS. Any traces of LAS and larger amounts of SPCs in the effluent from sewage works are exposed to degradative organisms in acclimated and pristine environments. These degradative reactions can now be studied in pure cultures.

Ethanedisulfonate is degraded via sulfoacetaldehyde in Ralstonia sp. strain EDS1.

Aerobic enrichment cultures (11) yielded three cultures able to utilise ethane-1,2-disulfonate as sole source of carbon and energy in salts medium. Two pure cultures were obtained and we worked with strain EDS1, which was assigned to the genus Ralstonia on the basis of its 16S rDNA sequence and simple taxonomic tests. Strain EDS1 utilised at least seven alkane(di)sulfonates, ethane-1,2-disulfonate, taurine, isethionate, sulfoacetate, sulfoacetaldehyde and propane-1,3-disulfonate, as well as methanesulfonate and formate. Growth with ethanedisulfonate was concomitant with substrate disappearance and the formation of 2 mol sulfate per mol substrate. The growth yield, 7 g protein (mol C)(-1), indicated quantitative utilisation of the substrate. Ethanedisulfonate-dependent oxygen uptake of whole cells during growth rose to a maximum before the end of growth and then sank rapidly; this was interpreted as evidence for an inducible desulfonative oxygenase that was not active in cell extracts. Inducible sulfoacetaldehyde sulfo-lyase was detected at high activity. Inducible degradation of taurine or isethionate or sulfoacetate via sulfoacetaldehyde sulfo-lyase is interpreted from the data.

Sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12) from Desulfonispora thiosulfatigenes: purification, properties and primary sequence.

The strictly anaerobic bacterium Desulfonispora thiosulfatigenes ferments taurine via sulphoacetaldehyde, which is hydrolysed to acetate and sulphite by sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12). The lyase was expressed at high levels and a two-step, 4.5-fold purification yielded an apparently homogeneous soluble protein, which was presumably a homodimer in its native form; the molecular mass of the subunit was about 61 kDa (by SDS/PAGE). The mass was determined to be 63.8 kDa by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) MS. The purified enzyme converted 1 mol of sulphoacetaldehyde to 1 mol each of sulphite and acetate, but no requirement for thiamine pyrophosphate (TPP) was detected. The N-terminal and two internal amino acid sequences were determined, which allowed us to generate PCR primers. The gene was amplified and sequenced. The DNA sequence had no significant homologue in the databases searched, whereas the derived amino acid sequence indicated an oxo-acid lyase, revealed a TPP-binding site and gave a derived molecular mass of 63.8 kDa.

An alpha-proteobacterium converts linear alkylbenzenesulfonate surfactants into sulfophenylcarboxylates and linear alkyldiphenyletherdisulfonate surfactants into sulfodiphenylethercarboxylates.

The surfactant linear alkylbenzenesulfonate (LAS; 0.5 mM) or linear monoalkyldiphenyletherdisulfonate (LADPEDS; 0.5 mM) in salts medium was easily degraded in laboratory trickling filters, whereas carbon-limited, aerobic enrichment cultures in suspended culture with the same inocula did not grow. We took portions of the trickling filters which degraded LADPEDS, shook the organisms from the solid support (polyester), and found that growth in suspended culture in LADPEDS-salts medium occurred only in the presence of some solid support (polyester fleece or glass wool), though little biomass was immobilized on the support. The end products in suspended culture were identical with those from the trickling filters. There was low plating efficiency of LADPEDS-grown cultures on complex medium, and no picked colony or mixture of colonies grew in LADPEDS-salts-glass wool medium. However, selective plates containing LADPEDS-salts medium solidified with agarose yielded LADPEDS-dependent, pinpoint colonies which could be picked singly and subcultured in selective liquid medium. Isolate DS-1 was a bacterium which showed 93% sequence homology (16S ribosomal DNA) to its nearest phylogenetic neighbor, an alpha-proteobacterium. Strain DS-1 grew heterotrophically in LADPEDS-salts-glass wool medium and converted the set of aryl-substituted alkanes to the corresponding aryl-substituted carboxylic acids of shorter chain length. Similarly, strain DS-1 grew heterotrophically with commercial LAS, converting it to a set of sulfophenylcarboxylates. Growth with a single isomer of LAS [3-(4-sulfophenyl)dodecane] was concomitant with excretion of 4-(4-sulfophenyl)hexanoate, which was identified by matrix-assisted laser desorption ionization mass spectrometry. The growth yield (6.4 g of protein/mol of C) indicated mass balance, which, with the specific growth rate (0.05 h(-1)), indicated a specific utilization rate of LAS of 2.2 mkat/kg of protein.

Desulfonispora thiosulfatigenes gen. nov., sp. nov., a taurine-fermenting, thiosulfate-producing anaerobic bacterium.

Strain GKNTAUT has been described as a bacterium able to ferment the organosulfonate taurine (2-aminoethanesulfonate) quantitatively to acetate, ammonia and thiosulfate, an unusual metabolic product. This novel fermentation has now also been observed in four independent isolates from two continents. All five organisms were strictly anaerobic, Gram-positive, motile, spore-forming bacteria. Enrichments with isethionate (2-hydroxyethanesulfonate) and cysteate (2-amino-3-sulfopropionate), in contrast, yielded bacteria that disproportionated the sulfonate to sulfate and sulfide. The phylogenetic location of the taurine fermenters was analysed on the basis of 16S rDNA sequences. Strain GKNTAUT (= DSM 11270T = ATCC 700533T) is described as the type strain of a new genus and species, for which the name Desulfonispora thiosulfatigenes gen. nov., sp. nov. is proposed.

Thiosulfate as a metabolic product: the bacterial fermentation of taurine.

Thiosulfate (S2O32-) is a natural product that is widely utilized in natural ecosystems as an electron sink or as an electron donor. However, the major biological source(s) of this thiosulfate is unknown. We present the first report that taurine (2-aminoethanesulfonate), the major mammalian solute, is subject to fermentation. This bacterial fermentation was found to be catalyzed by a new isolate, strain GKNTAU, a strictly anaerobic, gram-positive, motile rod that formed subterminal spores. Thiosulfate was a quantitative fermentation product. The other fermentation products were ammonia and acetate, and all could be formed by cell-free extracts.

Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU.

Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.

Assimilation of sulfur from alkyl- and arylsulfonates by Clostridium spp.

Organisms able to utilize one of several alkyl- and arylsulfonates as sole source of sulfur under anoxic conditions were enriched. Three fermenting bacteria, all putative Clostridium spp., were isolated in pure culture. All three organisms had wide substrate ranges for alkylsulfonates, taurine and arylsulfonates, presumably due to three different enzyme systems. One organism, strain KNNDS (DSM 10612) was selected for further characterization. The organism was possibly a new Clostridium sp., with Clostidium intestinalis as its nearest neighbor (97.6% similarity of rDNA). Strain KNNDS catalyzed complete sulfonate utilization concomitant with growth. Growth yields of approximtely 3 kg protein/mol sulfur were observed, independent of the sulfur source [e.g. sulfate, sulfide, 4-(phenyl)butyl-1-sulfonate, 2,6-naphthyldisulfonate or 4-nitrocatechol sulfate]. We failed to detect significant amounts of either an arylsulfonatase or an arylsulfatase, and we hypothesize different arylsulfatases [EC 3.1.6.1] in aerobes and in Clostridium spp.

Anaerobic Desulfonation of 4-Tolylsulfonate and 2-(4-Sulfophenyl) Butyrate by a Clostridium sp.

Alkyl- and arylsulfonates were tested as sole added sources of sulfur for the growth of enrichment cultures under strictly anaerobic denitrifying or fermentative conditions. Cultures that utilized taurine, ethylsulfonate, the dyestuffs orange II and acid red I, tolylsulfonate, 2-(4-sulfophenyl)butyrate (SPB), a dialkyltetralinesulfonate, and 1-(4-sulfophenyl)octane were readily obtained. We chose to work with the simple aromatic compounds and isolated a fermentative bacterium, strain EV4, which utilized SPB as the sole added source of sulfur in glucose-mineral medium. The organism was identified as a Clostridium sp. related to Clostridium beijerinckii. Clostridium sp. strain EV4 utilized seven of seven tested arylsulfonates quantitatively. The growth yield was about 3 kg of protein per mol of sulfur, whether sulfonate or sulfate was utilized. A major product specific to each sulfonate could be observed. Although no product was identified, the existence of anaerobic desulfonation has been established.

Energy conservation by succinate decarboxylation in Veillonella parvula.

Veillonella parvula cannot grow with succinate as sole energy source. However, succinate decarboxylation simultaneous with malate or lactate fermentation increased growth yields by 2.4-3.5 g (mol succinate)-1. Malate was fermented stoichiometrically to acetate and propionate whereas lactate fermentation produced more acetate and considerable amounts of H2. Aspartate was utilized only in the presence of succinate as co-substrate. Methylmalonyl-CoA decarboxylase and ATP-dependent pyruvate carboxylase, but not methylmalonyl-CoA:pyruvate transcarboxylase, were detected in cell-free extracts of malate- or lactate-grown cells. The energetic aspects of these fermentation patterns are discussed.