Global Regulatory Mechanisms Research Articles

Catabolite repression of SOS-dependent and SOS-independent spontaneous mutagenesis in stationary-phase Escherichia coli

Previous work in our laboratory established that a spontaneous mutagenesis process operating in stationary-phase Escherichia coli cells undergoing selection is subject to regulation by the global regulatory mechanism known as catabolite repression (formerly also called glucose-repression). Here, we set out to determine the identity of this hitherto unknown catabolite-repressible spontaneous mutation generation mechanism(s). We used two different spontaneous mutation detection assays, reversion of a Lac(-) (lacI33OmegalacZ) frameshift marker and forward mutation to valine-resistance, and tested the effects of varying the nature of the carbon source(s) present in the selective plating medium on the mutability of bacterial cells carrying known defects in the recA, umuDC and dinB genes, three well-known SOS response genes, whose products are important for mutagenesis in E. coli. Consistent with the results of our previous Lac(-)-->Lac(+) assay using otherwise SOS-proficient bacterial cells, we found that the overall numbers of spontaneous Lac(+)E. coli revertants were highest when the selective medium contained lactose and lowest when it contained lactose plus the non-utilizable but strongly catabolite-repressing glucose analogue, methyl-alpha-d-glucopyranoside (alphaMG). In contrast, we found that the numbers of Lac(+) revertants appearing on the lactose and lactose+alphaMG selection plates were greatly diminished and not significantly different when the bacterial cells concerned carried either a DeltarecA or DeltadinB mutation. Furthermore, introducing the DeltadinB mutant allele into bacterial cells over-expressing the recA gene reduced the numbers of Lac(+) mutations to those being recovered with the DeltadinB cells. These results appear to suggest that (i) the DinB-dependent mutation generation pathway is alone responsible for spontaneous reversion of the lacI33OmegalacZ frameshift marker, and (ii) the varying numbers of Lac(+) colonies that we recover on the lactose and lactose+alphaMG plates provide a direct measure of the differential effects of these particular carbon compounds on the overall expression of the dinB gene. Interestingly, the yields of spontaneous Val mutations arising in wild-type, DeltarecA, DeltadinB and DeltaumuDC cells were found to be similar, but always tended to be highest when the medium contained only a non-repressing carbon source (glycerol) and lowest when it had been supplemented with a strong catabolite repressor such as glucose or alphaMG. Together, our results would seem to establish that stationary-phase E. coli cells exposed to strong selection pressures can accumulate spontaneous mutations via SOS-dependent and SOS-independent mutation generation pathways whose levels of expression are regulated by catabolite repression.

Plasticity of transcriptional machinery in bacteria is increased by the repertoire of regulatory families

Escherichia coli K12 and Bacillus subtilis 168 are two of the best characterized bacterial organisms with a long history in molecular biology for understanding various mechanisms in prokaryotic species. However, at the level of transcriptional regulation little is known on a comparative scale. Here we address the question of the degree to which transcription factors (TFs) and their evolutionary families are shared between them. We found that 59 proteins and 28 families are shared between these two bacteria, whereas different subsets were lineage specific. We demonstrate that majority of the common families expand in a lineage-specific manner. More specifically, we found that AraC, ColD, Ebp, LuxR and LysR families are over-represented in E. coli, while ArsR, AsnC, MarR, MerR and TetR families have significantly expanded in B. subtilis. We introduce the notion of regulatory superfamilies based on an empirical number of functional categories regulated by them and show that these families are essentially different in the two bacteria. We further show that global regulators seem to be constrained to smaller regulatory families and generally originate from lineage-specific families. We find that although TF families may be conserved across genomes their functional roles might evolve in a lineage-specific manner and need not be conserved, indicating convergence to be an important phenomenon involved in the functional evolution of TFs of the same family. Although topologically the networks of transcriptional interactions among TF families are similar in both the genomes, we found that the players are different, suggesting different evolutionary origins for the transcriptional regulatory machinery in both bacteria. This study provides evidence from complete repertoires that not only novel families originate in different lineages but conserved TF families expand/contrast in a lineage-specific manner, and suggests that part of the global regulatory mechanisms might originate independently in different lineages.

Transcriptional profiling of the endosymbiont Blochmannia floridanus during different developmental stages of its holometabolous ant host

The transcriptome of Blochmannia floridanus, the endosymbiont of the carpenter ant Camponotus floridanus, is presented during various developmental stages of its holometabolous host by use of a whole-genome DNA macroarray. The detected transcription patterns indicate the presence of local transcription units as well as global regulatory mechanisms. Yet, the overall regulation scale is very modest, rarely exceeding a factor of three. A large number of genes show differential expression in different life stages and a distinct expression pattern of genes possibly involved in symbiotic function as compared with housekeeping genes is apparent. However, these transcriptional changes are small as compared with the changes in the number of bacteria during host development, which is the highest in pupae and in young imagines. Control of replication of the bacteria in certain life stages may therefore be the decisive parameter influencing the overall level of gene expression of Blochmannia in the animal. The few highly expressed genes like those encoding molecular chaperones exhibit a significantly higher G+C content than moderately expressed genes.

Proteomic Analysis of Stationary Phase in the Marine Bacterium “ Candidatus Pelagibacter ubique”

"Candidatus Pelagibacter ubique," an abundant marine alphaproteobacterium, subsists in nature at low ambient nutrient concentrations and may often be exposed to nutrient limitation, but its genome reveals no evidence of global regulatory mechanisms for adaptation to stationary phase. High-resolution capillary liquid chromatography coupled online to an LTQ mass spectrometer was used to build an accurate mass and time (AMT) tag library that enabled quantitative examination of proteomic differences between exponential- and stationary-phase "Ca. Pelagibacter ubique" cells cultivated in a seawater medium. The AMT tag library represented 65% of the predicted protein-encoding genes. "Ca. Pelagibacter ubique" appears to respond adaptively to stationary phase by increasing the abundance of a suite of proteins that contribute to homeostasis rather than undergoing a major remodeling of its proteome. Stationary-phase abundances increased significantly for OsmC and thioredoxin reductase, which may mitigate oxidative damage in "Ca. Pelagibacter," as well as for molecular chaperones, enzymes involved in methionine and cysteine biosynthesis, proteins involved in rho-dependent transcription termination, and the signal transduction enzyme CheY-FisH. We speculate that this limited response may enable "Ca. Pelagibacter ubique" to cope with ambient conditions that deprive it of nutrients for short periods and, furthermore, that the ability to resume growth overrides the need for a more comprehensive global stationary-phase response to create a capacity for long-term survival.

Transcriptional responses ofSaccharomyces cerevisiaeto preferred and nonpreferred nitrogen sources in glucose-limited chemostat cultures

Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae grown with six different nitrogen sources were subjected to transcriptome analysis. The use of chemostats enabled an analysis of nitrogen-source-dependent transcriptional regulation at a fixed specific growth rate. A selection of preferred (ammonium and asparagine) and nonpreferred (leucine, phenylalanine, methionine and proline) nitrogen sources was investigated. For each nitrogen source, distinct sets of genes were induced or repressed relative to the other five nitrogen sources. In total, 131 such 'signature transcripts' were identified in this study. In addition to signature transcripts, genes were identified that showed a transcriptional coresponse to two or more of the six nitrogen sources. For example, 33 genes were transcriptionally upregulated in leucine-grown, phenylalanine-grown and methionine-grown cultures; this was partly attributed to the involvement of common enzymes in the dissimilation of these amino acids. In addition to specific transcriptional responses elicited by individual nitrogen sources, their impact on global regulatory mechanisms such as nitrogen catabolite repression (NCR) were monitored. NCR-sensitive gene expression in the chemostat cultures showed that ammonium and asparagine were 'rich' nitrogen sources. By this criterion, leucine, proline and methionine were 'poor' nitrogen sources, and phenylalanine showed an 'intermediate' NCR response.

Transcriptome Analysis Reveals Global Expression Changes in an IndustrialL-Lysine Producer ofCorynebacterium glutamicum

Toward the elucidation of advanced mechanisms of L-lysine production by Corynebacterium glutamicum, a highly developed industrial strain B-6 was analyzed from the viewpoint of gene expression. Northern blot analysis showed that the lysC gene encoding aspartokinase, the key enzyme of L-lysine biosynthesis, was up-regulated by several folds in strain B-6, while no repression mechanism exists in L-lysine biosynthesis of this bacterium. To analyze the underlying mechanisms of the up-regulation, we compared the transcriptome between strain B-6 and its parental wild-type, finding that not only lysC but also many other amino acid-biosynthetic genes were up-regulated in the producer. These results suggest that a certain global regulatory mechanism is involved in the industrial levels of L-lysine production.

Germline genomics

The completion of the C. elegans genome sequence permits the comprehensive examination of the expression and function of genes. Annotation of virtually every encoded gene in the genome allows systematic analysis of those genes using high-throughput assays, such as microarrays and RNAi. This chapter will center on the use of microarrays to comprehensively identify genes with enriched expression in the germ line during development. This knowledge provides a database for further studies that focus on gene function during germline development or early embryogenesis. Additionally, a comprehensive overview of germline gene expression can uncover striking biases in how genes expressed in the germ line are distributed in the genome, leading to new discoveries of global regulatory mechanisms in the germ line.

Steady-state analysis of glucose repression reveals hierarchical expression of proteins under Mig1p control in Saccharomyces cerevisiae.

Glucose repression is a global transcriptional regulatory mechanism commonly observed in micro-organisms for the repression of enzymes that are not essential for glucose metabolism. In Saccharomyces cerevisiae, Mig1p, a homologue of Wilms' tumour protein, is a global repressor protein dedicated to glucose repression. Mig1p represses genes either by binding directly to the upstream repression sequence of structural genes or by indirectly repressing a transcriptional activator, such as Gal4p. In addition, some genes are repressed by both of the above mechanisms. This raises a fundamental question regarding the physiological relevance of the varied mechanisms of repression that exist involving Mig1p. We address this issue by comparing two well-known glucose-repression systems, that is, SUC2 and GAL gene expression systems, which encompass all the above three mechanisms. We demonstrate using steady-state analysis that these mechanisms lead to a hierarchical glucose repression profile of different family of genes. This switch over from one carbon source to another is well-calibrated as a function of glucose concentration through this hierarchical transcriptional response. The mechanisms prevailing in this repression system can achieve amplification and sensitivity, as observed in the well-characterized MAPK (mitogen-activated protein kinase) cascade system, albeit through a different structure. A critical feature of repression predicted by our steady-state model for the mutant strain of S. cerevisiae lacking Gal80p agrees well with the data reported here as well as that available in the literature.

Generation of p53 target database via integration of microarray and global p53 DNA-binding site analysis.

The completion of the human genome sequence and availability of cDNA microarray technology provide new approaches to explore global cellular regulatory mechanisms. Here we present a strategy to identify genes regulated by specific transcription factors in the human genome, and apply it to p53. We first collected promoters or introns of all genes available using two methods: GenBank annotation and a computationally derived transcript map. The "FindPatterns" program is then used to search sequences in regulatory regions that match the p53 DNA-binding consensus sequence, resulting in the p53 Target Database. This database collects human genes that have at least one p53 DNA-binding sequence in their regulatory region. cDNA microarray was also used to identify genes that respond to p53 at a genomic scale. Integration of the microarray data and the p53 Target Database should greatly enrich direct p53 target genes. Taqman analysis and quantitative chromatin immunoprecipitation analysis are used to validate the in silico prediction and microarray data. Enrichment factor analysis is used to demonstrate that in silico prediction greatly enriches for genes that are transcriptionally regulated by p53 and assists us to identify other signaling pathways that are potentially connected to p53. The approaches can be extended to other transcription factors. The methods shown here illustrate a novel approach to the analysis of global gene regulatory networks through the integration of human genomic sequence information and genome-wide gene expression analysis.

Kyrgyzstan: Central Asia's Island of Democracy? by John s> Anderson (review)

588 SEER, 8o, 3, 2002 Anderson, John. Kyrgyzstan.CentralAsia's Island of Democracy? Harwood Academic Publishers, Amsterdam, I999. xvi + 107 pp. Notes. Tables. Figures.Bibliography.Index. 2 I.00; [ I 2.95. ALTHOUGH severalbooks have been published in the last decade concerning the independent statesof CentralAsia, there are very few that have a country specific focus. John Anderson's book on Kyrgyzstan's transition since the dissolution of the Soviet Union should therefore be regarded as a welcome and timely additionto thisfamilyof academic literature. The book isdividedinto fourchapters.The firstchapterprovidesthe reader with an overview of Kyrgyzstan's history with particular emphasis on the period since Russia'sinterventionin the region.The second chapterexamines Kyrgyzstan'stransition to democracy, the initial enthusiasm for democratic reform and the later tempering of such support that was accompanied by certain set-backs to the democratic process. The chapter illustrates the author's in-depth 'on the ground' knowledge of the ebb and flow of politics within the countryand, as such, is one of the greateststrengthsof the book. In this regard the author is particularlyprescient in identifying a down-turn in the democratization of the country, a trend corroborated by the recent presidential electoral process. Anderson moves from the political to the economic realm in the third chapter. This section details the precipitous decline of the national economy since independence and the deleterious impact upon the welfareof the population. The economic transitionis placed within the context of Kyrgyzstan'sformerintegrationin the Soviet systemand the difficultiesthis raised after independence was achieved. The countrywas confronted simultaneously with three major problems: the de-linking of its economy fromthe formerSovietsystem;the terminationof economic transfers from the centre; and the transitionfrom a command economy to one based on free market principles. One weakness of this section is its reliance on slightlyoutdated economic informationfor national production and poverty. However, thismay be more to do with the time delaybetween the manuscript being completed and the publication of the book, a common problem that must be addressed by the various publishing houses. The final chapter examines the securitychallenges that confront the country in the post-Soviet environment. Anderson identifies two major tasksfor the defense establishment : how to maintain a credible defense force in the context of economic decline and limited resourcesand how to manage its relationswith the major regionalmilitarypowers, namely, Russia, China and Uzbekistan. The book might have benefited from greater engagement with the burgeoningliteratureon democratictransitionsand consolidation, allowing a more comparativeperspectiveto be applied. Similarly,much could be gained by analysing Kyrgyzstan's economic transition in terms of its exit from a traditionalempire in which the political and economic unit were congruent and its entrance into a world system controlled in an altogether different manner by the United States. Doing so might have produced greaterinsight into the global regulatorymechanisms that Kyrgyzstannow has to abide by and the political and economic impact of these constraints. In fairness the author'sobjectiveis not to applya particulartheoryto the case of Kyrgyzstan, REVIEWS 589 but to give the reader an insider'sview of what has happened in the country since independence. To this end the author succeeds. However, an introduction and conclusion developing connections between the economic, political and securityaspectsexamined within the book would have improved such an account. Department ofPolitics J. GLENN University ofSouthampton Holscher, Jens (ed.). FinancialTurbulence and CapitalMarketsin Transition Countries. Macmillan, Basingstoke and London, and St Martin's Press, New York,2000. XX+ 197 pp. Index. ?45.oo. WE now think that we know more about the key determinants of successful transition.Very high on the list is the financial sector. In a marketsystemthe main systemicfunction of the financialsystemis to collect savingsfroma very large number of people, and with them to provide investment funds for somewhat smallernumbersof people and projects.As a necessary adjunctto this processfinancialintermediariesmake assessmentsof the creditworthiness of borrowers,and the viability of projects.They also monitor the progressof theprojectsand the loan repayments.Ifthe financialinstitutionsareincapable of providingthese servicesthen firmsare thrownbackon self-financeor interfirm loans. But even if there are functioning banks in transition economies, then it is important to remember that the financial system is inherently unstable, because it is driven by the profit motive. So regulation is required. But in addition it is necessary to recall...

Impact of genomic technologies on studies of bacterial gene expression.

The ability to simultaneously monitor expression of all genes in any bacterium whose genome has been sequenced has only recently become available. This requires not only careful experimentation but also that voluminous data be organized and interpreted. Here we review the emerging technologies that are impacting the study of bacterial global regulatory mechanisms with a view toward discussing both perceived best practices and the current state of the art. To do this, we concentrate upon examples using Escherichia coli and Bacillus subtilis because prior work in these organisms provides a sound basis for comparison.

Analyses of p53 Target Genes in the Human Genome by Bioinformatic and Microarray Approaches

The completion of the human genome sequence (International Human Genome Sequence Consortium (2001) Nature 409, 860-921; Venter, J. C., et al. (2001) Science 291, 1304-1351) allows for new ways to analyze global cellular regulatory mechanisms. Here we present a strategy to identify genes regulated by specific transcription factors in the human genome, and apply it to p53. We first collected promoters or introns of all genes available using two methods: GenBank(TM) annotation and a computationally derived transcript map. 4,852 genes analyzed in this way contained at least one p53 consensus binding sequence. Of 13 genes randomly selected for mRNA analysis, 11 were shown to respond to p53 expression. Five promoters were analyzed by chromatin immunoprecipitation, which revealed that all were bound by p53 in vivo. We then analyzed 33,615 unique human genes on cDNA microarrays, identifying 1,501 genes that respond to p53 expression. A parameter was derived that demonstrates that in silico prediction greatly enriches for genes that are activated and repressed by p53 and assists us to suggest other signaling pathways that may be connected to p53. The methods shown here illustrate a novel approach to analysis of global gene regulatory network through the integration of human genomic sequence information and genome-wide gene expression analysis.

Quorum sensing is a global regulatory mechanism in enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 microm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with lambda-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

Co-ordinate regulation of genes involved in storage lipid mobilization in Arabidopsis thaliana

Molecular genetic approaches in the model plant Arabidopsis thaliana (ColO) are shedding new light on the role and control of the pathways associated with the mobilization of lipid reserves during oilseed germination and post-germinative growth. Numerous independent studies have reported on the expression of individual genes encoding enzymes from the three major pathways: β-oxidation, the glyoxylate cycle and gluconeogenesis. However, a single comprehensive study of representative genes and enzymes from the different pathways in a single plant species has not been done. Here we present results from Arabidopsis that demonstrate the co-ordinate regulation of gene expression and enzyme activities for the acyl-CoA oxidase- and 3-ketoacyl-CoA thiolasemediated steps of β-oxidation, the isocitrate lyase and malate synthase steps of the glyoxylate cycle and the phosphoenolpyruvate carboxykinase step of gluconeogenesis. The mRNA abundance and enzyme activities increase to a peak at stage 2, 48 h after the onset of seed germination, and decline thereafter either to undetectable levels (for malate synthase and isocitrate lyase) or low basal levels (for the genes of β-oxidation and gluconeogenesis). The co-ordinate induction of all these genes at the onset of germination raises the possibility that a global regulatory mechanism operates to induce the expression of genes associated with the mobilization of storage reserves during the heterotrophic growth period.

Co-ordinate regulation of genes involved in storage lipid mobilization in Arabidopsis thaliana.

Molecular genetic approaches in the model plant Arabidopsis thaliana (Col0) are shedding new light on the role and control of the pathways associated with the mobilization of lipid reserves during oilseed germination and post-germinative growth. Numerous independent studies have reported on the expression of individual genes encoding enzymes from the three major pathways: beta-oxidation, the glyoxylate cycle and gluconeogenesis. However, a single comprehensive study of representative genes and enzymes from the different pathways in a single plant species has not been done. Here we present results from Arabidopsis that demonstrate the co-ordinate regulation of gene expression and enzyme activities for the acyl-CoA oxidase- and 3-ketoacyl-CoA thiolase-mediated steps of beta-oxidation, the isocitrate lyase and malate synthase steps of the glyoxylate cycle and the phosphoenolpyruvate carboxykinase step of gluconeogenesis. The mRNA abundance and enzyme activities increase to a peak at stage 2, 48 h after the onset of seed germination, and decline thereafter either to undetectable levels (for malate synthase and isocitrate lyase) or low basal levels (for the genes of beta-oxidation and gluconeogenesis). The co-ordinate induction of all these genes at the onset of germination raises the possibility that a global regulatory mechanism operates to induce the expression of genes associated with the mobilization of storage reserves during the heterotrophic growth period.

The effects of DNA supercoiling on the expression of operons of the ilv regulon of Escherichia coli suggest a physiological rationale for divergently transcribed operons.

Transcriptional activities of closely spaced divergent promoters are affected by the accumulation of local negative superhelicity in the region between transcribing RNA polymerase molecules (transcriptional coupling). The effect of this transcription-induced DNA supercoiling on these promoters depends on their intrinsic properties. As the global superhelical density of the chromosome is controlled by the energy charge of the cell, which is affected by environmental stresses and transitions from one growth state to another, the transcriptional coupling that occurs between divergently transcribed promoters is likely to serve a physiological purpose. Here, we suggest that transcriptional coupling between the divergent promoters of the ilvYC operon of Escherichia coli serves to co-ordinate the expression of this operon with other operons of the ilv regulon during metabolic adjustments associated with growth state transitions. As DNA supercoiling-dependent transcriptional coupling between the promoters of other divergently transcribed operons is investigated, additional global gene regulatory mechanisms and physiological roles are sure to emerge.

Effects of global regulatory proteins and environmental conditionson fimbrial gene expression of F165 1 and F165 2 producedby Escherichia coli causing septicaemia in pigs

Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and possess at least two types of fimbriae. F165 1 fimbriae belong to the P fimbrial family and F165 2 fimbriae belong to the S fimbrial family. Regulatory regions of foo (F165 1) and fot (F165 2) fimbrial gene clusters from wild-type strain 4787 were sequenced and characterised. Expression of F165 1 and F165 2 fimbrial genes was analysed by using lacZ and/or luxAB as reporter genes under the control of the native fimbrial promoters. Differential expression of fimbrial genes was observed. Global regulatory mechanisms such as catabolite repression, leucine-responsive regulatory protein (Lrp), methylation and DNA supercoiling were demonstrated to influence foo and fot expression. foo and fot expression was optimal at 37 °C and under aerobic conditions. Expression of foo was higher on minimal medium, whereas fot expression was higher on complex Luria-Bertani medium. This could reflect an in vivo differential expression.

The Study of Guanosine 5′-Diphosphate 3′-Diphosphate-mediated Transcription Regulation in Vitro Using a Coupled Transcription-Translation System

The effects of the "alarmone" guanosine 5'-diphosphate 3'-diphosphate (ppGpp) on regulation of the Salmonella typhimurium histindine operon and the Escherichia coli tRNA(leu) operon were analyzed in vitro using a DNA-dependent transcription-translation system, S-30. The expression of the hisG promoter is positively regulated by ppGpp, whereas that of the leuV promoter (of tRNA(1eu)) is negatively regulated by ppGpp. In an attempt to understand the global regulatory mechanism of ppGpp control, interrelationship between ppGpp-dependent activation and repression of gene expression was examined using these promoters as models. It has been traditionally supposed that the ppGpp-dependent regulation, at least for the activation, is by a passive mode of control: the activation of gene expression by ppGpp is a consequence of the repression of stable RNA gene expression in the condition of RNA polymerase limiting. To test this model, the ppGpp-dependent regulations of both an activable promoter (hisGp) and a repressible promoter (leuVp) were determined in vitro simultaneously using a mixed template setup. The rationale for this exercise was to see whether the ppGpp-dependent activation and repression are inversely correlated in the in vitro condition in which RNA polymerase is limiting. No correlation was observed. It was concluded that the ppGpp-dependent activation is independent of the repression. Moreover, it was proposed that ppGpp-dependent activation and repression are mediated by titratable factors, each of which operate independently.

The bvr locus of Listeria monocytogenes mediates virulence gene repression by beta-glucosides.

The beta-glucoside cellobiose has been reported to specifically repress the PrfA-dependent virulence genes hly and plcA in Listeria monocytogenes NCTC 7973. This led to the hypothesis that beta-glucosides, sugars of plant origin, may act as signal molecules, preventing the expression of virulence genes if L. monocytogenes is living in its natural habitat (soil). In three other laboratory strains (EGD, L028, and 10403S), however, the effect of cellobiose was not unique, and all fermentable carbohydrates repressed hly. This suggested that the downregulation of virulence genes by beta-glucosides is not a specific phenomenon but, rather, an aspect of a global regulatory mechanism of catabolite repression (CR). We assessed the effect of carbohydrates on virulence gene expression in a panel of wild-type isolates of L. monocytogenes by using the PrfA-dependent phospholipase C gene plcB as a reporter. Utilization of any fermentable sugar caused plcB repression in wild-type L. monocytogenes. However, an EGD variant was identified in which, as in NCTC 7973, plcB was only repressed by beta-glucosides. Thus, the regulation of L. monocytogenes virulence genes by sugars appears to be mediated by two separate mechanisms, one presumably involving a CR pathway and another specifically responding to beta-glucosides. We have identified in L. monocytogenes a 4-kb operon, bvrABC, encoding an antiterminator of the BglG family (bvrA), a beta-glucoside-specific enzyme II permease component of the phosphoenolpyruvate-sugar phosphotransferase system (bvrB), and a putative ADP-ribosylglycohydrolase (bvrC). Low-stringency Southern blots showed that this locus is absent from other Listeria spp. Transcription of bvrB was induced by cellobiose and salicin but not by arbutin. Disruption of the bvr operon by replacing part of bvrAB with an interposon abolished the repression by cellobiose and salicin but not that by arbutin. Our data indicate that the bvr locus encodes a beta-glucoside-specific sensor that mediates virulence gene repression upon detection of cellobiose and salicin. Bvr is the first sensory system found in L. monocytogenes that is involved in environmental regulation of virulence genes.

New Global Regulatory Mechanisms and the Environment:The Emerging Linkage between the WTO and the ISO

Summary This article seeks to highlight the emerging linkage between the World Trade Organisation (WTO) and the International Organisation for Standardisation (ISO). This relationship is seen against a background of liberalisation, and more generally of profound changes in the structure of global governance. Indeed, after having promoted deregulation on a massive scale, the time seems to be ripe to reintroduce regulation, albeit in a new format. Standards, especially environmental standards, appear to provide new forms of global regulation. As a result, we witness the emergence of new forms of actor arrangements, dominated by private, as opposed to public actors.