bluuebunny/crossref_metadata_2025_split huggingface dataset:
Response of a yeast glycogen synthase gene to stress
Authors: Ni Hsiao‐Tzu , LaPorte David C. 
Published: October 2006

SummaryIn the yeast Saccharomyces cerevisiae, glycogen synthase is encoded by two genes: GSY1 and GSY2. The activity of the enzymes increases as cultures enter the stationary phase of growth. Using a GSy2::lacZ fusion gene, we have demonstrated that the increase in glycogen synthase activity resulted, at least in part, from an increase in the level of the protein rather than simply from a change in its phosphorylation state. Northern blot analysis showed a parallel increase in the level of the GSY2 mRNA, which is consistent with transcriptional activation of GSY2. Deletion analysis identified three regions upstream of GSY2 which are involved in GSy2 expression: regions A (‐390 to ‐347 relative to the start of translation), B (‐252 to ‐209) and C (‐209 to ‐167). Region A or C independently activated expression of GSY2. In contrast, region B alone yielded only modest expression. Expression of GSY2 is induced by growth to stationary phase, heat shock or nitrogen starvation. Response to these stressors is mediated by elements within regions A and C. These elements appear to be related to the stress‐response elements found in other stress‐responsive genes.
Role of the conserved C‐repeat region of the M protein of Streptococcus pyogenes
Authors: Perez‐Casal José , Okada Nobuhiko , Caparon Michael G. , Scott June R. 
Published: October 2006

SummaryThe surface‐located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C‐repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M‐protein‐mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C‐repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C‐repeat domain, a region of the M‐protein molecule distinct from the C‐repeat domain confers upon S. pyogenes its ability to resist phagocytosis.
Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora
Authors: Bugert Peter , Geider Klaus 
Published: October 2006

SummaryA16 kb transcript of the ams region, which is essential for biosynthesis of amylovoran, the acidic exopolysaccharide of Erwinia amylovora, was detected by Northern hybridization analysis. The positive regulator RcsA enhanced transcription of the targe mRNA from the ams operon. The nucleotide sequence of this area revealed 12 open reading frames (ORFs), which are all transcribed in the same direction. Five ORFs corresponded to the previously mapped genes amsA to amsE, Sequence analysis of the insertion sites of several Tn5 mutations confirmed these data. Tn5 or site‐directed mutagenesis of the ORFs 477, 377, 144, and 743 reveated an amytovoran‐deficient phenotype, and the newly identified genes were named amsG, amsH, amsI, and amsF, respectivety. The predicted amino acid sequence of AmsG is highly homologous to gatactosyl‐1 ‐phosphate undecaprenyl‐phosphate transferases. AmsB and AmsD are similar to other glycosyl transferases, and AmsH may be related to BexD. A significant homology to mammalian phosphatases was observed for Amsl. AmsA shows characteristic motifs for membrane association and ATP binding. AmsF carries a secretory signal sequence in the N‐terminus and could be involved in periplasmic processing of the repeating units. Complementation experiments located a promoter region required for gene expression as far as 500 bp upstream of amsG. It is preceded by a typical transcriptional termination sequence. A mutation upstream of the terminator did not affect amylovoran synthesis. Partial nucleotide sequences further upstream of the ams region showed homology to genes mapped at 45min on the Escherichia coli chromosome. A termination sequence was also found downstream of the ams operon at a distance of 16 kb from the promoter. Between amsF and this terminator, three additional ORFs were detected.
Sequence‐specific DNA binding by NIT4, the pathway‐specific regulatory protein that mediates nitrate induction in Neurospora
Authors: Fu Y. H. , Feng B. , Evans S. , Marzluf G. A. 
Published: October 2006

SummaryThe expression of the structural genes nit‐3 and nit‐6, which encode the nitrate assimilatory enzymes nitrate reductase and nitrite reductase, respectively, is highly regulated by the global‐acting NIT2 regulatory protein. These structural genes are also controlled by nitrogen catabolite repression and by specific induction via nitrate. A pathway‐specific regulatory protein, NIT4, appears to mediate nitrate induction of nit‐3 and of nit‐6. The NIT4 protein, composed of 1090 amino acids, contains a putative GAL4‐like Cys‐6 zinc cluster DNA‐binding motif, which is joined by a short segment to a stretch of amino acids that appear to constitute a coiled‐coil dimerization domain. Chemical crosslinking studies demonstrated that a truncated form of NIT4 forms homodimers. Mobility‐shift and DNA‐footprinting experiments have identified two NIT4‐binding sites of significantly different strengths in the promoter region of the nit‐3 gene. The stronger binding site contains a symmetrical octameric sequence, TCCGCGGA, whereas the weaker site has a related sequence. Sequences related to this palindromic element can be found upstream of the nit‐6 gene.
Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells
Authors: Rockey Daniel D. , Heinzen Robert A. , Hackstadt T. 
Published: October 2006

SummaryChlamydiae are obligate intracellular bacteria which occupy a non‐acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin‐killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C. psittaci elementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inclusion membrane protein A) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post‐translational modification. This pattern was not observed in immunoblots of RBs or in the E. coli expressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells.
Structural identification of the iipo‐chitin oligosaccharide nodulation signals of Rhizobium loti
Authors: López‐Lara Isabel M. , van den Berg Jorrit D. J. , Thomas‐Oates Jane E. , Glushka John , Lugtenberg Ben J. J. , Spaink Herman P. 
Published: October 2006

Summary Rhizobium loti is a fast‐growing Rhizobium species that has been described as a microsymbiont of plants of the genus Lotus. Nodulation studies show that Lotus plants are nodulated by R loti, but not by most other Rhizobium strains, indicating that R. loti produces specific lipo‐chitin oligosaccharides (LCOs) which are necessary for the nodulation of Lotus plants. The LCOs produced by five different Rhizobium ioti strains have been purified and were shown to be N‐acetylglucosamine pentasaccharides of which the non‐reducing residue is N‐methylated and N‐acylated with c/s‐vaccenic acid (C18:1) or stearic acid (C18:O) and carries a carbamoyl group. In one R. loti strain, NZP2037, an additional carbamoyl group is present on the non‐reducing terminal residue. The major class of LCO molecules is substituted on the reducing terminal residue with 4‐O‐acetylfucose. Addition of LCOs to the roots of Lotus plants results in abundant distortion, swelling and branching of the root hairs, whereas spot inoculation leads to the formation of nodule primordia.
The bacteriophage T4 middle promoter PuvsX: analysis of regions important for binding of the T4 transcriptional activator MotA and for activation of transcription
Authors: March‐Amegadzie Roslyn , Hinton Deborah M. 
Published: October 2006

SummaryBacteriophage T4 middle promoters, which are transcribed using phage‐modified host RNA polymerase and the T4 transcriptional activator, MotA, match the host σ70 consensus sequence at – 10, but they have a different consensus ((t/a)(t/a)TGCTT(t/c)A) (a MotA box) at – 30. While the T4 middle promoter Puvsx has these – 10 and –30 motifs, it also has matches to the MotA box at –35, –51, –70, and –87. We show that MotA binds to Puvsx DNA, footprinting a region that includes the MotA boxes at –30, –35, and –51. Very high levels of MotA are required for footprinting and gel‐shift experiments, and protein‐DNA complexes formed in the presence of both phage‐modified polymerase and MotA are more resistant to Hindlll cleavage than those formed with either protein alone. These results suggest that MotA‐DNA interactions may be stabilized by phage‐modified polymerase. Sequences between –18 and –38 are absolutely required for MotA activation of transcription, but sequences upstream of –38 are stimulatory, particularly when chloride instead of glutamate is the major anion. Our results dissect Puvsx into a core promoter, downstream of ‐38, which is required for MotA activation, and an upstream region that enhances transcription especially under conditions less favourable for protein‐DNA interactions.
Analysis of Vibrio cholierae ToxR function by construction of novel fusion proteins
Authors: Ottemann Karen M. , Mekalanos John J. 
Published: October 2006

SummaryThe ToxR protein is a transmembrane protein that regulates the expression of several virulence factors of Vibrio cholerae. Previous analysis of fusion proteins between ToxR and alkaline phosphatase (ToxR‐PhoA) suggested that ToxR was active as a dimer. In order to determine whether dimerization of the ToxR periplasmic domain was essential for activity, this domain was replaced by monomeric and dimeric protein domains. Surprisingly, PhoA (dimeric), β‐lactamase (monomeric, ToxR–Bla), or the leucine zipper of GCN4 (dimeric, ToxR‐GCN4‐M) could substitute functionally for the ToxR periplasmic domain. ToxR‐GCN4 fusion proteins, in which the ToxR trans‐membrane domain was eliminated (ToxR‐GCN4‐C), were inactive, but an additional fusion protein that contained a heterologous membrane‐spanning domain retained activity. Strains containing each of these ToxR fusion proteins were analysed for in vivo colonization properties and response to in vitro growth conditions that are known to affect expression of the ToxR regulon. Strains containing ToxR‐GCN4‐M and ToxR‐Bla responded like wild‐type strains to in vitro growth conditions. In the infant‐mouse colonization model, strains containing ToxR fusion proteins were all deficient in colonization relative to strains containing wild‐type ToxR, and strains containing monomeric ToxR‐Bla were most severely outcompeted. These results suggest that, under in vitro conditions, ToxR does not require a dimerized periplasmic domain, but that, under in vivo conditions, the correct conformation of the ToxR periplasmic domain may be more important for function.
Requirement for FlhA in flagella assembly and swarm‐cell differentiation by Proteus mirabilis
Authors: Gygi Daniel , Bailey Marc J. , Allison Clive , Hughes Colin 
Published: October 2006

SummarySwarming by Proteus mirabilis is characterized by cycles of rapid population migration across surfaces, following differentiation of typical rods into long, aseptate swarm cells that overexpress flagella and virulence factors, particularly haemolysin. A non‐swarming Tn5phoA mutant was unable to synthesize flagella, to fully elongate or to induce high levels of the toxin. The mutation lay within a 2091 bp gene encoding a homologue of the Escherichia coli FlhA belonging to a family of proteins that are required for assembly of flagella or virulence proteins and that are suggested to act either directly in membrane trans‐location and/or in regulating synthesis of the export apparatus. In trans expression of multicopy flhA restored cell elongation and migration and generated differentiation‐specific hyperexpression of flagellin and toxin genes to levels above those seen in the wild‐type strain. Transcription of flhA was strongly induced during differentiation, from its own putative σ28 promoter. The results suggest a mechanistic coupling of flagella assembly and swarm‐cell differentiation.
Complementation studies with the gas vesicle‐encoding p‐vac region of Halobacterium salinarium PHH1 reveal a regulatory role for the p‐gvpDE genes
Authors: Offner Sonja , Pfeifer Felicitas 
Published: October 2006

SummaryGas‐vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the p‐vac region consisting of 14 different gvp genes that are arranged in two clusters: p‐gvpACNO and, oppositely oriented, p‐gvpDEFGHIJKLM. The latter cluster of genes is transcribed as two units: p‐gvpDE and p‐gvpF–M. The 5′‐terminus of the p‐gvpF–M mRMA was located 169 nucleotides upstream of p‐gvpF within p‐gvpE. The p‐gvpG and p‐gvpK gene was expressed in Escherichia coli and antibodies to proteins obtained were raised in rabbits. Both proteins could be detected in halobacterial cell lysates; in gas‐vesicle preparations, however, neither GvpG nor GvpK could be found. The requirement for single p‐gvp gene expression for gas‐vesicle synthesis was determined by transformation experiments using the Vac− species Haloferax volcanii as recipient. Construct ΔA containing all p‐gvp genes except for p‐gvpA, encoding the major gas‐vesicle structural protein, produced Vac− transformants, but the addition of p‐gvpA on a second vector restored gas‐vesicle synthesis to wild‐type level (Vac++). Similarly, double transformants containing p‐gvpD–M plus p‐gvpACNO, or p‐gvpG–M (fused to the promoter of the halobacterial ferredoxin gene for expression) plus p‐gvpFED–ACNO were Vac++. Transformants containing the p‐vac region either lacking gvpA, gvpF, or gvpGHI were Vac−, indicating the absolute requirement of these gvp genes (or at least one in the case of gvpGHI) for gas‐vesicle formation. Double transformants containing the constructs p‐gvpF–M plus p‐gvpACNO (ΔDE) accumulated gas vesicles (Vac+) but synthesized fewer than the wild type, showing that the p‐gvpDE genes are not necessary for gas‐vesicle assembly. A repressor function affecting the synthesis of the p‐gvpF–M mRNA could be suggested for p‐gvpD and the 5′‐ region of its mRNA.
dewA encodes a fungal hydrophobin component of the Aspergillus spore wall
Authors: Stringer Mary A. , Timberlake William E. 
Published: October 2006

SummaryAn anonymous cDNA clone, pCAN4, was shown previously to correspond to an mRNA that accumulates preferentially during asexual sporulation of the filamentous fungus Aspergillus nidulans. The peptide encoded by pCAN4 is a fungal hydrophobin, a group of small, hydrophobic cell wall proteins. When the CAN4 gene was disrupted, conidia and conidiophores appeared to be normal, but sporulating colonies wetted more rapidly with detergent solutions than did the wild type. We renamed CAN4 dewA for the detergent wettable phenotype and mapped it to chromosome V, 24 map units from cysC. The A. nidulans rodA gene also encodes a sporulation‐specific fungal hydrophobin. Spores of a dewA rodA double mutant were less hydrophobic than those of either mutant alone, showing that dewA and rodA contribute independently to spore‐wall hydrophobicity, Immuno‐localization of DewA by epitope tagging demonstrated that DewA is present in the spore wall, but not in the walls of germ tubes, hyphae or cells of the spore‐producing conidiophore. We conclude that dewA encodes a new fungal hydrophobin component of the conidial wall.
Novel colicin 10: assignment of four domains to TonB‐and TolC dependent uptake via the Tsx receptor and to pore formation
Authors: Pilsl Holger , Braun Volkmar 
Published: October 2006

SummaryUptake of a new colicin, colicin 10(Col10), into cells of Escherichia coli required TonB, ExbBD (Ton system), but its cognate receptor, Tsx, functioned independently of Ton and TolQRAB (Tol system). Uptake of Col10 also required TolC which is unique for a Ton‐coupled translocation through the outer membrane. A 2470bp DNA fragment from the natural plasmid pCol10 encoding the Col10 activity (cta), immunity (cti) and lysis (ctl) genes was sequenced. The Cta, Cti and Ctl proteins, as deduced from the nucleotide sequences, consisted of 490 (Mr 53 342), 96 (Mr 11586) and 43 (Mr 4484) amino acid residues, respectively. Col10 (Cta) was highly homologous to colicin E1 in two regions which determined the common TolC requirement for uptake and the pore‐forming activity. Col10 and E1 differed entirely in the regions which are predicted to determine the Ton dependence of Col10 and the Ton dependence of E1, and binding to the receptors Tsx and BtuB, respectively. The region responsible for the Ton‐dependent uptake of Col10 was localized in the sequence ranging from residues 1 to 43 (Ton region), and the region responsible for the Tol‐dependent uptake of colicin E1 extended from residues 1 to 34 (Tol region). Each Tol‐dependent colicin contained a pentapeptide homologous to the sequence DGSGS in the Tol region of E1 which is proposed to be implicated in Tol‐dependent uptake (TolA box). After the exchange of the Ton and the Tol regions between Col10 and E1, the Col10‐E1 fusion protein was carried into cells via the Ton system and BtuB, whereas the E1–Col10 fusion protein was imported via the Tol system and Tsx. Although the immunity proteins of Col10 and E1 displayed a low homology, Cti conferred full immunity to E1, in contrast to the immunity protein of E1 which did not protect cells against Col10. It is proposed that Col10 belongs to the colicin E1, Ia, Ib group as opposed to the colicin A, B, N group of pore‐forming colicins. Col10 consists of 4 domains of which two are very similar and two are very different to E1, supporting our previous proposal that colicins evolved by recombination of DNA fragments which encode uptake and activity domains.
Helicobacter pylori nickel‐transport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions
Authors: Mobley Harry L. T. , Garner Rachel M. , Bauerfeind Peter 
Published: October 2006

SummaryUrease is a virulence determinant, a taxonomic and diagnostic marker, and immunogen for Helicobacter pylori, an aetiologic agent of gastritis and peptic ulceration. This enzyme requires Ni2+ ions in the active site for successful hydrolysis of urea. When expressed in Escherichia coli, recombinant urease is only weakly active unless urease structural subunits are overexpressed, exogenous NiCl2 is added, and the host strain is grown in medium that does not chelate free Ni2+. As wild‐type H. pylori does not require such conditions for very high levels of urease expression, we reasoned that additional genes were required to accumulate the metal ion. To isolate such genes, E. coll SE5000 (pHP808), which carries the H. pylori urease gene cluster, was complemented with a λ ZAP‐derived plasmid library of the H. pylori chromosome. One of 1000 ampicillin‐resistant clones, plated onto urea segregation agar, produced detectable urease. Urease activity of this co‐transformant, grown in Luria broth containing 1 μM NiCl2, was 36μmol NH3min−1 mg−1 protein. Urease‐enhancing activity, which is not directly linked to the urease gene cluster, was localized by subcloning and nucleotide sequencing. The largest open reading frame, designated nixA, predicted a polypeptide of 34317 Da that displayed characteristics of an integral membrane protein. In vitro transcription‐translation of nixA sequences yielded a polypeptide estimated to be 32 kDa in size. An in‐frame Bal31 deletion within nixA abolished urease‐enhancing activity. At 50 nM NiCl2, E. coli containing the nixA clone transported 1250±460 pmol Ni2+min−110−8 cells, whereas the vector control transported only 140±85pmol Ni+2 min−1 108 cells, i.e. significantly less (P=0.01). We conclude that NixA confers upon E. coli a high‐affinity nickel‐transport system (KT=11.3±2.4nM; Vmax=1750±220 pmol Ni2+ min−110−8 cells) and is necessary for expression of catalytically active urease, regardless of growth conditions.
UreR activates transcription at multiple promoters within the plasmid‐encoded urease locus of the Enterobacteriaceae
Authors: D'Orazio Sarah E. F. , Collins Carleen M. 
Published: October 2006

SummaryUrease activity is produced by members of the family Enterobacteriaceae that contain the plasmid‐encoded urease locus only when urea is present in the growth medium. The plasmid‐encoded urease locus contains seven tandem urease structural and accessory genes (ureDABCEFG). Previously we showed that transcription of the first gene in this cluster. ureD, is initiated at a urea‐dependent promoter (ureD p). Expression from ureD p requires the product of ure R, which is transcribed divergently from the plasmid‐encoded ureDABCEFG. From DNA sequence analysis, UreR is predicted to be a 34kDa protein with identity to the AraC family of transcriptional activators. In this report we demonstrate that there are two additional urea and UreR‐dependent promoters within the plasmid‐encoded urease locus: ureR p and ureG p. A low‐level constitutive promoter was also identified upstream of ureE (ureE p). Three major mRNA transcripts were induced when urea was present in the growth medium: a transcript containing ureDABCEF, a transcript corresponding to ureG, and a transcript corresponding to ureR. These results indicate that expression of each of the plasmid‐encoded urease genes is transcriptionally regulated in response to urea and suggest that there is autogenous regulation of ureR. Therefore UreR is one of three AraC family members described thus far that are positively auto‐regulated.