Characterization of a second Rhodococcus erythropolis SQ1 3-ketosteroid 9alpha-hydroxylase activity comprising a terminal oxygenase homologue, KshA2, active with oxygenase-reductase component KshB.

Previously we have characterized 3-ketosteroid 9alpha-hydroxylase (KSH), a key enzyme in microbial steroid degradation in Rhodococcus erythropolis strain SQ1, as a two-component iron-sulfur monooxygenase, comprised of the terminal oxygenase component KshA1 and the oxygenase-reductase component KshB. Deletion of the kshA1 gene resulted in the loss of the ability of mutant strain RG2 to grow on the steroid substrate 4-androstene-3,17-dione (AD). Here we report characteristics of a close KshA1 homologue, KshA2 of strain SQ1, sharing 60% identity at the amino acid level. Expression of the kshA2 gene in mutant strain RG2 restored growth on AD and ADD, indicating that kshA2 also encodes KSH activity. The functional complementation was shown to be dependent on the presence of kshB. Transcriptional analysis showed that expression of kshA2 is induced in parent strain R. erythropolis SQ1 in the presence of AD. However, promoter activity studies, using beta-lactamase of Escherichia coli as a convenient transcription reporter protein for Rhodococcus, revealed that the kshA2 promoter in fact is highly induced in the presence of 9alpha-hydroxy-4-androstene-3,17-dione (9OHAD) or a metabolite thereof. Inactivation of kshA2 in parent strain SQ1 by unmarked gene deletion did not affect growth on 9OHAD, cholesterol, or cholic acid. We speculate that KshA2 plays a role in preventing accumulation of toxic intracellular concentrations of ADD during steroid catabolism. A third kshA homologue was additionally identified in a kshA1 kshA2 double gene deletion mutant strain of R. erythropolis SQ1. The developed degenerate PCR primers for kshA may be useful for isolation of kshA homologues from other (actino) bacteria.

Fermentative bacteria from estuarine mud: phylogenetic position of Acidaminobacter hydrogenoformans and description of a new type of gram-negative, propionigenic bacterium as Propionibacter pelophilus gen. nov., sp. nov.

The phylogenetic positions of two strains of fermentative bacteria that had been isolated from the highest positive tubes inoculated with serial dilutions of estuarine mud in agar media with either glutamate or aspartate as substrate were determined by comparative sequence analysis of their 16S rRNA genes. The strain isolated with glutamate (glu 65) utilized several substrates, including a number of amino acids but no sugars. The degradation of certain substrates was enhanced by or dependent upon co-cultivation with a hydrogen-utilizing partner. In earlier work this strain was assigned to the new genus and species Acidaminobacter hydrogenoformans. On the basis of its 16S rRNA gene sequence Acidaminobacter hydrogenoformans has now been identified as a member of cluster XI of the Clostridium subphylum with Clostridium halophilum as its closest relative. The aspartate-fermenting strain asp 66T was a Gram-negative, rather aerotolerant anaerobe which utilized a wide range of substrates in a propionic fermentation and had the ability to fix molecular nitrogen. Strain asp 66T was shown to be a new member of the beta-subclass of the Proteobacteria with Azoarcus sp. strain 6a3 and Rhodocyclus tenuis as its closest relatives. It is described as Propionibacter pelophilus gen. nov., sp. nov., with the type strain asp 66T (= DSM 12018T).