The pentacyclic hopanoids, a class of eubacterial lipids, are synthesized by squalene-hopene cyclase and side chain-elongating enzymes. With the aid of DNA probes based on the amino-terminal sequence of purified squalene-hopene cyclase from Bacillus acidocaldarius, clones of Escherichia coi that express this enzy'me in the cytoplasmic membrane were isolated. According to the DNA sequence, the cyclase contained 627 amino acids with a molecular mass of 69,473 Da. A high percentage of the amino acids were basic. No significant similarity to existing sequenced proteins was found.Hopanoids are a class of pentacyclic triterpenoids that occur in all crude oils (18) and are also widespread eubacterial lipids (19,24). They have a structure similar to that of sterols (18) and condense phospholipids in mnodel membrane systems (2, 22) and supposedly in cellular membranes. Hopanoids are more effectively produced at higher growth temperatures by Bacillus acidocaldarius (21), Zymomonas mobilis (26), and Rhodospirillum acidophila (12) and at high ethanol levels by Z. mobilis (6). In Mycoplasma mycoides they are able to replace sterols (11). It was recently found that the N2-fixing symbiont Frankia sp. contains high levels of hopanoids (3).In contrast to sterol biosynthesis, the biosynthesis of hopanoids is independent of molecular oxygen as a substrate. Hopanoids are cyclized directly from squalene by squalene-hopene cyclase (28), which may be homologous to sterol cyclases and other triterpenoid cyclases from eucaryotic cells, synthesizing by a similar reaction a similar product with analogous membrane properties. It would therefore be of interest to compare the amino acid sequence of hopene cyclase with those of sterol cyclases, thereby increasing our understanding of the recruitment of these enzymes. If homologies are found, in vitro mutagenesis studies would be of value in understanding the evolution of sterol cyclases. So far no triterpenoid cyclase has been sequenced. As a first step in this direction, the squalene-hopene cyclase gene from B. acidocaldarius was cloned, expressed, and sequenced; this enzyme appears to represent a new gene family. MATERIALS AND METHODSBacterial strains and plasmids. The source of squalenehopene cyclase and genomic DNA was B. acidocaldarius ATCC 27009. Escherichia coli BMH71-18 (14) was used for transformation. Plasmid pUC19 was described previously (31).Culture conditions. B. acidocaldarius was grown on sporulation medium at pH 3 and 60°C (8). E. coli was grown at 37°C in LB medium (GIBCO, Neuisenburg, Germany) or on LB plates, both containing 30 mg of ampicillin per liter, when plasmid-containing cells were selected. * Corresponding author. DNA preparation. B. acidocaldarius grown to the early exponential phase was harvested by centrifugation and washed with 150 mM NaCl-10 mM EDTA (pH 8.0) (SE). The wet cells (1 g) were resuspended in 10 ml of SE. After lysozyme (1 mg/ml) was added, the suspension was incubated at 37°C. After 1 h, the cells were lysed by the addition of 200 ,ul of 20% (wt/v...

Aims: To investigate the inhibitory and lethal effects of triclosan against several micro-organisms at different stages of their phase of population growth. Methods and Results: Triclosan minimum inhibitory concentrations against several test organisms were determined in broth and agar using standard protocols. The bisphenol effect on bacterial population growth kinetics was studied using the Bioscreen C microbial growth analyser. Finally, the efficacy of triclosan on phases of bacterial growth was determined using a standard suspension test. The duration of the lag phase for all micro-organisms tested was increased by bisphenol in a concentration-dependent manner. The population growth kinetics of the micro-organisms was also altered after biocide exposure. At higher concentrations, triclosan was bactericidal regardless of their phase of population growth, although population in stationary phase and particularly, washed suspensions, were more resilient to the lethality of triclosan. This lethal activity was concentration and contact time dependent, and in some instances, bactericidal activity of bisphenol was observed within 15 s. Conclusions: Low concentrations of triclosan affected the growth of several bacteria, while higher concentrations were bactericidal regardless of the bacterial phase of population growth. Significance and Impact of the Study: Here, we presented clear evidence that the interaction of triclosan with the bacterial cell is complex and its lethality cannot be explained solely by the inhibition of metabolic pathways such as the enoyl acyl-reductase. However, the inhibition of such pathways cannot be ruled out as part of the lethal mechanism of the bisphenol at a low bactericidal concentration.

This study shows that the inhibition of fatty acid biosynthesis by the bisphenol might be involved in its growth-inhibitory action and that other mechanisms are involved in its lethal effect. In addition, although microorganisms with a high triclosan MIC were still susceptible to the inhibitory effect of the bisphenol on fatty acid biosynthesis, a higher concentration of the compound was required. This suggested that triclosan bioavailability was different in these strains.

No abstract

The squalene-hopene cyclase from Bacillus acidocaldarius cytoplasmic membrane, was purified to homogeneity by solubilization with Triton X-100, chromatography on DEAE-cellulose, phenyl Sepharose and two gel-filtration columns. The enzyme monomer had a molecular mass of 75 kDa. The sequence of the first 23 amino acids was determined by Edman degradation. The enzyme activity was efficiently inhibited by n-alkyldimethylammonium halides with alkyl chain lengths between 12 and 18 C atoms. Inhibition was also observed with (5-hydroxycarvacry1)trimethylammonium chloride I-piperidine carboxylate, dodecyldimethylamine N-oxide, azasqualene and farnesol. Competitive inhibition with dodecyltrimethylammonium bromide, (5-hydroxycarvacry1)trimethylammonium chloride I-piperidine carboxylate and dodecyldimethylamine N-oxide was demonstrated by Lineweaver-Burk plots.Tetracyclic and pentacyclic triterpenoids are widespread metabolites. Derivatives of hopan, lupan, fernan, olean, ursan, gammaceran and different sterols occur in plants [l]. Sterols (e.g. cholesterol, sitosterol, stigmasterol and ergosterol) are found in animals and plants as membrane lipids [l]. In contrast, the occurrence and especially the synthesis of sterols is very rare in bacteria [2]. However, hopanoids, exclusively those with an elongated side chain, are frequently found in bacterial membranes [2], and have a similar function to sterols, condensing phospholipids above the transition temperature in model membrane systems [3, 41 and presumably also in biological membranes [5].Hopanoids are cyclized from squalene [6], whereas sterols are synthesized from squalene epoxide [7, 81. The molecular oxygen dependence for the synthesis of squalene epoxide and for the demethylation reactions in membrane sterol synthesis, support the hypothesis that sterol biosynthesis is a modern metabolic evolutionary trait in comparison to hopanoid biosynthesis [S -101.We are interested in the question of homology between the squalene and squalene-epoxide cyclases and also in the exact amino acid alterations necessary for the postulated evolution of squalene cyclase to a modern squalene-epoxide cyclase. This issue is now possible since another group has purified squalene-epoxide cyclases for amyrins and cycloartenol from plants [I1 -131 and therefore a basis is laid for a future sequence comparison of the different cyclase genes.In this paper we describe the purification, N-terminal amino acid sequence, and the effect of new inhibitors of [4, 8, 12, 17, 21-3H]Squalene was purchased from New England Nuclear (Dreieich, FRG); unlabeled squalene, (5-hydroxycarvacry1)trimethylammonium chloride 1-piperidine carboxylate (AM0 161 8) dodecyldimethylamine N-oxide (DodMe2NO), dodecyltrimethylammonium bromide (DodMe3NBr), taurodeoxycholate, Chaps, N-lauroylsarcosine and trypsin inhibitor from Sigma; DEAE-cellulose, Sevacel SH-23 and Triton X-100 from Serva; phenyl-Sepharose CL4B, Sephacryl S-300 and Sephacryl S-500 from Pharmacia; sodium taurocholate from Aldrich. MATERIALS AND METHODS Chemical...