Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes in Bacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays. The direct targets of CodY newly identified by this approach included regulatory genes for sporulation, genes that are likely to encode transporters for amino acids and sugars, and the genes for biosynthesis of branched-chain amino acids.Bacteria have evolved a variety of mechanisms to accommodate gene expression to changes in nutritional availability. Some of these mechanisms are specific to a particular gene or operon. In other cases, regulatory proteins control large groups of genes of related function, such as the nitrogen metabolism genes regulated by the Ntr system in enteric bacteria (43) and by TnrA in Bacillus subtilis (17) and the carbon metabolism genes regulated by CcpA in gram-positive bacteria (13) and catabolic gene activator protein-cyclic AMP complex in gram-negative bacteria (59). Even broader forms of regulation are mediated by the leucine-responsive protein (Lrp) of gram-negative bacteria and the sigma-B protein of B. subtilis. Lrp and sigma-B control the transcription of operons that have diverse functions but have a common need to be expressed under a particular set of environmental conditions (50, 54). Lrp regulates the biosynthesis of leucine, isoleucine, valine, serine, glycine, and glutamate; the degradation of serine and threonine; transport of peptides, amino acids, and sugars; and production of fimbriae in response to the availability of leucine and serine (50). Sigma-B activates transcription of a host of genes when cells are exposed to excessive heat, ethanol, salt, or acid (54). Sigma-B responds through a complex, multibranched signal transduction pathway.The B. subtilis CodY protein also has broad effects on gene expression. CodY is a GTP-binding repressor of several genes that are normally quiescent when cells are growing in a rich medium (57). A high concentration of GTP activates CodY as a repressor (57). When the growth rate of B. subtilis slows down because of limitation of the carbon or nitrogen or phosphorus source, the GTP level drops (39, 40), CodY loses repressing activity, and targets of CodY repression are transcribed. The known targets of CodY in B. subtilis include the genes that encode transport systems for dipeptides (dpp) (65) and ␥-aminobutyrate (gabP) (16); catabolic pathways for acetate (acsA) (S. H. Fisher, personal communication), urea (ureABC) (71), histidine (hut) (18), arginine (rocABC and roc-DEF) (B. Belitsky, personal communication), and branchedchain keto acids (the bkd operon) (12); an enzyme of surfactin synthesis (srfAA) (63); the transcription factor for DNA uptake genes (comK) (63); a ComA aspartyl phosphate phosphatase and its inhibitor (rapC-phrC) (37); motility and chemotaxis (hag, fla/che) (45; F. Bergara, C. Ibarra, J. Iwamasa, R. Aguilera, and L. M. Màrquez-Magaña, submitted for publication); and aconitase (citB) (3...

SummaryBacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen-limited growth. In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome-wide screening for TnrAregulated genes associated with a TnrA box. A combination of DNA microarray hybridization and a genome-wide search for TnrA boxes allowed us to find 36 TnrA-regulated transcription units associated with a putative TnrA box. Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which ( nasA and nasBCDEF ) possessed a common TnrA box. The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA. The TnrA targets detected in this study were nrgAB , pucJKLM , gln-QHMP , nasDEF , oppABCDF , nasA , nasBCDEF and ywrD for positive regulation, and gltAB , pel , ywdIJK , yycCB , yttA , yxkC , ywlFG , yodF and alsT for negative regulation, nrgAB and gltAB being well-studied TnrA targets. It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets. The physiological role of the TnrA regulon is discussed.

Fine Structure, Thermal and Viscoelastic Properties of Starches Separated from Indica Rice CultivarsStarches were separated from indica rice cultivars and evaluated using gel permeation chromatography (GPC), X-ray diffraction, differential scanning calorimetry (DSC) and dynamic viscoelasticity. Debranching of starch with isoamylase and subsequent fractionation by GPC revealed 9.7-28.3% apparent amylose content, 3.7-5.0% intermediate fraction (mixture of short amylose and long side-chains of amylopectin), 20.6-26.6% long sidechains of amylopectin and 45.8-59.4% short side-chains of amylopectin). IR-64 starch with the highest crystallinity had the highest gelatinization temperatures and enthalpy, T o , T p , T c , and DH gel being 71.8, 75.9, 82.47C and 5.1 J/g, respectively, whereas PR-113 starch with lower crystallinity showed the lowest gelatinization temperatures (T o , T p , T c , of 60.8, 65.7 and 72.27C, respectively). Basmati-386 starch exhibited two endotherms during heating, the first and second endotherm being associated with the melting of crystallites and amylose-lipid complexes, respectively. T o, T p , T c and DH gel of the second endotherm of Basmati-386 starch were 99.0, 100.1, 101.17C and 2.0 J/g, respectively. During cooling, Basmati-386 also showed an exotherm at a peak temperature of 877C. PR-113 starch with the highest amylose content and the lowest content of short side-chains of amylopectin had the highest peak storage modulus (G 0 = 1.6610 4 Pa). The granules of PR-113 starch were the least disintegrated after heating. The effects of heating starch suspensions at different temperatures (927C, 1307C and 1707C) on intrinsic viscosity [Z], transmittance and viscoelasticity were also studied to evaluate the extent of breakdown of the molecular structure. The intrinsic viscosity of starch suspensions heated at 92, 130 and 1707C ranged between 103-114, 96-110 and 28-93 mL/g. Transmittance value of starches cooked at 927C decreased with increase in storage duration. All starches except PR103, cooked at 1307C also showed decrease in transmittance during storage, however, at lower rate. PR103 starch heated at 1307C did not show any change in transmittance up to a storage time of 48 h. The changes in viscoelasticity of starch pastes cooked at different temperatures during cooling and reheating were also evaluated. G 0 and G 00 increased with decrease in temperature during cooling cycle. Starches heated at 1307C with apparent amylose content 21.2% showed an improvement in G 0 and G 00 in comparison to the corresponding starches heated at 927C, this improvement was observed to be higher in starches with lower amylose content. All starches heated at 1707C had a higher proportion of breakdown in molecular structure as indicated by lower G 0 and G 00 than the same starches heated at 130 and 927C.

A comparison between the morphological, structural, thermal and viscoelastic properties of starches separated from pigeon pea, chickpea, field pea, kidney bean and blackgram was made. The shape of the starch granules in the different legumes varied from oval to elliptical or spherical. X-ray diffraction of the legume starches indicated a typical C-pattern (mixture of A-and B-type). Granules of blackgram and pigeon pea starch had a higher degree of crystallinity than those of field pea and kidney bean starches. Apparent amylose content of field pea, kidney bean, chickpea, blackgram and pigeon pea starch was 37.9%, 36.0%, 34.4-35.5%, 32.9-35.6% and 31.8%, respectively. Distribution of isoamylase-branched materials among the starches revealed that the proportions of long and short side chains of amylopectin ranged between 13.6-18.5% and 41.7-46.5%, respectively. Field pea and kidney bean starch had the highest apparent amylose content and the lowest amount of long side chains of amylopectin, respectively. Blackgram and pigeon pea starch possessed higher proportions of both long and short side chains of amylopectin than field pea and chickpea starches. The onset, peak and conclusion temperatures of gelatinization (T o T p and T c , respectively) were determined by differential scanning calorimetry. T o and T c ranged from 59.3 to 77.37C, 66.8 to 79.67C, 55.4 to 67.67C and 68.3 to 69.37C, respectively, for chickpea, blackgram, field pea and kidney bean starch. The enthalpy of gelatinization (DH gel ) of field pea, kidney bean, chickpea, blackgram and pigeon pea starches was 3.6, 3.0, 2.6-4.2, 1.6-1.7 and 2.6 J/g, respectively. Pastes of blackgram and pigeon pea starches showed lower storage and loss shear moduli G 0 than field pea, kidney bean and chickpea starches. The changes in moduli during 10 h at 107C revealed retrogradation in the order of: field pea. kidney bean. chickpea. blackgram. pigeon pea starch. In blackgram and pigeon pea starches, the lower proportion of amylose plus intermediate fraction and higher proportion of short and long side chains of amylopectin are considered responsible for the higher crystallinity, gelatinization temperature and enthalpy of gelatinization.

Four types of corn starch (waxy corn starch (WC), normal corn starch (NC), high-amylose corn starch class 5 (HAC class 5) and high-amylose corn starch class 7 (HAC class 7)) were hydrolyzed with 1.5% hydrochloric acid and the resistant starch (RS) content was measured. The acid-hydrolyzed HAC class 5 and class 7 show significantly higher RS content. The change in RS content, X-ray crystallinity, molecular size distribution, thermal property and appearance of HAC class 7 after up to 100 h acid hydrolysis were analyzed. The RS content increased to 69.3 from 40.5% at 16 h and then decreased gradually, while crystallinity continued to increase during acid hydrolysis. The fractionation profiles indicated that with the decrease of the amylose fraction correlating well with the increase in RS content. Granules hydrolyzed for 24 h retained their natural shape, whereas after 100 h hydrolysis granules were damaged. The enzyme-digested residues of native and acid-hydrolyzed HAC class 7 were recovered. The crystalline regions of HAC class 7 acid-hydrolyzed for 24 h were only slightly digested by enzymatic treatment, whereas native and HAC class 7 acid-hydrolyzed for 100 h were extensively digested. Thermal analysis showed the transition peak in high temperature region increased with acid hydrolysis and this peak was also shown in enzyme-digested residues. Acid hydrolysis may influence RS content by two mechanisms: (1) moderate acid hydrolysis increases the crystalline regions and thus resistance to enzymatic digestion of the hydrolyzed granules and (2) excess acid hydrolysis damages granular structure and decreases resistance to enzymatic digestion.