Two rpoN-linked delta Tn10-kan insertions suppress the conditionally lethal erats allele. One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation. Neither alter expression of era indicating that suppression is post-translational. Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by erats. rpoN and six neighboring genes were sequenced and compared with sequences in the database. Two of these genes encode proteins homologous to Enzyme IIAFru and HPr of the phosphoenolpyruvate:sugar phosphotransferase system. We designate these proteins IIANtr (ptsN) and NPr (npr). Purified IIANtr and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate: sugar phosphotransferase system. Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy. This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP. These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIANtr to the unknown process regulated by the essential GTPase Era.

(TMG). These in vivo studies led to the discovery of an ATP-dependent metabolite-activated HPr kinase and an HPr(serine phosphate) IHPr(Ser-P)] phosphatase that reversibly phosphorylate HPr (6). This kinase was shown to phosphorylate serine46 in HPr, and it and the HPr(Ser-P) phosphatase were shown to be present in partial association with the cytoplasmic membrane in a wide variety of low-G+C Gram-positive bacteria (7).Lactobacillus brevis has been shown to possess a lactose/H+ symport permease that exhibits the glucose-promoted phenomenon of inducer efflux (8). This organism possesses HPr and the kinase/phosphatase system that reversibly phosphorylates serine-46 in this protein, but enzyme I and the various sugar-specific enzyme II complexes of the PTS are apparently lacking (9). The function of HPr and its reversible phosphorylation in L. brevis might be supposed to be regulation of non-PTS carbohydrate transport, but no direct evidence for this postulate has been forthcoming.In this paper we describe the development and use of a vesicular system that allowed us to test the postulate that HPr plays a direct role in the regulation of the L. brevis lactose permease. We show that both inducer exclusion and inducer effiux are dependent on intravesicular HPr and a metabolic intermediate such as fructose 1,6-bisphosphate (Fru-P2) or gluconate 6-phosphate. The results provide compelling evidence regarding the mechanism of non-PTS transport regulation in Gram-positive bacteria. MATERIALS AND METHODSOrganisms, Growth, and Vesicle Preparations. Growth conditions and the bacterial strain used, L. brevis strain ATCC367, were described previously (8). Cells were grown for 18 hr at 300C in the presence of 25 mM galactose, harvested, washed, and used directly for transport experiments or for membrane vesicle preparation following the method described by Kaback for Escherichia coli (10) with the following modifications. In the spheroplast formation step, lysozyme (2 mg/ml) in 10 mM potassium EDTA (pH 7.0) was incubated with cells for 180 min at room tempera-

Leaf senescence represents the final stage of leaf growth and development, and its onset and progression are strictly regulated; however, the underlying regulatory mechanisms remain largely unknown. In this study we found that WRKY42 was highly induced during leaf senescence. Loss-of-function wrky42 mutants showed delayed leaf senescence whereas the overexpression of WRKY42 accelerated senescence. Transcriptome analysis revealed 2721 differentially expressed genes between wild-type and WRKY42-overexpressing plants, including genes involved in salicylic acid (SA) and reactive oxygen species (ROS) synthesis as well as several senescence-associated genes (SAGs). Moreover, WRKY42 activated the transcription of isochorismate synthase 1 (ICS1), respiratory burst oxidase homolog F (RbohF) and a few SAG genes. Consistently, the expression of these genes was reduced in wrky42 mutants but was markedly increased in transgenic Arabidopsis overexpressing WRKY42. Both in vitro electrophoretic mobility shift assays (EMSAs) and in vivo chromatin immunoprecipitation and dual luciferase assays demonstrated that WRKY42 directly bound to the promoters of ICS1 and RbohF, as well as a few SAGs, to activate their expression. Genetic analysis further showed that mutations of ICS1 and RbohF suppressed the early senescence phenotype evoked by WRKY42 overexpression. Thus, we have identified WRKY42 as a novel transcription factor positively regulating leaf senescence by directly activating the transcription of ICS1, RbohF and SAGs, without any seed yield penalty.

Lactobacillus brevis takes up glucose and the nonmetabolizable glucose analog 2-deoxyglucose (2DG), as well as lactose and the nonmetabolizable lactose analoge thiomethyl I-galactoside (TMG), via proton symport. Our earlier studies showed that TMG, previously accumulated in L. brevis cells via the lactose:H+ symporter, rapidly efiluxes from L. brevis cells or vesicles upon addition of glucose and that glucose inhibits further accumulation of TMG. This regulation was shown to be mediated by a metabolite-activated protein kinase that phosphorylates serine 46 in the HPr protein. We have now analyzed the regulation of 2DG uptake and efflux and compared it with that of TMG. Uptake of 2DG was dependent on an energy source, effectively provided by intravesicular ATP or by extravesicular arginine which provides ATP via an ATP-generating system involving the arginine deiminase pathway. 2DG uptake into these vesicles was not inhibited, and preaccumulated 2DG did not efflux from them upon electroporation of fructose 1,6-diphosphate or gluconate 6-phosphate into the vesicles. Intravesicular but not extravesicular wild-type or H15A mutant HPr of BaciUlus subtilis promoted inhibition (53 and 46%, respectively) of the permease in the presence of these metabolites. Counterflow experiments indicated that inhibition of 2DG uptake is due to the partial uncoupling of proton symport from sugar transport. Intravesicular S46A mutant HPr could not promote regulation of glucose permease activity when electroporated into the vesicles with or without the phosphorylated metabolites, but the S46D mutant protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km values for both TMG and 2DG uptake were affected. Uptake of the natural, metabolizable substrates of the lactose, glucose, mannose, and ribose permeases was inhibited by wild-type HPr in the presence of fructose 1,6-diphosphate or by S46D mutant HPr. These results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase regulates glucose and lactose permease activities in L. brevis and suggest that other permeases may also be subject to this mode of regulation.Many but not all low-GC gram-positive bacteria possess the phosphoenolpyruvate:sugar phosphotransferase system (PTS) that catalyzes the concomitant uptake and phosphorylation of its sugar substrates. The PTS-catalyzed process requires the sequential phosphorylation of four proteins or protein domains, enzyme I, HPr, IlAsugar and IIBsusgar, before sugar phosphorylation and concomitant transport can occur (8,20). The PTS proteins function in numerous biochemical and genetic regulatory capacities (17)(18)(19)21). In early studies, it was shown that addition of a rapidly metabolizable sugar such as glucose to streptococci, lactococci, or lactobacilli resulted in inhibition of the uptake of other sugars (inducer exclusion) as well as rapid efflux of preaccumulated sugars or sugar metabolites (inducer expulsion). For example, lactose and its nonmetabolizable analog...

Streptomyces liwidans, S. coelicolor and S. griseohscus were examined for the presence of the enzymes of the phosphoenolpyruvate : sugar phosphotransferase system (PTS). All three species were shown to possess Enzyme I, HPr and fructose-specific Enzyme II (IIFN) activities. In 5. liwidans and 5. coelicolor, all three PTS enzymes were fructose-inducible, but in 5. griseokrscus the system was expressed constitutively. These organisms apparently lack the HPr(Ser) kinase and HPr(Ser-P) phosphatase that characterize low-GC Gram-positive bacteria.