Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis.

We initiated a study of pIM13, a multicopy, macrolide-lincosamide-streptogramin B (MLS) plasmid first isolated from a strain of Bacillus subtilis and described by Mahler and Halvorson (J. Gen. Microbiol. 120:259-263, 1980). The copy number of this plasmid was about 200 in B. subtilis and 30 in Staphylococcus aureus. The MLS resistance determinant of pIM13 was shown to be highly homologous to ermC, an inducible element on the S. aureus plasmid pE194. The product of the pIM13 determinant was similar in size to that of ermC and immunologically cross-reactive with it. The MLS resistance of pIM13 was expressed constitutively. The complete base sequence of pIM13 is presented. The plasmid consisted of 2,246 base pairs and contained two open reading frames that specified products identified in minicell extracts. One was a protein of 16,000 molecular weight, possibly required for replication. The second was the 29,000-molecular-weight MLS resistance methylase. The regulatory region responsible for ermC inducibility was missing from pIM13, explaining its constitutivity. The remainder of the pIM13 MLS determinant was nearly identical to ermC. The ends of the region of homology between pIM13 and pE194 were associated with hyphenated dyad symmetries. A segment partially homologous to one of these termini on pIM13 and also associated with a dyad was found in pUB110 near the end of a region of homology between that plasmid and pBC16. The entire sequence of pIM13 was highly homologous to that of pE5, an inducible MLS resistance plasmid from S. aureus that differs from pIM13 in copy control.     

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.     

A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).     

Fatty-acid biosynthesis in a branched-chain alpha-keto acid dehydrogenase mutant of Streptomyces avermitilis

Fatty-acid biosynthesis by a branched-chain alpha-keto acid dehydrogenase (bkd) mutant of Streptomyces avermitilis was analyzed. This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant, was shown not to have an obligate growth requirement for these precursors. The bkd mutant produced only straight-chain fatty acids (SCFAs) with membrane fluidity provided entirely by unsaturated fatty acids (UFAs), the levels of which increased dramatically compared to the wild-type strain. The levels of UFAs increased in both the wild-type and bkd mutant strains as the growth temperature was lowered from 37 degrees C to 24 degrees C, suggesting that a regulatory mechanism exists to alter the proportion of UFAs in response either to a loss of BCFA biosynthesis, or a decreased growth temperature. No evidence of a regulatory mechanism for BCFAs was observed, as the types of these fatty acids, which contribute significantly to membrane fluidity, did not alter when the wild-type S. avermitilis was grown at different temperatures. The principal UFA produced by S. avermitilis was shown to be delta 9-hexadecenoate, the same fatty acid produced by Escherichia coli. This observation, and the inability of S. avermitilis to convert exogenous labeled palmitate to the corresponding UFA, was shown to be consistent with an anaerobic pathway for UFA biosynthesis. Incorporation studies with the S. avermitilis bkd mutant demonstrated that the fatty acid synthase has a remarkably broad substrate specificity and is able to process a wide range of exogenous branched chain carboxylic acids into unusual BCFAs.     

Processing of naked 45-S ribosomal RNA precursor in vitro by an RNA-associated endoribonuclease

A processing endoribonuclease was isolated from the cytoplasm of chick embryos. The enzyme was easily obtained using an RNA extraction procedure based on a mild deproteinization with Sarkosyl and cold phenol/chloroform. This technique assured the recovery of several proteins and the endoribonuclease in association with the RNA. It was demonstrated that this endoribonuclease was capable of promoting, in vitro, a precise processing of naked 45-S ribosomal RNA precursor to molecules resembling the intermediates as well as the 28-S and 18-S cytoplasmic RNAs found in vivo. The presence of magnesium ions was required for the correct processing function of the enzyme. In addition, under the same conditions, the mature ribosomal RNA substrates were degraded at a slower rate by this RNA-associated RNase. It was possible to fractionate the enzymatic preparation into two different populations by means of a sucrose gradient: one associated and the other partially free of an RNA component. The effect of the intrinsic RNA associated with the endoribonuclease on the enzymatic activity was tested by analyzing both the enzymatic populations and the total enzymatic preparation treated with pronase or with immobilized pancreatic RNase. In all cases in which the RNA component was present, the enzyme showed processing activity. On the other hand, when the RNA component was absent or at least partially degraded the enzyme proved to be more active in processing precursor molecules and in promoting extensive degradation of mature RNA species. Although the presence of RNA in association with the enzyme was demonstrated, its role in the regulation of the enzymatic activity is yet not clear.