Control of utilization of L-arginine, L-ornithine, agmatine, and putrescine as nitrogen sources in Escherichia coli K-12.

The regulation of the synthesis of the enzymes involved in the utilization of L-arginine, L-ornithine, agmatine, and putrescine as a sole nitrogen source in Escherichia coli K-12 was examined. The synthesis of agmatine ureohydrolase, putrescine aminotransferase, and pyrroline dehydrogenase is dually controlled by catabolite repression and nitrogen availability. Catabolite repression of agmatine ureohydrolase, but not that of putrescine aminotransferase or pyrroline dehydrogenase, is relieved by the addition of cAMP. Agmatine ureohydrolase synthesis in addition is subject to induction by L-arginine and agmatine. Arginine decarboxylase and ornithine decarboxylase synthesis is not sensitive to catabolite repression or to stimulation by nitrogen limitation or subject to substrate induction.     

Gamma-glutamyl-gamma-aminobutyrate hydrolase in the putrescine utilization pathway of Escherichia coli K-12

gamma-Glutamyl-gamma-aminobutyrate hydrolase (PuuD) was purified and the properties of the enzyme were characterized. The active center of PuuD was identified as Cys-114 by site-directed mutagenesis. The expression of PuuD was induced by putrescine and O2 (substrates of the Puu pathway), while the addition of succinate or NH4Cl (products of the Puu pathway) to the medium reduced the expression of PuuD. The findings that the puuD-deficient strain accumulated gamma-glutamyl-gamma-aminobutyrate (gamma-Glu-GABA) and could not grow on putrescine as a sole nitrogen source indicate that PuuD is physiologically important as a gamma-Glu-GABA hydrolase.     

Metabolic engineering of Escherichia coli for agmatine production

Agmatine is a kind of important biogenic amine. The chemical synthesis route is not a desirable choice for industrial production of agmatine. To date, there are no reports on the fermentative production of agmatine by microorganism. In this study, the base Escherichia coli strain AUX4 (JM109 ?speC ?speF ?speB ?argR) capable of excreting agmatine into the culture medium was first constructed by sequential deletions of the speC and speF genes encoding the ornithine decarboxylase isoenzymes, the speB gene encoding agmatine ureohydrolase and the regulation gene argR responsible for the negative control of the arg regulon. The speA gene encoding arginine decarboxylase harboured by the pKK223‐3 plasmid was overexpressed in AUX4, resulting in the engineered strain AUX5. The batch and fed‐batch fermentations of the AUX5 strain were conducted in a 3‐L bioreactor, and the results showed that the AUX5 strain was able to produce 1.13 g agmatine L^?1 with the yield of 0.11 g agmatine g^?1 glucose in the batch fermentation and the fed‐batch fermentation of AUX5 allowed the production of 15.32 g agmatine L^?1 with the productivity of 0.48 g agmatine L^?1 h^?1, demonstrating the potential of E. coli as an industrial producer of agmatine.     

Regulation of D-arabinose utilization in Escherichia coli K-12.

Studies involving lambda phage transduction of the D-arabinose utilization gene (dar+) in Escherichia coli K-12 indicated the product of this gene to be a transdominant activator. An apparent anomaly regarding this hypothesis exists in that a diploid recessive lysogen (lambda dar-/dar-) can spontaneously become capable of growth on D-arabinose.     

A pathway for putrescine catabolism in Escherichia coli

Escherichia coli mutants able to grow in putrescine have been isolated from gamma-aminobutyrate mutants. These mutants show putrescine-alpha-ketoglutarate transaminase and gamma-aminobutyraldehyde dehydrogenase activities. Both enzymes have been characterized, the first of them showing an apparent Km for putrescine of 22.5 microM and the second an apparent Km of 37 microM for NAD and 18 microM for delta-1-pyrroline; the optimum pH values were 7.2 and 5.4, respectively, for the two enzymes.     

Chromosomal mutation for citrate utilization by Escherichia coli K-12.

A mutant strain of Escherichia coli K-12 that utilizes citrate as a sole source of carbon and energy was isolated. Citrate utilization arose as the consequence of two mutations in genes citA and citB, which are linked to the gal operon. The mutant strain expresses a semiconstitutive citrate transport system, and it utilizes both citrate and isocitrate as carbon and energy sources. It is capable of utilizing cis- and trans-aconitate, but only if it is preinduced by growth on citrate.     

A novel putrescine importer required for type 1 pili-driven surface motility induced by extracellular putrescine in Escherichia coli K-12

Recently, many studies have reported that polyamines play a role in bacterial cell-to-cell signaling processes. The present study describes a novel putrescine importer required for induction of type 1 pili-driven surface motility. The surface motility of the Escherichia coli ΔspeAB ΔspeC ΔpotABCD strain, which cannot produce putrescine and cannot import spermidine from the medium, was induced by extracellular putrescine. Introduction of the gene deletions for known polyamine importers (ΔpotE, ΔpotFGHI, and ΔpuuP) or a putative polyamine importer (ΔydcSTUV) into the ΔspeAB ΔspeC ΔpotABCD strain did not affect putrescine-induced surface motility. The deletion of yeeF, an annotated putative putrescine importer, in the ΔspeAB ΔspeC ΔpotABCD ΔydcSTUV strain abolished surface motility in putrescine-supplemented medium. Complementation of yeeF by a plasmid vector restored surface motility. The surface motility observed in the present study was abolished by the deletion of fimA, suggesting that the surface motility is type 1 pili-driven. A transport assay using the yeeF(+) or ΔyeeF strains revealed that YeeF is a novel putrescine importer. The K(m) of YeeF (155 μM) is 40 to 300 times higher than that of other importers reported previously. On the other hand, the V(max) of YeeF (9.3 nmol/min/mg) is comparable to that of PotABCD, PotFGHI, and PuuP. The low affinity of YeeF for putrescine may allow E. coli to sense the cell density depending on the concentration of extracellular putrescine.     

Purification and properties of agmatine ureohydrolyase, a putrescine biosynthetic enzyme in Escherichia coli.

The putrescine biosynthetic enzyme agmatine ureohydrolase (AUH) (EC 3.5.3.11) catalyzes the conversion of agmatine to putrescine in Escherichia coli. AUH was purified approximately 1,600-fold from an E. coli strain transformed with the plasmid pKA5 bearing the speB gene encoding the enzyme. The purification procedure included ammonium sulfate precipitation, heat treatment, and DEAE-sephacel column chromatography. The molecular mass of nondenatured AUH is approximately 80,000 daltons as determined by gel-sieving column chromatography, while on denaturing polyacrylamide gels, the molecular mass is approximately 38,000 daltons; thus, native AUH is most likely a dimer. A radiolabeled protein extracted from minicells carrying the pKA5 plasmid comigrated with the purified AUH in both sodium dodecyl sulfate-polyacrylamide and native polyacrylamide gels. The pI of purified AUH is between 8.2 and 8.4, as determined by either chromatofocusing or isoelectric focusing. The Km of purified AUH for agmatine is 1.2 mM; the pH optimum is 7.3. Neither the numerous ions and nucleotides tested nor polyamines affected AUH activity in vitro. EDTA and EGTA [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] at 1 mM inactivated AUH activity by 53 and 74%, respectively; none of numerous divalent cations tested restored AUH activity. Ornithine inhibited AUH activity noncompetitively (Ki = 6 X 10(-3) M), while arginine inhibited AUH activity competitively (Ki = 9 X 10(-3) M).     

YjjG, a dUMP phosphatase, is critical for thymine utilization by Escherichia coli K-12

Exogenous thymine must be converted to thymidine to enable a thyA (thymidylate synthase) mutant to grow. The deoxyribose in the thymidine comes from dUMP, which must first be dephosphorylated. The nucleotidase YjjG is critical for this step. A yjjG thyA mutant cannot use thymine for growth on a glucose minimal medium.     

Occurrence of agmatine pathway for putrescine synthesis in Selenomonas ruminatium

Selenomonas ruminantium synthesizes cadaverine and putrescine from L-lysine and L-ornithine as the essential constituents of its peptidoglycan by a constitutive lysine/ornithine decarboxylase (LDC/ODC). S. ruminantium grew normally in the presence of the specific inhibitor for LDC/ODC, DL-alpha-difluoromethylornithine, when arginine was supplied in the medium. In this study, we discovered the presence of arginine decarboxylase (ADC), the key enzyme in agmatine pathway for putrescine synthesis, in S. ruminantium. We purified and characterized ADC and cloned its gene (adc) from S. ruminantium chromosomal DNA. ADC showed more than 60% identity with those of LDC/ODC/ADCs from Gram-positive bacteria, but no similarity to that from Gram-negative bacteria. In this study, we also cloned the aguA and aguB genes, encoding agmatine deiminase (AguA) and N-carbamoyl-putrescine amidohydrolase (AguB), both of which are involved in conversion from agmatine into putrescine. AguA and AguB were expressed in S. ruminantium. Hence, we concluded that S. ruminantium has both ornithine and agmatine pathways for the synthesis of putrescine.     

Construction of an improved D-arabinose pathway in Escherichia coli K-12.

A ribitol catabolic pathway was transduced into Escherichia coli K-12 in an effort to determine whether the ribitol pathway would confer an advantage to D-arabinose-positive mutants growing on D-arabinose as the sole carbon source. Competition studies in chemostats showed that ribitol-positive strains, with a selection coefficient of 9%/h, have a significant competitive advantage over ribitol-negative strains. Ribitol-positive strains grown in batch culture also exhibited a shorter lag period than did ribitol-negative strains when transferred from glucose to D-arabinose. Repeated transfer of a ribitol-positive strain of E. coli K-12 on D-arabinose yielded a strain with further improved growth on D-arabinose. This "evolved" strain was found to constitutively synthesize L-fucose permease, isomerase, and kinase but had lost the ability to grow on L-fucose, apparently owing to the loss of a functional aldolase. This constitutive mutation is not linked to the fucose gene cluster and may be similar to an unlinked constitutive mutation described by Chen et al. (J. Bacteriol. 159:725-729, 1984).     

Stereospecificity of tripeptide utilization in a methionine auxotroph of Escherichia coli K-12

The stereospecificity of peptide utilization in Escherichia coli K-12 4212, a methionine auxotroph, was investigated using diastereomers of trimethionine and trimethionine methyl ester. Of the eight stereoisomers examined, only l-Met-l-Met-l-Met, l-Met-l-Met-d-Met, and d-Met-l-Met-l-Met and the corresponding methyl esters serve as growth substrates. Triornithine-resistant mutants of strain 4212 were isolated which failed to transport d-Met-l-Met-l-Met. These results provide evidence that an oligopeptide containing a d residue at its amine terminus can enter E. coli by the oligopeptide transport system.