Evaluation of the antibiotic biosynthetic potential of the genus Amycolatopsis and description of Amycolatopsis circi sp. nov., Amycolatopsis equina sp. nov. and Amycolatopsis hippodromi sp. nov.

AIMS: To describe three new Amycolatopsis strains and assess the antibiotic biosynthetic potential of the genus. METHODS AND RESULTS: Three strains, designated S1·3(T) , S3·6(T) and SE(8)3(T) , belonging to the genus Amycolatopsis were isolated and found to cluster together by 16S rRNA and gyrB gene-based phylogenetic analysis. Genetic distance values, based on the gyrB gene, were calculated between the strains and their closest relatives and were all above the threshold value of 0·02 that has been proposed to distinguish Amycolatopsis type strains. DNA-DNA hybridization experiments against related type strains confirmed that strain S3·6(T) represents a unique genomic species. Strain S3·6(T) was also found to be distinct from strains S1·3(T) and SE(8)3(T) , the latter two of which were also shown to be distinct from each other. Antibiotic biosynthetic genes were identified from multiple Amycolatopsis strains, and their presence was found to be phylogenetically associated. CONCLUSIONS: The data presented in this study indicate that strains S1·3(T) , SE(8)3(T) and S3·6(T) belong to three novel species, for which the names Amycolatopsis circi sp. nov. (= DSM 45561(T) = NRRL B-24841(T) ), Amycolatopsis equina sp. nov. (= DSM 45563(T) = NRRL B-24842(T) ) and Amycolatopsis hippodromi sp. nov. (= DSM 45562(T) = NRRL B-24843(T) ) are proposed. SIGNIFICANCE AND IMPACT OF THE STUDY: Three new species of Amycolatopsis are described, and the knowledge of the antibiotic biosynthetic potential of the genus has been extended.

PCR screening reveals unexpected antibiotic biosynthetic potential in Amycolatopsis sp. strain UM16.

AIMS: To assess the antibiotic biosynthetic potential of Amycolatopsis sp. strain UM16 and eight other Amycolatopsis species. METHODS AND RESULTS: Amycolatopsis genomic DNA was screened by PCR for the glycopeptide, Type-II (aromatic) polyketide and ansamycin biosynthetic gene clusters. Amycolatopsis sp. strain UM16, which exhibits weak antitubercular activity, was shown to have the glycopeptide oxyB gene and the Type-II (aromatic) polyketide-synthase KSalpha-KSbeta tandem gene pair, but not the AHBA synthase gene. The ristocetin (glycopeptide) producer, Amycolatopsis lurida NRRL 2430(T), was shown to have the oxyB gene and the Type-II polyketide-synthase KSalpha-KSbeta tandem gene pair. Amycolatopsis alba NRRL 18532(T) was shown to have the glycopeptide oxyB gene and the AHBA synthase gene. Phylogenetic analyses using Amycolatopsis oxyB and KSalpha-KSbeta gene sequences were conducted. CONCLUSIONS: Amycolatopsis sp. strain UM16 appears to have the biosynthetic potential to produce glycopeptide and Type-II polyketide antibiotics, but not ansamycins. The potential to synthesize aromatic polyketides may be more widely distributed in Amycolatopsis than is currently recognized. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR screening is a very useful tool for rapidly identifying the biosynthetic potential of an antibiotic-producing actinomycete isolate. Advanced knowledge of the type of antibiotic(s) produced will allow appropriate methods to be selected for antibiotic purification.

The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral.

The quaternary structure of the cyanide dihydratase from Pseudomonas stutzeri AK61 was determined by negative stain electron microscopy and three-dimensional reconstruction using the single particle technique. The structure is a spiral comprising 14 subunits with 2-fold symmetry. Interactions across the groove cause a decrease in the radius of the spiral at the ends and the resulting steric hindrance prevents the addition of further subunits. Similarity to two members of the nitrilase superfamily, the Nit domain of NitFhit and N-carbamyl-D-amino acid amidohydrolase, enabled the construction of a partial atomic model that could be unambiguously fitted to the stain envelope. The model suggests that interactions involving two significant insertions in the sequence relative to these structures leads to the left-handed spiral assembly.

Novel method for rapid measurement of growth of mycobacteria in detergent-free media.

We describe a novel, rapid, and inexpensive method for the measurement of growth of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium smegmatis in the presence or absence of detergent. The method, which employs hot NaOH treatment of mycobacterial cells to release total cellular protein, compares favorably with other methods for monitoring mycobacterial growth but is particularly useful for heavily clumped cultures grown in defined minimal medium.

Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1.

A cyanide-degrading enzyme from Bacillus pumilus C1 has been purified and characterized. This enzyme consisted of three polypeptides of 45.6, 44.6, and 41.2 kDa; the molecular mass by gel filtration was 417 kDa. Electron microscopy revealed a multimeric, rod-shaped protein approximately 9 by 50 nm. Cyanide was rapidly degraded to formate and ammonia. Enzyme activity was optimal at 37 degrees C and pH 7.8 to 8.0. Activity was enhanced by Sc3+, Cr3+, Fe3+, and Tb3+; enhancement was independent of metal ion concentration at concentrations above 5 microM. Reversible enhancement of enzymatic activity by azide was maximal at 4.5 mM azide and increased with time. No activity was recorded with the cyanide substrate analogs CNO-, SCN-, CH3CN, and N3- and the possible degradation intermediate HCONH2. Kinetic studies indicated a Km of 2.56 +/- 0.48 mM for cyanide and a Vmax of 88.03 +/- 4.67 mmol of cyanide per min/mg/liter. The Km increased approximately twofold in the presence of 10 microM Cr3+ to 5.28 +/- 0.38 mM for cyanide, and the Vmax increased to 197.11 +/- 8.51 mmol of cyanide per min/mg/liter. We propose naming this enzyme cyanide dihydratase.

Segmental stenosis of the renal artery: pattern recognition of tardus and parvus abnormalities with duplex sonography.

Segmental renal artery branches within the renal sinus were prospectively evaluated with color Doppler imaging and pulsed-Doppler spectral analysis in 56 patients before angiography. Waveforms were evaluated for the tardus and parvus abnormalities of prolonged acceleration time, diminished acceleration index, and loss of the normal early systolic compliance peak/reflective-wave complex (ESP). Findings obtained with these parameters were compared with the subsequent findings on angiograms to ascertain their efficacy in detection of hemodynamically significant (greater than or equal to 60%) renal arterial stenosis (RAS), which was present in 32 kidneys in 26 patients. Simple pattern-recognition analysis of ESP proved to be the best of the three parameters. Loss of ESP enabled identification of RAS with 95% sensitivity, 97% specificity, a 92% positive predictive value, a 98% negative predictive value, a 96% overall accuracy. On the basis of the high technical success rate, high sensitivity and specificity, and short examination time, waveform analysis for detection of tardus-parvus abnormalities, especially loss of ESP, of the segmental artery is recommended as an alternative to direct examination of the main renal arteries for evaluation of RAS.

An efficient cyanide-degrading Bacillus pumilus strain.

A Gram-positive, aerobic, endospore-forming bacterium was isolated by an enrichment technique for the ability to degrade cyanide and was identified as a Bacillus pumilus strain. The bacterium rapidly degraded 100 mg l-1 of free cyanide in the absence of added inorganic and organic substances. The ability to degrade cyanide was linked to the growth phase and was not exhibited before late exponential/early stationary phase. Cyanide-degrading activity could not be induced before this time by the addition of 20 mg cyanide l-1. Production of the cyanide-degrading activity required 0.01 mg Mn2+ l-1 and did not occur at Mn2+ concentrations below 0.002 mg l-1. Cyanide-degrading activity was intracellular and cell-free extracts rapidly degraded cyanide.