Ralph A.D. Williams

Thermus oshimai sp. nov., isolated from hot springs in Portugal, Iceland, and the Azores, and comment on the concept of a limited geographical distribution of Thermus species

We examined aerobic, thermophilic, gram-negative bacteria that were isolated from hot springs in Portugal and were identified as Thermus strains and placed in phenetic groups on the basis of their phenotypic characteristics. We determined the composition of the peptidoglycan, identified the respiratory quinones, and determined the mean base composition of the DNA, and the levels of DNA-DNA homology were determined by both the filter hybridization and reassociation rate methods. Thermus aquaticus, Thermus brockianus, and Thermus filiformis were not detected in this collection of organisms, although three Thermus thermophilus strains were identified. We propose that the isolates that belonged to phenetic clusters E and F are members of a new species, Thermus oshimai; the type strain of T. oshimai is strain SPS17.

Microbial transformations of steroids—VIII. Transformation of progesterone by whole cells and microsomes of Aspergillus fumigatus

The filamentous fungus, Aspergillus fumigatus, efficiently hydroxylated exogenous progesterone producing, after 3 h of incubation, 11 alpha- and 15 beta-hydroxyprogesterone as major products, 7 beta-hydroxyprogesterone as a minor product and trace amounts of 7 beta, 15 beta- and 11 alpha, 15 beta-dihydroxyprogesterone. After 72 h the dihydroxyprogesterones were the sole metabolites in the culture medium. Microsomes, prepared by Ca2+ precipitation, catalysed only monohydroxylation of progesterone at the same sites as whole cells. Hydroxylation was dependent on NADPH (but not NADH) which was replaceable by NaIO4. Hydroxylation was inhibited by carbon monoxide and by the azole fungicide, ketoconazole. Microsomes gave a dithionite-reduced, carbon monoxide difference absorbance spectrum with a peak at 448 nm and a Type-I progesterone-binding spectrum typical of cytochrome P450 interaction with substrate. Ketoconazole inhibition studies suggest the presence of two non-inducible cytochrome P450 progesterone hydroxylases, one possessing 7 beta site-selectivity, the other 11 alpha/15 beta site-selectivity.

Microbial transformations of steroids—X. Cytochromes P-450 11α-hydroxylase and C17–C20 lyase and a 1-ene dehydrogenase transform steroids in Nectria haematococca

Nectria haematococca contains four enzymes that metabolise exogenous steroids. Two of these are microsomal cytochromes P-450 which act sequentially on progesterone producing firstly, by side-chain cleavage, the C19 steroid androstenedione (C17-C20 lyase), and then, in a subsequent set of transformations, 11 alpha-hydroxylated derivatives (11 alpha-hydroxylase). Two other conversions occur after side-chain cleavage. Unsaturation, in the form of a double bond at C1-C2, is introduced into the A ring by a catalytically self-sufficient microsomal 1-ene dehydrogenase. This enzyme is specific for C19 substrates. A C17-specific oxidoreductase is also involved in the production of androstenedione and testosterone from progesterone. The lyase, 11 alpha-hydroxylase and 1-ene dehydrogenase were purified to homogeneity.

Progesterone 6-hydroxylation is catalysed by cytochrome P-450 in the moderate thermophile Bacillus thermoglucosidasius strain 12060

The moderate thermophile, Bacillus thermoglucosidasius, transforms progesterone into four metabolites. These are 6alpha- and 6beta-hydroxyprogesterone, androstenedione and testosterone. This is the first report of bacterial 6alpha-hydroxylation of steroids. The identity of the progesterone metabolites shows that there are three major types of transforming activity in this organism; C-17-C-20 lyase that cleaves the pregnane side chain of the substrate, C-17 oxidoreductase that interconverts the metabolites androstenedione and testosterone, and 6-hydroxylation. 6-hydroxylation activity was purified virtually to homogeneity and was shown to be catalysed by a cytochrome P-450 monooxygenase enzyme. This is the first report of a thermostable cytochrome P-450.

The Taxonomy and Identification of Thermus

Bacteria of the genus Thermus were first isolated in the late 1960s from hot springs in Japan and the USA. Thermus strains also appear to be widespread in neutral, hot, aqueous artificial environments such as domestic and industrial hot water systems and thermally polluted streams. The reference strains that have been used in taxonomic studies are listed in Table I. Isolates from sources in Yellowstone National Park were originally allocated to the type species, Thermus aquaticus (Brock and Freeze, 1969), of this new genus. Subsequently, sites in Yellowstone National Park were sampled for Thermus, but isolates were only obtained from sources between 55 °C and 80 °C and pH 6.0 to 10.5 (Munster et al., 1986). In Japan, early isolates were named “Flavobacterium thermophilum” (Oshima and Imahori, 1971), but validly redescribed as “Thermus thermophilus” (Oshima and Imahori, 1974). Other Japanese strains were given the invalid species names “Thermus flavus” (Saiki et al., 1972), and “Thermus caldophilus” (Taguchi et al., 1982), but these strains seem to belong to the T. thermophilus genospecies (see sections 5.2. and 6.1). Other terrestrial hot springs in Iceland were sampled (Cornetta et al., 1982b; Hudson et al., 1987a; Kristjansson and Alfredsson, 1983; Pask-Hughes and Williams, 1977). Extensive studies were also made of many New Zealand isolates, (Hudson et al., 1987a) and the validly named strain Thermus filiformis (Hudson et al., 1987b).

Microbial transformations of steroids-XII. Progesterone hydroxylation profiles are modulated by post-translational modification of an electron transfer protein in Streptomyces roseochromogenes

When Streptomyces roseochromogenes strain 10984 was incubated with exogenous progesterone for 25 h the major monohydroxylated metabolite, 16alpha-hydroxyprogesterone was produced in 3.6 fold excess to the minor metabolite 2beta,16alpha-dihydroxyprogesterone. In a reconstituted system containing highly purified progesterone 16alpha-hydroxylase cytochrome P-450, and electron transfer proteins ferredoxin-like redoxin (roseoredoxin) and redoxin reductase (roseoredoxin reductase), both metabolites were produced but in a 10:1 ratio. When S. roseochromogenes was pre-incubated for 8 h with 0.32 mM progesterone and the purified components of the hydroxylase system incubated as before, the ratio of 16alpha-hydroxyprogesterone to 2beta,16alpha-dihydroxyprogesterone produced decreased to 2.8:1, virtually identical to the ratio in whole cell transformations. Reconstitution assays containing all combinations of hydroxylase proteins purified from progesterone pre-incubated and control cells showed that the roseoredoxin was solely responsible for the observed changes in in vitro metabolite ratios. The fact that the lower 16alpha-hydroxyprogesterone to 2beta,16alpha-dihydroxyprogesterone ratio was also obtained when S. roseochromogenes was exposed to 0.335 mM cycloheximide for 8 h prior to the progesterone pre-incubation, pointed to post-translation modification of the roseoredoxin. Separation of two isoforms of roseoredoxin by isoelectric focusing supported this proposition.

Microbial transformation of steroids—IX. Purification of progesterone hydroxylase cytochrome P-450 from Phycomyces blakesleeanus

Progesterone hydroxylase cytochrome P-450 was purified to homogeneity from Phycomyces blakesleeanus microsomes by a four step procedure. An M(r) value of 60,000 was determined for this protein by SDS-PAGE. The DEAE-cellulose and Blue-1 MIMETIC affinity fractions gave major peaks at 452 nm in a dithionite-reduced, carbon monoxide, difference spectrum. NaIO4-dependent progesterone hydroxylation was obtained by the pure enzyme without NADPH and NADPH-cytochrome P-450 reductase. NADPH-dependent hydroxylation required the addition of other Phycomyces microsomal proteins present in the Blue-1 fraction.

Physiology and Metabolism of Thermus

The metabolism of thermophilic bacteria was extensively reviewed in 1979 by Ljungdahl. More recent books, monographs, and reviews that bear on the physiology of Thermus include those of Gould and Corry (1986), Brock (1986), Herbert and Codd (1986), Bergquist et al. (1987), Krulwich and Ivey (1990), Edwards (1990), Kristjansson (1992), and Herbert and Sharp (1992).

Sodium-stimulated transport of glutamate by Thermus thermophilus strain B

SUMMARY: Thermus thermophilus B is one of the many Thermus strains able to utilize glutamate as a sole source of carbon. Sodium is an obligate requirement for glutamate transport by washed whole cells, but the affinity for sodium (K m = 23 mM) is low. At pH 7.6, uptake of glutamate is rapid in 50 mM-sodium at 65 °C and obeys saturation kinetics with an apparent K m of 0.23 μ;M-glutamate and a V max of 12 nmol glutamate min-1 (mg protein)-1. The transport system is insensitive to osmotic shock and is specific for glutamate, with both the L- and D-isomers being transported. Uptake is very sensitive to inhibitors that collapse the membrane potential (Δψ) or the chemical gradient of sodium ions (ΔpNa), but a transmembrane pH gradient (ΔpH) plays no role in the transport of glutamate. These results are therefore consistent with a membrane sodium/glutamate symport system.

Isoschizomers of the restriction endonuclease Taql (T/CGA) requiring different metal ion concentrations and having a range of thermal stabilities from Thermus species from different continents

One-hundred-and-fifty-two isolates of the genus Thermus, collected from hot springs on four continents, were screened for evidence of the presence of the thermophilic Type II restriction endonuclease TaqI (T/CGA). The presence of isoschizomers of TaqI in 27 of the isolates, originating from hot springs in New Zealand, Iceland, USA, Japan, mainland Portugal and the island of São Miguel in the Azores, is reported. Six of the TaqI-containing isolates from diverse geographical locations, identified by means of DNA/DNA homology and 16S rRNA sequence alignment as belonging to the Thermus species T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus and T. brockianus, were selected for comparative studies. The TaqI isoschizomers from each of the six isolates were partially purified. They differed in their magnesium ion requirements, isoelectric points, subunit molecular masses and thermal stability.