Cell volume change of Escherichia coli under stringent control. Apparent increase of K+ in rel- cells.

With several pairs of rel+ and rel- strains of Escherichia coli, the effects of amino acid starvation on the intracellular concentration of K+ and the rate of uptake of 42K+ were investigated. In the early phase of the experiments, the intracellular concentration of K+ was estimated by the conventional method in which the cell volume per A660 value of the culture was assumed to be constant, being not influenced by the variation of growth condition and strain. Apparently, the K+ concentration of rel+ cells was kept almost constant, while that of rel- cells increased about 1.5-fold 2 h after the exposure to amino acid starvation. Unexpectedly, however, the above assumption was found not to be valid in the present study. The cell volume per A660 changed only slightly in CP78 (rel+) cells, while it increased markedly in CP79 (rel-) cells after the exposure to amino acid starvation. Reestimation of the K+ concentrations based on the estimated respective values of cell volumes per A660 revealed no significant difference between both strains. After all, the above apparent phenomenon was found to be due to the fact that the increase in cell volume of the rel+ cells was arrested upon amino acid starvation whereas that in the rel- cells was not. The 42K+ uptake by the rel+ cells was depressed upon amino acid starvation, whereas that by the rel- cells increased. Some regulatory mechanism was suggested to operate in both strains to keep their K+ concentrations constant. When intracellular concentration of a metabolite is to be determined, importance of measurement of cell volume under the respective conditions, without assuming the constancy of the cell volume per A660 of the culture, was pointed out.

cDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases.

Using degenerate oligonucleotide primers corresponding to conserved regions of the calcium-dependent protein kinase (CDPK) family, we carried out a polymerase chain reaction and obtained four distinct partial-length cDNAs from a maize leaf library. We then used these clones as probes for conventional screening and isolated 19 longer clones from another cDNA library of maize seedlings. These clones were classified into four groups based on their DNA cross-hybridization. Two full-length cDNAs, designated as ZmCDPK9 and ZmCDPK7, were sequenced and characterized. The predicted protein of each clone was a typical CDPK with eleven canonical subdomains of protein kinases, and four EF-hand calcium-binding motifs in its N-terminal and C-terminal halves, respectively. The catalytic and regulatory domains were linked by a well-conserved junction domain. The N-terminus of the protein also contained a consensus sequence for an N-myristoylation signal. Northern blot analysis showed that the transcription level of each gene was higher in roots and etiolated leaves than in green leaves. To confirm the calcium dependency of the maize enzymes, the entire coding region of ZmCDPK9 was subcloned into an expression vector so that it was in frame with the vector-encoded peptide tags. A cell-free extract of Escherichia coli transformed with the recombinant plasmid exhibited calcium-dependent phosphorylation activity, using casein as a substrate.

cDNA cloning and expression analysis of a non-photosynthetic ferredoxin gene in morning glory (Pharbitis nil).

A full-length cDNA encoding a non-photosynthetic ferredoxin was isolated from apical buds of morning glory (Pharbitis nil), a short-day plant, by differential screening under flower-inducing and non-inducing conditions. Northern analysis and in situ hybridization showed that the transcript was abundant in shoot apices and root tips. The transcript level in the apical buds decreased with the flower-inducing light treatment.

Phosphatidylethanolamine molecular species of fatty acid auxotroph of Escherichia coli grown with elaidate.

Monoacetyldiglycerides derived from the phosphatidylethanolamine molecular species of the fatty acid auxotroph of Escherichia coli grown with elaidate at 37 degrees C were fractionated on thin-layer plates of silica impregnated with silver nitrate and were identified by gas chromatography-mass spectrometry with an OV-17 column and gas chromatography with a Silar-10C column. Phosphatidylethanolamine was made up of the following molecular species: 1-16 : 0-2-16 : 0 (1.2%), 1-14 : 0-2-trans-16 : 1 (1%), 1-16 : 0-2 trans-16 : 1 (3.5%), 1-16 : 0-2-trans-18 : 1 (26.4%), 1-16 : 0-2-cis-16 : 1 (3.8%), 1-trans-18 : 1-2-trans-16 : 1 (13.2%), 1-trans-18 : 1-2-trans-18 : 1 (44.9%), 1-trans-18 : 1-2-cis-16 : 1 (4.5%) and trans-18 : 1-cis-18 : 1 (1.5%).

First crystallization of a phosphoenolpyruvate carboxylase from Escherichia coli.

Two different forms of crystal for a phosphoenolpyruvate carboxylase from Escherichia coli were obtained by the hanging-drop vapor diffusion technique, using polyethylene glycol 4000 as precipitant. The hexagonal crystal in space group P6(2)22 (or P6(4)22) has cell dimensions of a = 131 A and c = 325 A, whereas the orthorhombic crystal in space group I222 has a = 119 A, b = 252 A and c = 83 A. A tetrameric molecule (396,244 Mr), a subunit of which contains 883 amino residues, has a crystallographic 2 symmetry in the hexagonal crystal or 222 symmetry in the orthorhombic crystal, respectively.

Lotus japonicus forms early senescent root nodules with Rhizobium etli.

Mesorhizobium loti and Rhizobium etli are microsymbionts of the Lotus and Phaseolus spp., respectively, and secrete essentially the same Nod factors. Lotus japonicus efficiently formed root nodules with R. etli CE3, irrespective of the presence or absence of a flavonoid-independent transcription activator nodD gene. On a nitrogen-free medium, however, the host plant inoculated with R. etli showed a severe nitrogen deficiency symptom. Initially, the nodules formed with R. etli were pale pink and leghemoglobin mRNA was detectable at significant levels. Nevertheless, the nodules became greenish with time. Acetylene-reduction activity of nodules formed with R. etli was comparable with that formed by M. loti 3 weeks postinoculation, but thereafter it decreased rapidly. The nodules formed with R. etli contained much more starch granules than those formed with M. loti. R. etli developed into bacteroids in the L. japonicus nodules, although the density of bacteroids in the infected cells was lower than that in the nodules formed with M. loti. The nodules formed with R. etli were of the early senescence type, in that membrane structures were drastically disintegrated in the infected cells of the greenish nodules. Thus, L. japonicus started and then ceased a symbiotic relationship with R. etli at the final stage.

Nucleotide sequence of the phosphoenolpyruvate carboxylase gene of the cyanobacterium Anacystis nidulans.

Nucleotide sequence of the open reading frame (ORF) for the phosphoenolpyruvate carboxylase gene (ppc) of the cyanobacterium Anacystis nidulans was determined. The ORF consists of 3159 bp and codes for 1053 amino acid (aa) residues. The codon usage of the ppc of A. nidulans is not so markedly different from that of the Escherichia coli ppc, yet, in A. nidulans the preferred codons are AAG for lysine and CCC for proline, whereas those are seldom used in the E. coli ppc.

Molecular cloning of the phosphoenolpyruvate carboxylase gene, ppc, of Escherichia coli.

The ColE1 hybrid plasmid, pLC20-10, carrying the ppc gene and the argECBH gene cluster of Escherichia coli K-12, was characterized. The ppc gene coding for phosphoenolpyruvate carboxylase (EC 4.1.1.31), was subcloned into the plasmid pBR322 to give the plasmids pS2 and pS3. These plasmids carried a 4.4-kb SalI segment containing the ppc gene, in both orientations. The specific activity of the enzyme was increased approx. 20-fold by these plasmids. Experiments with maxicells harboring pS2 showed that the 90-kDal enzyme subunit was encoded by the plasmid. The location of the ppc gene in pS2 and the direction of transcription of the gene were determined. In DNA-DNA hybridization experiments using pS2 as a probe, significant hybridizations were observed with DNAs from E. coli strains K-12 and W, and from Salmonella typhimurium, but not with those from Chlorella regularis, Anacystis nidulans, Rhodospirillum rubrum, and Pseudomonas AM-1.

Regulatory phosphorylation of plant phosphoenolpyruvate carboxylase: role of a conserved basic residue upstream of the phosphorylation site.

In order to mimic regulatory phosphorylation of the Ser-15 of maize C4-form phosphoenolpyruvate carboxylase (PEPC), we replaced Ser-15 and Lys-12 with Asp (S15D) and Asn (K12N), respectively, by site-directed mutagenesis. Although both mutant enzymes were catalytically as active as the wild-type PEPC, they showed much less sensitivity to malate, an allosteric inhibitor, similarly to the phosphorylated wild-type PEPC. A maize protein kinase of 30 kDa which is known to be specific to PEPC (PEPC-PK), phosphorylated K12N as well as the wild-type PEPC but not S15D. The phosphorylation of K12N further diminished the sensitivity to malate. Thus, a positive charge of the conserved Lys-12 is not required for the recognition by PEPC-PK but contributes to the intrinsic sensitivity to malate inhibition.

A Ca(2+)-dependent protein kinase phosphorylates phosphoenolpyruvate carboxylase in maize.

In C4 plants the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is regulated by phosphorylation/dephosphorylation which is mediated by light/dark signals. The study using protein kinase inhibitors showed that the inhibition pattern of maize PEPC-protein kinase (PEPC-PK) is similar to that of myosin light chain kinase, a Ca(2+)-calmodulin-dependent PK. The kinase activity was also inhibited by EGTA and the inhibition was relieved by Ca2+. These results suggest that PEPC-PK is Ca(2+)-dependent in contrast with previous observations by other research groups.

Plant calcium-dependent protein kinase-related kinases (CRKs) do not require calcium for their activities.

In plants, calcium-dependent protein kinases (CDPKs) make up a large family that is characterized by a C-terminal calmodulin(CaM)-like domain. Recently, a novel carrot cDNA clone encoding an atypical CDPK, which has a significantly degenerate sequence in the CaM-like domain, was found and named CDPK-related protein kinase (CRK) [Lindzen, E. and Choi, J.H. (1995) Plant Mol. Biol. 28, 785-797]. We obtained two different cDNA clones from maize which encode CRKs. For the first enzymatic characterization of CRK, a maize cDNA clone was expressed in E. coli. The recombinant protein efficiently phosphorylated casein, a conventional protein substrate. Notably, in this in vitro phosphorylation assay, the kinase activity did not require calcium as an activator. Thus, CRKs were suggested to be novel calcium-independent protein kinases having a degenerate CaM domain, the function of which remains to be elucidated.

Phosphoenolpyruvate carboxylase kinase involved in C(4) photosynthesis in Flaveria trinervia: cDNA cloning and characterization.

In C(4) plants, phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), a key enzyme in C(4) photosynthesis, is controlled by reversible phosphorylation of a conserved Ser residue near the N-terminus. We now report the first cloning of a cDNA from a C(4) plant, Flaveria trinervia, which encodes the specific protein kinase (FtPEPC-PK) involved in the phosphorylation of C(4)-form PEPC. Several lines of supportive evidence are: strict substrate specificity of the recombinant enzyme, prominent light/dark response of the transcript level and abundant expression in leaves of C(4) plant (F. trinervia) but very low expression in a C(3) plant of the same genus (Flaveria pringlei). We also discuss the possibility that the FtPEPC-PK gene has co-evolved with the PEPC gene to participate in C(4) photosynthesis.

Further analysis of cDNA clones for maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis. Nucleotide sequence of entire open reading frame and evidence for polyadenylation of mRNA at multiple sites in vivo.

Four clones of cDNA for phosphoenolpyruvate carboxylase [EC 4.1.1.31] were obtained from a maize green leaf cDNA library by colony hybridization. The largest cDNA was of full-length (3335 nucleotides), being 243 nucleotides longer than the cDNA cloned previously [(1986) Nucleic Acids Res. 14, 1615-1628]. Alignment of the sequence for the N-terminal coding region found in two of the four clones with the sequence reported previously, established the sequence of the entire coding region for the enzyme. The sequencing of 3'-untranslated region of the clones revealed that the poly(A) tract is attached at multiple sites in vivo.

Plausible phosphoenolpyruvate binding site revealed by 2.6 A structure of Mn2+-bound phosphoenolpyruvate carboxylase from Escherichia coli.

We have determined the crystal structure of Mn2+-bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 A resolution, and specified the location of enzyme-bound Mn2+, which is essential for catalytic activity. The electron density map reveals that Mn2+ is bound to the side chain oxygens of Glu-506 and Asp-543, and located at the top of the alpha/beta barrel in PEPC. The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure. The model study of Mn2+-bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg-396, Arg-581 and Arg-713 could interact with PEP.

Maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis: nucleotide sequence analysis of the 5' flanking region of the gene.

To clone the genomic DNA fragment containing the putative promoter region of the gene for phosphoenolpyruvate carboxylase [EC 4.1.1.31] involved in C4 photosynthesis (C4-type PEPC), maize genomic libraries were screened. On probing with a 384-bp fragment from the N-terminal coding region of the maize cDNA for C4-type PEPC, four EcoRI-fragments differing in the restriction map were cloned, reflecting the presence of a small gene family. Southern blot analyses were carried out on the genomic DNA and the cloned DNA fragments using several segments of the cDNA for C4-type PEPC as probes. The results indicated that the C4-type PEPC is encoded by a single gene and the cloned 7.0-kb fragment was derived from this gene. The transcription start site, as determined in a primer extension experiment, was located at about 4 kb downstream of the 5' end of the cloned fragment. The nucleotide sequence was determined for the region which extended about 1 kb upstream from the transcription start site and possible signal sequences related to gene expression were found, including four classes of direct repeats. The sequences of the corresponding regions of the other two cloned fragments (8.9 and 12.9 kb) which strongly hybridized with the 384-bp probe were very similar to each other, but they were quite different in the 5' upstream region from the sequence of the gene for C4-type PEPC.

Site-directed mutagenesis of phosphoenolpyruvate carboxylase from E. coli: the role of His579 in the catalytic and regulatory functions.

Phosphoenolpyruvate carboxylases (PEPC) [EC 4.1.1.31] from a wide variety of organisms contain a unique and highly conserved sequence, 578FHGRGGSIGRGGAP591 (coordinates for the Escherichia coli enzyme), which has been presumed to participate in the binding of phosphoenolpyruvate (PEP). Since previous chemical modification studies had suggested the importance of His for the catalytic activity, the role of His579 was investigated by constructing variants of E. coli PEPC, in which this residue was substituted to Asn (H579N) or Pro (H579P). Kinetic studies with partially purified enzymes revealed the following: (1) The apparent maximal velocities in the presence of acetyl-CoA (CoASAc, one of the allosteric activators) were 29% and 5.4% of the wild-type enzyme, for H579N and H579P, respectively. (2) The half-saturation concentration for PEP was increased about 40-fold by the substitutions, while those for another substrate (HCO3-) and the metal cofactor (Mg2+) were increased only 2- to 4-fold. (3) The half-saturation concentrations of four kinds of allosteric activators and of dioxane, an artificial activator, were also changed to various extents. Among them the most remarkable increase was observed for CoASAc (28-fold). (4) The concentration of an allosteric inhibitor, aspartate, required for 50% inhibition remained substantially unchanged. It was concluded that the imidazole group of His579 is not obligatory for the enzyme catalysis, but plays important roles in catalytic and regulatory functions.

Augmentation of cyclopropane fatty acid synthesis under stringent control in Escherichia coli.

An abrupt increase of cyclopropane fatty acid (CFA) occurred concomitant with a decrease of the corresponding unsaturated fatty acids in CP78 (rel+) of Escherichia coli at the onset of the stationary growth phase, whereas such variations were slight in CP79 (rel-). When the cells were starved for isoleucine, the CFA content increased in CP78 but not in CP79. The rate of 14C-incorporation from [methyl-14C]methionine into CFAs increased in CP78 abut two-fold due to the starvation. The apparent level of CFA synthase also increased due to the starvation. These results that the CFA formation is augmented under stringent control.

Augmentation of glycogen synthesis under stringent control in Escherichia coli.

When Escherichia coli strain CP78 (rel+) was starved for isoleucine by the addition of valine, the amount of glucose in polymeric form in the cells increased markedly compared to that of the control cells. In contrast, this phenomenon was not seen in strain CP79 (rel-). The increase in CP78 was shown to be due to the increase of glycogen. These results indicate that glycogen synthesis was augmented under stringent control. This was confirmed using other isogenic pairs of rel+ and rel- strains starved for other amino acids. When the cultivation temperature of strains 10B601 (rel+) and 10B602 (rel-) possessing temperature-sensitive valyl-tRNA synthetase was shifted from 30 degrees C to 40 degrees C, no difference was observed in the response of glycogen synthesis between the two strains. These results indicate that protein synthesis was necessary for the augmentation of glycogen synthesis and that guanosine 5'-diphosphate 3'-diphosphate did not exert its effect through stimulation of the activity of pre-existing enzyme(s) involved in glycogen synthesis. These conclusions were supported by the results of experiments using chloramphenicol and rifampicin. The rates of glucose utilization of CP78 and CP79 were decreased to nearly the same extent by valine addition. This suggests that the regulation site of glycogen synthesis under stringent control resides in a step after the transport of glucose by the phosphotransferase system.

Phosphoenolpyruvate carboxylase of Escherichia coli K-12. N- and C-terminal sequences and tentative assignment of the catalytically essential cysteine residue.

The N- and C-terminal amino acid sequences of phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were determined to establish the primary structure deduced from the nucleotide sequence of the cloned gene for the enzyme (Fujita, N., Miwa, T., Ishijima, S., Izui, K., & Katsuki, H. (1984) J. Biochem. 95, 909-916). As predicted from the nucleotide sequence, two polypeptides were produced upon treatment with hydroxylamine, which specifically cleaves the Asn-Gly bond, and their amino acid compositions were also in accordance with those predicted. The tryptic peptides which contained cysteine residues labeled with a fluorescent reagent, N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide, were isolated by high-performance liquid chromatography and partially sequenced. All of them could be assigned on the deduced primary structure. The modified cysteine residues were Cys-157, Cys-385, Cys-458, Cys-568, Cys-665, and Cys-754. Furthermore, the essential cysteine residue which is presumably located at or near the active site was tentatively identified as Cys-568, since it was consistently protected against the modification by 2-phospholactate, a substrate analog.

Purification and characterization of recombinant phosphoenolpyruvate carboxylase of Thermus sp.

Recombinant phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) of an extreme thermophile, Thermus sp., which was expressed in Escherichia coli cells, was purified and its enzymological properties were investigated and compared with native Thermus PEPC. The enzyme activity was strongly dependent on acetyl-CoA, an allosteric activator, and inhibited by malate or aspartate. Contrary to the other known PEPCs, Thermus PEPC was not activated but rather inhibited by phosphorylated compounds such as fructose 1,6-bisphosphate and GTP. The specific activity in the presence of 0.3 mM acetyl-CoA and 2 mM phosphoenolpyruvate was highest at 70 degrees C. The half-saturation concentrations for both substrates at 70 degrees C were about twice those at 30 degrees C. Half-lives of the enzyme at 85, 90, and 95 degrees C were 220, 110, and 50 min, respectively. Thermus PEPC was highly tolerant also to guanidine hydrochloride (Gdn-HCl): the concentrations required for complete inactivation of Thermus and E. coli PEPCs after incubation at 30 degrees C for 20 h were 3.5 and 0.6 M, respectively. The properties of recombinant and native enzyme were similar to each other except for the catalytic activity after incubation with 1-2 M Gdn-HCl.