Genetic and biochemical characterization of the Escherichia coli K-12 fhuB mutation.

The fhuB region of Escherichia coli K-12 was subcloned from pLC4-44 into pP lac to obtain pCPN1. Deletions of this recombinant plasmid were made, and a 1.4-kilobase PstI fragment was further subcloned into the vector plasmid pKK177-2 to obtain pCPN12. The response of tonA and tonB strains and fhuB strains containing the plasmids to 15 hydroxamate siderophores were assayed. Results showed that tonA strains were deficient only in the utilization of ferrichrome-type siderophores, whereas fhuB strains were deficient in the utilization of all hydroxamate-type siderophores. The response of the plasmid-containing fhuB strains to the siderophores showed that the fhuB gene resides on a 1.4-kilobase PstI fragment of DNA. The proteins synthesized by these plasmids were examined in maxicells of strain CSR603. Plasmid pCPN1 expressed five proteins of molecular weights 78,000, 40,000, 30,000, 24,000, and 13,700. By the use of deletions of pCPN1, the approximate order of the genes for these proteins was determined. Plasmid pCPN12 expressed no proteins other than the beta-lactamase proteins in maxicell strain CSR603. However, in maxicell strain BN660, a lon mutant, it expressed a 20,000-molecular-weight protein. Inner membrane vesicles made from tonB and fhuB strains were able to transport [55Fe]ferrichrome and [55Fe]rhodotorulate at rates similar to those obtained in vesicles from tonB+ and fhuB+ strains.     

Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio–product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.     

Increased unsaturated fatty acid production associated with a suppressor of the fabA6(Ts) mutation in Escherichia coli.

Plasmids that corrected the temperature-sensitive unsaturated fatty acid auxotrophy of strain M6 [fabA6 (Ts)] were isolated from an Escherichia coli genomic library. Subcloning and physical mapping localized the new gene (called sfa for suppressor of fabA) at 1,070 kb on the E. coli chromosome. DNA sequencing revealed the presence of a 227-bp open reading frame which directed the synthesis of a peptide of approximately 8 kDa, which correlated with the correction of the fabA6(Ts) phenotype. However, the sfa gene was an allele-specific suppressor since plasmids harboring the sfa gene corrected the growth phenotype of fabA6(Ts) mutants but did not correct the growth of fabA2(Ts) or fabB15(Ts) unsaturated fatty acid auxotrophs. Overexpression of the sfa gene in fabA6(Ts) mutants restored unsaturated fatty acid content at 42 degrees C, and overexpression in wild-type cells resulted in a substantial increase in the unsaturated fatty acid content of the membrane. Thus, the suppression of the fabA6(Ts) mutation by sfa was attributed to its ability to increase the biosynthesis of unsaturated fatty acids.     

Genetic mapping and biochemical characterization of suppressor mutations sukA and sukB for a dnaK7(Ts) mutation of Escherichia coli K-12.

Temperature-resistant pseudorevertants were isolated from a dnaK7(Ts) mutant of Escherichia coli K-12. Two of these pseudorevertants were shown to carry suppressor mutations, sukA and sukB, respectively. Genetic mapping by conjugation and P1-transduction revealed that these suppressor mutations were located at two distinct sites between 76 and 77 min close to the suhA and rpoH genes. Labeled cellular proteins were extracted from suppressor mutants grown at various temperatures and subjected to SDS-gel electrophoresis. Autoradiograms of the gels indicated that these suppressor mutations each resulted in increased synthesis of the heat shock protein Lon (an ATP-dependent protease, La) at both permissive and nonpermissive temperatures.     

Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity.

A trans unsaturated fatty acid was found as a major constituent in the lipids of Pseudomonas putida P8. The fatty acid was identified as 9-trans-hexadecenoic acid by gas chromatography, argentation thin-layer chromatography, and infrared absorption spectrometry. Growing cells of P. putida P8 reacted to the presence of sublethal concentrations of phenol in the medium with changes in the fatty acid composition of the lipids, thereby increasing the degree of saturation. At phenol concentrations which completely inhibited the growth of P. putida, the cells were still able to increase the content of the trans unsaturated fatty acid and simultaneously to decrease the proportion of the corresponding 9-cis-hexadecenoic acid. This conversion of fatty acids was also induced by 4-chlorophenol in nongrowing cells in which the de novo synthesis of lipids had stopped, as shown by incorporation experiments with labeled acetate. The isomerization of the double bond in the presence of chloramphenicol indicates a constitutively operating enzyme system. The cis-to-trans modification of the fatty acids studied here apparently is a new way of adapting the membrane fluidity to the presence of phenols, thereby compensating for the elevation of membrane permeability induced by these toxic substances.     

Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity.

A trans unsaturated fatty acid was found as a major constituent in the lipids of Pseudomonas putida P8. The fatty acid was identified as 9-trans-hexadecenoic acid by gas chromatography, argentation thin-layer chromatography, and infrared absorption spectrometry. Growing cells of P. putida P8 reacted to the presence of sublethal concentrations of phenol in the medium with changes in the fatty acid composition of the lipids, thereby increasing the degree of saturation. At phenol concentrations which completely inhibited the growth of P. putida, the cells were still able to increase the content of the trans unsaturated fatty acid and simultaneously to decrease the proportion of the corresponding 9-cis-hexadecenoic acid. This conversion of fatty acids was also induced by 4-chlorophenol in nongrowing cells in which the de novo synthesis of lipids had stopped, as shown by incorporation experiments with labeled acetate. The isomerization of the double bond in the presence of chloramphenicol indicates a constitutively operating enzyme system. The cis-to-trans modification of the fatty acids studied here apparently is a new way of adapting the membrane fluidity to the presence of phenols, thereby compensating for the elevation of membrane permeability induced by these toxic substances.     

Genetic characterization of the Escherichia coli cyclopropane fatty acid (cfa) locus and neighboring loci

Summary The phenotypically silent cyclopropane fatty acid synthesis (cfa) gene of Escherichia coli K-12 has been located on the genetic linkage map. This was accomplished by integrating (via homologous recombination) the selectable marker of a recombinant plasmid into the host chromosome near the cfa locus. This integration allowed the subsequent isolation of a cfa-linked transposon Tn10 insertion. Genetic mapping of the Tn10 insertion, using conventional techniques, placed the cfa locus at min 36.5 on the linkage map in the vicinity of several other non-selectable markers. We ordered cfa and these other loci by three-factor transductional analyses. Selection for excision of the Tn10 element resulted in several types of mutants which harbor mutations of cfa and of neighboring genes, presumably as a consequence of Tn10-catalyzed chromosomal rearrangements.     

Thermodynamic interactions of a cis and trans benzanilide with Escherichia coli bacterial membranes

The activity of a membrane interactive cis and trans benzanilide against Escherichia coli membrane mimics was investigated using Langmuir monolayers. It was found that in the presence of E. coli lipid mix monolayers, cis-benzanilide induced maximal surface pressure changes of 1?mN?m(-1), whereas a reduced interaction was observed with trans-benzanilide. Compression isotherm analysis of these monolayers showed ?G (mix)?相似文献   

Genetic mapping of a mutation that causes ribonucleases III deficiency in Escherichia coli.

the mutation that causes ribonuclease III (RNase III) deficiency in strain AB301-105 of Kindler et al. (1973) has been mapped by use of F' merodiploids, Hfr matings, and P1 transduction. This mutation, rnc-105, lies close to nadB, near 49 min on the genetic map of Escherichia coli. The rnc-105 mutation has been transferred from its original genetic background by transduction and conjugation, and these new strains have the same defects in ribonucleic acid processing reported previously for AB301-105. Strains that carry rnc-105 grow more slowly than parental rnc+ strains, but the difference in growth rate seems to depend on the genetic background of each strain. Bacteriophage T7 grows about equally well in RNase III+ and III- female strains of E. coli, even though the specific cuts that RNase III makes in T7 ribonucleic acid are not made in the RNase III- strains. A low-phosphate defined medium in which most E. coli strains seem to grow well was developed. This medium is equally useful for labeling ribonucleic acids with 32PO4 and as a selective medium for genetic manipulations. It was used to determine the growth requirements of strain AB301-105, which are biotin and succinate in addition to the methionine and histidine requirements of the parental strain. The biotin mutation lies near the position expected from known mutations of E. coli, but the succinate mutation apparently does not. The possibility that the succinate requirement could be due to the RNase III deficiency is discussed. A uraP mutation was isolated for use in transferring rnc-105 between strains by conjugation. It lies near 47 min, somewhat removed from the commonly accepted position for uraP.     

Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12.

Periplasmic-leaky mutants of Escherichia coli K-12 were isolated after nitrosoguanidine-induced mutagenesis. They released periplasmic enzymes into the extracellular medium. Excretion of alkaline phosphatase, which started immediately in the early exponential phase of growth, could reach up to 90% of the total enzyme production in the stationary phase. Leaky mutants were sensitive to ethylenediaminetetraacetic acid, cholic acid, and the antibiotics rifampin, chloramphenicol, mitomycin C, and ampicillin. Furthermore, they were resistant to colicin E1 and partially resistant to phage TuLa. Their genetic characterization showed that the lky mutations mapped between the suc and gal markers, near or in the tolPAB locus. A biochemical analysis of cell envelope components showed that periplasmic-leaky mutants contained reduced amounts of major outer membrane protein OmpF and increased amounts of a 16,000-dalton outer membrane protein.     

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli , has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42°C (non-permissive temperature) and at 37°C (semi-permissive temperature), but not at 28°C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which overinitiation of DNA replication occurs at low temperature (28°C), showed a higher level of unsaturation of fatty acids at 28°C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.     

cis/trans isomerase of unsaturated fatty acids of Pseudomonas putida P8: evidence for a heme protein of the cytochrome c type.

From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.     

Isolation and characterization of a mutation that alters the substrate specificity of the Escherichia coli glucose permease.

We isolated 10 mannitol-positive mutants from a mannitol-negative Escherichia coli strain. These mutations mapped within ptsG, encoding the glucose permease (EIIGlc), and resulted in a G-320-to-V substitution that allows EIIGlc to transport mannitol. Gly-320 lies within a putative transmembrane helix of EIIGlc that may be involved in substrate recognition.     

Effect of dietary cis and trans fatty acids on serum lipoprotein[a] levels in humans.

Serum lipoprotein[a] (Lp[a]) is a strong risk factor for coronary heart disease. We therefore examined the effect of dietary fatty acid composition on serum Lp[a] levels in three strictly controlled experiments with healthy normocholesterolemic men and women. In Expt. I, 58 subjects consumed a control diet high in saturated fatty acids for 17 days. For the next 36 days, 6.5% of total energy intake from saturated fatty acids was replaced by monounsaturates plus polyunsaturates (monounsaturated fatty acid diet; n = 29) or by polyunsaturates alone (polyunsaturated fatty acid diet; n = 29). Both diets caused a slight, nonsignificant, increase in median Lp[a] levels, with no difference between diets. In Expt. II, 10% of energy from the cholesterol-raising saturated fatty acids (lauric, myristic, and palmitic acid) was replaced by oleic acid or by trans-monounsaturated fatty acids. Each of the 59 participants received each diet for 3 weeks in random order. The median level of Lp[a] was 26 mg/l on the saturated fatty acid diet; it increased to 32 mg/l (P less than 0.020) on the oleic acid diet and to 45 mg/l (P less than 0.001) on the trans-fatty acid diet. The difference in Lp[a] between the trans-fatty acid and the oleic acid diets was also highly significant (P less than 0.001). Expt. III involved 56 subjects; all received 8% of energy from stearic acid, from linoleic acid, or from trans-monounsaturates, for 3 weeks each. All other nutrients were equal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the involvement of unsaturated fatty acids in initiating chromosome replication in Escherichia coli

The analogue 3-decynoyl-N-acetylcysteamine inhibits the synthesis of unsaturated fatty acids in Escherichia coli, resulting in the accumulation of saturated fatty acids in the membrane (Kass, 1968).In the presence of this analogue, DNA, RNA and protein synthesis continue at a linear rate for approximately two doubling times, and then cease. On the other hand, the analogue will inhibit the formation of new replication forks (premature initiation), which normally arise as a result of thymine starvation.Unlike other temperature-sensitive DNA mutants, mutants that are defective in initiating DNA replication (dnaA or dnaC) are unable to replicate DNA at a permissive temperature if they terminate replication at 42 °C in the presence of 3-decynoyl-N-acetylcysteamine.When replication is terminated at 42 °C, cultures of dnaA or dnaC mutants normally will reinitiate replication upon lowering the temperature to 30 °C. For each mutant this reinitiation is characterized by a particular temperature sensitivity. Such mutants become more temperature sensitive if the temperature is lowered in the presence of 3-decynoyl-N-acetylcysteamine. All the effects of this analogue can be reversed by the addition of unsaturated fatty acids.These results are interpreted using a model in which replication is initiated at a particular lipid site on the membrane. In the absence of unsaturated fatty acids functional lipid sites are not made. Functional sites, however, can be used again provided they are not inactivated by interaction with an inactive dnaA or dnaC product.     

Mapping of fabC, a locus for the biosynthesis of unsaturated fatty acids in Escherichia coli K12

Summary A mutation affecting the biosynthesis of unsaturated fatty acids in Escherichia coli K12 has been mapped. This mutation called fabC, which is cotransducible with nalA, lies between the nalA and his loci.This paper is taken from a thesis to be submitted by J. H. F. F. Broekman to the faculty of Science of the State University in Utrecht in partial fulfilment of the requirements for the Ph. D. degree.