bluuebunny/crossref_metadata_2025_split huggingface dataset:
Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis
Authors: Larsen Michelle H. , Vilchèze Catherine , Kremer Laurent , Besra Gurdyal S. , Parsons Linda , Salfinger Max , Heifets Leonid , Hazbon Manzour H. , Alland David , Sacchettini James C. , Jacobs William R. 
Published: March 2003

SummaryThe inhA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed inhA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either inhA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy inhA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing inhA plasmids uniformly exhibited 20‐fold or greater increased resistance to INH and 10‐fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH‐susceptible M. tuberculosis strains. Using molecular beacons, quantified inhA and kasA mRNA levels showed that increased inhA mRNA levels corre­‐lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring inhA or kasA plasmids yielded the same conclusion: overexpressed inhA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, InhA is the primary target of action of INH and ETH in all three species.
Glutathione regulates the expression of γ‐glutamylcysteine synthetase via the Met4 transcription factor
Authors: Wheeler Glen L. , Quinn Kathryn A. , Perrone Gabriel , Dawes Ian W. , Grant Chris M. 
Published: March 2003

SummaryOur previous studies have shown that glutathione is an essential metabolite in the yeast Saccharomyces cerevisiae because a mutant deleted for GSH1, encoding the first enzyme in γ‐l‐glutamyl‐l‐cysteinylglycine (GSH) biosynthesis, cannot grow in its absence. In contrast, strains deleted for GSH2, encoding the second step in GSH synthesis, grow poorly as the dipeptide intermediate, γ‐glutamylcysteine, can partially substitute for GSH. In this present study, we identify two high copy suppressors that rescue the poor growth of the gsh2 mutant in the absence of GSH. The first contains GSH1, indicating that γ‐glutamylcysteine can functionally replace GSH if it is present in sufficiently high quantities. The second contains CDC34, encoding a ubiquitin conjugating enzyme, indicating a link between the ubiquitin and GSH stress protective systems. We show that CDC34 rescues the growth of the gsh2 mutant by inducing the Met4‐dependent expression of GSH1 and elevating the cellular levels of γ‐glutamylcysteine. Furthermore, this mechanism normally operates to regulate GSH biosynthesis in the cell, as GSH1 promoter activity is induced in a Met4‐dependent manner in a gsh1 mutant which is devoid of GSH, and the addition of exogenous GSH represses GSH1 expression. Analysis of a cis2 mutant, which cannot breakdown GSH, confirmed that GSH and not a metabolic product, serves as the regulatory molecule. However, this is not a general mechanism affecting all Met4‐regulated genes, as MET16 expression is unaffected in a gsh1 mutant, and GSH acts as a poor repressor of MET16 expression compared with methionine. In summary, GSH biosynthesis is regulated in parallel with sulphate assimilation by activity of the Met4 protein, but GSH1‐specific mechanisms exist that respond to GSH availability.
TheHaemophilus influenzaeHia autotransporter harbours two adhesive pockets that reside in the passenger domain and recognize the same host cell receptor
Authors: Laarmann Sven , Cutter David , Juehne Twyla , Barenkamp Stephen J. , St Geme Joseph W. 
Published: March 2003

SummaryHaemophilus influenzaeis a human‐specific pathogen and a major source of morbidity worldwide. Infection with this organism begins with colonization of the nasopharynx, a process that probably depends on adherence to respiratory epithelium. The Hia autotransporter protein is the major adhesin ex‐pressed by a subset of non‐typeableH. influenzaestrains and promotes high‐level adherence to a variety of human epithelial cell lines. In the current study, we discovered that the Hia passenger domain contains two distinct binding pockets, including one at the C‐terminal end and a second at the N‐terminal end. Competition assays revealed that the two binding pockets interact with the same host cell receptor structure, although with differing affinities. Additional experiments demonstrated that both binding domains are required for full‐level bacterial adherence. These observations are reminiscent of eukaryotic cell adhesion molecules and highlight the first example of a bacterial adhesin with two domains that participate in a bivalent interaction with identical host cell receptors. Such an interaction increases avidity, thus stabilizing bacterial adherence to the epithelial surface, despite physical forces such as coughing, sneezing and mucociliary clearance.
Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression
Authors: Aas Finn Erik , Wolfgang Matthew , Frye Stephan , Dunham Steven , Løvold Cecilia , Koomey Michael 
Published: March 2003

SummaryThe mechanisms by which DNA is taken up into the bacterial cell during natural genetic transformation are poorly understood. Although related components essential to the uptake of DNA during transforma‐tion have been defined in Gram‐negative species, it remains unclear whether DNA binding and uptake are dissociable events. Therefore, DNA uptake has been the earliest definable step in any Gram‐negative transformation pathway. In the human pathogen Neisseria gonorrhoeae, sequence‐specific DNA uptake requires an intact type IV pili (Tfp) biogenesis machinery along with three molecules that are dispensable for Tfp expression: ComP (a pilin subunit‐like molecule), PilT (a cytoplasmic protein involved in pilus retraction) and ComE (a periplasmic protein with intrinsic DNA‐binding activity). By conditionally altering the levels of ComP and PilT expression, we show here that DNA binding and uptake are resolvable events. Consequently, we are able to demonstrate that PilT is largely dispensable for functional DNA binding and, therefore, contributes specifically to uptake. Furthermore, sequence specificity in this system is imposed at the level of DNA binding, a process that is influenced by both ComP and PilE. However, sequence‐specific DNA binding is not attributable to an intrinsic property of the Tfp subunit protein. Finally, we demonstrate the existence of a robust, non‐specific DNA‐binding activity associated with the expression of both Tfp and PilT, which is unrelated to transformation but obscures the observation of specific binding events.
Overexpression of ccl1−2 can bypass the need for the putative apocytochrome chaperone CycH during the biogenesis of c‐type cytochromes
Authors: Deshmukh Meenal , May Matthew , Zhang Yan , Gabbert Karen K. , Karberg Katherine A. , Kranz Robert G. , Daldal Fevzi 
Published: March 2003

SummaryIn Gram‐negative bacteria, including Rhodobacter capsulatus, the membrane protein CycH acts as a putative apocytochrome chaperone during the biogenesis of c‐type cytochromes. CycH‐null mutants are unable to produce various c‐type cytochromes and sustain photosynthetic (Ps) growth that requires the cytochromes c1 and c2 or cy. However, Ps+ revertants are readily obtained only on minimal, but not on enriched, medium. To obtain further information about the biogenesis of c‐type cytochromes, these suppressor mutants were studied. Complementation of a CycH‐null mutant for Ps+ growth by a genomic library constructed using DNA from a Ps+ suppressor yielded a plasmid carrying the ccl1–2 operon, the products of which, Ccl1 and Ccl2, are also involved in the biogenesis of c‐type cytochromes. DNA sequence analysis revealed that the complementing activity resulted from a single point mutation, G488A, located upstream of the coding region of ccl1–2. This mutation changed the −35 region of the ccl1–2 promoter from TTGGCC to TTGACC, improving its similarity to the consensus sequence of Escherichia coliσ70‐dependent promoters. That the G488A mutation indeed enhanced transcription of ccl1–2 was demonstrated by the use of reporter gene fusions. An appropriate ccl1‐2::lacZ transcriptional–translational fusion carrying the G488A mutation produced in R. capsulatus over 30‐fold higher β‐galactosidase activity than a wild‐type construct. Immunoblot analyses confirmed that Ccl1 and Ccl2 were overproduced in the Ps+ suppressors. Deletion of either ccl1 or ccl2, from the ccl1–2 cluster carrying the G488A mutation abolished the complementing ability, indicating that overexpression of both ccl1 and ccl2 was required to confer the Ps+ phenotype on a CycH‐null mutant. These findings therefore demonstrate that, during R. capsulatus growth on minimal medium, the requirement for CycH in c‐type cytochrome biogenesis could be bypassed by overexpressing the ccl1–2 operon.
The Zygosaccharomyces bailii antifungal virus toxin zygocin: cloning and expression in a heterologous fungal host
Authors: Weiler Frank , Rehfeldt Klaus , Bautz Frank , Schmitt Manfred J. 
Published: March 2003

SummaryZygocin, a monomeric protein toxin secreted by a virus‐infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double‐stranded (ds)RNA killer virus (ZbV‐M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N‐terminal amino acid sequence was determined, and a full‐length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238‐amino‐acid preprotoxin precursor (pptox) consisting of a hydrophobic N‐terminal secretion signal, followed by a potentially N‐glycosylated pro‐region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N‐terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix‐assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post‐translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin‐sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.
Expression of the glutamyl‐tRNA synthetase gene from the cyanobacterium Synechococcus sp. PCC 7942 depends on nitrogen availability and the global regulator NtcA
Authors: Luque Ignacio , Contreras Asunción , Zabulon Gérald , Herrero Antonia , Houmard Jean 
Published: March 2003

SummaryWe report here transcriptional analyses of a cyanobacterial gene encoding an aminoacyl‐tRNA synthetase (aaRS), the gltX gene from Synechoccocus sp. PCC 7942, coding for the glutamyl‐tRNA synthetase. We show that the transcript levels of gltX in Synechococcus depend on nitrogen availability and do not increase with the growth rate, which is at odds with observations from other bacteria. We also demonstrate the involvement of the cyanobacterial global regulator NtcA in transcriptional control of gltX according to nitrogen status. Our results support a regulatory model in which the gltX transcript level is finely tuned by a dynamic equilibrium between activation and repression relying upon the cellular concentration of NtcA.
Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)‐anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae
Authors: Kitagaki Hiroshi , Wu Hong , Shimoi Hitoshi , Ito Kiyoshi 
Published: March 2003

SummaryThe cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)‐anchored proteins and are then transferred to cell wall β‐1,6‐glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI‐anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cΔ was hypersensitive to a cell wall‐digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope‐tagged Dcw1p is an N‐glycosylated, GPI‐anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI‐anchored membrane proteins and are required for normal biosynthesis of the cell wall.
TlpC, a novel chemotaxis protein in Rhodobacter sphaeroides, localizes to a discrete region in the cytoplasm
Authors: Wadhams G. H. , Martin A. C. , Porter S. L. , Maddock J. R. , Mantotta J. C. , King H. M. , Armitage J. P. 
Published: March 2003

SummaryTlpC is encoded in the second chemotaxis operon of Rhodobacter sphaeroides. This protein shows some homology to membrane‐spanning chemoreceptors of many bacterial species but, unlike these, is essential for R. sphaeroides chemotaxis to all compounds tested. Genomic replacement of tlpC with a C‐terminal gfp fusion demonstrated that TlpC localized to a discrete cluster within the cytoplasm. Immunogold electron microscopy also showed that TlpC localized to a cytoplasmic electron‐dense region. Correct TlpC–GFP localization depended on the downstream signalling proteins, CheW3, CheW4 and CheA2, and was tightly linked to cell division. Newly divided cells contained a single cluster but, as the cell cycle progressed, a second cluster appeared close to the initial cluster. As elongation continued, these clusters moved apart so that, on septation, each daughter cell contained a single TlpC cluster. The data presented suggest that TlpC is either a cytoplasmic chemoreceptor responding to or integrating global signals of metabolic state or a novel and essential component of the chemotaxis signalling pathway. These data also suggest that clustering is essential for signalling and that a mechanism may exist for targeting and localizing proteins within the bacterial cytoplasm.
Mss11p is a transcription factor regulating pseudohyphal differentiation, invasive growth and starch metabolism in Saccharomyces cerevisiae in response to nutrient availability
Authors: Gagiano Marco , Bester Michael , Van Dyk Dewald , Franken Jaco , Bauer Florian F. , Pretorius Isak S. 
Published: March 2003

SummaryIn Saccharomyces cerevisiae, the cell surface protein, Muc1p, was shown to be critical for invasive growth and pseudohyphal differentiation. The transcription of MUC1 and of the co‐regulated STA2 glucoamylase gene is controlled by the interplay of a multitude of regulators, including Ste12p, Tec1p, Flo8p, Msn1p and Mss11p. Genetic analysis suggests that Mss11p plays an essential role in this regulatory process and that it functions at the convergence of at least two signalling cascades, the filamentous growth MAPK cascade and the cAMP‐PKA pathway. Despite this central role in the control of filamentous growth and starch metabolism, the exact molecular function of Mss11p is unknown. We subjected Mss11p to a detailed molecular analysis and report here on its role in transcriptional regulation, as well as on the identification of specific domains required to confer transcriptional activation in response to nutritional signals. We show that Mss11p contains two independent transactivation domains, one of which is a highly conserved sequence that is found in several proteins with unidentified function in mammalian and invertebrate organisms. We also identify conserved amino acids that are required for the activation function.
Structure and function of the Mur enzymes: development of novel inhibitors
Authors: El Zoeiby Ahmed , Sanschagrin François , Levesque Roger C. 
Published: March 2003

SummaryOne of the biggest challenges for recent medical research is the continuous development of new antibiotics interacting with bacterial essential mechanisms. The machinery for peptidoglycan biosynthesis is a rich source of crucial targets for antibacterial chemotherapy. The cytoplasmic steps of the biosynthesis of peptidoglycan precursor, catalysed by a series of Mur enzymes, are excellent candidates for drug development. There has been growing interest in these bacterial enzymes over the last decade. Many studies attempted to understand the detailed mechanisms and structural features of the key enzymes MurA to MurF. Only MurA is inhibited by a known antibiotic, fosfomycin. Several attempts made to develop novel inhibitors of this pathway are discussed in this review. Three novel inhibitors of MurA were identified recently. 4‐Thiazolidinone compounds were designed as MurB inhibitors. Many phosphinic acid derivatives and substrate analogues were identified as inhibitors of the MurC to MurF amino acid ligases.
Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium‐metabolizing enzymes (GS, NADP‐GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum
Authors: Javelle Arnaud , Morel Mélanie , Rodríguez‐Pastrana Blanca‐Rosa , Botton Bernard , André Bruno , Marini Anne‐Marie , Brun Annick , Chalot Michel 
Published: March 2003

SummaryExternal hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP‐dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum. We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA. In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum. Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N‐starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH4+. AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.
A novel regulatory gene for light‐induced carotenoid synthesis in the bacteriumMyxococcus xanthus
Authors: Fontes Marta , Galbis‐Martínez Lilian , Murillo Francisco J. 
Published: March 2003

SummaryMyxococcus xanthuscells respond to blue light by producing carotenoids. Light triggers a network of regulatory actions that lead to the transcriptional activation of the carotenoid genes. By screening the colour phenotype of a collection of Tn5‐lacinsertion mutants, we have isolated a new mutant devoid of carotenoid synthesis. We map the transposon insertion, which co‐segregates with the mutant phenotype, to a previously unknown gene designated here ascarF. An in frame deletion withincarFcauses the same phenotype as the Tn5‐lacinsertion. ThecarFdeletion prevents the activation of the normally light‐inducible genes, without affecting the expression of any of the regulatory genes known to be expressed in a light‐independent manner. Until now, the switch that sets off the regulatory cascade had been identified with light‐driven inactivation of protein CarR, an antisigma factor. The exact mechanism of this inactivation has remained elusive. We show by epistatic analysis that thecarFgene product participates in the light‐dependent inactivation of CarR. The predicted CarF amino acid sequence reveals no known prokaryotic homologues. On the other hand, CarF is remarkably similar to Kua, a family of proteins of unknown function that is widely distributed among eukaryotes.
Mechanisms of iron regulation in mycobacteria: role in physiology and virulence
Authors: Rodriguez G. Marcela , Smith Issar 
Published: March 2003

SummaryThe role of iron in mycobacteria as in other bacteria goes beyond the need for this essential cofactor. Limitation of this metal triggers an extensive response aimed at increasing iron acquisition while coping with iron deficiency. In contrast, iron‐rich environments prompt these prokaryotes to induce synthesis of iron storage molecules and to increase mechanisms of protection against iron‐mediated oxidative damage. The response to changes in iron availability is strictly regulated in order to maintain sufficient but not excessive and potentially toxic levels of iron in the cell. This response is also linked to other important processes such as protection against oxidative stress and virulence. In bacteria, iron metabolism is regulated by controlling transcription of genes involved in iron uptake, transport and storage. In mycobacteria, this role is fulfilled by the iron‐dependent regulator IdeR. IdeR is an essential protein in Mycobacterium tuberculosis, the causative agent of human tuberculosis. It functions as a repressor of iron acquisition genes, but is also an activator of iron storage genes and a positive regulator of oxidative stress responses.