bluuebunny/crossref_metadata_2025_split huggingface dataset:
The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum: their expression in other genera requires ECF σ factor RpoI
Authors: Carter R. A. , Worsley P. S. , Sawers G. , Challis G. L. , Dilworth M. J. , Carson K. C. , Lawrence J. A. , Wexler M. , Johnston A. W. B. , Yeoman K. H. 
Published: March 2003

SummaryA cluster of eight genes, vbsGSO, vbsADL, vbsC and vbsP,
are involved in the synthesis of vicibactin, a cyclic, trihydroxamate siderophore
made by the symbiotic bacterium Rhizobium leguminosarum. None of these vbs
genes was required for symbiotic N2 fixation on peas or Vicia.
Transcription of vbsC, vbsGSO and vbsADL (but not vbsP)
was enhanced by growth in low levels of Fe. Transcription of vbsGSO and vbsADL,
but not vbsP or vbsC, required the closely linked gene rpoI,
which encodes an ECF σ factor of RNA polymerase. Transfer of the cloned
vbs genes, plus rpoI, to Rhodobacter, Paracoccus and
Sinorhizobium conferred the ability to make vicibactin on these other genera.
We present a biochemical genetic model of vicibactin synthesis, which accommodates
the phenotypes of different vbs mutants and the homologies of the vbs
gene products. In this model, VbsS, which is similar to many non‐ribosomal peptide
synthetase multienzymes, has a central role. It is proposed that VbsS activates L‐N5‐hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain. Subsequent VbsA‐catalysed acylation of the hydroxyornithine, followed by VbsL‐mediated epimerization and acetylation catalysed by VbsC, yields the vicibactin subunit, which is then trimerized and cyclized by the thioesterase domain of VbsS to give the completed siderophore.
Regulation of cell separation in the dimorphic fungus Ustilago maydis
Authors: Weinzierl Gerhard , Leveleki Leonora , Hassel Annette , Kost Gerhard , Wanner Gerhard , Bölker Michael 
Published: March 2003

SummaryDuring its haploid phase the dimorphic fungus Ustilago maydis grows vegetatively by budding. We have identified two genes, don1 and don3, which control the separation of mother and daughter cells. Mutant cells form tree‐like clusters in liquid culture and grow as ring‐like (donut‐shaped) colonies on solid medium. In wild‐type U. maydis cells, two distinct septa are formed during cytokinesis and delimit a fragmentation zone. Cells defective for either don1 or don3 display only a single septum and fail to complete cell separation. don1 encodes a guanine nucleotide exchange factor (GEF) of the Dbl family specific for Rho/Rac GTPases. Don3 belongs to the germinal‐centre‐kinase (GC) subfamily of Ste20‐like protein kinases. We have isolated the U. maydis homologues of the small GTP binding proteins Rho2, Rho3, Rac1 and Cdc42. Out of these, only Cdc42 interacts specifically with Don1 and Don3 in the yeast two‐hybrid system. We propose that Don1 and Don3 regulate the initiation of the secondary septum, which is required for proper cell separation.
Novel mode of transcription regulation of divergently overlapping promoters by PhoP, the regulator of two‐component system sensing external magnesium availability
Authors: Yamamoto Kaneyoshi , Ogasawara Hiroshi , Fujita Nobuyuki , Utsumi Ryutaro , Ishihama Akira 
Published: March 2003

SummaryPhoP is a response regulator of the PhoQ‐PhoP two‐component system controlling a set of the Mg(II)‐response genes in Escherichia coli. Here we demonstrate the mode of transcription regulation by phosphorylated PhoP of divergently transcribed mgtA and treR genes, each encoding a putative Mg(II) transporter and a repressor for the trehalose utilization operon respectively. Under Mg(II)‐limiting conditions in vivo, two promoters, the upstream constitutive P2 and the downstream inducible P1, were detected for the mgtA gene. Gel‐shift analysis in vitro using purified PhoP indicates its binding to a single DNA target, centred between –43 and –24 of the mgtAP1 promoter. This region includes the PhoP box, which consists of a direct repeat of the heptanucleotide sequence (T)G(T)TT(AA). Site‐directed mutagenesis studies indicate the critical roles for T (position 3), T (position 4) and A (position 6) for PhoP‐dependent transcription from mgtAP1. DNase I footprinting assays reveal weak binding of PhoP to this PhoP box, but the binding becomes stronger in the simultaneous presence of RNA polymerase. Likewise the RNA polymerase binding to the P1 promoter becomes stronger in the presence of PhoP. For the PhoP‐assisted formation of open complex at the mgtAP1 promoter, however, the carboxy‐terminal domain of α subunit (αCTD) is not needed. For transcription in vivo of the treR gene, four promoters were identified. The most upstream promoter treRP4 divergently overlaps with the mgtAP1 promoter, sharing the same sequence as the respective –10 signal in the opposite direction. In vitro transcription using mutant promoters support this prediction. In the presence of PhoP, transcription from the promoter treRP3 was repressed with concomitant activation of mgtAP1 transcription. The PhoP box is located between −46 and –30 with respect to treRP3, and the αCTD is needed for this repression.
DNA replication initiation is required for mid‐cell positioning of FtsZ rings in Caulobacter crescentus
Authors: Quardokus Ellen M. , Brun Yves V. 
Published: March 2003

SummaryPolymerization of the GTPase FtsZ to form a structure called the Z‐ring is the earliest known step in bac‐terial cell division. Mid‐cell Z‐ring assembly coincides with the beginning of the replication cycle in the differentiating bacterium Caulobacter crescentus. Z‐ring disassembly occurs at the end of the division cycle, resulting in the complete degradation of FtsZ from both stalked and swarmer progeny cells. New Z‐rings can only form in the replicative stalked cell. Conditional mutants in DNA replication were used to determine what role DNA replication events play in the process of Z‐ring assembly at different stages in the cell cycle. Z‐ring assembly occurred even when early stages of DNA replication were blocked; however, the Z‐rings were localized at a subpolar region of the cell. Z‐rings only assembled at the proper mid‐cell location if DNA replication had initiated. Z‐ring assembly coincided with areas containing little or no DNA, and Z‐rings could not form over an unreplicated chromosome. Overexpressed FtsZ in the absence of DNA replication did not stimulate productive mid‐cell Z‐ring assembly but, instead, caused the ends of cells to constrict over an extended area away from the nucleoid. These results indicate that the state of chromosome replication is a major determinant of Z‐ring localization in Caulobacter.
Characterization of a novel phenazine antibiotic gene cluster in Erwinia herbicola Eh1087
Authors: Giddens Stephen R. , Feng Yunjiang , Mahanty H. Khris 
Published: March 2003

Summary Erwinia herbicola
strain Eh1087 produces the broad‐spectrum phenazine antibiotic
D
‐alanylgriseoluteic acid (AGA). In this report, a cluster of 16
ehp
(

E
rwinia
h
erbicola

p
henazine) plasmid genes required for the production of AGA by Eh1087 is described. The extent of the gene cluster was revealed by the isolation of 82 different Eh1087 AGA

−

mutants, all found to possess single mini‐Tn
5lacZ2
insertions within a 14 kbp DNA region. Additional transposon insertions that did not affect antibiotic production by Eh1087 were created to define the boundaries of the gene cluster. The size and location of genes between these boundaries were derived from a combination of DNA sequence analyses, minicell protein analyses and the correlation between mutation position and the production of coloured AGA intermediates by many
ehp
mutants. Precursor‐feeding and complementation experiments resulted in 15
ehp
genes being assigned to one of four functional groups according to their role in the synthesis of AGA. Group 1 is required for the synthesis of the phenazine nucleus in the form of antibiotic precursor one (AP1, phenazine‐1,6‐dicarboxylic acid). Group 2 is responsible for conversion of AP1 to AP2, which is subsequently modified to AP3 (griseoluteic acid) and exported by the group 3 gene products. Group 4 catalyses the addition of
D
‐alanine to AP3 to create AGA, independently of groups 1, 2 and 3. A gene that is divergently transcribed from the 15 AGA synthesis
ehp genes confers resistance to AGA.
Amplification of Hot DNA segments in Escherichia coli
Authors: Kodama Ken‐ichi , Kobayashi Takehiko , Niki Hironori , Hiraga Sota , Oshima Taku , Mori Hirotada , Horiuchi Takashi 
Published: March 2003

SummaryIn Escherichia coli, a replication fork blocking event at a DNA replication terminus (Ter) enhances homologous recombination at the nearby sister chromosomal region, converting the region into a recombination hotspot, Hot, site. Using a RNaseH negative (rnhA–) mutant, we identified eight kinds of Hot DNAs (HotA–H). Among these, enhanced recombination of three kinds of Hot DNAs (HotA–C) was dependent on fork blocking events at Ter sites. In the present study, we examined whether HotA DNAs are amplified when circular DNA (HotA plus a drug‐resistance DNA) is inserted into the homologous region on the chromosome of a rnhA– mutant. The resulting HotA DNA transformants were analysed using pulsed‐field gel electrophoresis, fluorescence in situ hybridization and DNA microarray technique. The following results were obtained: (i) HotA DNA is amplified by about 40‐fold on average; (ii) whereas 90% of the cells contain about 6–10 copies of HotA DNA, the remaining 10% of cells have as many as several hundred HotA copies; and (iii) amplification is detected in all other Hot DNAs, among which HotB and HotG DNAs are amplified to the same level as HotA. Furthermore, HotL DNA, which is activated by blocking the clockwise oriC‐starting replication fork at the artificially inserted TerL site in the fork‐blocked strain with a rnhA+ background, is also amplified, but is not amplified in the non‐blocked strain. From these data, we propose a model that can explain production of three distinct forms of Hot DNA molecules by the following three recombination pathways: (i) unequal intersister recombination; (ii) intrasister recombination, followed by rolling‐circle replication; and (iii) intrasister recombination, producing circular DNA molecules.
The functions of OmpATb, a pore‐forming protein of Mycobacterium tuberculosis
Authors: Raynaud Catherine , Papavinasasundaram K. G. , Speight Richard A. , Springer Burkhard , Sander Peter , Böttger Erik C. , Colston M. Joseph , Draper Philip 
Published: March 2003

SummaryThe functions of OmpATb, the product of the ompATb gene of Mycobacterium tuberculosis and a putative porin, were investigated by studying a mutant with a targeted deletion of the gene, and by observing expression of the gene in wild‐type M. tuberculosis H37Rv by real‐time polymerase chain reaction (PCR) and immunoblotting. The loss of ompATb had no effect on growth under normal conditions, but caused a major reduction in ability to grow at reduced pH. The gene was substantially upregulated in wild‐type bacteria exposed to these conditions. The mutant was impaired in its ability to grow in macrophages and in normal mice, although it was as virulent as the wild type in mice that lack T cells. Deletion of the ompATb gene reduced permeability to several small water‐soluble substances. This was particularly evident at pH 5.5; at this pH, uptake of serine was minimal, suggesting that, at this pH, OmpATb might be the only functioning porin. These data indicate that OmpATb has two functions: as a pore‐forming protein with properties of a porin, and in enabling M. tuberculosis to respond to reduced environmental pH. It is not known whether this second function is related to the porin‐like activity at low pH or involves a completely separate role for OmpATB. The involvement with pH is likely to contribute to the ability of M. tuberculosis to overcome host defence mechanisms and grow in a mammalian host.
Intracellular translation initiation factor levels in Saccharomyces cerevisiae and their role in cap‐complex function
Authors: von der Haar Tobias , McCarthy John E. G. 
Published: March 2003

SummaryKnowledge of the balance of activities of eukaryotic initiation factors (eIFs) is critical to our understanding of the mechanisms underlying translational control. We have therefore estimated the intracellular levels of 11 eIFs in logarithmically growing cells of Saccharomyces cerevisiae using polyclonal antibodies raised in rabbits against recombinant proteins. Those factors involved in 43S complex formation occur at levels comparable (i.e. within a 0.5‐ to 2.0‐fold range) to those published for ribosomes. In contrast, the subunits of the cap‐binding complex eIF4F showed considerable variation in their abundance. The helicase eIF4A was the most abundant eIF of the yeast cell, followed by eIF4E at multiple copies per ribosome, and eIF4B at approximately one copy per ribosome. The adaptor protein eIF4G was the least abundant of the eIF4 factors, with a copy number per cell that is substoichiometric to the ribosome and similar to the abundance of mRNA. The observed excess of eIF4E over its functional partner eIF4G is not strictly required during exponential growth: at eIF4E levels artificially reduced to 30% of those in wild‐type yeast, growth rates and the capacity for general protein synthesis are only minimally affected. This demonstrates that eIF4E does not exercise a higher level of rate control over translation than other eIFs. However, other features of the yeast life cycle, such as the control of cell size, are more sensitive to changes in eIF4E abundance. Overall, these data constitute an important basis for developing a quantitative model of the workings of the eukaryotic translation apparatus.
A fibrinogen receptor from group B Streptococcus interacts with fibrinogen by repetitive units with novel ligand binding sites
Authors: Schubert Axel , Zakikhany Katherina , Schreiner Mark , Frank Ronald , Spellerberg Barbara , Eikmanns Bernhard J. , Reinscheid Dieter J. 
Published: March 2003

SummaryGroup B Streptococcus (GBS) is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, GBS colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present report, we describe the isolation of the fbsA gene, which encodes a fibrinogen receptor from GBS. The deduced FbsA protein is characterized by repetitive units, each 16 amino acids in length. Sequencing of the fbsA gene from five different GBS strains revealed significant variation in the number of repeat‐encoding units. The deletion of the fbsA gene in the genome of GBS 6313 completely abolished fibrinogen binding, suggesting that FbsA is the major fibrinogen receptor in this strain. Growth of the fbsA deletion mutant in human blood was significantly impaired, indicating that FbsA protects GBS from opsonophagocytosis. In Western blot experiments with truncated FbsA ­proteins, the repeat region of FbsA was identified as mediating fibrinogen binding. Using synthetic peptides, even a single repeat unit of FbsA was demonstrated to bind to fibrinogen. Spot membrane analysis and competitive binding experiments with peptides carrying single amino acid substitutions allowed the prediction of a fibrinogen‐binding motif with the consensus sequence G‐N/S/T‐V‐L‐A/E/M/Q‐R‐R‐X‐K/R/W‐A/D/E/N/Q‐A/F/I/L/V/Y‐X‐X‐K/R‐X‐X.
Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase
Authors: Jung Un Sung , Sobering Andrew K. , Romeo Martin J. , Levin David E. 
Published: March 2003

SummaryThe Mpk1 MAP kinase of the Saccharomyces cerevisiae cell wall integrity signalling pathway phosphorylates and activates the Rlm1 transcription factor in response to cell wall stress. Rlm1 is related to mammalian MEF2 isoforms, and shares a similar DNA‐binding specificity. Signalling through Rlm1 regulates the expression of at least 25 genes, most of which have been implicated in cell wall biogenesis. We report here the transcriptional induction by agents of cell wall stress of a set of lacZ reporter plasmids derived from several Rlm1‐responsive genes. Analysis of substitution mutations at putative Mpk1 phosphorylation sites within Rlm1 revealed that Ser427 and Thr439 are important for its stress‐induced transcriptional activation of these reporter plasmids. Assessment of Rlm1 activation potency when fused to a heterologous DNA‐binding domain showed that the identified seryl and threonyl residues are necessary for the Rlm1 transcriptional activation function independently of its DNA binding. We also demonstrate that a MAP kinase docking site, shown recently to mediate activation of MEF2A and MEF2C, is conserved in Rlm1 and is required for its ability to mediate transcriptional activation in response to agents that induce cell wall stress. Finally, intracellular localization analyses show that Rlm1 resides in the nucleus regardless of its activation and phosphorylation status. Together these observations support the inference that Mpk1 regulates the Rlm1 transcriptional activation function by phosphorylation of Ser427 and Thr439.
The third chemotaxis locus of Rhodobacter sphaeroides is essential for chemotaxis
Authors: Porter Steven L. , Warren Anna V. , Martin Angela C. , Armitage Judith P. 
Published: March 2003

SummaryThe purple photosynthetic bacterium Rhodobacter sphaeroides has three loci encoding multiple homologues of the bacterial chemosensory proteins: 13 putative chemoreceptors, four CheW, four CheA, six CheY, two CheB and three CheR. Previously, studies have shown that, although deletion of cheOp1 led to only minor changes in behaviour, deletion of cheOp2 led to a loss of taxis. The third locus encodes two CheA, one CheR, one CheB, one CheW, one CheY, a putative cytoplasmic chemoreceptor (TlpT) and a protein showing homology to the chromosomal partitioning factor Soj (designated Slp). Here, we show that every protein encoded by this locus is essential for normal chemotaxis. Phototaxis is also dependent upon all the components of this locus, except CheB2 and Slp. The two putative CheA proteins encoded in this locus are unusual. CheA3 has only the P1 domain and the P5 regulatory domain linked by a large internal domain, whereas CheA4 lacks the P1 and P2 domains required for phosphorylation and response regulator binding. These data indicate that the minimal set of proteins required for normal chemotaxis in R. sphaeroides is all the proteins encoded by cheOp2 and the third chemotaxis locus, and that the multiple chemosensory protein homologues found in R. sphaeroides are not redundant.
Mutants in flaI and flaJ of the archaeon Methanococcus voltae are deficient in flagellum assembly
Authors: Thomas Nikhil A. , Mueller Steffen , Klein Albrecht , Jarrell Ken F. 
Published: March 2003

SummaryThe fla gene locus of Methanococcus voltae encodes the major structural components of the flagellum as well as other flagellar‐related proteins. The flaHIJ genes have been found in all flagellated archaea, suggesting a central role in flagella biogenesis. FlaI shares similarity with the type II and type IV secretion NTPases (such as PilB, VirB11 and TadA), and FlaJ exhibits similarity to putative bacterial integral membrane proteins involved in type IV pilus biogenesis such as TadB. In this study, reverse transcription polymerase chain reaction (RT‐PCR) and Northern blotting data revealed that flaHIJ are co‐transcribed with the upstream structural flagellin genes, thus demonstrating the expression of the entire fla gene cluster in vivo. Non‐polar mutants in flaI and flaJ of M. voltae were isolated using insertional inactivation via a novel mutagenic vector. These mutants were non‐motile and non‐flagellated by microscopy, demonstrating the involvement of FlaI and FlaJ in flagella biogenesis. Interestingly, all the mutants maintained the ability to produce and localize flagellins to the cytoplasmic membrane. Amino‐terminal sequencing of flagellins produced by the flaJ mutant strain revealed that the flagellins did not have their cognate leader peptides, thus indicating that preflagellin processing had occurred in vivo. This result was confirmed using an in vitro processing assay. The fla− phenotype and protein secretion characteristics of the flaI and flaJ mutants therefore implicate these respective genes in archaeal flagellin secretion and assembly. These findings further support a model describing the archaeal flagellum as a novel prokaryotic motility structure.
Autoregulation of Helicobacter pylori Fur revealed by functional analysis of the iron‐binding site
Authors: Delany Isabel , Spohn Gunther , Pacheco Ana‐Beatriz F. , Ieva Raffaele , Alaimo Cristina , Rappuoli Rino , Scarlato Vincenzo 
Published: March 2003

SummaryThe ferric uptake regulator protein Fur regulates iron‐dependent gene expression in bacteria. In Helicobacter pylori it has been shown to regulate iron‐activated and iron‐repressed genes. In this study, we show that H. pylori Fur protein regulates transcription from its own σ80 promoter Pfur in response to iron. Footprinting analysis shows that Fur binds at three distinct operators at Pfur overlapping and proximal to the promoter elements. Site‐directed mutagenesis of the proposed iron‐binding site of the protein results in derepression of Pfur and the loss of iron regulation. In vivo oligomerization assays reveals that the C‐terminus of Fur is necessary for multimerization of the protein and that the mutations do not affect this activity. Molecular and phenotypic analysis of the mutant proteins provides evidence that the iron‐binding site controls the specific affinity of Fur for the operators at Pfur and hence its repressive ability. In summary, the data presented are consistent with a model in which Fur acts as a rheostat of transcription to autoregulate its own expression in response to iron, which in turn controls expression of iron‐induced and iron‐repressed genes, providing maintenance of homeostasis.
Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane
Authors: Grahn A. Marika , Daugelavičius Rimantas , Bamford Dennis H. 
Published: March 2003

SummaryDNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double‐stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build‐up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.