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).     

Evidence that putrescine acts as an extracellular signal required for swarming in Proteus mirabilis

In a search for Proteus mirabilis genes that were regulated by cell-to-cell signalling, a lacZ fusion (cmr437::mini-Tn5lacZ) was identified that was repressed 10-fold by a self-produced extracellular signal from wild-type cells. However, the cmr437::mini-Tn5lacZ insertion itself led to a marked reduction in this extracellular repressing signal. The cmr437::mini-Tn5lacZ insertion was mapped to a speA homologue in P. mirabilis. Sequence analysis indicated that a speB homologue was encoded downstream of speA. Products of the SpeA and SpeB enzymes (agmatine and putrescine) were tested for repression of cmr437::lacZ. Agmatine did not have repressing activity. However, putrescine was an effective repressing molecule at concentrations down to 30 microM. A second prominent phenotype of the cmr437 (speA)::mini-Tn5lacZ insertion was a severe defect in swarming motility. This swarming defect was also observed in a strain containing a disruption of the downstream speB gene. Differentiation of the speB mutant to swarmer cells was delayed by two hours relative to wild-type cells. Furthermore, the speB mutant was unable to migrate effectively across agar surfaces and formed very closely spaced swarming rings. Exogenous putrescine restored both the normal timing of swarmer cell differentiation and the ability to migrate to speB mutants.     

Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK)

The speA, speB and speC genes, which code for arginine decarboxylase (ADCase), agmatine ureohydrolase (AUHase) and ornithine decarboxylase (ODCase), respectively, and the metK gene, which encodes methionine adenosyltransferase (MATase), have been cloned. The genes were isolated from hybrid ColE1 plasmids of the Clarke-Carbon collection and were ligated into plasmid pBR322. Escherichia coli strains transformed with the recombinant plasmids exhibit a 7- to 17-fold overproduction of the various enzymes, as estimated from increases in the specific activities of the enzymes assayed in crude extracts. Minicells bearing the pBR322 hybrid plasmids and labeled with radioactive lysine synthesize radiolabeled proteins with M_rs corresponding to those reported for purified ODCase, ADCase and MATase. Restriction enzyme analysis of the plasmids, combined with measurements of specific activities of the enzymes in crude extracts of cells bearing recombinant plasmids, clarified the relative position of speA and speB. The gene order in the 62- to 64-min region is serA speB speA metK speC glc.     

Isolation, characterization, and mapping of Escherichia coli mutants blocked in the synthesis of ornithine decarboxylase.

Several Escherichia coli K-12 mutants blocked in the synthesis of ornithine decarboxylase (OD) were isolated after transduction for serA+ in a strain (MA197) blocked in agmatine ureohydrolase (AUH) with a mutagenized phage lysate of P1. The new double-polyamine mutants were characterized by an unconditional polyamine dependence; either putrescine or spermidine was required for normal growth. The mutational block was varified by the demonstration of a virtual absence of OD activity in cellular extracts. The mutation, designated speC, was mapped by P1 transduction in several strains and was shown to have a cotransduction frequency of 17.2% with serA. Map order was established as serA speB speC metK. A derivative of one of the OD mutants having wild-type levels of AUH and blocked in OD was utilized along with an OD AUH mutant and an OD+ AUH strain to explore the phenomenon of "pathway selection" using growth rate as a parameter. Polyamine pool studies were carried out simultaneously. The results presented here support the hypothesis of pathway selection, implying a preferential utilization of exogenous arginine rather than endogenously produced arginine in polyamine biosynthesis.     

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.     

Identification of a recA homolog (recALP) on the conjugative lactococcal phage resistance plasmid pNP40: evidence of a role for chromosomally encoded recAL in abortive infection.

The determinants for two bacteriophage resistance mechanisms, AbiE and AbiF, are separated by approximately 3,300 nucleotides on the lactococcal plasmid pNP40 (P. Garvey, G.F. Fitzgerald, and C. Hill, Appl. Environ. Microbiol. 61:4321-4328, 1995). DNA sequence analysis of the intervening region led to the identification of two open reading frames (ORFs) which are transcribed in the opposite direction to the Abi determinants. One of these ORFs encodes a recA homolog (designated recALP). This is the first report of a recA-like determinant located to a plasmid. The second ORF (orfU) shares homology with the umuC gene of the SOS response. Analysis of a number of lactococcal strains confirmed the presence of recALP-like sequences in at least two other lactococcal strains. The proximity of the recA and umuC homologs suggested a possible role in the phase resistance encoded by the Abi determinants. However, no evidence was obtained to demonstrate a function for either ORF in the expression of either AbiE or AbiF. Nor could the recALP gene restore resistance to mitomycin in a recA-deficient lactococcal strain, VEL1122. Interestingly, it was shown that the chromosomally encoded recA is necessary for complete expression of the AbiF phenotype, confirming a role for RecA in this abortive infection system.     

N1-aminopropylagmatine, a new polyamine produced as a key intermediate in polyamine biosynthesis of an extreme thermophile, Thermus thermophilus

In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N1-Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N1-Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N1-Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N1-aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N1-aminopropylagmatine to spermidine but does not act on agmatine.     

Frequency of genes speA,speB, and speC in Streptococcus pyogenes strains and the identification of the infective agent by polymerase chain reaction

In cultures of S. pyogenes isolated from patients and carriers in different territories of the Russian Federation the genes of erythorogenic toxins A, B and C (speA, speB and specC) were detected. The possibility of the identification of S. pyogenes by means of PCR on the basis of primers to erythrogenic toxin B was determined. Gene speB was detected in all S. pyogenes cultures under study and proved to be species specific. Genes speA and speC were detected, respectively, in 29.4% and 9.35% of the S. pyogenes cultures under study. A test system for the identification of S. pyogenes on the basis of primers to gene speB was developed. The prospects for the detection of genes speA and speC for intraspecific typing of this infective agent were evaluated.     

Independent translation of ORFs in dicistronic operons,synthetic building blocks for polycistronic chloroplast gene expression

We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5′‐untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High‐level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.     

Maize Glutamine Synthetase Cdnas: Isolation by Direct Genetic Selection in Escherichia Coli

Maize glutamine synthetase cDNA clones were isolated by genetic selection for functional rescue of an Escherichia coli delta glnA mutant growing on medium lacking glutamine. The Black Mexican Sweet cDNA library used in this study was constructed in pUC13 such that cDNA sense strands were transcribed under the control of the lac promoter. E. coli delta glnA cells were transformed with cDNA library plasmid DNA, grown briefly in rich medium to allow phenotypic expression of the cDNAs and the pUC13 ampr gene, and challenged to grow on agar medium lacking glutamine. Large numbers of glutamine synthetase cDNA clones have been identified in individual 150-mm Petri dishes; all characterized cDNA clones carry complete coding sequences. Two cDNAs identical except for different 5' and 3' termini have been sequenced. The major open reading frame predicts a protein with an amino acid sequence that exhibits striking similarity to the amino acid sequences of the predicted products of previously sequenced eukaryotic glutamine synthetase cDNAs and genes. In addition, the maize glutamine synthetase cDNAs were shown to contain a 5' mini-ORF of 29 codons separated by 37 nucleotide pairs from the major ORF. This mini-ORF was shown not to be essential for the functional rescue of the E. coli delta glnA mutant. Expression of the cDNAs in E. coli is presumed to be due to the function of a polycistronic hybrid lac messenger RNA or translational fusions encoded by the pUC plasmids. Proteins of the expected sizes encoded by two different pUC clones were shown to react with antibodies to tobacco glutamine synthetase.     

Nucleotide sequence and analysis of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli.

The DNA sequence of a 3.23-kilobase fragment of the Escherichia coli chromosome encoding biosynthetic arginine decarboxylase (ADC) was determined. This sequence contained the speA open reading frame (ORF) as well as partial speB and metK ORFs. The ADC ORF is 1,974 nucleotides long; the deduced polypeptide contains 658 amino acids with a molecular size of 73,980 daltons. The molecular weight and predicted ADC amino acid composition are nearly identical to the amino acid analysis of purified ADC performed by Wu and Morris (J. Biol. Chem. 248:1687-1695, 1973). A translational speA-lacZ fusion, pRM65, including 1,389 base pairs (463 amino acids) of the 5' end of speA was constructed. Western blots (immunoblots) with beta-galactosidase antisera revealed two ADC::beta-galactosidase fusion proteins in E. coli bearing pRM65: 160,000 and 156,000 daltons representing precursor and mature hybrid proteins, respectively. The predicted amino acid sequence of ADC contains a region of six amino acid residues found in two bacterial diaminopimelic acid decarboxylases and three eucaryotic ornithine decarboxylases. This conserved sequence is located approximately eight amino acids from the putative pyridoxal phosphate-binding site of ADC and is predicted to be involved in substrate binding.