The alkaline phosphatase of Synechococcus sp. strain PCC 7942 is 145 kDa, which is larger than any alkaline phosphatase previously characterized and approximately three times the size of the analogous enzyme in Escherichia coli. The gene for the alkaline phosphatase, phoA, was cloned and sequenced, and the protein that it encodes was found to have little similarity to other phosphatases. Some sequence similarities were observed between the Synechococcus sp. strain PCC 7942 alkaline phosphatase, the ot subunit of the ATPase from bacteria and chloroplasts, and the UshA sugar hydrolase of E. coli. Also, limited sequence similarity was observed between a region of the phosphatase and a motif implicated in nucleotide binding. Interestingly, although the alkaline phosphatase is transported across the inner cytoplasmic membrane and into the periplasmic space, it does not appear to have a cleavable signal sequence at its amino terminus. The half-life of the mRNA encoding the alkaline phosphatase, measured after inhibition of RNA synthesis, is approximately 5 min. Similar kinetics for the loss of alkaline phosphatase mRNA occur upon the addition of phosphate to phosphate-depleted cultures, suggesting that high levels of this nutrient inhibit transcription from phoA almost immediately. The phoA gene also appears to be the first gene of an operon; the largest detectable transcript that hybridizes to a phoA gene-specific probe is 11 kb, over twice the size needed to encode the mature protein.Other phosphate-regulated mRNAs are also transcribed upstream of the phoA gene. Insertional inactivation of phoA results in the loss of extracellular, phosphate-regulated phosphatase activity but does not alter the capacity of the cell for phosphate uptake.

We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotidedirected mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histide-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.Poliovirus, a small RNA virus, is a member of the picornavirus family, which includes rhinovirus, encephalomyocarditis virus, and foot and mouth disease virus. During the viral life cycle, the genomic RNA is initially translated into a >200-kDa polyprotein, which, while nascent, is cleaved to a series of polypeptide intermediates, termed P1, P2, and P3. Further processing yields the majority of mature viral products (structural and nonstructural) and is dependent on a viral protease(s). The poliovirus P3 intermediate consists of three nonstructural proteins-the genome-linked virion protein (VPg), the protease (3C), and the replicase (3D)-and two proteins of unknown function (3C' and 3D') (1, 2). Hanecak et al. (3) have demonstrated that the 3C region of the poliovirus genome represents the core sequence responsible for the proteolytic processing (at Gln-Gly) of the precursor polypeptides. In addition, the poliovirus 3C protease has been reported to be autocatalytic (4), in agreement with results for encephalomyocarditis virus (5) and foot and mouth disease virus (6).The enzymatic mechanism of these viral proteases has not been well characterized. Inhibitors such as iodoacetamide, N-ethyl maleimide, and para-chloromercuribenzoate have implicated a cysteine residue in the active site (7,8). However, diisopropylphosphofluoridate, an inhibitor of serine proteases, has also been reported as an effective inhibitor of poliovirus protein processing (9). A comparative study (10) of the derived protease proteins from several picornaviruses as well as the related plant virus cowpea mosaic virus (11) revealed one region of significant homology located in the carboxyl third ofthe protein. Within this region a cysteine and a histidine residue have been strictly conserved. These observations suggest that the picornavirus proteases most probably belong to the class of cysteine proteases.To obtain sufficient quantities of the protease proteins for study, we have engineered a cDNA clone of the poliovirus 3C-3D region into an Escherichia coli expression vector, which produced the protease polypeptides in high yield...

The nucleotide sequence of aroH, the structural gene for the tryptophan-sensitive 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase [DAHPS(Trp)], is presented, and the deduced amino acid sequence of AroH is compared with that of the tyrosine-sensitive (AroF) and phenylalanine-sensitive (AroG) DAHPS isoenzymes, The high degree of sequence similarity among the three isoenzymes strongly indicates that they have a common evolutionary origin. In vitro chemical mutagenesis of the cloned aroH gene was used to identify residues and regions of the polypeptide essential for catalytic activity and for tryptophan feedback regulation. Missense mutations leading either to loss of catalytic activity or to feedback resistance were found interspersed throughout the polypeptide, suggesting overlapping catalytic and regulatory sites in DAHPS(Trp). We conclude that the specfficity of feedback regulation of the isoenzymes was probably acquired by the duplication and divergent evolution of an ancestral gene, rather than by domain recruitment.In bacteria and plants, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) (EC 4.1.2.15) catalyzes the first committed step in the pathway that leads to the biosynthesis of aromatic acids and vitamins. In Escherichia coli and other enteric bacteria there are three DAHPS isoenzymes (5). Although catalyzing the same reaction (i.e., the condensation of erythrose-4-phosphate and phosphoenolpyruvate [PEP] to form 3-deoxy-D-arabino-heptulosonate 7-phosphate [DAHP]), each isoenzyme is feedback regulated by a different aromatic amino acid. The structural genes aroF, aroG, and aroH encode the tyrosine (Tyr)-, phenylalanine (Phe)-, and tryptophan (Trp

The effect of a mutation in luxI (autoinducer synthetase gene) on transcription of luxR in the cloned Vibrio fischeri lux system (luxR, luxICDABE) was examined in Escherichia coli. For the luxI mutant, transcription from the luxR promoter (monitored with 0-galactosidase levels from a luxR::lacZ fusion, with LuxR supplied in trans) decreased fivefold, to levels of the luxI strain, only in the presence of added autoinducer. The results demonstrate that, as has been shown at the translational level, autoinducer is required for negative autoregulation of luxR at the transcriptional level.