Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5 end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.Branched-chain amino acids are the most abundant amino acids in proteins and form the hydrophobic cores of the proteins. Moreover, these amino acids are precursors for the biosynthesis of iso-and anteiso-branched fatty acids, which represent the major fatty acid species of the membrane lipids in Bacillus species (5). The initial step of isoleucine or valine synthesis is the condensation of 2-oxobutanoate derived from threonine and pyruvate or two pyruvates, leading to the formation of branched-chain keto acids (8). Leucine is synthesized from one of the branched-chain keto acids, i.e., ␣-ketoisovalerate. The Bacillus subtilis ilv-leu operon comprises seven genes (ilvBHC and leuABCD) necessary for the biosynthesis of branched-chain amino acids (12). The expression of the ilv-leu operon is under positive regulation involving the CcpA protein (36, 41), which is involved in carbon catabolite control of not only hundreds of the catabolic operons and genes but also many cellular processes (6, 11). This CcpAdependent positive regulation of ilv-leu links carbon metabolism to amino acid anabolism. Recent global gene expression studies of amino acid availability (23) and CodY regulation (25), as well as studies of metabolic linking of ilv-leu expression to nitrogen metabolism (40), revealed that the ilv-leu operon is under direct negative transcriptional control through two major global regulators of nitrogen metabolism (TnrA and CodY). TnrA is known to both activate and repress nitrogenregulated genes during nitrogen-limited growth (43). The CodY protein is a GTP-binding repressor of several operons, including ilv-leu, that...

Bacillus subtilis LmrA is known to be a repressor that regulates the lmrAB and yxaGH operons; lmrB and yxaG encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same cis sequences, LmrA/YxaF boxes, located in the promoter regions of the lmrAB operon, the yxaF gene, and the yxaGH operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the yxaGH promoter region. The lacZ fusion experiments essentially supported the above-mentioned in vitro results, except that galangin did not activate the lmrAB and yxaGH promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the lmrAB operon, the yxaF gene, and the yxaGH operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter β-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the lmrAB and yxaGH operons but that YxaF represses yxaGH more preferentially.

In Bacillus subtilis cells, the GTP level decreases and the ATP level increases upon a stringent response. This reciprocal change in the concentrations of the substrates of RNA polymerase affects the rate of transcription initiation of certain stringent genes depending on the purine species at their transcription initiation sites. DNA microarray analysis suggested that not only the rrn and ilv-leu genes encoding rRNAs and the enzymes for synthesis of branched-chain amino acids, respectively, but also many genes, including genes involved in glucose and pyruvate metabolism, might be subject to this kind of stringent transcription control. Actually, the ptsGHI and pdhABCD operons encoding the glucose-specific phosphoenolpyruvate:sugar phosphotransferase system and the pyruvate dehydrogenase complex were found to be negatively regulated, like rrn, whereas the pycA gene encoding pyruvate carboxylase and the alsSD operon for synthesis of acetoin from pyruvate were positively regulated, like ilv-leu. Replacement of the guanine at position 1 and/or position 2 of ptsGHI and at position 1 of pdhABCD (transcription initiation base at position 1) by adenine changed the negative stringent control of these operons in the positive direction. The initiation bases for transcription of pdhABCD and pycA were newly determined. Then the promoter sequences of these stringent operons were aligned, and the results suggested that the presence of a guanine(s) and the presence of an adenine(s) at position 1 and/or position 2 might be indispensable for negative and positive stringent control, respectively. Such stringent transcription control that affects the transcription initiation rate through reciprocal changes in the GTP and ATP levels likely occurs for numerous genes of B. subtilis.Stringent control is one of the most important adaptations which help bacteria survive under harsh conditions. Of the various occasions when stringent control results from the synthesis of GDP 3Ј-diphosphate (ppGpp) from GTP, which is catalyzed by the RelA protein associated with ribosomes, the most prominent is the repression of stable RNA synthesis (4). This control also affects the expression of certain genes, including genes involved in amino acid biosynthesis. However, Bacillus subtilis and Escherichia coli use different strategies for stringent transcription control (19). ppGpp may not inhibit B. subtilis RNA polymerase directly, whereas ppGpp decreases rRNA promoter activity by directly inhibiting E. coli RNA polymerase.GTP and ATP are well-known gauges of the general energetic capacity and energy charge of cells, respectively. In B. subtilis, the GTP and ATP levels decrease and increase during a stringent response (e.g., amino acid starvation), respectively (20,24,31,48), as shown in Fig. S1 in the supplemental material. The changes are mediated by ppGpp synthesized by RelA (52) during a stringent response, probably through its inhibition of IMP dehydrogenase, the first enzyme in the pathway leading to the biosynthesis of GTP (24). The inhibition o...

Abstract. The objective of this experiment was to investigate whether the motility parameters and acrosome integrity of goat ejaculated spermatozoa are affected by collecting semen into tubes containing an extender, and thereby determine the significance of reducing contact between seminal plasma and the sperm membrane at ejaculation. Semen were collected from three goats into tubes containing 0, 1 or 10 ml extender, or collected into tubes containing 10 ml extender supplemented with 0.1, 1 or 5% BSA. Sperm motion parameters were evaluated immediately after collection, after washing, and during a 3-h thermal resistance test. Acrosome integrity was assessed using FITC-PNA staining. Semen collection into tubes containing 10 ml extender produced higher sperm motility, progressive motility, and acrosome integrity than that using a smaller volume of extender. Furthermore, collection into 5% BSA-containing extender exhibited higher sperm characteristics and maintained high sperm motility and progressive motility throughout incubation. In conclusion, semen collection into tubes with a large volume of extender, especially extender containing higher concentrations of BSA, improved the quality of ejaculated spermatozoa, strongly suggesting that the in vitro functional characteristics of the spermatozoa were abruptly modified by flash sperm contact with accessory sex gland fluid at ejaculation.

Background: The objective of this study was to investigate whether the steroid hormone(s) secreted from cumulus-oocyte complexes (COCs) is a prerequisite for bovine oocyte maturation and cumulus expansion using aminoglutethimide (AGT), a P450 cholesterol side-chain cleavage inhibitor.