Squalene-hopene cyclase from Methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids.

We report the cloning and characterisation of the Methylococcus capsulatus shc gene, which encodes the squalene-hopene cyclase (SHC). This enzyme catalyses the complex cyclization of squalene to the pentacyclic triterpene skeleton of hopanoids and represents the key reaction in this biosynthesis. Using a combination of PCR amplification and DNA hybridization, two overlapping 2.6 kb PstI and 3.3 kb SalI DNA fragments were cloned bearing a 1962 bp open reading frame encoding a 74 kDa protein with 654 amino acids and a predicted isoelectric point at about pH 6.3. The deduced amino acid sequence of the M. capsulatus shc gene showed significant similarity to known prokaryotic SHCs and to a lesser degree to the related eukaryotic oxidosqualene cyclases (OSCs). Like other triterpene cyclases, the M. capsulatus SHC contains seven so-called QW-motifs as well as an aspartate-rich domain. The recombinant M. capsulatus SHC was expressed in Escherichia coli and in vitro activity of the recombinant cyclase was demonstrated using crude cell-free lysate or solubilized membrane preparation. The cyclization products hop-22-ene and hopan-22-ol (diplopterol) were identified by GC and GC-MS.

Phase I/II study of combined granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor administration for the mobilization of hematopoietic progenitor cells.

PURPOSE: To study the toxicity and efficacy of combined granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) administration for mobilization of hematopoietic progenitor cells (HPCs). MATERIALS AND METHODS: Cohorts of a minimum of five patients each were treated subcutaneously as follows: G-CSF 5 micrograms/kg on days 1 to 12 and GM-CSF at .5, 1, or 5 micrograms/kg on days 7 to 12 (cohorts 1, 2, and 3); GM-CSF 5 micrograms/kg on days 1 to 12 and G-CSF 5 micrograms/kg on days 7 to 12 (cohort 4); and G-CSF and GM-CSF 5 micrograms/kg each on days 1 to 12 (cohort 5). Ten-liter aphereses were performed on days 1 (baseline, pre-CSF), 5, 7, 11, and 13. Colony assays for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) were performed on each harvest. RESULTS: The principal toxicities were myalgias, bone pain, fever, nausea, and mild thrombocytopenia, but none was dose-limiting. Four days of treatment with either G-CSF or GM-CSF resulted in dramatic and sustained increases in the numbers of CFU-GM per kilogram collected per harvest that represented 35.6 +/- 8.9- and 33.7 +/- 13.0-fold increases over baseline, respectively. This increment was attributable both to increased numbers of mononuclear cells collected per 10-L apheresis and to increased concentrations of progenitors within each collection. The administration of G-CSF to patients already receiving GM-CSF (cohort 4) caused the HPC content to surge to nearly 80-fold the baseline (P = .024); the reverse sequence, ie, the addition of GM-CSF to G-CSF, was less effective. The CFU-GM content of the baseline aphereses correlated with the maximal mobilization achieved (r = .74, P = .001). CONCLUSION: Combined G-CSF and GM-CSF administration effectively and predictably mobilizes HPCs and facilitates apheresis.

High-dose chemotherapy, autologous bone marrow or stem cell transplantation and post-transplant consolidation chemotherapy in patients with advanced breast cancer.

This study was designed to determine the complete response (CR) rate, event-free survival (EFS) and overall survival (OS) in patients with metastatic breast cancer treated with an adriamycin-based induction regimen, high-dose chemotherapy consisting of cyclophosphamide and thiotepa with autologous bone marrow or stem cell reinfusion, followed by post-transplant 5-fluorouracil and cisplatin. Forty-eight consecutive patients were enrolled and 35 received two to four cycles of a cytoreductive chemotherapy regimen followed by high-dose chemotherapy which included cyclophosphamide and thiotepa. Thirty-three patients with non-progressive disease received at least one cycle of post-transplant 5-fluorouracil and cisplatin. Fifty percent of patients with evaluable disease responded to induction chemotherapy. Three of the 34 patients (9%) evaluable for response to high-dose chemotherapy achieved CR, eight (24%) achieved partial response (PR), 12 (35%) had stable disease (SD) and 11 (32%) had progressive disease (PD). The median time to neutrophil recovery was 11.5 days (range, 8 to 40 days) post- reinfusion. The median time to platelet independence was 14.5 days (range, 8 to 44 days). The median follow-up is 24.5 months (range, 1 to 96 months). The actuarial probability of EFS for all patients is 17% at 4 years. The EFS for patients receiving all four cycles of post-transplant chemotherapy is 27% at 4 years, compared to 36% at 1 year for patients not receiving any post-transplant chemotherapy. Ten of the 48 patients (21%) are alive, and seven of these (15%) have no evidence of disease. High-dose chemotherapy with autologous bone marrow or peripheral blood-derived stem cell transplantation followed by post-transplant consolidation chemotherapy in patients with metastatic breast cancer results in a proportion of patients without evidence of disease at 4 years.

The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath).

Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 degrees C resulted in changes to the whole cell lipid constituents. As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the delta 9, delta 10 and delta 11 C16:1 double bond positional isomers changed. Methyl sterol content also increased as the growth temperature was lowered. The highest amounts of methyl sterol were found in 30 degrees C cells and the lowest in 50 degrees C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively). The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism.

Evidence for the synthesis of the multi-positional isomers of monounsaturated fatty acid in Methylococcus capsusatus by the anaerobic pathway.

The biosynthesis of the positional isomers of the monounsaturated fatty acids of Methylococcus capsulatus (Bath) has been investigated by studying the incorporation of [2-14C]malonyl CoA into long-chain fatty acids in vitro. The major unsaturated products were delta 9 16 : 1 and delta 11 18 : 1; however, delta 8, delta 10, and delta 11, 16 : 1, as well as, delta 10, delta 12 and delta 13 18 : 1 were also synthesized. The exclusion of O2 from the reaction vessel did not affect the synthesis of unsaturated fatty acids or the double bonds positions. Cerulenin inhibited the synthesis of unsaturated fatty acid more than saturated fatty acid. The use of both [1-14C] octanoate and [1-14C] decanote as substrate resulted in the synthesis of long-chain fatty acids, however, unsaturates were only synthesized from octanoate. These results imply that the unique positional isomers of M. capsulatus are not synthesized by an aerobic mechanism.

Molecular and lipid biomarker analysis of a gypsum-hosted endoevaporitic microbial community.

Modern evaporitic microbial ecosystems are important analogs for understanding the record of earliest life on Earth. Although mineral-depositing shallow-marine environments were prevalent during the Precambrian, few such environments are now available today for study. We investigated the molecular and lipid biomarker composition of an endoevaporitic gypsarenite microbial mat community in Guerrero Negro, Mexico. The 16S ribosomal RNA gene-based phylogenetic analyses of this mat corroborate prior observations indicating that characteristic layered microbial communities colonize gypsum deposits world-wide despite considerable textural and morphological variability. Membrane fatty acid analysis of the surface tan/orange and lower green mat crust layers indicated cell densities of 1.6 × 10(9) and 4.2 × 10(9)  cells cm(-3) , respectively. Several biomarker fatty acids, ∆7,10-hexadecadienoic, iso-heptadecenoic, 10-methylhexadecanoic, and a ∆12-methyloctadecenoic, correlated well with distributions of Euhalothece, Stenotrophomonas, Desulfohalobium, and Rhodobacterales, respectively, revealed by the phylogenetic analyses. Chlorophyll (Chl) a and cyanobacterial phylotypes were present at all depths in the mat. Bacteriochlorophyl (Bchl) a and Bchl c were first detected in the oxic-anoxic transition zone and increased with depth. A series of monomethylalkanes (MMA), 8-methylhexadecane, 8-methylheptadecane, and 9-methyloctadecane were present in the surface crust but increased in abundance in the lower anoxic layers. The MMA structures are similar to those identified previously in cultures of the marine Chloroflexus-like organism 'Candidatus Chlorothrix halophila' gen. nov., sp. nov., and may represent the Bchl c community. Novel 3-methylhopanoids were identified in cultures of marine purple non-sulfur bacteria and serve as a probable biomarker for this group in the lower anoxic purple and olive-black layers. Together microbial culture and environmental analyses support novel sources for lipid biomarkers in gypsum crust mats.

Characterization and spatial distribution of methanogens and methanogenic biosignatures in hypersaline microbial mats of Baja California.

Well-developed hypersaline cyanobacterial mats from Guerrero Negro, Baja California Sur, sustain active methanogenesis in the presence of high rates of sulfate reduction. Very little is known about the diversity and distribution of the microorganisms responsible for methane production in these unique ecosystems. Applying a combination of 16S rRNA and metabolic gene surveys, fluorescence in situ hybridization, and lipid biomarker analysis, we characterized the diversity and spatial relationships of methanogens and other archaea in the mat incubation experiments stimulated with methanogenic substrates. The phylogenetic and chemotaxonomic diversity established within mat microcosms was compared with the archaeal diversity and lipid biomarker profiles associated with different depth horizons in the in situ mat. Both archaeal 16S rRNA and methyl coenzyme M reductase gene (mcrA) analysis revealed an enrichment of diverse methanogens belonging to the Methanosarcinales in response to trimethylamine addition. Corresponding with DNA-based detection methods, an increase in lipid biomarkers commonly synthesized by methanogenic archaea was observed, including archaeol and sn-2-hydroxyarchaeol polar lipids, and the free, irregular acyclic isoprenoids, 2,6,10,15,19-pentamethylicosene (PMI) and 2,6,11,15-tetramethylhexadecane (crocetane). Hydrogen enrichment of a novel putative archaeal polar C(30) isoprenoid, a dehydrosqualane, was also documented. Both DNA and lipid biomarker evidence indicate a shift in the dominant methanogenic genera corresponding with depth in the mat. Specifically, incubations of surface layers near the photic zone predominantly supported Methanolobus spp. and PMI, while Methanococcoides and hydroxyarchaeol were preferentially recovered from microcosms of unconsolidated sediments underlying the mat. Together, this work supports the existence of small but robust methylotrophic methanogen assemblages that are vertically stratified within the benthic hypersaline mat and can be distinguished by both their DNA signatures and unique isoprenoid biomarkers.

Lipid biomarker and phylogenetic analyses to reveal archaeal biodiversity and distribution in hypersaline microbial mat and underlying sediment.

This study has utilized the tools of lipid biomarker chemistry and molecular phylogenetic analyses to assess the archaeal contribution to diversity and abundance within a microbial mat and underlying sediment from a hypersaline lagoon in Baja California. Based on abundance of ether-linked isoprenoids, archaea made up from 1 to 4% of the cell numbers throughout the upper 100 mm of mat and sediment core. Below this depth archaeal lipid was two times more abundant than bacterial. Archaeol was the primary archaeal lipid in all layers. Relatively small amounts of caldarchaeol (dibiphytanyl glyceroltetraether) were present at most depths with phytanyl to biphytanyl molar ratios lowest (approximately 10 : 1) in the 4-17 mm and 100-130 mm horizons, and highest (132 : 1) in the surface 0-2 mm. Lipids with cyclic biphytanyl cores were only detected below 100 mm. A novel polar lipid containing a C(30) isoprenoid (squalane) moiety was isolated from the upper anoxic portion of the core and partially characterized. Hydrocarbon biomarker lipids included pentamethylicosane (2-10 mm) and crocetane (primarily below 10 mm). Archaeal molecular diversity varied somewhat with depth. With the exception of samples at 0-2 mm and 35-65 mm, Thermoplasmatales of marine benthic group D dominated clone libraries. A significant number of phylotypes representing the Crenarchaeota from marine benthic group B were generally present below 17 mm and dominated the 35-65 mm sample. Halobacteriaceae family made up 80% of the clone library of the surface 2 mm, and consisted primarily of sequences affiliated with the haloalkaliphilic Natronomonas pharaonis.

Measurement of hydroxyl radical-generated methane sulfinic acid by high-performance liquid chromatography and electrochemical detection.

A liquid chromatographic (HPLC) method has been developed for direct quantitative determination of methane sulfinic acid (MSA) produced by hydroxyl radical oxidation of dimethyl sulfoxide. This method measures MSA directly by HPLC separation and electrochemical oxidation following rapid extraction from intact cells. MSA can be measured in tissue extracts at 0.04 nmol (equivalent to 2 microM). Using this technique, MSA production in paraquat-treated bean leaves is demonstrated. When compared with the widely used dye-binding technique, this method simplifies the preparation of the extract by eliminating two steps required in the dye-binding method: removal of interfering lipophilic compounds and the derivitization (color reaction) of the MSA.

Identification of the methylhopanes in sediments and petroleum.

Three C31 methylhopanes have been prepared by partial synthesis from appropriate diplopterol precursors. 2 alpha-Methyldiplopterol (prepared from 22-hydroxyhopan-3-one), 2 beta-methyldiplopterol (isolated from Methylobacterium organophilum), and a mixture of diplopterol and 3 beta-methyldiplopterol (isolated from Methylococcus capsulatus) were each converted to the corresponding 17 alpha(H), 21 beta(H)-hopane. Comparison of these standards, using gas chromatography--mass spectrometry with multiple reaction monitoring, with the hopanoids from a variety of bitumens showed that all three C31 hydrocarbons may occur in sediments and that they are members of C28 and C30-C36 pseudohomologous series. 2 alpha-Methyl-17 alpha(H), 21 beta(H)-hopane, and 3 beta-methyl-17 alpha(H), 21 beta(H)-hopane are most commonly encountered in mature bitumens. 2 beta-Methyl-17 alpha(H), 21 beta(H)-hopane occurs in some immature bitumens, is much less abundant in others of intermediate maturity, and appears to be absent from mature samples. This, and the similarity of the distribution patterns of homohopane and methylhomohopane isomers, indicates that the common sedimentary methylhopanes are probably derived from biogenic precursors via diagenetic processes analogous to those which give rise to hopanes. In the case of the 2 alpha-methyl series, common to petroleum and mature sediments, derivation from the 2 beta-methyl hopanoids found in certain bacteria implies a maturity-related change in the configuration at C-2.

Methyl sterol and cyclopropane fatty acid composition of Methylococcus capsulatus grown at low oxygen tensions.

Methylococcus capsulatus contained extensive intracytoplasmic membranes when grown in fed-batch cultures over a wide range of oxygen tensions (0.1 to 10.6%, vol/vol) and at a constant methane level. Although the biomass decreased as oxygen levels were lowered, consistently high amounts of phospholipid and methyl sterol were synthesized. The greatest amounts of sterol and phospholipid were found in cells grown between 0.5 and 1.1% oxygen (7.2 and 203 mumol/g [dry weight], respectively). While sterol was still synthesized in significant amounts in cells grown at 0.1% oxygen, the major sterol product was the dimethyl form. Analysis by capillary gas chromatography-mass spectrophotometry showed that the phospholipid esterified fatty acids were predominantly 16:0 and 16:1 and that the hexadecenoates consisted of cis delta 9, delta 10, and delta 11 isomers. At low oxygen tensions, the presence of large amounts (25%) of cyclopropane fatty acids (cy 17:0) with the methylene groups at the delta 9, delta 10, and delta 11 positions was detected. Although the delta 9 monoenoic isomer was predominant, growth at low oxygen levels enhanced the synthesis of the delta 10 isomers of 16:1 and cy 17:0. As the oxygen level was increased, the amount of cyclopropanes decreased, such that only a trace of cy 17:0 could be detected in cells grown at 10.6% oxygen. Although M. capsulatus grew at very low oxygen tensions, this growth was accompanied by changes in the membrane lipids.

Toward better access to health insurance coverage for U.S. retirees in Mexico.

Many retirees from the United States of America have limited health insurance coverage while living in Mexico. Medicare and Medicaid benefits are not portable to other countries and Medigap (private insurance that supplements Medicare) is very limited. This causes economic and medical hardships and serves as a barrier to retirement to Mexico. Increasing numbers of U.S. retirees will be interested in moving to Mexico in the future because of the climate, the culture, and the lower cost of living. The numbers are increasing as a result of several factors such as aging "baby boomers" and the rapidly growing Mexican-origin population in the U.S.A. who are citizens or permanent residents but would like to return to their communities of origin after working in the U.S.A. There are several policy initiatives that could provide opportunities for improving health insurance coverage for these retirees that could be cost-effective.

Variations in the localization of acetyl-coenzyme A synthetase in aerobic yeast cells.

In cells of Saccharomyces cerevisiae growing aerobically for 24 hr, acetyl-coenzyme A synthetase [acetate: CoA ligase (AMP), EC 6.2.1.1] was localized principally in the microsomal fraction. On density gradients, the enzyme in such cells behaved as a low-density particle, readily separable from the soluble proteins. After 48 hr of incubation, the cells showed a bimodal distribution of enzyme, with most of the activity now sedimenting with the mitochondrial fraction and only a smaller amount with the microsomal fraction. By using density gradients, two forms of synthetase were obtained from these cells: one band denser and the other band less dense than the intact mitochondria. In all preparations containing synthetase activity, appreciable levels of phospholipids were also detected.

Cellular localization of acetyl-coenzyme A synthetase in yeast.

In cells of Saccharomyces cerevisiae grown with glucose in standing cultures, the microsomal fraction had the highest specific activity for acetyl-coenzyme A synthetase and contained the greatest fraction of the total activity regardless of when the cells were harvested during growth. The addition of acetate did not affect the distribution of the enzyme, nor did subsequent aeration of such cells in phosphate buffer even in the presence of glucose, acetate, or succinate. In cells grown aerobically, however, the microsomal fraction had the highest specific activity and the greatest fraction of the total activity only until the cells reached the stationary phase. After this time, most of the activity was associated with the mitochondrial fraction. Finally, 3 or 4 days after inoculation, this fraction appeared to lose most of the enzyme to the microsomal and soluble fractions. Chloramphenicol, at concentrations that interfered with respiration but not with fermentation, prevented the association of acetyl-coenzyme A synthetase with the mitochondrial fraction in aerated cells, but it did not appreciably affect the large increases in enzyme activity observed during aerobic incubation. Cells grown with glucose under strict anaerobic conditions contained barely detectable amounts of acetyl-coenzyme A synthetase.

Oxygen as a factor in eukaryote evolution: some effects of low levels of oxygen on Saccharomyces cerevisiae.

A comparative study of the effects of varying levels of oxygen on some of the metabolic functions of the primitive eukaryote, Saccharomyces cerevisiae, has shown that these cells are responsive to very low levels of oxygen: the level of palmitoyl-Co A desaturase was greatly enhanced by only 0.03% (v/v) oxygen. Similarly, an acetyl-CoA synthetase associated predominantly with anaerobic growth, was stimulated by as little as 0.1% oxygen, while an isoenzyme correlated with aerobic growth, was maximally active at much higher oxygen levels (greater than 1%). Closely following this latter pattern were three mitochondrial enzymes that attained maximal activity only under atmospheric levels of oxygen.

Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae.

A method is shown to be effective over a wide range of enzyme ratios for the simultaneous detection of the two isoenzymes of acetyl coenzyme A synthetase [acetate:coenzyme A ligase (AMP-forming); EC 6.2.1.1] in homogenates and cellular fractions of Saccharomyces cerevisiae. When this method was used, it was found that cells grown under anaerobic conditions contained only one variety of this enzyme, designated the nonaerobic synthetase, whereas cells grown with vigorous aeration contained principally the other, aerobic, synthetase. In cells grown as standing cultures (i.e., semi-aerobically), both enzymes were present and were found mainly in the extramitochondrial material of homogenates. When anaerobic cultures were aerated, the amount of aerobic enzyme increased steadily over a 24-h period, so that at the end of this time, aerated cells contained predominantly aerobic enzyme. During this same period, the amount of nonaerobic enzyme decreased. The percentage of aerobic enzyme that sedimented with the mitochondria increased steadily during this period of aeration, so that, at the end of 24 h of aeration, essentially all of the aerobic enzyme sedimented with the mitochondria. The nonaerobic enzyme was never found in this cellular compartment.

Oxygen requirements for formation and activity of the squalene epoxidase in Saccharomyces cerevisiae.

The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the "mitochondrial" fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the "microsomal" fraction. At 0.03% (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent Km for oxygen of 0.38% (vol/vol) was determined from a crude particulate preparation for the epoxidase.

Photooxidative reactions in chloroplast thylakoids. Evidence for a Fenton-type reaction promoted by superoxide or ascorbate.

A methyl viologen (MV)(*) mediated Mehler reaction was studied using Type C and D chloroplasts (thylakoids) from spinach. The extent of photooxidative reactions were measured as (a) rate of ethylene formation from methional oxidation indicating the production of oxygen radicals, and (b) rate of malondialdehyde (MDA) formation as a measure of lipid peroxidation. Without added ascorbate, 1 µM FerricEDTA increased ethylene formation by greater than 4-fold, but had no effect on MDA production. Ascorbate (1 mM) produced a tripling of ethylene while it reduced MDA formation in the presence of iron. Radical scavengers diethyldithiocarbamate (DDTC), formate, 1,4-diazabicyclo (2.2.2octane) (DABCO), inhibited ethylene formation. Using 0,4 M mannitol to scavenge hydroxyl radicals, the rates of ethylene formation were reduced 40 to 60% with or without 1 µM Fe(III) EDTA. The strong oxidant(s) not scavenged by mannitol are hypothesized to be either alkoxyl radicals from lipid peroxidation, or 'site specific' formation of hydroxyl radicals in a lipophillic environment not exposed to mannitol. Singlet oxygen does not appear to be a significant factor in this system. Catalase strongly inhibited both ethylene and MDA synthesis under all conditions; 1 mM ascorbate did not reverse this inhibition. However, the strong superoxide dismutase (SOD) inhibition of ethylene and MDA formation was completely reversed by 1 mM ascorbate. This suggests that superoxide was functioning as an iron reducing agent and that in its absence, ascorbate was similarly promoting oxidations. Therefore, these oxidative processes were dependent on the presence of H2O2 and a reducing agent, suggesting the involvement of a Fenton-type reaction.

Carbon isotopic fractionation in lipids from methanotrophic bacteria: relevance for interpretation of the geochemical record of biomarkers.

Experiments with cultured aerobic methane oxidising bacteria confirm that their biomarker lipids will be significantly depleted in 13C compared to the substrate. The methanotrophic bacteria Methylococcus capsulatus and Methylomonas methanica, grown on methane and using the RuMP cycle for carbon assimilation, show maximum 13C fractionation of approximately 30% in the resultant biomass. In M. capsulatus, the maximum fractionation is observed in the earliest part of the exponential growth stage and decreases to approximately 16% as cells approach stationary phase. This change may be associated with a shift from the particulate form to the soluble form of the methane monooxygenase enzyme. Less than maximum fractionation is observed when cells are grown with reduced methane availability. Biomass of M. capsulatus grown on methanol was depleted by 9% compared to the substrate. Additional strong 13C fractionation takes place during polyisoprenoid biosynthesis in methanotrophs. The delta 13C values of individual hopanoid and steroid biomarkers produced by these organisms were as much as l0% more negative than total biomass. In individual cultures, squalene was 13C-enriched by as much as 14% compared to the triterpane skeleton of bacteriohopaneaminopentol. Much of the isotopic dispersion in lipid metabolites could be attributed to shifts in their relative abundances, combined with an overall reduction in fractionation during the growth cycle. In cells grown on methanol, where there was no apparent effect of growth stage on overall fractionation there were still significant isotopic differences between closely related lipids including a 5.3% difference between the hopane and 3 beta-methylhopane skeletons. Hopane and sterane polyisoprenoids were also 13C-depleted compared to fatty acids. These observations have significant implications for the interpretation of specific compound isotopic signatures now being measured for hydrocarbons and other lipids present in sediments and petroleum. In particular, biomarker lipids produced by a single organism do not necessarily have the same carbon isotopic composition.

Halobacterium denitrificans sp. nov., an extremely halophilic denitrifying bacterium.

Halobacterium denitrificans was one of several carbohydrate-utilizing, denitrifying, extremely halophilic bacteria isolated by anaerobic enrichment in the presence of nitrate. Anaerobic growth took place only when nitrate (or nitrite) was present and was accompanied by the production of dinitrogen. In the presence of high concentrations of nitrate (i.e., 0.5%), nitrous oxide and nitrite were also detected. When grown aerobically in a mineral-salts medium containing 0.005% yeast extract, H. denitrificans utilized a variety of carbohydrates as sources of carbon and energy. In every case, carbohydrate utilization was accompanied by acid production. A type culture has been deposited with the American Type Culture Collection, Rockville, Md. (ATCC 35960).