bluuebunny/crossref_metadata_2025 huggingface dataset:
The phenolic vir gene inducer ferulic acid is O‐demethylated by the VirH2 protein of an Agrobacterium tumefaciens Ti plasmid
Authors: Kalogeraki Virginia S. , Zhu Jun , Eberhard Anatol , Madsen Eugene L. , Winans Stephen C.
Published: March 2003

Some or possibly all Ti plasmids of Agrobacterium tumefaciens encode a bicistronic operon designated virH, which encodes two proteins, VirH1 and VirH2, that resemble a family of cytochrome P450‐type monooxygenases. Expression of this operon is induced by a family of phenolic compounds that induce all other operons within the vir regulon. We hypothesized that either or both of these proteins might metabolize some or all of these phenolic compounds. We therefore tested induction of a vir promoter by a variety of phenolic compounds in isogenic strains that express or lack virH1 and virH2. Although some compounds were equally effective inducers regardless of the virH status, other compounds induced vir expression far more effectively in the virH mutant than in the virH‐proficient host. For all tested compounds, VirH2 appeared to be solely responsible for this effect. One such compound, ferulic acid, was chosen for biochemical analysis. Ferulic acid was degraded by a VirH‐proficient host but not by a VirH mutant. The wild‐type strain released large amounts of a more hydrophilic compound into the cell supernatant. This compound was tested by mass spectroscopy, nuclear magnetic resonance and UV spectroscopy and found to consist of caffeic acid. This indicates that wild‐type strains convert virtually all added ferulic acid to caffeic acid, and that VirH2 is essential for this O‐demethylation reaction. Ferulic acid was far more toxic than caffeic acid to the wild‐type strain, although the wild‐type strain was more resistant to ferulic acid than was the virH mutant. Caffeic acid was slowly removed from the broth, suggesting further metabolic reactions.
Functional analysis of the FimE integrase of Escherichia coli K‐12: isolation of mutant derivatives with altered DNA inversion preferences
Authors: Smith Stephen G. J. , Dorman Charles J.
Published: March 2003

Phase variable expression of type 1 fimbriae in Escherichia coli arises from a site‐specific recombination event that inverts a short segment of chromosomal DNA carrying the promoter for transcription of the gene encoding the fimbrial subunit protein. Two integrase‐like recombinases are involved in switching. The FimB recombinase inverts the DNA segment in either orientation, whereas the FimE protein inverts it predominantly in the ON‐to‐OFF direction. In this paper, we report the isolation of a FimE mutant protein that has enhanced bidirectional switching activity. This protein has an arginine‐to‐lysine substitution at position 59, and this confers a FimB‐like switching character on FimE without altering its ability to bind to DNA. The arginine was not a member of the arginine‐histidine‐arginine‐tyrosine catalytic tetrad that is common to all integrase‐like recombinases. The catalytic tetrad members of FimE were identified at positions 41, 136, 139 and 171 and shown to be essential for FimE function. In addition, other amino acid residues that make important contributions to the DNA binding activity of FimE or its ON‐to‐OFF inversion efficiency were identified.
CheB is required for behavioural responses to negative stimuli during chemotaxis in Bacillus subtilis
Authors: Kirby John R. , Niewold Timothy B. , Maloy Stanley , Ordal George W.
Published: March 2003

The methyl‐accepting chemotaxis protein, McpB, is the sole receptor mediating asparagine chemotaxis in Bacillus subtilis. In this study, we show that wild‐type B. subtilis cells contain ≈ 2000 copies of McpB per cell, that these receptors are localized polarly, and that titration of only a few receptors is sufficient to generate a detectable behavioural response. In contrast to the wild type, a cheB mutant was incapable of tumbling in response to decreasing concentrations of asparagine, but the cheB mutant was able to accumulate to low concentrations of asparagine in the capillary assay, as observed previously in response to azetidine‐2‐carboxylate. Furthermore, net demethylation of McpB is logarithmically dependent on asparagine concentration, with half‐maximal demethylation of McpB occurring when only 3% of the receptors are titrated. Because the corresponding methanol production is exponentially dependent on attractant concentration, net methylation changes and increased turnover of methyl groups must occur on McpB at high concentrations of asparagine. Together, the data support the hypothesis that methylation changes occur on asparagine‐bound McpB to enhance the dynamic range of the receptor complex and to enable the cell to respond to a negative stimulus, such as removal of asparagine.
A developmentally regulated catalase required for proper differentiation and osmoprotection of Streptomyces coelicolor
Authors: Cho You‐Hee , Lee Eun‐Jin , Roe Jung‐Hye
Published: March 2003

Streptomyces coelicolor produces at least three catalases, the expression of which varies under different conditions. We characterized a gene (catB) for developmentally controlled catalase of 779 amino acids (83408 Da), homologous to KatE of Escherichia coli and Bacillus subtilis. Expression of the catB gene increased at the stationary phase in liquid culture and after the onset of differentiation on solid culture. It was also increased by osmotic treatments. Transcription was initiated from a promoter (catBp), whose sequence (ATGCCTCG‐N13‐GGGTAC) resembled promoters recognized by σB of B. subtilis. CatB protein underwent proteolytic cleavage of its N‐terminal 95 amino acids and was secreted to the medium when cells sporulated. Disruption of the catB gene caused impairment in the formation of aerial mycelium and reduction in the synthesis of undecylprodigiosin. On the contrary, hyperproduction of actinorhodin was observed in accordance with the increase in actII‐ORF4 transcription. In addition, catB mutant became hypersensitive to osmotic stresses. These results suggest that regulated synthesis of CatB protein is necessary to ensure proper differentiation as well as to protect S. coelicolor cells against osmotic stresses.
Mth1 receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae‡
Authors: Lafuente María J. , Gancedo Carlos , Jauniaux Jean‐Claude , Gancedo Juana M.
Published: March 2003

We have determined that the mutant genes DGT1‐1 and BPC1‐1, which impair glucose transport and catabolite repression in Saccharomyces cerevisiae, are allelic forms of MTH1. Deletion of MTH1 had only slight effects on the expression of HXT1 or SNF3, but increased expression of HXT2 in the absence of glucose. A two‐hybrid screen revealed that the Mth1 protein interacts with the cytoplasmic tails of the glucose sensors Snf3 and Rgt2. This interaction was affected by mutations in Mth1 and by the concentration of glucose in the medium. A double mutant, snf3 rgt2, recovered sensitivity to glucose when MTH1 was deleted, thus showing that glucose signalling may occur independently of Snf3 and Rgt2. A model for the possible mode of action of Snf3 and Rgt2 is presented.
Transfer RNA modification, temperature and DNA superhelicity have a common target in the regulatory network of the virulence of Shigella flexneri: the expression of the virF gene
Authors: Durand Jérôme M. B. , Dagberg Björn , Uhlin Bernt Eric , Björk Glenn R.
Published: March 2003

Full expression of the virulence genes of Shigella flexneri requires the presence of two modified nucleosides in the tRNA [queuosine, Q34, present in the wobble position (position 34) and 2‐methylthio‐N6‐isopentenyladenosine (ms2i6A37, adjacent to and 3′ of the anticodon)]. The synthesis of these two nucleosides depends on the products of the tgt and miaA genes respectively. We have shown that the intracellular concentration of the virulence‐related transcriptional regulator VirF is reduced in the absence of either of these modified nucleosides. The intracellular concentration of VirF is correlated with the expression of the virulence genes. Overproduction of VirF in the tgt and the miaA mutants suppressed the less virulent (tgt) or the avirulent (miaA) phenotypes respectively, caused by the tRNA modification deficiency. This suggests that the primary result of undermodification of the tRNA is a poor translation of virF mRNA and not of any other mRNA whose product acts downstream of the action of VirF. Shigella showed no virulence phenotypes at 30°C, but forced synthesis of VirF at 30°C induced the virulence phenotype at this low temperature. In addition, removal of the known gene silencer H‐NS by a mutation in its structural gene hns increased the synthesis of VirF at low temperature and thus induced a virulent phenotype at 30°C. Conversely, decreased expression of VirF at 37°C induced by the addition of novobiocin, a known inhibitor of gyrase, led to an avirulent phenotype. We conclude that tRNA modification, temperature and superhelicity have the same target – the expression of VirF – to influence the expression of the central regulatory gene virB and thereby the virulence of Shigella. These results further strengthen the suggestion that the concentration of VirF is the critical factor in the regulation of virulence in Shigella. In addition, they emphasize the role of the bacterial translational machinery in the regulation of the expression of virulence genes which appears here quantitatively as important as the well‐established regulation on the transcriptional level.
Characterization of ylbF, a new gene involved in competence development and sporulation in Bacillus subtilis
Authors: Tortosa Pablo , Albano Mark , Dubnau David
Published: March 2003

We used mini Tn10 transposition to generate a library of Bacillus subtilis insertion mutants, with the goal of identifying and characterizing new competence genes. Two new regulatory genes were identified in our screen: ypuN (also known as rsiX, the anti‐σX factor) and ylbF. The disruption of ylbF leads to a dramatic decrease in the expression of comK, encoding the competence transcription factor. Our data show that ylbF positively controls ComK at a post‐transcriptional level. It has been reported previously that ComK is degraded in vivo and in vitro by a multimeric protein complex composed of ClpP, ClpC and MecA. This proteolysis is inhibited by the ComS peptide. We show that both the overexpression of comS and the inactivation of mecA individually suffice to bypass the competence phenotype of the ylbF mutation. This mutation does not seem to alter the cellular concentrations of MecA or ClpP, and we propose a role for YlbF in modulating the translation, stability or activity of ComS. In addition to its role in competence, ylbF also appears to regulate sporulation by acting before stage II.
RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans
Authors: Alspaugh J. Andrew , Cavallo Lora M. , Perfect John R. , Heitman Joseph
Published: March 2003

Cryptococcus neoformans is a basidiomycete yeast and opportunistic human pathogen of increasing clinical importance due to the increasing population of immunocompromised patients. To further investigate signal transduction cascades regulating fungal pathogenesis, we have identified the gene encoding a RAS homologue in this organism. The RAS1 gene was disrupted by transformation and homologous recombination. The resulting ras1 mutant strain was viable, but failed to grow at 37°C, and exhibited significant defects in mating and agar adherence. The ras1 mutant strain was also avirulent in an animal model of cryptococcal meningitis. Reintroduction of the wild‐type RAS1 gene complemented these ras1 mutant phenotypes and restored virulence in animals. A dominantly active RAS1 mutant allele, RAS1Q67L, induced a differentiation phenotype known as haploid fruiting, which involves filamentation, agar invasion and sporulation in response to nitrogen deprivation. The ras1 mutant mating defect was suppressed by overexpression of MAP kinase signalling elements and partially suppressed by exogenous cAMP. Additionally, cAMP also suppressed the agar adherence defect of the ras1 mutant. However, the ability of the ras1 mutant strain to grow at elevated temperature was not restored by cAMP or MAP kinase overexpression. Our findings support a model in which RAS1 signals in C. neoformans through cAMP‐dependent, MAP kinase, and RAS‐specific signalling cascades to regulate mating and filamentation, as well as growth at high temperature which is necessary for maintenance of infection.
Specificity determinants for bacteriophage Hong Kong 022 integrase: analysis of mutants with relaxed core‐binding specificities
Authors: Cheng Qiong , Swalla Brian M. , Beck Michael , Alcaraz Renato , Gumport Richard I. , Gardner Jeffrey F.
Published: March 2003

The integrase (Int) proteins encoded by bacteriophages HK022 and λ catalyse similar site‐specific integration and excision reactions between specific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and λ are unable to catalyse recombination between non‐cognate att sites. The att sites of both phages contain weak binding sites for Int, known as ‘core‐type’ sites. Negatively acting nucleotide determinants associated with specific core sites (λ B′, HK022 B′, HK022 C) are responsible for the barrier to non‐cognate recombination. In this study, we used challenge phages to demonstrate that the λ and HK022 Ints cannot bind to core sites containing non‐cognate specificity determinants in vivo. We isolated mutants of the HK022 Int, which bind the λ B′ core site. Two mutants, D99N and D99A, have changed a residue in the core‐binding (CB) domain, which may be directly contacting the core site DNA. We suggest that binding to the λ B′ site was accomplished by removing the negatively charged aspartate residue, which normally participates in a conflicting interaction with the G4 nucleotide of the λ B′ site. We showed that, although our mutants retain the ability to recombine their cognate att sites, they are unable to recombine λatt sites.
New functions for the ancient globin family: bacterial responses to nitric oxide and nitrosative stress
Authors: Poole Robert K. , Hughes Martin N.
Published: March 2003

Globin‐like oxygen‐binding proteins occur in bacteria, yeasts and other fungi, and protozoa. The simplest contain protohaem as sole prosthetic group, but show considerable variation in their similarity to the classical animal globins and plant globins. Flavohaemoglobins comprise a haem domain homologous to classical globins and a ferredoxin‐NADP+ reductase (FNR)‐like domain that converts the globin into an NAD(P)H‐oxidizing protein with diverse reductase activities. In Escherichia coli, the prototype flavohaemoglobin (Hmp) is clearly involved in responses to nitric oxide (NO) and nitrosative stress: (i) the structural gene hmp is upregulated by NO and nitrosating agents; (ii) purified Hmp binds NO avidly, but also converts it to nitrate (aerobically) or nitrous oxide (anaerobically); (iii) hmp mutants are hypersensitive to NO and nitrosative stresses. Here, we review recent advances in E. coli and the growing number of microbes in which globins are known, draw particular attention to the essential chemistry of NO and related reactive species and their interactions with globins, and suggest that microbial globins have additional functions unrelated to ‘NO’ stresses.
Salmonella SsrB activates a global regulon of horizontally acquired genes
Authors: Worley Micah J. , Ching Katherine H. L. , Heffron Fred
Published: March 2003

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. This ability requires an S. enterica specific locus termed Salmonella pathogenicity island 2 (SPI‐2). SPI‐2 encodes a type III secretion system that injects effectors encoded within the island into host cell cytosol to promote virulence. SsrAB is a two‐component regulator encoded within SPI‐2 that was assumed to activate SPI‐2 genes exclusively. Here, it is shown that SsrB in fact activates a global regulon. At least 10 genes outside SPI‐2 are SsrB regulated within epithelial and macrophage cells. Nine of these 10 SsrB‐regulated genes outside SPI‐2 reside within previously undescribed regions of the Salmonella genome. Most share no sequence homology with current database entries. However, one is remarkably homologous to human glucosyl ceramidase, an enzyme involved in the ceramide signalling pathway. The SsrB regulon is modulated by the two‐component regulatory systems PhoP/PhoQ and OmpR/EnvZ, and is upregulated in the intracellular microenvironment.
Upstream nucleosomes and Rgr1p are required for nucleosomal repression of transcription
Authors: Moss David R. , Laybourn Paul J.
Published: March 2003

The mechanisms of transcription repression and derepressionin vivoare not fully understood. We have obtained evidence that begins to clarify the minimum requirements for counteracting nucleosomal repressionin vivo. Location of the TATA element near the nucleosome dyad does not block RNA polymerase II transcriptionin vivoif there is a nucleosome‐free region located immediately upstream. However, location of the TATA element similarly within the nucleosome does block transcription if the region upstream of it is nucleosome bound. Histone H4 depletion derepresses transcription in the latter case, supporting the idea that the nucleosomes are responsible for the repression. These results raise the intriguing possibility that the minimum requirement for derepression of transcriptionin vivois a nucleosome‐free region upstream of the core promoter. Importantly, we find that a C‐terminal deletion inRGR1, a component of the mediator/holoenzyme complex and a global repressor, can also derepress transcription.
The Escherichia coli SeqA protein binds specifically and co‐operatively to two sites in hemimethylated and fully methylated oriC
Authors: Skarstad Kirsten , Lueder Gerhild , Lurz Rudi , Speck Christian , Messer Walter
Published: March 2003

The Escherichia coli SeqA protein has been found to affect initiation of replication negatively, both in vivo and in vitro. The mechanism of inhibition is, however, not known. SeqA has been suggested to affect the formation and activity of the initiation complex at oriC, either by binding to DNA or by interacting with the DnaA protein. We have investigated the binding of SeqA to oriC by electron microscopy and found that SeqA binds specifically to two sites in oriC, one on each side of the DnaA binding site R1. Specific binding was found for fully and hemimethylated but not unmethylated oriC in good agreement with earlier mobility shift studies. The affinity of SeqA for hemimethylated oriC was higher than for fully methylated oriC. The binding was in both cases strongly co‐operative. We suggest that SeqA binds to two nucleation sites in oriC, and by the aid of protein–protein interaction spreads to adjacent regions in the same oriC as well as recruiting additional oriC molecules and/or complexes into larger aggregates.
UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognition
Authors: Gourse Richard L. , Ross Wilma , Gaal Tamas
Published: March 2003

In recent years, it has become clear that promoter recognition by bacterial RNA polymerase involves interactions not only between core promoter elements and the σ subunit, but also between a DNA element upstream of the core promoter and the α subunit. DNA binding by α can increase transcription dramatically. Here we review the current state of our understanding of the α interaction with DNA during basal transcription initiation (i.e. in the absence of proteins other than RNA polymerase) and activated transcription initiation (i.e. when stimulated by transcription factors).
The ClpX protein of Bacillus subtilis indirectly influences RNA polymerase holoenzyme composition and directly stimulates σH‐dependent transcription
Authors: Liu Jiajian , Zuber Peter
Published: March 2003

In Bacillus subtilis, several processes associated with the onset of stationary phase, including the initiation of sporulation, require the activity of the minor σH form of RNA polymerase (RNAP). The induction of σH‐dependent gene transcription requires the regulatory ATPase, ClpX. The ClpX‐dependent post‐exponential increase in σH activity is not dependent on the activator of sporulation gene expression, Spo0A. By determining the level of σH and σA in whole‐cell extracts and RNAP preparations, evidence is presented that clpX does not influence the concentration of sigma subunits, but is required for the stationary phase reduction in σA‐RNAP holoenzyme. This is probably an indirect consequence of ClpX activity, because the ClpX‐dependent decrease in σA‐RNAP concentration does not occur in a spo0A abrB mutant. The addition of ClpX to in vitro transcription reactions resulted in the stimulation of RNAP holoenzyme activity, but σH‐RNAP was observed to be more sensitive to ClpX‐dependent stimulation than σA‐RNAP. No difference in transcriptional activity was observed in single‐cycle in vitro transcription reactions, suggesting that ClpX acted at a step in transcription initiation after closed‐ and open‐promoter complex formation. ClpX is proposed to function indirectly in the displacement of σA from core RNAP and to act directly in the stimulation of σH‐dependent transcription in sporulating B. subtilis cells.
The CitST two‐component system regulates the expression of the Mg‐citrate transporter in Bacillus subtilis
Authors: Yamamoto Hiroki , Murata Masayoshi , Sekiguchi Junichi
Published: March 2003

citS and citT genes encoding a new two‐component system were identified in the 71° region between the pel and citM loci on the Bacillus subtilis chromosome. citS‐ and citT‐deficient strains were unable to grow on minimal plates including citrate as a sole carbon source. In addition, a strain deficient in citM, which encodes the secondary transporter of the Mg‐citrate complex, exhibited the same phenotype on this medium. Northern blot analysis revealed that citM was polycistronically transcribed with the downstream yflN gene, and that CitS and CitT were necessary for transcription of the citM–yflN operon. Upon addition of 2 mM citrate to DSM, this operon was strongly induced after the middle of the exponential growth phase in the wild type, but not in the citST double null mutant. Moreover, the transcription of this operon was completely repressed in the presence of 1% glucose. We found a sequence exhibiting homology to a catabolite‐responsive element (cre) in the citM promoter region. Glucose repression was lost in ccpA and citM–cre mutants. From the result of a citM–promoter deletion experiment, putative CitT target sequences were found to be located around two regions, from −62 to −74 and from −149 to −189, relative to the citM start point. Furthermore, DNase I footprinting assays revealed that these two CitT target regions extended maximally from −36 to −84 and from −168 to −194. From these findings, we concluded that the expression of citM is positively regulated by the CitST system and negatively regulated by CcpA.
Suppression of FNR‐dependent transcription activation at the Escherichia coli nir promoter by Fis, IHF and H‐NS: modulation of transcription initiation by a complex nucleo‐protein assembly
Authors: Browning Douglas F. , Cole Jeffrey A. , Busby Stephen J. W.
Published: March 2003

Expression from the Escherichia coli nir promoter is co‐dependent on both the FNR protein (an anaerobically triggered transcription activator) and the NarL or NarP proteins (transcription activators triggered by nitrite and nitrate). Under anaerobic conditions, FNR binds to a site centred between positions −41 and −42, activating transcription of the nir operon. In previous work, we showed that this activation is suppressed by the binding of Fis protein, and at least one other protein, to sequence elements located upstream of the nir promoter. We proposed that the binding of NarL or NarP to a site centred between positions −69 and −70 counteracts this suppression, resulting in increased transcription in response to nitrite or nitrate. Here we have further investigated the different proteins that downregulate the nir promoter. We show that the nir promoter is repressed by three DNA binding proteins, Fis, IHF and H‐NS. We demonstrate that, in addition to binding to its previously characterized upstream site located at position −142, Fis also binds to a second downstream site located at position +23. A second suppressing factor is IHF, that binds to a site located at position −88. Finally, the nucleoid associated protein, H‐NS, preferentially binds to upstream sequences at the nir promoter and represses promoter activity. The association of Fis, IHF and H‐NS suggests that nir promoter DNA is sequestrated into a highly ordered nucleo‐protein structure that represses FNR‐dependent transcription activation. NarL and NarP can relieve both IHF‐ and Fis‐mediated repression, but are unable to counteract H‐NS‐mediated repression.
Identification of Streptococcus agalactiae virulence genes in the neonatal rat sepsis model using signature‐tagged mutagenesis
Authors: Jones Amanda L. , Knoll Katherine M. , Rubens Craig E.
Published: March 2003

Group B streptococcal (GBS) infections are the most common cause of bacterial sepsis in the immediate newborn period. Apart from the capsule, the factors required for survival of GBS in the host are not well defined. In this study, signature‐tagged transposon mutagenesis (STM) was used to identify genes required for growth and survival of GBS in a neonatal rat sepsis infection model. Approximately 1600 transposon mutants were screened in pools of 80 mutants, and approximately 120 mutants defective for survival in the animal host were identified. We successfully cloned and sequenced DNA flanking the transposon insertions from 92 of the mutants. Fifty per cent of the mutants had transposon insertions in genes with homologues in the public databases, whereas the remaining 50% had transposon insertions in genes with unknown function. A significant proportion of the avirulent mutants had transposon insertions in genes encoding transport‐associated or regulatory proteins or in genes involved in cell surface metabolism, emphasizing the significance of these functions for in vivo survival of GBS. Overall, STM analysis revealed GBS genomic loci that encode a wide variety of functional gene classes, underscoring the diversity of bacterial processes required for the infection process. Currently, the function of the genes identified during the screening can only be inferred by homology to previously described genes. However, a number of the genes identified in this study have been shown to correlate with virulence in other pathogens. Avirulence of a subset of mutants identified during the screening was confirmed by performing competitive index assays and lethal dose assays. This represents the first report of a genome‐wide scan for virulence factors in GBS. The identified genes will further our understanding of the pathogenesis of GBS infections and may represent targets for intervention or lead to the development of novel therapies.
FliH, a soluble component of the type III flagellar export apparatus of Salmonella, forms a complex with FliI and inhibits its ATPase activity
Authors: Minamino Tohru , Macnab Robert M.
Published: March 2003

Both FliH and the ATPase FliI are cytoplasmic components of the Salmonella type III flagellar export apparatus. Dominance and inhibition data have suggested that the N‐terminus of FliI interacts with FliH and that this interaction is important for the ATPase function of the C‐terminal domain of FliI. N‐terminally histidine‐tagged, wild‐type FliI retarded untagged FliH in a Ni‐NTA affinity chromatography assay, as did N‐His‐tagged versions of FliI carrying catalytic mutations. In contrast, N‐His‐tagged FliI carrying the double mutation R7C/L12P did not, further indicating that the N‐terminus of FliI is responsible for interaction with FliH. Native agarose gel electrophoresis confirmed that FliH and FliI form a complex. Analytical gel filtration with in‐line multiangle light scattering indicated that FliH alone forms a dimer, FliI alone remains as a monomer, and FliH and FliI together form a (FliH)2FliI complex. Ni‐NTA affinity chromatography using N‐His‐tagged FliH and a large excess of untagged FliH confirmed that FliH forms a homodimer. The ATPase activity of the FliH–FliI complex was about 10‐fold lower than that of FliI alone; the presence or absence of ATP did not affect the formation of the complex. We propose that FliH functions as a negative regulator to prevent FliI from hydrolysing ATP until the flagellar export apparatus is competent to link this hydrolysis to the translocation of export substrates across the plane of the cytoplasmic membrane into the lumen of the nascent flagellar structure.
Oxygen detoxification in the strict anaerobic archaeon Archaeoglobus fulgidus: superoxide scavenging by Neelaredoxin
Authors: Abreu Isabel A. , Saraiva Lígia M. , Carita João , Huber H. , Stetter K. O. , Cabelli Diane , Teixeira Miguel
Published: March 2003

Archaeoglobus fulgidus is a hyperthermophilic sulphate‐reducing archaeon. It has an optimum growth temperature of 83°C and is described as a strict anaerobe. Its genome lacks any homologue of canonical superoxide (O2·−) dismutases. In this work, we show that neelaredoxin (Nlr) is the main O2·− scavenger in A. fulgidus, by studying both the wild‐type and recombinant proteins. Nlr is a 125‐amino‐acid blue‐coloured protein containing a single iron atom/molecule, which in the oxidized state is high spin ferric. This iron centre has a reduction potential of +230 mV at pH 7.0. Nitroblue tetrazolium‐stained gel assays of cell‐soluble extracts show that Nlr is the main protein from A. fulgidus which is reactive towards O2·−. Furthermore, it is shown that Nlr is able to both reduce and dismutate O2·−, thus having a bifunctional reactivity towards O2·−. Kinetic and spectroscopic studies indicate that Nlr's superoxide reductase activity may allow the cell to eliminate O2·− quickly in a NAD(P)H‐dependent pathway. On the other hand, Nlr's superoxide dismutation activity will allow the cell to detoxify O2·− independently of the cell redox status. Its superoxide dismutase activity was estimated to be 59 U mg−1 by the xanthine/xanthine oxidase assay at 25°C. Pulse radiolysis studies with the isolated and reduced Nlr proved unambiguously that it has superoxide dismutase activity; at pH 7.1 and 83°C, the rate constant is 5 × 106 M−1 s−1. Besides the superoxide dismutase activity, soluble cell extracts of A. fulgidus also exhibit catalase and NAD(P)H/oxygen oxidoreductase activities. By putting these findings together with the entire genomic data available, a possible oxygen detoxification mechanism in A. fulgidus is discussed.
The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA
Authors: Schneider Robert , Travers Andrew , Muskhelishvili Georgi
Published: March 2003

The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA. Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling. Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling. Any deviations from these optimal levels decrease fis promoter activity. This strict dependence of fis expression on the superhelical density suggests that fis may be involved in ‘fine‐tuning’ the homeostatic control mechanism of DNA supercoiling in E. coli.
Regulation of the Pcl7–Pho85 cyclin–cdk complex by Pho81
Authors: Lee M. , O'regan S. , Moreau J.‐L. , Johnson A. L. , Johnston L. H. , Goding C. R.
Published: March 2003

Saccharomyces cerevisiae strains lacking a functional Pho85 cyclin‐dependent kinase (cdk) exhibit a complex phenotype, including deregulation of phosphatase genes controlled by the transcription factor Pho4, slow growth on rich media, failure to grow using galactose, lactate or glycerol as a carbon source and hyperaccumulation of glycogen. The ability of Pho85 to regulate the transcription factor Pho4 is mediated by its association the Pho80 cyclin. Some other regulatory functions of the Pho85 cdk have been shown to be mediated via its interaction with a recently identified family of Pho80‐related cyclins (Pcls). Here, we show that the poorly characterized Pho80‐like protein Pcl7 forms a functional kinase complex with the Pho85 cdk, and that the activity of this complex is inhibited in response to phosphate starvation. Additionally, we show that Pcl7 interacts with the phosphate‐regulated cyclin–cdk inhibitor Pho81, and that the regulation of the Pcl7–Pho85 complex in response to changes in phosphate levels is dependent on Pho81. Thus, we demonstrate for the first time that the Pho81 regulator is not dedicated to regulating Pho80, but may act to co‐ordinate the activity of both the Pho80–Pho85 and Pcl7–Pho85 cyclin–cdk complexes in response to phosphate levels. We also demonstrate that expression of Pcl7 is cell cycle regulated, with maximal activity occurring in mid to late S‐phase, perhaps suggesting a role for Pcl7 in cell cycle progression. Finally, we describe the phenotype of pcl7Δ and pcl6Δ yeast strains that have defects in carbon source utilization.
Multiple histidine kinases regulate entry into stationary phase and sporulation in Bacillus subtilis
Authors: Jiang Min , Shao Weilan , Perego Marta , Hoch James A.
Published: March 2003

Protein homology studies identified five kinases potentially capable of phosphorylating the Spo0F response regulator and initiating sporulation in Bacillus subtilis. Two of these kinases, KinA and KinB, were known from previous studies to be responsible for sporulation in laboratory media. In vivo studies of the activity of four of the kinases, KinA, KinC, KinD (ykvD) and KinE (ykrQ), using abrB transcription as an indicator of Spo0A∼P level, revealed that KinC and KinD were responsible for Spo0A∼P production during the exponential phase of growth in the absence of KinA and KinB. In vitro, all four kinases dephosphorylated Spo0F∼P with the production of ATP at approximately the same rate, indicating that they possess approximately equal affinity for Spo0F. All the kinases were expressed during growth and early stationary phase, suggesting that the differential activity observed in growth and sporulation results from differential activation by signal ligands unique to each kinase.
The clpP multigenic family in Streptomyces lividans: conditional expression of the clpP3 clpP4 operon is controlled by PopR, a novel transcriptional activator
Authors: Viala Julie , Rapoport Georges , Mazodier Philippe
Published: March 2003

The clpP genes are widespread among living organisms and encode the proteolytic subunit of the Clp ATP‐dependent protease. These genes are present in a single copy in most eubacteria. However, five clpP genes were identified in Streptomyces coelicolor. The clpP1 clpP2 operon was studied: mutations affected the growth cycle in various Streptomyces. Here, we report studies of the expression of the clpP3 clpP4 operon in Streptomyces lividans. The clpP3 operon was induced in a clpP1 mutant strain, and the regulation of expression was investigated in detail. The product of the putative regulator gene, downstream from clpP4, was purified. Gel migration shift assays and DNase I footprinting showed that this protein binds to the clpP3 promoter and recognizes a tandem 6 bp palindromic repeat (TCTGCC‐3N‐GGCAGA). In vivo, this DNA‐binding protein, named PopR, acts as an activator of the clpP3 operon. Studies of popR expression indicate that the regulator is probably controlled at the post‐transcriptional level.
A Rox1‐independent hypoxic pathway in yeast. Antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene
Authors: Bourdineaud Jean‐Paul , De Sampaïo Guillaume , Lauquin Guy J.‐M.
Published: March 2003

Hypoxic SRP1/TIR1 gene expression depends on the absence of haem but is independent of Rox1‐mediated repression. We have found a new hypoxic pathway involving an antagonistic interaction between the Ixr1/Ord1 repressor and the Yap1 factor, a transcriptional activator involved in oxidative stress response. Here, we show that Ord1 repressed SRP1 gene expression under normoxia and hypoxia, whereas Yap1 activated it. Ord1 and Yap1 have been shown to bind the SRP1 promoter in a region extending from −299 to −156 bp upstream of the start codon. A typical AP‐1 responsive element lying from −247 to −240 bp allows Yap1 binding. Internal deletion of sequences within the SRP1 promoter were introduced. Two regions were characterized at positions −299/−251 and −218/−156 that, once removed, resulted in a constitutive expression of SRP1 in a wild‐type strain under normoxic conditions. Deletion of both these two sequences allowed the bypass of YAP1 requirement in a Δyap1 strain, whereas these two internal deletions did not yield increased expression in a Δord1 strain compared with the full‐length promoter. Both a single Δord1 mutant and a doubly disrupted Δyap1 Δord1 strain yielded normoxic constitutive SRP1 expression and increased hypoxic SRP1 induction, thereby demonstrating that ord1 is epistatic to yap1. Thus, Yap1 is not directly involved in SRP1 induction by hypoxia, but is necessary to counteract the Ord1 effect.
Modulation of KdpD phosphatase implicated in the physiological expression of the Kdp ATPase of Escherichia coli
Authors: Brandon Lauren , Dorus Steve , Epstein Wolfgang , Altendorf Karlheinz , Jung Kirsten
Published: March 2003

The KdpD sensor kinase and the KdpE response regulator control the expression of the kdpFABC operon, encoding the KdpFABC high‐affinity K+ transport system of Escherichia coli. Low turgor pressure has been postulated to be the environmental stimulus to express KdpFABC. KdpD has autokinase, phosphotransferase and, like many sensor kinases, response regulator (phospho‐KdpE) specific phosphatase activity. To determine which of these activities are altered in response to the environmental stimulus, we isolated and analysed six kdpD mutants that cause constitutive expression of KdpFABC. In three of the mutants, phosphatase activity was undetectable and, in two, phosphatase was reduced. Kinase activity was unaffected in four of the mutants, but elevated in one. In one mutant, a pseudorevertant of a kdpD null mutation, kinase and phosphatase were both reduced to 20% of the wild‐type level. These findings suggest that initiation of signal transduction by KdpD is mediated by the inhibition of the phospho‐KdpE‐specific phosphatase activity of KdpD, leading to an accumulation of phospho‐KdpE, which in turn activates the expression of the KdpFABC system. The data also suggest that levels of activity in vitro may differ from what occurs in vivo, because in vitro conditions cannot replicate those in vivo.
Fis, a DNA nucleoid‐associated protein, is involved in Salmonella typhimurium SPI‐1 invasion gene expression
Authors: Wilson Rebecca L. , Libby Stephen J. , Freet Anne M. , Boddicker Jennifer D. , Fahlen Thomas F. , Jones Bradley D.
Published: March 2003

The ability of Salmonella enterica serovar Typhimurium to cause disease depends upon the co‐ordinated expression of many genes located around the Salmonella chromosome. Specific pathogenicity loci, termed Salmonella pathogenicity islands, have been shown to be crucial for the invasion and survival of Salmonella within host cells. Salmonella pathogenicity island 1 (SPI‐1) harbours the genes required for the stimulation of Salmonella uptake across the intestinal epithelia of the infected host. Regulation of SPI‐1 genes is complex, as invasion gene expression responds to a number of different signals, presumably signals similar to those found within the environment of the intestinal tract. As a result of our continued studies of SPI‐1 gene regulation, we have discovered that the nucleoid‐binding protein Fis plays a pivotal role in the expression of HilA and InvF, two activators of SPI‐1 genes. A S. typhimurium fis mutant demonstrates a two‐ to threefold reduction in hilA::Tn5lacZY and a 10‐fold reduction in invF::Tn5lacZY expression, as well as a 50‐fold decreased ability to invade HEp‐2 tissue culture cells. This decreased expression of hilA and invF resulted in an altered secreted invasion protein profile in the fis mutant. Furthermore, the virulence of a S. typhimurium fis mutant is attenuated 100‐fold when administered orally, but has wild‐type virulence when administered intraperitoneally. Expression of hilA::Tn5lacZY and invF::Tn5lacZY in the fis mutant could be restored by introducing a plasmid containing the S. typhimurium fis gene or a plasmid containing hilD, a gene encoding an AraC‐like regulator of Salmonella invasion genes.
The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139
Authors: Watnick Paula I. , Lauriano Crystal M. , Klose Karl E. , Croal Laura , Kolter Roberto
Published: March 2003

Throughout most of history, epidemic and pandemic cholera was caused by Vibrio cholerae of the serogroup O1. In 1992, however, a V. cholerae strain of the serogroup O139 emerged as a new agent of epidemic cholera. Interestingly, V. cholerae O139 forms biofilms on abiotic surfaces more rapidly than V. cholerae O1 biotype El Tor, perhaps because regulation of exopolysaccharide synthesis in V. cholerae O139 differs from that in O1 El Tor. Here, we show that all flagellar mutants of V. cholerae O139 have a rugose colony morphology that is dependent on the vps genes. This suggests that the absence of the flagellar structure constitutes a signal to increase exopolysaccharide synthesis. Furthermore, although exopolysaccharide production is required for the development of a three‐dimensional biofilm, inappropriate exopolysaccharide production leads to inefficient colonization of the infant mouse intestinal epithelium by flagellar mutants. Thus, precise regulation of exopolysaccharide synthesis is an important factor in the survival of V. cholerae O139 in both aquatic environments and the mammalian intestine.
Induction of ppGpp synthesis in Streptomyces coelicolor A3(2) grown under conditions of nutritional sufficiency elicits actII‐ORF4 transcription and actinorhodin biosynthesis
Authors: Hesketh Andrew , Sun Jongho , Bibb Mervyn
Published: March 2003

Production of ppGpp in Streptomyces coelicolor A3(2) was achieved independently of amino acid limitation by placing N‐terminal segments of the ppGpp synthetase gene, relA, under the control of a thiostrepton‐inducible promoter (tipAp). S1 nuclease protection experiments indicated that induced ppGpp concentrations of 6–12 pmol mg−1 dry weight in late‐exponential phase cultures caused activation of transcription of actII‐ORF4, the pathway‐specific activator gene for actinorhodin production. This level of ppGpp had no effect on growth rate, implying a causal role for ppGpp in activating actII‐ORF4 transcription. No effect was observed on the transcription of the corresponding and homologous activator gene for undecylprodigiosin production, redD, reflecting a requirement for additional regulatory factors for activation of its transcription. This work provides the most compelling evidence yet for the activation of an antibiotic biosynthetic pathway by the stringent factor ppGpp.
The microsatellites of Escherichia coli: rapidly evolving repetitive DNAs in a non‐pathogenic prokaryote
Authors: Metzgar David , Thomas Elizabeth , Davis Clara , Field Dawn , Wills Christopher
Published: March 2003

We have demonstrated hypervariability of native short‐motif repeats (microsatellites) in Escherichia coli. Twenty‐five of the longest microsatellites in the E. coli genome were identified. These were analysed for length variability among 22 wild‐type (non‐mutator) isolates from the E. coli collection of reference (ECOR). Non‐coding mononucleotide repeats are consistently polymorphic among these genetically diverse E. coli. Length differences in variable microsatellites allowed all E. coli strains examined to be uniquely differentiated. Phylogenetic analysis of the variable repeats shows ubiquitous homoplasy at the level of divergence represented by the sample set, suggesting that these markers are hypermutable and should prove valuable for the discrimination of closely related strains that are not otherwise genetically differentiable. Genomic analyses suggest that similar markers are also likely to be found in all other prokaryotes.
Extensive alanine scanning reveals protein–protein and protein–DNA interaction surfaces in the global regulator FlhD from Escherichia coli
Authors: Campos Andrés , Matsumura Philip
Published: March 2003

FlhD and FlhC are the transcriptional activators of the flagellar regulon. The heterotetrameric complex formed by these two proteins activates the transcription of the class II flagellar genes. The flagellar regulon consists not only of flagellar genes, but also of the chemotactic genes and some receptor proteins. Recently, a connection between the flagellar regulon and some virulence genes has been found in some species. Furthermore, FlhD, but not FlhC, regulates another non‐flagellar target. As a first attempt to understand the mechanism of the flagellar transcriptional activation by FlhD and FlhC, the structure of FlhD has been solved. In order to understand the mechanism of the action of FlhD when it regulates the flagellar genes, we conducted site‐directed mutagenesis based on its three‐dimensional structure. Six interaction surfaces in the FlhD dimer were mapped by alanine scanning mutagenesis. Two of them are surface clusters formed by residues His‐2, Asp‐28, Arg‐35, Phe‐34 and Asn‐61 located at each side of the dimer core. The other four are located in the flexible arms of the dimer. The residues Ser‐82, Arg‐83, Val‐84, His‐91, Thr‐92, Ile‐94 and Leu‐96 are located at this region. All these residues are involved in the FlhD/FlhC interaction with the exception of Ser‐82, Arg‐83 and Val‐84. These three residues affect the DNA‐binding ability of the complex. The three‐dimensional topology of FlhD and the site‐directed mutagenesis results support the hypothesis of FlhC as an allosteric effector that activates FlhD for the recognition of the DNA.
CueR (YbbI) of Escherichia coli is a MerR family regulator controlling expression of the copper exporter CopA
Authors: Stoyanov Jivko V. , Hobman Jon L. , Brown Nigel L.
Published: March 2003

We have shown that the open reading frame ybbI in the genomic sequence of Escherichia coli K‐12 encodes the regulator of expression of the copper‐exporting ATPase, CopA. In vivo studies showed that ybbI (designated cueR for copper export regulator gene) was required for copper tolerance during growth, that disruption of cueR caused loss of copA expression and that copA gene expression was regulated by cueR and by copper or silver ions. Expression of a lacZ reporter gene under the control of the copA promoter was approximately proportional to the concentration of cupric ions in the medium, but increased more rapidly in response to silver ion concentrations. The start of the copA transcript was located by primer extension mapping, and DNase I protection assays showed that the CueR protein binds in vitro to a dyad symmetrical sequence within a 19 bp spacer sequence in the copA promoter. CueR binding occurs in vitro in both the presence and the absence of RNA polymerase with or without copper ions present but, in the presence of CueR, RNA polymerase and copper ions, permanganate‐sensitive transcription complexes were formed. CueR is predicted to have an N‐terminal helix–turn–helix sequence and shows similarity to MerR family regulators.