The rates of formation of the two alternative products of acetohydroxy acid synthase (AHAS) have been determined by a new analytical method (N. Gollop, Z. Barak, and D. M. Chipman, Anal. Biochem., 160:323-331, 1987 (AHB) at approximately 180-and 60-fold faster rates, respectively, than acetolactate (AL) at equal pyruvate and 2-ketobutyrate concentrations. R values higher than 60 represent remarkably high specificity in favor of the substrate with one extra methylene group. In exponentially growing E. coli cells (under aerobic growth on glucose), which contain about 300 ,uM pyruvate and only 3 ,uM 2-ketobutyrate, AHAS I would produce almost entirely AL and only 1 to 2% AHB. However, isozymes II and III would synthesize AHB (on the pathway to Ile) and AL (on the pathway to valine-leucine) in essentially the ratio required for protein synthesis. The specificity ratio R of any AHAS isozyme was affected neither by the natural feedback inhibitors (Val, Ile) nor by the pH. On the basis of the specificities of the isozymes, the known regulation of AHAS I expression by the catabolite repression system, and the reported behavior of bacterial mutants containing single AHAS isozymes, we suggest that AHAS I enables a bacterium to cope with poor carbon sources, which lead to low endogenous pyruvate concentrations. Although AHAS II and III are well suited to producing the branched-chain amino acid precursors during growth on glucose, they would fail to provide appropriate quantities of AL when the concentration of pyruvate is relatively low.Acetohydroxy acid synthase (AHAS; EC 4.1.3.18, also known as acetolactate synthase) catalyzes the condensation of an active acetaldehyde moiety derived from pyruvate with either another molecule of pyruvate, to form 2-acetolactate (AL), or with 2-ketobutyrate to form 2-aceto-2-hydroxybutyrate (AHB) (28). The synthesis of the acetohydroxy acids is the first of a series of common steps in the biosynthesis of the branched-chain amino acids isoleucine, leucine, and valine ( Fig. 1) (5, 28, 29). The two AHAS reactions are the key steps in this pathway, as they are irreversible and committed steps toward the synthesis of two different sets of products; furthermore, the formation of AL is the first committed step on the valine pathway. There are several conceivable strategies for the regulation of such separate but interrelated biosynthetic pathways. (i) A single enzyme might operate with variable, metabolically controlled specificity for the two reactions (e.g., as in the case of ribonucleotide diphosphate reductase [27]). (ii) Different enzymes might specialize almost exclusively in one or another reaction, so that each could be separately controlled. (iii) The relative rates of the two reactions might be controlled by the relative concentrations of the precursors. The information available on AHAS has not been sufficient to determine to what extent each of these strategies has been adopted, even in the well-studied cases of Escherichia coli and Salmonella typhimurium (5, 29).

An investigation aimed at a better understanding of the molecular adaptation mechanisms of salt stress was carried out in 7-d-old tomato Solanum lycopersicum (L.) Mill cultivars Patio and ‘F144’, using a proteomic approach. Total proteins were extracted from radicles and hypocotyls collected from both non-saline control and salt-stressed seedlings, and separated by two-dimensional gel electrophoresis. Liqud chromatography-electron spray ionization tandem mass spectrometry (LC-ESI-MS/MS) identified 23 salt stress response proteins, classified into six functional categories. The effect of exogenously applied glycinebetaine (GB) on the salt stress-induced inhibition of growth in tomato seedlings of cultivars Patio and ‘F144’ and on the protein profile was investigated. It was found that GB could alleviate the inhibition of tomato growth induced by salt stress through changing the expression abundance of six proteins in Patio and two proteins in ‘F144’ more than twice compared with salt-stressed seedlings. Furthermore, the interaction analysis based on computational bioinformatics reveals major regulating networks: photosystem II (PSII), Rubisco, and superoxide dismutase (SOD). The results suggest that it is likely that improvement of salt tolerance in tomato might be achieved through the application of exogenous compatible solutes, such as GB. Moreover, quantitative and qualitative analysis of the differentially expressed proteins of tomato under salt stress is an important step towards further elucidation of mechanisms of salt stress resistance.

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) isozyme III from Escherichia coli has been studied in steady-state kinetic experiments in which the rates of formation of acetolactate (AL) and acetohydroxybutyrate (AHB) have been determined simultaneously. The ratio between the rates of production of the two alternative products and the concentrations of the substrates pyruvate and 2-ketobutyrate (2KB) leading to them, R, VAHB/VAL = R[( 2KB]/[pyruvate]), was found to be 40 +/- 3 under a wide variety of conditions. Because pyruvate is a common substrate in the reactions leading to both products and competes with 2-ketobutyrate to determine whether AL or AHB is formed, steady-state kinetic studies are unusually informative for this enzyme. At a given pyruvate concentration, the sum of the rates of formation of AL and AHB was nearly independent of the 2-ketobutyrate concentration. On the basis of these results, a mechanism is proposed for the enzyme that involves irreversible and rate-determining reaction of pyruvate, at a site which accepts 2-ketobutyrate poorly, if at all, to form an intermediate common to all the reactions. In the second phase of the reaction, various 2-keto acids can compete for this intermediate to form the respective acetohydroxy acids. 2-Keto acids other than the natural substrates pyruvate and 2-ketobutyrate may also compete, to a greater or lesser extent, in the second phase of the reaction to yield alternative products, e.g., 2-ketovalerate is preferred by about 2.5-fold over pyruvate. However, the presence of an additional keto acid does not affect the relative specificity of the enzyme for pyruvate and 2-ketobutyrate; this further supports the proposed mechanism. The substrate specificity in the second phase is an intrinsic property of the enzyme, unaffected by pH or feedback inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Era is a membrane-associated GTP-binding protein which is essential for cell growth in Escherichia coli. In order to examine the physiological role of Era, strains in which Era was expressed at 40°C but completely repressed at 27°C were constructed. The growth of these strains was inhibited at the nonpermissive temperature, and cells became elongated. Under such conditions, no constrictions or septum formation could be detected by phase-contrast microscopy, and DNA segregation was apparently normal as revealed by fluorescence staining. These data demonstrate that Era has an essential function in cell growth rate control in liquid media and that depletion of Era blocks cell division either directly or indirectly. Thus, the role of GTPbinding proteins as important regulators of cell growth and division may be ubiquitous in nature.It is now widely recognized that a large family of similar proteins possessing GTPase activity are key regulators of cell growth and division. These proteins include factors required for protein synthesis, G proteins, and the ras oncogenes, among others. Only a few such GTP-binding proteins are known in Escherichia coli, including a membrane-associated GTP-binding protein (Era) which was shown to possess low-level intrinsic GTPase activity (1,8,16). The gene (era) for this protein has been cloned, sequenced, and located at min 55 on the E. coli chromosome within the rnc operon (1). Mutational analysis has shown that an intact era allele is required for growth on plates, but analysis of conditional mutants has not led to the delineation of any phenotype that suggests a function for Era (11,16,21). In order to understand the function of Era in vivo, we constructed several strains in which transcription of era is controlled by the phage X promoter PR. The strategy employed in mutant construction allows one to obtain a new strain by simply transforming bacteria with the vector harboring the disrupted gene. era mutants incubated at the nonpermissive temperature are nonviable and exhibit an elongated cell phenotype.MATERIALS AND METHODS Strains. The bacterial strains employed to construct conditional era mutants have been described elsewhere and are referred to in Table 1.Growth conditions. Cells were grown in Luria broth media (5 g of yeast extract, 10 g of tryptone [Difco], and 5 g of NaCl per liter). The media were supplemented with antibiotics at the following concentrations in micrograms per milliliter: ampicillin, 50; kanamycin sulfate, 50; and tetracycline, 15.Shift-down experiments were carried out in two water bath shakers with platforms rotating at the same speed. Growth was monitored turbidimetrically by using a KlettSummerson calorimeter containing a red 66 filter.Strategy of mutant construction. The basic strategy involved constructing a temperature-sensitive vector which would permit survival of recipients only when there was a single homologous recombination between the plasmid and * Corresponding author. the targeted gene while at the same time disrupting the target.The plasmid ...

Low molecular weight compounds were isolated by high-performance liquid chromatography from the maggot or haemolymph extracts of Lucilia sericata (Meigen) (Diptera: Calliphoridae). Using gas chromatography-mass spectrometry analysis, three compounds were obtained: p-hydroxybenzoic acid (molecular weight 138 Da), p-hydroxyphenylacetic acid (molecular weight 152 Da) and octahydro-dipyrrolo[1,2-a;1',2'-d] pyrazine-5,10-dione (molecular weight 194 Da), also known as the cyclic dimer of proline (or proline diketopiperazine or cyclo[Pro,Pro]). All three molecules revealed antibacterial activity when tested against Micrococcus luteus and/or Pseudomonas aeruginosa, and the effect was even more pronounced when these molecules were tested in combination and caused lysis of these bacteria.

Aims: To determine antibacterial activity in lactic acid bacteria (LAB) silage inoculants and in wheat and corn silages which were treated with these inoculants. Methods and Results: Wheat and two corn silages were prepared in 0AE25 l sealed glass jars. Inoculant treatments were prepared for each type of silage with each of 10 LAB silage inoculants at inoculation rate of 10 6 CFU g )1 . Untreated silages served as controls. Antibacterial activity was determined in the inoculants and in their respective silages with Micrococcus luteus and Pseudomonas aeruginosa. Antibacterial activity was detected in nine of the 10 inoculants whereas such activity in the silages varied. Control silages did not have antibacterial activity. Conclusions: Many LAB silage inoculants have antibacterial activity and in some cases this activity is imparted on inoculated silages. Significance and Impact of the Study: This study was conducted as part of a broader research objective, which is to find out how LAB silage inoculants enhance ruminant performance. The results of this study indicate that LAB silage inoculants produce antibacterial activity, and therefore, have a potential to inhibit detrimental microorganisms in the silage or in the rumen.

The nature of the antibacterial materials extracted from maggots not only indicates their ability to ingest the necrotic tissue on the wound, but also their potential significance in wound healing,

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