Conversion of Unsaturated Fatty Acids by Bacteria Isolated from Compost

A compost mixture amended with soybean oil was enriched in microorganisms that transformed unsaturated fatty acids (UFAs). When oleic acid or 10-ketostearic acid was the selective fatty acid, Sphingobacterium thalpophilum (NRRL B-23206, NRRL B-23208, NRRL B-23209, NRRL B-23210, NRRL B-23211, NRRL B-23212), Acinetobacter spp. (NRRL B-23207, NRRL B-23213), and Enterobacter cloacae (NRRL B-23264, NRRL B-23265, NRRL B-23266) represented isolates that produced either hydroxystearic acid, ketostearic acid, or incomplete decarboxylations. When ricinoleic (12-hydroxy-9-octadecenoic) acid was the selective UFA, Enterobacter cloacae (NRRL B-23257, NRRL B-23267) and Escherichia sp. (NRRL B-23259) produced 12-C and 14-C homologous compounds, and Pseudomonas aeruginosa (NRRL B-23256, NRRL B-23260) converted ricinoleate to a trihydroxyoctadecenoate product. Also, various Enterobacter, Pseudomonas, and Serratia spp. appeared to decarboxylate linoleate substrate incompletely. These saprophytic, compost bacteria were aerobic or facultative anaerobic Gram-negative and decomposed UFAs through decarboxylation, hydroxylation, and hydroperoxidation mechanisms. Received: 3 November 1998 / Accepted: 30 November 1998     

Genetic Analysis of DNA Replication in Bacteria: dna B Mutations That Suppress dnaC Mutations and dnaQ Mutations That Suppress dnaE Mutations in SALMONELLA TYPHIMURIUM

We have isolated and characterized extragenic suppressors of mutations in two different target genes that affect DNA replication in Salmonella typhimurium. Both the target and the suppressor genes are functional homologues of known replication genes of E. coli that were identified in intergeneric complementation tests. Our results point to interactions in vivo involving the dnaB and dnaC proteins in one case and the dnaQ and dnaE proteins in the other case. The suppressor mutations, which were isolated as derivatives of lambda-Salmonella in vitro recombinants, were detected by an adaptation of the red plaque complementation assay. This method was applicable even when the locus of suppressor mutations was not chosen in advance.     

海洋细菌产生的多不饱和脂肪酸

鱼油的主要成分为二十碳五烯酸和二十二碳六烯酸,这些多不饱和脂肪酸具有多方面的生理活性。近年来在海洋细菌中发现这些多不饱和脂肪酸的存在,海洋细菌很可能是这些多不饱和脂肪酸的原始生产之一。对其生物合成的深入研究表明,海洋细菌多不饱和脂肪酸的合成不同于其他生物的不饱和脂肪酸的合成机制,合成过程中不涉及重要的脂肪酸脱氢和延长机制,其合成由一种多聚乙酰合成酶(PKS)催化。     

Control of Fatty Acid Synthesis in Bacteria

When glycerol-requiring auxotrophs of Bacillus subtilis are deprived of glycerol, the synthesis of fatty acids continues at an apparent rate of 20 to 50% that of supplemented cultures. The newly synthesized fatty acids are not incorporated into phospholipid and accumulate as free fatty acids. These molecules undergo a much more rapid turnover than phospholipid fatty acids, and the rate of turnover is sufficient to indicate that the rate of fatty acid synthesis in glycerol-deprived cultures is similar to that in supplemented ones. The average chain length of the free fatty acids is greater than that of the phospholipid fatty acids. Cells deprived of required amino acids also show a diminution in the apparent rate of fatty acid synthesis; however, in this case, the fatty acids accumulate in phospholipid, and no increase of the free fatty acid fraction is observed. It is argued on the basis of these findings that the control of lipid synthesis does not operate at the level of transacylation but must act on one or more of the reactions of the fatty acid synthetase.     

Fatty Acid Competition as a Mechanism by Which Enterobacter cloacae Suppresses Pythium ultimum Sporangium Germination and Damping-Off

Interactions between plant-associated microorganisms play important roles in suppressing plant diseases and enhancing plant growth and development. While competition between plant-associated bacteria and plant pathogens has long been thought to be an important means of suppressing plant diseases microbiologically, unequivocal evidence supporting such a mechanism has been lacking. We present evidence here that competition for plant-derived unsaturated long-chain fatty acids between the biological control bacterium Enterobacter cloacae and the seed-rotting oomycete, Pythium ultimum, results in disease suppression. Since fatty acids from seeds and roots are required to elicit germination responses of P. ultimum, we generated mutants of E. cloacae to evaluate the role of E. cloacae fatty acid metabolism on the suppression of Pythium sporangium germination and subsequent plant infection. Two mutants of E. cloacae EcCT-501R3, Ec31 (fadB) and EcL1 (fadL), were reduced in β-oxidation and fatty acid uptake, respectively. Both strains failed to metabolize linoleic acid, to inactivate the germination-stimulating activity of cottonseed exudate and linoleic acid, and to suppress Pythium seed rot in cotton seedling bioassays. Subclones containing fadBA or fadL complemented each of these phenotypes in Ec31 and EcL1, respectively. These data provide strong evidence for a competitive exclusion mechanism for the biological control of P. ultimum-incited seed infections by E. cloacae where E. cloacae prevents the germination of P. ultimum sporangia by the efficient metabolism of fatty acid components of seed exudate and thus prevents seed infections.     

Isolation and Characterization of Polyester-Based Plastics-Degrading Bacteria from Compost Soils

Four potential polyester-degrading bacterial strains were isolated from compost soils in Thailand. These bacteria exhibited strong degradation activity for polyester biodegradable plastics, such as polylactic acid (PLA), polycaprolactone (PCL), poly-(butylene succinate) (PBS) and polybutylene succinate-co-adipate (PBSA) as substrates. The strains, classified according to phenotypic characteristics and 16S rDNA sequence, belonging to the genera Actinomadura, Streptomyces and Laceyella, demonstrated the best polyester- degrading activities. All strains utilized polyesters as a carbon source, and yeast extract with ammonium sulphate was utilized as a nitrogen source for enzyme production. Optimization for polyester-degrading enzyme production by Actinomadura sp. S14, Actinomadura sp. TF1, Streptomyces sp. APL3 and Laceyella sp. TP4 revealed the highest polyester-degrading activity in culture broth when 1% (w/v) PCL (18 U/mL), 0.5% (w/v) PLA (22.3 U/mL), 1% (w/v) PBS (19.4 U/mL) and 0.5% (w/v) PBSA (6.3 U/mL) were used as carbon sources, respectively. All strains exhibited the highest depolymerase activities between pH 6.0–8.0 and temperature 40–60°C. Partial nucleotides of the polyester depolymerase gene from strain S14, TF1 and APL3 were studied. We determined the amino acids making up the depolymerase enzymes had a highly conserved pentapeptide catalytic triad (Gly-His-Ser-Met-Gly), which has been shown to be part of the esterase-lipase superfamily (serine hydrolase).     

Volatile Fatty Acid Requirements of Cellulolytic Rumen Bacteria

A gas chromatographic method was developed which could separate the isomers isovaleric and 2-methylbutyric acid. Subsequent analyses revealed that most commercially available samples of these acids were cross-contaminated; however, one sample of each acid was found to be pure by this criterion. The growth response of seven strains of cellulolytic rumen bacteria (three strains of Bacteroides succinogenes, three strains of Ruminococcus flavefaciens, and one strain of R. albus) to additions of isobutyric, isovaleric, 2-methylbutyric, valeric, and combinations of valeric and a branched-chain acid was determined. Strains of B. succinogenes required a combination of valeric plus either isobutyric or 2-methylbutyric acid. Isovaleric acid was completely inactive. Either isobutyric or 2-methylbutyric acid was required for the growth of R. albus 7. Strain C-94 of R. flavefaciens grew slowly in the presence of any one of the three branched-chain acids, but a combination of isobutyric and 2-methylbutyric acids appeared to satisfy this organism's growth requirements. None of the individual acids or mixtures of straight- and branched-chain acids allowed growth of R. flavefaciens strain C1a which would approach the response obtained from the total mixture of acids. Further work indicated that all three branched-chain acids were required for optimal growth by this strain, although isovaleric acid only influenced the rate of maximal growth. Either 2-methylbutyric or isovaleric acid allowed growth of nearly the same magnitude as that of the positive control for R. flavefaciens B34b. The presence of acetic acid had little influence on the rate or extent of growth of any of the strains except R. albus 7, for which the extent of growth was markedly increased. Determination of the quantitative fatty acid requirements for the three B. succinogenes strains indicated that 0.1 μmole of valeric per ml and 0.05 μmole of 2-methylbutyric per ml permitted maximal growth. However, with isobutyric acid as the branched-chain component, strains A3c and B21a required 0.1 μmole/ml in contrast to S-85 which exhibited optimal growth at the 0.05 μmole/ml level. By use of mixtures of isobutyric and 2-methylbutyric acids, good growth of C-94 was obtained at concentrations of 0.1 and 0.01 μmole/ml, respectively. About 0.3 μmole/ml of each acid was required for satisfactory growth of C1a.     

Lactic Acid Bacteria Biomass Monitoring in Highly Conductive Media by Permittivity Measurements

Recent technological improvements have extended the application range of permittivity biomass measurements to bacterial fermentations in highly conductive media. With Lactobacillus casei, the effective biomass detection sensitivity of the FOGALE Biomass System is around 0.2 g/l (0.01 pF/cm). Fermentations growth kinetics of Lactobacillus casei can be recorded with good reproducibility and accuracy despite the high medium conductivity varying between 15 and 75 mS/cm, and the low cell concentration (<6 g/l).     

Cellular Fatty Acid Composition and Identification of Rumen Bacteria

The fatty acid compositions of 21 pure cultures of rumen bacteria, representing 12 genera and 14 species, were compared as methyl esters. Each organism possessed a consistent and reproducible fatty acid profile. Overlapping similarities and differences in composition did not allow differentiation between families or genera. Although species differentiation was possible, fatty acid composition appeared to be only an aid in the identification of bacteria.     

Isolation and Genetic Characterization of Bacteria That Degrade Chloroaromatic Compounds

Bacteria were isolated from a landfill site previously used for disposal of chlorinated organic wastes. These soil isolates were capable of utilizing various chloroaromatic compounds. One such bacterial strain, designated Pseudomonas cepacia HCV (2,6-DCT) and growing on 2,6-dichlorotoluene, transferred this trait to a catechol-1,2-oxygenase mutant of Pseudomonas aeruginosa.     

Stimulation of Bacteriocin Production by Dialyzed Culture Media from Different Lactic Acid Bacteria

The cross-effects of dialyzed postincubates (with a cut-off at 1000 Da) on the biomass and bacteriocin production of six strains of lactic acid bacteria were studied, and a predominance of stimulating responses was found, the characteristics of which suggested merely nutritional effects or the presence of precursor fragments of the bacteriocins. Additionally, cluster analysis of the detected responses provides an approach to define groups of highly compatible (potential consortia) or doubtfully compatible strains of lactic acid bacteria. Such a definition, which does not claim taxonomic value, has practical interest, however, in cases (e.g., silage production) in which it is convenient to use mixed inocula including strains able to establish positive interactions.     

Enrichment and Isolation of Rumen Bacteria That Reduce trans- Aconitic Acid to Tricarballylic Acid

Bacteria from the bovine rumen capable of reducing trans-aconitate to tricarballylate were enriched in an anaerobic chemostat containing rumen fluid medium and aconitate. After 9 days at a dilution rate of 0.07 h⁻¹, the medium was diluted and plated in an anaerobic glove box. Three types of isolates were obtained from the plates (a crescent-shaped organism, a pleomorphic rod, and a spiral-shaped organism), and all three produced tricarballylate in batch cultures that contained glucose and trans-aconitate. In glucose-limited chemostats (0.10 h⁻¹), trans-aconitate reduction was associated with a decrease in the amount of reduced products formed from glucose. The crescent-shaped organism produced less propionate, the pleomorphic rod produced less ethanol, and the spiral made less succinate and possibly H₂. Aconitate reduction by the pleomorphic rod and the spiral organism was associated with a significant increase in cellular dry matter. Experiments with stock cultures of predominant rumen bacteria indicated that Selenomonas ruminantium, a species taxonomically similar to the crescent-shaped isolate, was an active reducer of trans-aconitate. Strains of Bacteroides ruminicola, Butyrivibrio fibrisolvens, and Megasphaera elsdenii produced little if any tricarballylate. Wolinella succinogenes produced some tricarballylate. Based on its stability constant for magnesium (K_eq = 115), tricarballylate could be a factor in the hypomagnesemia that leads to grass tetany.     

Stability of Lactic Acid Bacteria to Freezing as Related to Their Fatty Acid Composition

The viability of Streptococcus lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h was better preserved when the cells were grown in medium supplemented with oleic acid or Tween 80 (polyoxyethylene sorbitan monooleate). A pronounced change in the cellular fatty acid composition was noted when the bacteria were grown in the presence of Tween 80. In S. lactis the ratio of unsaturated to saturated fatty acids increased from 1.18 to 2.55 and in Lactobacillus sp. A-12 it increased from 0.85 to 1.67 when Tween 80 was added to the growth medium. The antibiotic cerulenin markedly inhibited the growth of lactic acid bacteria in tomato juice (TJ) medium but had almost no effect on the growth of the bacteria in TJ medium containing Tween 80 (or oleic acid). The antibiotic inhibited markedly the incorporation of [1-¹⁴C]acetate but had no inhibitory effect on the incorporation of exogenous [1-¹⁴C]oleate (or [1-¹⁴C]palmitate) into the lipid fractions of lactic acid bacteria. Thus, the fatty acid composition of lactic acid bacteria, inhibited by the antibiotic cerulenin, can be modulated by exogenously added oleic acid (or Tween 80) without the concurrent endogenous fatty acid synthesis from acetate. The data obtained suggest that cerulenin inhibits neither cyclopropane fatty acid synthesis nor elongation of fatty acid acyl intermediates. The radioactivity of cells grown in the presence of [1-¹⁴C]oleate and cerulenin was associated mainly with cyclopropane Δ19:0, 20:0 + 20:1, and 21:0 acids. As a consequence, cerulenin caused a decrease in the ratio of unsaturated to saturated fatty acids in lactic acid bacteria as compared with cells grown in TJ medium plus Tween 80 but without cerulenin. Cerulenin caused a decrease in the viability of S. lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h only when Tween 80 was present in the growth medium. We conclude that the sensitivity of lactic acid bacteria to damage from freezing can be correlated with specific alterations in the cellular fatty acids.     

Characterization of Fatty Acid biosynthesis in isolated pea root plastids

Fatty acid biosynthesis from Na[1-¹⁴C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl₂, 1 millimolar each of MnCl₂ and glycerol-3-phosphate, 15 millimolar KHCO₃, 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35°C. At the standard incubation temperature of 25°C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [¹⁴C]acetate into fatty acids. On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.     

Comparison of rRNA and Polar-Lipid-Derived Fatty Acid Biomarkers for Assessment of 13C-Substrate Incorporation by Microorganisms in Marine Sediments

We determined whether a recently developed method to isolate specific small-subunit (SSU) rRNAs can be used in ¹³C-labeling studies to directly link community structure and function in natural ecosystems. Replicate North Sea sediment cores were incubated at the in situ temperature following addition of ¹³C-labeled acetate, propionate, amino acids, or glucose. Eukaryotic and bacterial SSU rRNAs were separated from total RNA by means of biotin-labeled oligonucleotide probes and streptavidin-coated paramagnetic beads, and the ¹³C content of the isolated rRNA was determined by elemental analysis-isotope ratio mass spectrometry. The SSU rRNA yield with the bead-capture protocol was improved by using helper probes. Incorporation of label into bacterial SSU rRNA was detectable after 2 h of incubation. The labeling was always much greater in bacterial SSU rRNA than in eukaryotic SSU rRNA, suggesting that bacteria were the main consumers of the ¹³C-labeled compounds. Similar results were obtained with the ¹³C-labeled polar-lipid-derived fatty acid (PLFA) approach, except that more label was detected in bacterial PLFA than in bacterial SSU rRNA. This may be attributable to the generally slow growth of sediment microbial populations, which results in low ribosome synthesis rates and relatively few ribosomes per cell. We discuss possible ways to improve the probe-capture protocol and the sensitivity of the ¹³C analysis of the captured SSU rRNA.     

Comparison of Whole-Cell Fatty Acid (MIDI) or Phospholipid Fatty Acid (PLFA) Extractants as Biomarkers to Profile Soil Microbial Communities

The whole-cell lipid extraction to profile microbial communities on soils using fatty acid (FA) biomarkers is commonly done with the two extractants associated with the phospholipid fatty acid (PLFA) or Microbial IDentification Inc. (MIDI) methods. These extractants have very different chemistry and lipid separation procedures, but often shown a similar ability to discriminate soils from various management and vegetation systems. However, the mechanism and the chemistry of the exact suite of FAs extracted by these two methods are poorly understood. Therefore, the objective was to qualitatively and quantitatively compare the MIDI and PLFA microbial profiling methods for detecting microbial community shifts due to soil type or management. Twenty-nine soil samples were collected from a wide range of soil types across Oregon and extracted FAs by each method were analyzed by gas chromatography (GC) and GC-mass spectrometry. Unlike PLFA profiles, which were highly related to microbial FAs, the overall MIDI-FA profiles were highly related to the plant-derived FAs. Plant-associated compounds were quantitatively related to particulate organic matter (POM) and qualitatively related to the standing vegetation at sampling. These FAs were negatively correlated to respiration rate normalized to POM (Resp_POM), which increased in systems under more intensive management. A strong negative correlation was found between MIDI-FA to PLFA ratios and total organic carbon (TOC). When the reagents used in MIDI procedure were tested for the limited recovery of MIDI-FAs from soil with high organic matter, the recovery of MIDI-FA microbial signatures sharply decreased with increasing ratios of soil to extractant. Hence, the MIDI method should be used with great caution for interpreting changes in FA profiles due to shifts in microbial communities.     

Characterization of Microbial Community Structure in the Surface Sediment of Osaka Bay, Japan, by Phospholipid Fatty Acid Analysis

Twenty-eight sediment samples collected from Osaka Bay, Japan, were analyzed for phospholipid ester-linked fatty acids (PLFA) to determine regional differences in microbial community structure of the bay. The abundance of three major groups of C₁₀ to C₁₉ PLFA (saturated, branched, and monounsaturated PLFA), which accounted for 84 to 97% of the total PLFA, indicated the predominance of prokaryotes in the sediment. The distribution of six clusters obtained by similarity analysis in the bay revealed a marked regional distribution in the PLFA profiles. Total PLFA concentrations (0.56 to 2.97 μg/g [dry weight] of the sediment) in sediments also showed marked variation among the stations, with higher concentrations of total PLFA in the central part of the bay. The biomass, calculated on the basis of total PLFA concentration, ranged from 0.25 × 10⁸ to 1.35 × 10⁸ cells per g (dry weight) of the sediment. The relative dominance of microbial groups in sediments was described by using the reported bacterial biomarker fatty acids. Very small amounts of the characteristic PLFA of microeukaryotes in sediments indicated the restricted distribution of microeukaryotes. By examining the distribution of clusters and groups of microorganisms in the bay, there were two characteristics of the distribution pattern: (i) the predominance of anaerobic bacteria and gram-positive prokaryotes, characterized by the high proportions of branched PLFA in the eastern and northeastern sides of the bay, where the reported concentrations of pollutants were also high, and (ii) the predominance of aerobic prokaryotes and eukaryotes, except for a few stations, in the western and southwestern sides of the bay, as evidenced by the large amounts of monounsaturated PLFA. Such significant regional differences in microbial community structure of the bay indicate shifts in microbial community structure.     

Purification and Characterization of Fatty Acid Cyclooxygenase from Human Platelets

Abstract

The fatty acid cyclooxygenase (EC 1.14.99.1) that produces the prostaglandin, thromboxane, and prostacyclin precursor (PGHp), was solubilized from human platelet microsomes in 20 sucrose and 1.0% Triton X-100. The enzyme was purified 300-fold by electrofocusing, Sephadex G-200 gel filtration, and hydrophobic chromatography on ethyl agarose. The cyclooxygenase catalyzed the conversion of arachidonic acid to prostaglandin endoperioxide, PGH₂, that was trapped at ?25°C and separated on TLC at ?20°C. PGH₂ was hydrolyzed to HHT in acidic pH, or was chemically converted to PGE₂ in slightly alkaline pH in the absence of cofactors. The enzyme showed a broad pH optimum in the range of 7–9. Hemin containing substances such as methemoglobin were absolutely required as cofactors, while tryptophan, epinephrine, phenol, and hydro-quinone stimulated the PGH₂ formation. Metal ions, such as Zn²⁺ and Cd²⁺ inhibited the enzyme reaction at 0.1 to 1 mM.

The molecular weight of the purified enzyme was estimated at 79,432 by sodium dodecyl sulfate disc gel electrophoresis at pH 8.0. The properties of the human platelet enzyme was generally similar to the sheep vesicular enzyme in the method of solubilization, pH optimum, and molecular weight.