Bifunctional Regulatory Protein Research Articles

LasX, a transcriptional regulator of the lactocin S biosynthetic genes in Lactobacillus sakei L45, acts both as an activator and a repressor

The 11 kb las locus, present on the 50 kb plasmid pCIM1, specifies the production of the lantibiotic lactocin S in Lactobacillus sakei L45. The gene cluster is organized into two oppositely orientated operons, lasAMNTUVPJW ( lasA–W) and lasXY, the former of which contains the biosynthetic, immunity and transport genes. We have previously shown that inactivation of lasX abolishes lactocin S production and causes a drastic reduction in lasA-specific transcripts (encoding pre-lactocin S). The aim of this study was to determine whether or not the product of lasX, which is significantly similar to Rgg-like regulators, was directly involved in transcriptional regulation of the lactocin S biosynthetic genes. The divergently orientated and overlapping promoters, P lasA – W and P lasXY , were transcriptionally fused to the Escherichia coli gusA gene, and the activity of the fusions was assayed in the presence and absence of lasX, which was expressed on a separate plasmid. A significant stimulation of expression (5–6-fold) of the P lasA-W – gusA fusion was observed in the presence of lasX, whereas expression of the P lasXY –gusA construct was reduced 1.5–2-fold. Our results strongly suggest that LasX is a bifunctional regulatory protein, acting both as an activator of lasA– W transcription and as a repressor of lasXY transcription. While a transcription stimulation activity has been described for several of the Rgg-like proteins, the present study is the first to report an autorepressor function for a member of this protein group.

Further Analysis of Maize C4 Pyruvate,Orthophosphate Dikinase Phosphorylation by Its Bifunctional Regulatory Protein Using Selective Substitutions of the Regulatory Thr-456 and Catalytic His-458 Residues

In C4 plants such as maize, pyruvate,orthophosphate dikinase (PPDK) catalyzes the regeneration of the initial carboxylation substrate during C4 photosynthesis. The primary catalytic residue, His-458 (maize C4 PPDK), is involved in the ultimate transfer of the β-phosphate from ATP to pyruvate. C4 PPDK activity undergoes light–dark regulation in vivo by reversible phosphorylation of a nearby active-site residue (Thr-456) by a single bifunctional regulatory protein (RP). Using site-directed mutagenesis of maize recombinant C4 dikinase, we made substitutions at the catalytic His residue (H458N) and at this regulatory target Thr (T456E, T456Y, T456F). Each of these affinity-purified mutant enzymes was assayed for changes in dikinase activity. As expected, substituting His-458 with Asn results in a catalytically incompetent enzyme. Substitutions of the Thr-456 residue with Tyr and Phe reduced activity by about 94 and 99%, respectively. Insertion of Glu at this position completely abolished activity, presumably by the introduction of negative charge proximal to the catalytic His. Furthermore, neither the T456Y nor inactive H458N mutant enzyme was phosphorylated in vitro by RP. The inability of the former to serve as a phosphorylation substrate indicates that RP is functionally a member of the Ser/Thr family of protein kinases rather than a “dual-specificity” Ser-Thr/Tyr kinase, since our previous work showed that RP effectively phosphorylated Ser inserted at position 456. The inability of RP to phosphorylate its native target Thr residue when Asn is substituted for His-458 documents that RP requires the His-P catalytic intermediate form of PPDK as its protein substrate. For these latter studies, synthetic phosphopeptide-directed antibodies specific for the Thr456-P form of maize C4 PPDK were developed and characterized.

Site-directed mutagenesis of maize recombinant C 4-pyruvate,orthophosphate dikinase at the phosphorylatable target threonine residue

A key regulatory enzyme of the C 4-photosynthetic pathway is stromal pyruvate,orthophosphate dikinase (PPDK, EC 2.7.9.1). As a pivotal enzyme in the C 4 pathway, it undergoes diurnal light–dark regulation of activity which is mediated by a single bifunctional regulatory protein (RP). RP specifically inactivates PPDK in the dark by an ADP-dependent phosphorylation of an active-site Thr residue (Thr-456 in maize). Conversely, RP activates inactive PPDK in the light by phosphorolytic dephosphorylation of this target Thr-P residue. We have employed a His-tagged maize recombinant C 4 PPDK for directed mutagenesis of this active-site regulatory Thr. Three such mutants (T456V, T456S, T456D) were analyzed with respect to overall catalysis and regulation by exogenous maize RP. Substitution with Val and Ser at this position does not affect overall catalysis, whereas Asp abolishes enzyme activity. With respect to regulation by RP, it was found that Ser can effectively substitute for the wild-type Thr residue in that mutant enzyme is phosphorylated and inactivated by RP. The T456V mutant, however, could not be phosphorylated and was, thus, resistant to ADP-dependent inactivation by RP.

Maize recombinant C4-pyruvate,orthophosphate dikinase: Expression in Escherichia coli, partial purification, and characterization of the phosphorylatable protein

The gene for C4-pyruvate,orthophosphate dikinase (PPDK) from maize (Zea mays) was cloned into an Escherichia coli expression vector and recombinant PPDK produced in E. coli cells. Recombinant enzyme was found to be expressed in high amounts (5.3 U purified enzyme-activity liter(-1) of induced cells) as a predominantly soluble and active protein. Biochemical analysis of partially purified recombinant PPDK showed this enzyme to be equivalent to enzyme extracted from illuminated maize leaves with respect to (i) molecular mass, (ii) specific activity, (iii) substrate requirements, and (iv) phosphorylation/inactivation by its bifunctional regulatory protein.

A simple, single-tube radioisotopic assay for the phosphorylation/inactivation activity of the pyruvate,orthophosphate dikinase regulatory protein

A simple, single-tube radiolsotopic method has been developed to assay the relative phosphorylation (inaetivation) activity of the bifunctional regulatory protein (RP) of C4-leaf pyruvate,orthophosphate dikinase (PPDK) in desalted leaf homogenates and partially purified preparations. RP catalyzes the inactivation of maize PPDK by phosphorylation of Thr-456, utilizing [β-P]ADP as the specific phosphoryl donor. Existing spectrophotometric and radioisotopic assays for the detection of RP activity are either relatively insensitive or labor-intensive and timeconsuming. We describe a modified radioisotopic assay that couples the synthesis of [β-(32)P]ADP by exogenous adenylate kinase with the subsequent RP-catalyzed [β-(32)P]ADP-dependent phosphorylation of exogenous maize PPDK. The incorporation of [β-(32)P] is dependent on the initial concentrations of ATP and PPDK, as well as the presence of active RP. Desalted leaf homogenates of C3 species fail to catalyze (32)P incorporation into exogenous maize PPDK. Conversely, heterologous systems containing the maize target enzyme and leaf homogenats of other C4 species result in PPDK-specific (32)P-incorporation. This simple radioisotopic assay is at least 40-times more sensitive than the routine spectrophotometric assay, and qualitatively exhibits comparable sensitivity and requires significantly less time than the currently available radioisotopic RP assay. The present assay reliably generates [β-(32)P]ADP and as such may be useful for studies of other systems requiring β-labeled ADP, which is not commercially available.

Role of Metabolites in the Reversible Light Activation of Pyruvate, Orthophosphate Dikinase in Zea mays Mesophyll Cells in Vivo

Whole leaf and mesophyll cell concentrations of pyruvate, phosphoenolpyruvate (PEP), ATP, and ADP were determined in Zea mays during the reversible light activation of pyruvate, orthophosphate dikinase in vivo. Mesophyll cell levels of the four metabolites were estimated by extrapolation from values in freeze-quenched leaf samples that were fractionated by differential filtration through nylon mesh nets (adapted from M Stitt, HW Heldt [1985] Planta 164: 179-188). During the 3 minutes required for complete light activation of dikinase, pyruvate levels in the mesophyll cell decreased (from 166 +/- 15 to 64 +/- 10 nanomoles per milligram of chlorophyll [nmol/mg Chl]) while PEP levels increased (from 31 +/- 4 to 68 +/- 4 nmol/mg Chl, with a transient burst of 133 +/- 16 nmol/mg Chl at 1 minute). Mesophyll cell levels of ATP increased (from 22 +/- 4 to 48 +/- 3 nmol/mg Chl) and ADP levels decreased (from 16 +/- 4 to 7 +/- 6 nmol/mg Chl) during the first minute of illumination. Upon darkening of the leaf and inactivation of dikinase, pyruvate levels initially increased in the mesophyll (from 160 +/- 30 to a maximum of 625 +/- 40 nmol/mg Chl), and then slowly decreased to about the initial value in the light over an hour. PEP levels dropped (from 176 +/- 5 to 47 +/- 3 nmol/mg Chl) in the first 3 minutes and remained low for the remainder of the dark period. Mesophyll levels of ATP and ADP rapidly decreased and increased, respectively, about twofold upon darkening. The trends observed for these metabolite levels in the mesophyll cell during the light/dark regulation of pyruvate, orthophosphate dikinase activity suggest that pyruvate and PEP do not play a major role in vivo in regulating the extent of light activation (dephosphorylation) or dark inactivation (ADP-dependent threonyl phosphorylation) of dikinase by its bifunctional regulatory protein. While the changes in ADP levels appear qualitatively consistent with a regulatory role for this metabolite in the light activation and dark inactivation of dikinase, they are not of a sufficient magnitude to account completely for the tenfold change in enzyme activity observed in vivo.

Chemical modification of the bifunctional regulatory protein of maize leaf pyruvate, orthophosphate dikinase. Evidence for two distinct active sites.

The active site(s) of the bifunctional regulatory protein of pyruvate,orthophosphate dikinase catalyze(s) the Pi-dependent activation (dephosphorylation) and ADP-dependent inactivation (phosphorylation) of maize leaf dikinase. The chemical modification studies of the regulatory protein active sites presented in this paper are interpreted as showing the two sites to be physically distinct. Pyridoxal 5'-phosphate and 2-nitro-5-thiocyanatobenzoate (NTCB) selectively inhibit the dikinase activating site, which is protected by the nonprotein substrate, Pi. Phenylglyoxal blocks both the activation and inactivation sites; the former is protected selectively by Pi and the latter by both the nonprotein substrate, ADP, and Pi. The Pi that protects the inactivation site is distinct from the activation substrate. Inhibition studies show Pi to be a parabolic competitive inhibitor of the ADP-dependent inactivation of dikinase, implying that besides substrate Pi, a second phosphate also binds to the regulatory protein. The above chemical modifications are not mutually exclusive; neither NTCB, 5,5'-dithiobis-(2-nitrobenzoate), nor pyridoxal 5'-phosphate blocks subsequent modification of the activation site by phenylglyoxal. Similarly, prior modification with NTCB does not affect modification by pyridoxal 5'-phosphate.

Regulation of NAD metabolism in Salmonella typhimurium: genetic analysis and cloning of the nadR repressor locus.

The nadR locus (99 min) controls the transcription of several genes involved with either the biosynthesis (nadAB) or recycling (pncB) of NAD in Salmonella typhimurium. Point mutations in this locus were found to cause defects either in the transport of nicotinamide mononucleotide (PnuA-), the regulation of nadAB (NadR-) or both transport and regulation (PnuA-NadR-). Deletions or insertions into nadR always resulted in the PnuA- NadR- phenotypes. Merodiploids constructed with various combinations of PnuA-, NadR- or PnuA- NadR- strains indicate a single complementation group. The results suggest the NadR product is a bifunctional regulatory protein. Operon fusions to lacZ (nadR :: Mud1-8) were used to show that nadR is not autoregulated and is transcribed in a clockwise direction. The gene was also cloned and located within a 2 kb EcoR1-Bg/II fragment.

Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins.

We have determined the nucleotide sequences of two genes from Klebsiella pneumoniae, nifA, the nif-specific activator of transcription and ntrC, the bifunctional regulatory protein involved in 'nitrogen control'. These sequences differ significantly from those previously published. In particular, nifA extends 40 codons beyond the stop codon reported earlier. This extension encodes a putative DNA-binding domain strongly homologous to the Rhizobium meliloti nifA protein and to some extent to the ntrC protein. In all three proteins this domain is linked by a segment of variable length to a strongly conserved central domain of 240 residues. A short segment having the properties of an interdomain linker joins the central region to an N-terminal domain, which is weakly related in the case of the two nifA proteins. This homology is not shared by the N-terminal domain of ntrC, which is clearly but unexpectedly related to the N-terminal domains of a diverse set of procaryotic pleiotropic control proteins, including ompR, dye and nusA from Escherichia coli and spoOA and spoOF from Bacillus subtilis.

Genetic analysis of the ompB locus in Escherichia coli K-12

Fine-structure mapping and complementation analysis of the ompB locus indicate that this locus is comprised of two genes, envZ and ompR. These genes are ordered on the chromosome as follows: aroB-envZ-ompR-asd. A model is proposed for the ompB-mediated regulation of the major outer membrane porin genes, ompF and ompC. According to this model, the ompR gene product is a bifunctional regulatory protein. The envZ gene product is an envelope protein that senses the cell's external environment.