Growth of Escherichia coli on Short-Chain Fatty Acids: Nature of the Uptake System

Mutants of Escherichia coli K-12 which grow on butyrate and valerate were studied with respect to uptake of these substrates. To utilize short-chain and medium-chain fatty acids, E. coli must synthesize the beta-oxidation enzymes constitutively. In addition, growth on the C(4) and C(5) acids requires a second mutation which permits entry of these substrates. At pH 5, both in the parent and mutant strains, butyrate and valerate penetrate as the undissociated acids but appear not to be activated and thus inhibit growth. At pH 7, the parent strain is not permeable to the anions, whereas the mutant concentrates these substrates. There appear to be two components of the uptake system, a nonspecific diffusion component and an energy-linked activating enzyme. Two mutant types which take up short-chain fatty acids are described. One synthesizes the uptake system constitutively and is inhibited by 4-pentenoate when cultured on acetate. In the other, the uptake system is inducible, and the strain is pentenoate-resistant when grown on acetate but pentenoate-sensitive when cultured on butyrate or valerate.     

Isolation and Characterization of Mutants of Escherichia coli with Cellular Division Selectively Affected by Growth on Fatty Acids

Isolation and characterization of mutants of Escherichia coli that beta-oxidize fatty acids at normal rates, but which divide very slowly when grown on fatty acids, are described. These mutants grow normally on other carbon sources. By growth on oleate, experiments with radioactive precursors showed that the rates of incorporation into ribonucleic acid, protein, and cell wall were comparable to those observed with the parent, whereas the rate of incorporation into phospholipids was slightly decreased. Under these conditions the rate of incorporation of ³²P-orthophosphate into deoxyribonucleic acid was low. On the other hand, by growth on oleate, neither gross mass increase in the different macromolecules nor loss of viability was observed, whereas in the presence of inducer the derepression of the lac operon enzymes occurred. Therefore, extensive turnover of the macromolecules is involved when these mutants are grown on fatty acids. Studies of the crypticity and of the binding of 1-anilino-8-naphthalene sulfonate show differences in membrane structure between the mutants and the constitutive parent. Properties of these mutants, which are affected in the process of cellular division, are discussed.     

Volatile Fatty Acids and the Inhibition of Escherichia coli Growth by Rumen Fluid

Concentrations of volatile fatty acids (VFA) normally found in bovine rumen fluid inhibited growth of Escherichia coli in Antibiotic Medium 3. Acetic, propionic, and butyric acids each produced growth inhibition which was markedly pH-dependent. Little inhibition was observed at pH 7.0, and inhibition increased with decreasing pH. A combination of 60 mumoles of acetate, 20 mumoles of propionate, and 15 mumoles of butyrate per ml gave 96, 69, and 2% inhibition at pH 6.0, 6.5, and 7.0, respectively. Rumen fluid (50%) gave 89 and 48% inhibition at pH 6.0 and 6.5, respectively, and growth stimulation (22%) at pH 7.0. Rumen fluid inhibitory activity was heat-stable, was not precipitated by 63% ethyl alcohol, and was lost by dialysis and by treatment with anion-exchange resins but not with cation-exchange resins. These results are consistent with the idea that VFA are the inhibitory substances in rumen fluid. Previous results which indicated that rumen fluid VFA did not inhibit E. coli growth were due to lack of careful control of the final pH of the growth medium. The E. coli strain used does not grow in rumen fluid alone at pH 7.0.     

工程大肠杆菌合成游离脂肪酸的研究进展

游离脂肪酸作为一种重要的平台化合物,其衍生产品被广泛应用到能源、化学工业中。作为更加可持续、绿色的生产策略,利用工程微生物合成游离脂肪酸是以石油基和动植物为原料生产脂肪酸类产品的重要补充。大肠杆菌作为经典的模式微生物,通过对其进行代谢工程改造,脂肪酸的积累已经从痕量提高到了约9g/L,展示了其作为脂肪酸合成菌株的巨大应用潜力。随着合成生物学技术的涌现,“感应-调控器”、体外重构、β氧化逆循环、异源合成途径的整合等思路的引入极大地加快了工程大肠杆菌脂肪酸合成的进化速率,并赋予大肠杆菌合成多种脂肪酸产品的能力。对近年来通过代谢工程和合成生物学手段改造大肠杆菌合成游离脂肪酸的研究进展进行综述,对其发展前景进行展望。     

Toxicity of Short-Chain Fatty Acids and Alcohols Towards Cladosporium resinae

Long-chain saturated fatty acids (C(13) to C(18)) and fatty alcohols (C(12) to C(18)) were well utilized by three different soil isolates of Cladosporium resinae as the sole carbon and energy sources in static liquid cultures. Shorter-chain compounds, down to C(5), did not support growth and were in fact toxic towards the fungus growing on glucose. Rapid and considerable potassium efflux, protein leakage, and inhibition of endogenous respiration were observed in the presence of the shorter fatty acids and alcohols. Possible mechanisms and significance of the toxicity are discussed.     

Chilling Cells Enhances the Bactericidal Action of Fatty Acids on Escherichia coli

[This corrects the article on p. 153 in vol. 31.].     

Effect of Short-Chain Fatty Acids and Soil Atmosphere on Tylenchorhynchus spp.

Short-chain fatty acids can be produced under anaerobic conditions by fermentative soil microbes and have nematicidal properties. We evaluated the effects of butyric and propionic acids on death and recovery of stunt nematodes (Tylenchorhynchus spp.), a common parasite of turfgrass. Nematodes in a sand-soil mix (80:20) were treated with butyric or propionic acid and incubated under air or N₂ for 7 days at 25 °C. Amendment of soil with 0.1 and 1.0 µmol (8.8 and 88 µg) butyric acid/g soil or 1.0 µmol (74 µg) propionic acid/g soil resulted in the death of all nematodes. The composition of the soil atmosphere had no effect on the nematicidal activity of the acids. Addition of hydrochloric acid to adjust soil pH to 4.4 and 3.5 resulted in nematode mortality relative to controls (41% to 86%) but to a lesser degree than short-chain fatty acids at the same pH. Nematodes did not recover after a 28-day period following addition of 10 µmol butyric acid/g soil under air or N₂. Carbon mineralization decreased during this period, whereas levels of inorganic N and microbial biomass-N remained constant. Short-chain fatty acids appear to be effective in killing Tylenchorhynchus spp. independent of atmospheric composition. Nematode mortality appears to be a function of the type and concentration of fatty acid and soil pH.     

Escherichia coli K-12 Mutants Altered in the Transport of Branched-Chain Amino Acids

Two mutants of Escherichia coli K-12 are described which are resistant to the inhibition that valine exerts on the growth of E. coli. These mutants have lesions at two different loci on the chromosome. One of them, brnP, is linked to leu (87% cotransduction) and is located between leu and azi represented on the map at 1 min; the other, brnQ, is linked to phoA (96% cotransduction), probably between proC and phoA and represented at 10 min. These mutants are resistant to valine inhibition but are sensitive to dipeptides containing valine. Since it is known that dipeptides are taken up by E. coli through a transport system(s) different from those used by amino acids, this sensitivity to the peptides suggests an alteration in the active transport of valine. The mutants are resistant to valine only if leucine is present in the growth medium; the uptake of valine is less in both mutants than it is in wild-type E. coli, and it is reduced even further if leucine is present. Under these conditions the total uptake of valine is almost completely abolished in the brnQ mutant. The brnP mutant takes up about 60% as much valine as does the wild type, but no exogenous valine is incorporated into proteins. The apparent K(m) and V(max) of isoleucine, leucine, and valine for the transport system are reported; the brnP mutant, when compared to the wild type, has a sevenfold higher K(m) for isoleucine and a 17-fold lower K(m) for leucine; the V(max) for the three amino acids is reduced in the brnQ mutant, up to 20-fold for valine. The transport of arginine, aspartic acid, glycine, histidine, and threonine is not altered in the brnQ mutant under conditions in which that of the branched amino acids is. Evidence is reported that O-methyl-threonine enters E. coli through the transport system for branched amino acids, and that thiaisoleucine does not.     

Transketolase Mutants of Escherichia coli

Transketolase mutants have been selected after ethyl methane sulfonate mutagenesis of Escherichia coli. These strains are unable to grow on any pentose and, in addition, require a supplement of aromatic amino acids or shikimic acid for normal growth on any other carbon source. Revertants are normal in both respects and also contain transketolase. Transketolase mutants do not require exogenous pentose for growth. Preliminary genetic mapping of the locus is presented.     

Effects of Unsaturated Fatty Acids on the Morphogenesis of an Unsaturated Fatty Acid Auxotroph of Escherichia coli

Supplementation of linoleate or linolenate in a culture medium caused abnormal morphogenesis in an unsaturated fatty acid auxotroph of Escherichia coli K-12.     

Nonchemotactic Mutants of Escherichia coli

We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged.     

Porphyrin-Accumulating Mutants of Escherichia coli

Four mutants (pop-1, pop-6, pop-10, and pop-14) which accumulate a red water-insoluble pigment were obtained in Escherichia coli K-12 AB1621. For each mutant, the red pigment was shown to be protoporphyrin IX, a late precursor of heme. Mutagenic treatment of mutant pop-1 yielded a secondary mutant, pop-1 sec-20, which accumulated a brown water-soluble pigment. The brown pigment was shown to be coproporphyrin III. Mutant pop-1 resembled the parental strain in its cytochrome absorption spectrum, catalase activity, and ability to grow on nonfermentable carbon and energy sources; therefore, its ability to produce and utilize heme was unimpaired. Judged on the same criteria, the secondary mutant, pop-1 sec-20, was partially heme and respiratory deficient. Growth in anaerobic conditions decreased by 25% the accumulation of protoporphyrin by pop-1; under the same conditions, pop-1 sec-20 did not accumulate coproporphyrin or coproporphyrinogen. The mutations causing protoporphyrin accumulation in all four pop mutants were found to map in the lac to purE (10-13 min) region of the E. coli chromosome. In the case of mutant pop-1, the mutation was shown to be strongly linked to the tsx locus (12 min). In mutant pop-1 sec-20, the second mutation causing coproporphyrin accumulation was co-transducible with the gal locus at a frequency of 88 to 96%. The mechanism of porphyrin accumulation by the mutants is discussed.     

Mutants of Escherichia coli with High Minimal Temperatures of Growth.

O'Donovan, Gerard A. (University of California, Davis), Catherine L. Kearney, and John L. Ingraham. Mutants of Escherichia coli with high minimal temperatures of growth. J. Bacteriol. 90:611-616. 1965.-Three general classes of mutants showing increased minimal temperatures of growth have been isolated from Escherichia coli. These mutants do not grow at temperatures below 20 C, although their parents can grow at temperatures as low as 8 C. The first class of mutants (K-I) cannot grow below 20 C in either complex or minimal medium, but grows at nearly normal rates at 37 C on both types of media. Normal growth rate at 20 C can be conferred on these mutants by infection at a low multiplicity with a transducing phage grown on the parent. The second class of mutants (K-II) fails to grow only in minimal medium at 20 C. These mutants are characterized by their singular response to specific nutrients in minimal medium at 20 C. The third class of mutants (K-III) grows normally in minimal medium at all temperatures with either glucose or glycerol as the carbon source, but does not grow at 20 C with lactose as the carbon source.     

Passive Diffusion Technique for Concentration of Short-Chain Volatile Fatty Acids from Seawater

A diffusion chamber is described which concentrates short-chain, volatile fatty acids from seawater while simultaneously separating them from interfering salts. The procedure relies on the passive diffusion of volatile compounds from acidified seawater samples and their subsequent absorption onto a base-impregnated filter. The method is simple, efficient, and adaptable to most commonly used methods of volatile acid analysis.     

Induction of Acid Resistance of Salmonella typhimurium by Exposure to Short-Chain Fatty Acids

Exposure to short-chain fatty acids (SCFA) is one of the stress conditions Salmonella typhimurium encounters during its life cycle, because SCFA have been widely used as food preservatives and SCFA are also present at high concentrations in the gastrointestinal tracts of host animals. The effects of SCFA on the acid resistance of the organism were examined in an attempt to understand the potential role of SCFA in the pathogenesis of S. typhimurium. The percent survival of S. typhimurium at pH 3.0 was determined after exposure to SCFA for 1 h at pH 7.0. The percent acid survival, which varied depending on the SCFA species and the concentration used, was 42 after exposure to 100 mM propionate at pH 7.0 under aerobic incubation conditions, while less than 1% could survive without exposure. The SCFA-induced acid resistance was markedly enhanced by anaerobiosis (64%), lowering pH conditions (138% at pH 5.0), or increasing incubation time (165% with 4 h) during exposure to propionic acid. When protein synthesis during exposure to propionate was blocked by chloramphenicol, the percent acid survival was less than 1, indicating that the protein synthesis induced by exposure to propionate is required for the induction of the acid resistance. The percent acid survival determined with the isogenic mutant strains defective in acid tolerance response revealed that AtrB protein is necessary for the full induction of acid resistance by exposure to propionate, while unexpectedly, inactivation of PhoP significantly increased acid resistance over that of the wild type (P < 0.05). The results suggest that the virulence of S. typhimurium may be enhanced by increasing acid resistance upon exposure to SCFA during its life cycle and further enhanced by anaerobiosis, low pH, and prolonged exposure time.     

Effect of Short-Chain Fatty Acids Extracted from Beef Cattle Manure on Germination and Seedling Development

Composted and fresh beef cattle manure samples were extracted with distilled water, acetone, methanol, 2 N sodium hydroxide, 2 N hydrochloric acid, and ether. Bioassay techniques, using the extracts, showed that composted manure extracts had limited effect on seed germination and seeding development of wheat and sorghum. All the extracts of fresh manure, other than distilled water, retarded germination. Acetic, butyric, propionic, valeric, and isovaleric acids were found in ether extracts of fresh manure at average concentrations of 348, 876, 578, 34, and 19 μg/g, respectively, on a dry-weight basis. However, only trace amounts of these acids were present in composted manure. Propionic acid up to the 200-μg/ml level stimulated seedling growth. Acetic and butyric below the 200-μg/ml level had no detrimental or beneficial effects on seedling development. When acetic, butyric, and propionic acids were mixed in equal parts, germination and seedling growth were reduced at all levels (50 to 500 μg/ml).     

Effect of the Osmotic Pressure of the Growth Medium on fabB Mutants of Escherichia coli

Temperature-sensitive, unsaturated fatty acid (fabB) auxotrophs of Escherichia coli can grow at the restrictive temperature in the absence of unsaturated fatty acid in a medium with a high osmotic pressure. If a mutant culture was starved for unsaturated fatty acids and harvested just before the lysis started, the fatty acid composition of the cells was the same as that of cells grown until late log phase in a high-osmotic medium. Evidence is presented that the in vivo unsaturated fatty acid biosynthesis is significantly increased in a high osmotic medium. The increase is probably due to a partial activation of the temperature-sensitive fabB product. Besides the stimulation of the temperature-sensitive fabB product, a minimal osmotic pressure of the medium appeared to be necessary to allow growth of cells containing lipids with a changed fatty acid composition. fabA mutants are unable to grow in a high-osmotic medium in the absence of unsaturated fatty acids. No increase in the in vivo unsaturated fatty acid biosynthesis could be detected in the temperature-sensitive fabA mutants.     

Rapid Growth Mutants of Escherichia Coli

If rapid growth (rap) mutants of Escherichia coli could be obtained, these might prove a valuable contribution to fields as diverse as growth rate control, biotechnology and the regulation of the bacterial cell cycle. To obtain rap mutants, a dnaQ mutator strain was grown for four and a half days continuously in batch culture. At the end of the selection period, there was no significant change in growth rate. This result means that selecting rap mutants may require an alternative strategy and a number of such alternatives are discussed.     

Temperature-Sensitive Osmotic Remedial Mutants of Escherichia coli

A collection of temperature-sensitive mutants of Escherichia coli K-12 was examined for ability to grow at the restrictive temperature when the osmotic pressure of the medium was increased. Five of the fourteen mutants were found to be osmotic remedial. Four strains containing temperature-sensitive, osmotic-remedial mutations affecting aminoacyl-transfer ribonucleic acid synthetases were found to have altered permeability characteristics which may be attributable to changes in the lipopolysaccharide layer of the cell envelope at restrictive temperatures.