Acetyl coenzyme A synthetase (Acs) activates acetate to acetyl coenzyme A through an acetyladenylate intermediate; two other enzymes, acetate kinase (Ack) and phosphotransacetylase (Pta), activate acetate through an acetyl phosphate intermediate. We subcloned acs, the Escherichia coli open reading frame purported to encode Acs (F. R. Blattner, V. Burland, G. Plunkett III, H. J. Sofia, and D. L. Daniels, Nucleic Acids Res. 21:5408-5417, 1993). We constructed a mutant allele, ⌬acs::Km, with the central 0.72-kb BclI-BclI portion of acs deleted, and recombined it into the chromosome. Whereas wild-type cells grew well on acetate across a wide range of concentrations (2.5 to 50 mM), those deleted for acs grew poorly on low concentrations (Յ10 mM), those deleted for ackA and pta (which encode Ack and Pta, respectively) grew poorly on high concentrations (Ն25 mM), and those deleted for acs, ackA, and pta did not grow on acetate at any concentration tested. Expression of acs from a multicopy plasmid restored growth to cells deleted for all three genes. Relative to wild-type cells, those deleted for acs did not activate acetate as well, those deleted for ackA and pta displayed even less activity, and those deleted for all three genes did not activate acetate at any concentration tested. Induction of acs resulted in expression of a 72-kDa protein, as predicted by the reported sequence. This protein immunoreacted with antiserum raised against purified Acs isolated from an unrelated species, Methanothrix soehngenii. The purified E. coli Acs then was used to raise anti-E. coli Acs antiserum, which immunoreacted with a 72-kDa protein expressed by wild-type cells but not by those deleted for acs. When purified in the presence, but not in the absence, of coenzyme A, the E. coli enzyme activated acetate across a wide range of concentrations in a coenzyme A-dependent manner. On the basis of these and other observations, we conclude that this open reading frame encodes the acetate-activating enzyme, Acs.Escherichia coli cells activate acetate to acetyl coenzyme A (acetyl-CoA) by two distinct pathways (Fig. 1) Brown et al. (6) hypothesized that the Acs pathway functions as a catabolite-repressible, acetate-inducible, high-affinity acetate uptake system that scavenges acetate present extracellularly at relatively low concentrations. They also proposed that the Ack-Pta pathway functions primarily in a catabolic role, excreting acetate and generating ATP during mixed-acid fermentation and aerobic growth on excess glucose or other glycolytic intermediates. Finally, they argued that the low-affinity Ack-Pta pathway activates acetate only when that molecule is present extracellularly in large quantity.In addition to their role in acetate metabolism, the acetate activation pathways have been implicated in the regulation of signal transduction by two-component regulatory systems in several bacterial species (reviewed in references 32 and 54; see also references 2, 9, 39, and 55), the regulation of the glucose starvation stimulon of E. coli ...

Cells of Escherichia coli growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. Here, we present evidence that this switch occurs primarily through the induction of acs and that the timing and magnitude of this induction depend, in part, on the direct action of the carbon regulator cyclic AMP receptor protein (CRP) and the oxygen regulator FNR. It also depends, probably indirectly, upon the glyoxylate shunt repressor IclR, its activator FadR, and many enzymes involved in acetate metabolism. On the basis of these results, we propose that cells induce acs, and thus their ability to assimilate acetate, in response to rising cyclic AMP levels, falling oxygen partial pressure, and the flux of carbon through acetate-associated pathways.

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Murine carcinoembryonic antigens serve as receptors for the binding and entry of the enveloped coronavirus mouse hepatitis virus (MHV) into cells. Numerous receptor isoforms are now known, and each has extensive differences in its amino terminal immunoglobulin-like domain (NTD) to which MHV binds via its protruding spike proteins. Some of these receptor alterations may affect the ability to bind viral spikes. To identify individual residues controlling virus binding differences, we have used plasmid and vaccinia virus vectors to express two forms of MHV receptor differing only in their NTD. The two receptors, designated biliary glycoproteins (Bgp) 1a and 1bNTD, varied by 29 residues in the 107 amino acid NTD. When expressed from cDNAs in receptor-negative HeLa cells, these two Bgp molecules were displayed on cell surfaces to equivalent levels, as both were equally modified by a membrane-impermeant biotinylation reagent. Infectious center assays revealed that the 1a isoform was 10 to 100 times more effective than 1bNTD in its ability to confer sensitivity to MHV (strain A59) infection. Bgp1a was also more effective than Bgp1bNTD in comparative virus absorption assays, binding 6 times-more MHV (strain A59) and 2.5 times more MHV (strain JHMX). Bgp1a was similarly more effective in promoting the capacity of viral spikes to mediate intercellular membrane fusion as judged by quantitation of syncytia following cocultivation of spike and receptor-bearing cells. To identify residues influencing these differences, we inserted varying numbers of 1b residues into the Bgp1a background via restriction fragment exchange and site-directed mutagenesis. Analysis of the resulting chimeric receptors showed that residues 38 to 43 of the NTD were key determinants of the binding and fusion differences between the two receptors. These residues map to an exposed loop (C-C' loop) in a structural model of the closely related human carcinoembryonic antigen.

In this era, electronic devices such as mobile phones, computers, laptops, sensors, and many more have become a necessity in healthcare, for a pleasant lifestyle, and for carrying out tasks quickly and easily. Different types of temperature sensors, biosensors, photosensors, etc., have been developed to meet the necessities of people. All these devices have chips inside them fabricated using diodes, transistors, logic gates, and ICs. The patterning of the substrate which is used for the further development of these devices is done with the help of a technique known as lithography. In the present work, we have carried out a review on different types of lithographic techniques such as optical lithography, extreme ultraviolet lithography, electron beam lithography, X-ray lithography, and ion beam lithography. The evolution of these techniques with time and their application in device fabrication are discussed. The different exposure tools developed in the past decade to enhance the resolution of these devices are also discussed. Chemically amplified and non-chemically amplified resists with their bonding and thickness are discussed. Mask and maskless lithography techniques are discussed along with their merits and demerits. Device fabrication at micro and nano scale has been discussed. Advancements that can be made to improve the performance of these techniques are also suggested.

Cells of Escherichia coli undergo a metabolic switch associated with the production and utilization of acetate. During exponential growth on tryptone broth, these cells excrete acetate via the phosphotransacetylase-acetate kinase (Pta-AckA) pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. This metabolic switch depends upon the induction of Acs. As part of our effort to dissect the mechanism(s) underlying induction and to identify the signal(s) that triggers that induction, we sought the sigma factor most responsible for acs expression. Using isogenic strains that carry a temperature sensitivity allele of the gene that encodes 70 and either a wild-type or null allele of the gene that encodes S , we determined by immunoblotting, reverse transcriptase PCR, and acs::lacZ transcriptional fusion analyses that 70 is the sigma factor primarily responsible for the acs transcription that cells induce during mid-exponential phase. In contrast, S partially inhibits that transcription as cells enter stationary phase.

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