Paternity testing using the poisonous sting in captive white-spotted eagle rays Aetobatus narinari: a non-invasive tool for captive sustainability programmes.

A group of captive white-spotted eagle rays Aetobatus narinari produced 20 offspring, with an unknown father. Part of the poisonous sting was removed from each fish and DNA was extracted from the epidermis for paternity research using eight microsatellite markers of which four were from another species Aetobatus flagellum. This non-invasive sampling technique can be applied on all members of Myliobatiformes.

Syntrophobacter fumaroxidans sp. nov., a syntrophic propionate-degrading sulfate-reducing bacterium.

A syntrophic propionate-oxidizing bacterium, strain MPOBT, was isolated from a culture enriched from anaerobic granular sludge. It oxidized propionate syntrophically in co-culture with the hydrogen- and formate-utilizing Methanospirillum hungateii, and was able to oxidize propionate and other organic compounds in pure culture with sulfate or fumarate as the electron acceptor. Additionally, it fermented fumarate. 16S rRNA sequence analysis revealed a relationship with Syntrophobacter wolinii and Syntrophobacter pfennigii. The G + C content of its DNA was 60.6 mol%, which is in the same range as that of other Syntrophobacter species. DNA-DNA hybridization studies showed less than 26% hybridization among the different genomes of Syntrophobacter species and strain MPOBT. This justifies the assignment of strain MPOBT to the genus Syntrophobacter as a new species. The name Syntrophobacter fumaroxidans is proposed; strain MPOBT (= DSM 10017T) is the type strain.

Investigation of the fumarate metabolism of the syntrophic propionate-oxidizing bacterium strain MPOB.

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.

Purification and characterization of malate dehydrogenase from the syntrophic propionate-oxidizing bacterium strain MPOB.

Malate dehydrogenase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 42-fold. The native enzyme had an apparent molecular mass of 68 kDa and consisted of two subunits of 35 kDa. The enzyme exhibited maximum activity with oxaloacetate at pH 8.5 and 60 degrees C. The Ka for oxaloacetate was 50 microM and for NADH 30 microM. The Km values for L-malate and NAD were 4 and 1.1 mM, respectively. Substrate inhibition was found at oxaloacetate concentrations higher than 250 microM. The N-terminal amino acid sequence of the enzyme was similar to the sequences of a variety of other malate dehydrogenases from plants, animals and micro-organisms.

Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB.

Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions. The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa. The enzyme exhibited maximum activity at pH 8.5 and approximately 54 degrees C. The Km values for fumarate and L-malate were 0.25 mM and 2.38 mM, respectively. Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe2+ and β-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a [3Fe-4S] cluster. Under reducing conditions, only a trace amount of a [4Fe-4S] cluster was detected. Addition of fumarate resulted in a significant increase of this [4Fe-4S] signal. The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%).

Sulfate reduction by a syntrophic propionate-oxidizing bacterium.

The syntrophic propionate-oxidizing bacterium MPOB was able to grow in the absence of methanogens by coupling the oxidation of propionate to the reduction of sulfate. Growth on propionate plus sulfate was very slow (mu = 0.024 day-1). An average growth yield was found of 1.5 g (dry weight) per mol of propionate. MPOB grew even slower than other sulfate-reducing syntrophic propionate-oxidizing bacteria. The growth rates and yields of strict sulfate-reducing bacteria (Desulfobulbus sp.) grown on propionate plus sulfate are considerably higher.