bluuebunny/crossref_metadata_2025_split huggingface dataset:
Co‐evolution of the agrocinopine opines and the agrocinopine‐mediated control of TraR, the quorum‐sensing activator of the Ti plasmid conjugation system
Authors: Oger Philippe , Farrand Stephen K. 
Published: March 2003

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is controlled by a hierarchical system in which opines, substrates produced by crown gall tumours, induce a quorum‐sensing system. The cascade results from the control of expression of traR, the quorum‐sensing activator, by a regulator responsive to the opine. In the two cases studied to date, the gene arrangements responsible for the cascade differ remarkably, suggesting that considerable diversity exists among the many Ti‐like plasmids in the agrobacteria. In this study, we demonstrated that the novel Ti plasmid pTiChry5 is induced to transfer at high frequency by extracts from tumours initiated by strain Chry5. The purified inducer had the chemical and biological properties of agrocinopines C and D, a set of sugar phosphodiester opines known to induce transfer of another Ti plasmid, pTiBo542. The T‐region of pTiChry5 contained a gene whose product, called AcsChry5, is virtually identical to the agrocinopine C+D synthase from the T‐region of pTiBo542. The two genes are less closely related to acs of pTiC58, which is responsible for the production of agrocinopines A+B, a similar but not identical set of phosphodiester opines by tumours induced by strain C58. Agrocinopines A+B induce transfer of pTiC58 but did not induce transfer of pTiChry5. A single copy of traR was identified at the 11 o'clock region of pTiChry5, where it is part of a two‐gene operon called arcChry5. Although altered by deletions, arcChry5 is related to the five‐gene arc operon that controls the expression of traR on pTiC58. Expression of traRChry5 was induced by agrocinopines C+D and the opines isolated from Chry5 tumours but not by agrocinopines A+B. A mutation in traRChry5 abolished transfer, and transfer was restored by complementation in trans. We conclude that the agrocinopine opines and the corresponding opine‐meditated conjugal regulatory regions of pTiChry5 and pTiC58 share a common origin, but that the opine signals for the two Ti plasmids have evolved divergently through changes in the opine synthase enzymes. The alterations in the opines, in turn, necessitated a co‐evolutionary change in the opine recognition systems responsible for controlling expression of the traR genes on these two types of Ti plasmids.
Regulation of gene expression in Vibrio cholerae by ToxT involves both antirepression and RNA polymerase stimulation
Authors: Yu Rosa R. , DiRita Victor J. 
Published: March 2003

SummaryCo‐ordinate expression of many virulence genes in the human pathogen Vibrio
cholerae is under the direct control of the ToxT protein, including genes whose
products are required for the biogenesis of the toxin‐co‐regulated pilus (TCP) and
cholera toxin (CTX). This work examined interactions between ToxT and the promoters
of ctx and tcpA genes. We found that a minimum of three direct repeats
of the sequence TTTTGAT is required for ToxT‐dependent activation of the ctx
promoter, and that the region from –85 to –41 of the tcpA promoter
contains elements that are responsive to ToxT‐dependent activation. The role of H‐NS
in transcription of ctx and tcpA was also analysed. The level of activation
of ctx–lacZ in an E. coli hns– strain was
greatly increased even in the absence of ToxT, and was further enhanced in the presence
of ToxT. In contrast, H‐NS plays a lesser role in the regulation of the tcpA
promoter. Electrophoretic mobility shift assays demonstrated that 6×
His‐tagged ToxT directly, and specifically, interacts with both the ctx and
tcpA promoters. DNase I footprinting analysis suggests that there may be two
ToxT binding sites with different affinity in the ctx promoter and that ToxT
binds to –84 to –41 of the tcpA promoter. In vitro transcription experiments demonstrated that ToxT alone is able to activate transcription from both promoters. We hypothesize that under conditions appropriate for ToxT‐dependent gene expression, ToxT binds to AT‐rich promoters that may have a specific secondary conformation, displaces H‐NS and stimulates RNA polymerase resulting in transcription activation.
A fadD mutant of Sinorhizobium meliloti shows multicellular swarming migration and is impaired in nodulation efficiency on alfalfa roots
Authors: Soto María José , Fernández‐Pascual Mercedes , Sanjuan Juan , Olivares José 
Published: March 2003

SummarySwarming is a form of bacterial translocation that involves cell differentiation and is characterized by a rapid and co‐ordinated population migration across solid surfaces. We have isolated a Tn5 mutant of Sinorhizobium meliloti GR4 showing conditional swarming. Swarm cells from the mutant strain QS77 induced on semi‐solid minimal medium in response to different signals are hyperflagellated and about twice as long as wild‐type cells. Genetic and physiological characterization of the mutant strain indicates that QS77 is altered in a gene encoding a homologue of the FadD protein (long‐chain fatty acyl‐CoA ligase) of several microorganisms. Interestingly and similar to a less virulent Xanthomonas campestris fadD(rpfB) mutant, QS77 is impaired in establishing an association with its host plant. In trans expression of multicopy fadD restored growth on oleate, control of motility and the symbiotic phenotype of QS77, as well as acyl‐CoA synthetase activity of an Escherichia coli fadD mutant. The S. meliloti QS77 strain shows a reduction in nod gene expression as well as a differential regulation of motility genes in response to environmental conditions. These data suggest that, in S. meliloti, fatty acid derivatives may act as intracellular signals controlling motility and symbiotic performance through gene expression.
Independent regulation of the divergent Escherichia coli nrfA and acsP1 promoters by a nucleoprotein assembly at a shared regulatory region
Authors: Browning Douglas F. , Beatty Christine M. , Wolfe Alan J. , Cole Jeffrey A. , Busby Stephen J. W. 
Published: March 2003

SummaryExpression from the Escherichia coli nrfA promoter (pnrfA) is activated by 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 at position –41.5 at pnrfA and activates transcription. Further activation, induced by the presence of nitrite, results from the binding of NarL and NarP to a site centred at position –74.5. A second promoter (pacsP1), which directs transcription into the adjacent gene encoding acetyl coenzyme A synthetase (acs), is overlapping and divergent to pnrfA. Despite extensive overlap of regulatory elements, pnrfA and pacsP1 are regulated independently. We demonstrate that at least two nucleoid‐associated factors bind to the nrfA–acs intergenic region. The Fis protein binds to a site centred at position –15 (in relation to pnrfA transcription), whereas the IHF protein binds to a site centred at position –54. Both Fis and IHF repress in vivo expression from pacsP1, but have smaller repressive effects on expression from pnrfA. Gel retardation assays were used to investigate the pairwise binding of FNR, NarL, Fis and IHF proteins to the nrfA–acs intergenic region. The binding of NarL and IHF is mutually exclusive, whereas all other combinations can bind simultaneously. Experiments in which deletions and point mutations were introduced into the upstream region of pnrfA demonstrated that an additional factor must bind upstream to inhibit FNR‐dependent transcription. We conclude that the nrfA–acs intergenic region is folded into an ordered nucleoprotein structure that permits the two divergent promoters to be regulated independently in response to different physiological signals.
H‐NS‐dependent activation of pectate lyases synthesis in the phytopathogenic bacterium Erwinia chrysanthemi is mediated by the PecT repressor
Authors: Nasser William , Reverchon Sylvie 
Published: March 2003

SummaryProduction of the main virulence determinant pectate lyases (Pels) of the phytopathogenic bacterium Erwinia chrysanthemi is modulated by a complex regulatory network involving the repressor proteins KdgR, PecS and PecT and the activator systems Pir, ExpI–ExpR and CRP. Of these regulators, CRP and PecT are particularly important since the absence of CRP or a slight overproduction of PecT leads to a drastic reduction in synthesis of Pel species. Recently, it has been shown that production of Pel species is strongly reduced in an E. chrysanthemi hns mutant, suggesting an activator function of the nucleoid‐associated protein H‐NS in the expression of the pel genes. Here, we report that the reduced synthesis of Pel species in the hns mutant results from a negative control, exerted by H‐NS, on the transcription of the regulatory gene pecT. This H‐NS/PecT cascade regulation is one of the first elucidations of a positive effect of H‐NS on target gene expression. Moreover, we found that H‐NS also represses the expression of expI, expR and pel genes. H‐NS control is the result of H‐NS binding to extended regions within the pecT, expI, expR and pel genes. Investigation of the simultaneous binding of CRP, RNA polymerase (RNAP) and H‐NS on the pelD gene revealed that these three proteins form a nucleoprotein com‐plex. Together, these data indicate that, by exerting a negative control at multiple levels, H‐NS plays a crucial role in the E. chrysanthemi pel regulatory network.
The alternative sigma factor σE controls antioxidant defences required for Salmonella virulence and stationary‐phase survival
Authors: Testerman Traci L. , Vazquez‐Torres Andrés , Xu Yisheng , Jones‐Carson Jessica , Libby Stephen J. , Fang Ferric C. 
Published: March 2003

SummaryBacteria must contend with conditions of nutrient limitation in all natural environments. Complex programmes of gene expression, controlled in part by the alternative sigma factors σS (σ38, RpoS) and σH (σ32, RpoH), allow a number of bacterial species to survive conditions of partial or complete starvation. We show here that the alternative sigma factor σE (σ24, RpoE) also facilitates the survival of Salmonella typhimurium under conditions of nutrient deprivation. Expression of the σE regulon is strongly induced upon entry of Salmonella into stationary phase. A Salmonella mutant lacking σE has reduced survival during stationary phase as well as increased susceptibility to oxidative stress. A Salmonella strain lacking both σE and σS is non‐viable after just 24 h in stationary phase, but survival of these mutants is completely preserved under anaerobic stationary‐phase conditions, suggesting that oxidative injury is one of the major mechanisms of reduced microbial viability during periods of nutrient deprivation. Moreover, the attenuated virulence of σE‐deficient Salmonella for mice can be largely restored by genetic abrogation of the host phagocyte respiratory burst, suggesting that the σE regulon plays an important antioxidant role during Salmonella infection of mammalian hosts.
Role of thioredoxins in the response of Saccharomyces cerevisiae to oxidative stress induced by hydroperoxides
Authors: Garrido Ester Ocón , Grant Chris M. 
Published: March 2003

SummaryGlutaredoxins and thioredoxins are highly conserved, small, heat‐stable oxidoreductases. The yeast Saccharomyces cerevisiae contains two gene pairs encoding cytoplasmic

glutaredoxins (GRX1, GRX2) and thioredoxins (TRX1, TRX2),

and we have used multiple mutants to determine their roles in medi‐ating resistance

to oxidative stress caused by hydroperoxides. Our data indicate that TRX2

plays the predominant role, as mutants lacking TRX2 are hypersensitive, and

mutants containing TRX2 are resistant to these oxidants. However, the requirement

for TRX2 is only apparent during stationary phase growth, and we present three

lines of evidence that the thioredoxin isoenzymes actually have redundant activities

as antioxidants. First, the trx1 and trx2 mutants show wild‐type resistance

to hydroperoxide during exponential phase growth; secondly, overexpression of either

TRX1 or TRX2 leads to increased resistance to hydroperoxides; and,

thirdly, both Trx1 and Trx2 are equally able to act as cofactors for the thioredoxin

peroxidase, Tsa1. The antioxidant activity of thioredoxins is required for both the

survival of yeast cells as well as protection against oxidative stress during stationary

phase growth, and correlates with an increase in the expression of both TRX1

and TRX2. We show that the requirement for thioredoxins during this growth phase is dependent on their activity as cofactors for the antioxidant enzyme Tsa1, and for regulation of the redox state and protein‐bound levels of the low‐molecular‐weight antioxidant glutathione.
Behaviour of topological marker proteins targeted to the Tat protein transport pathway
Authors: Stanley Nicola R. , Sargent Frank , Buchanan Grant , Shi Jiarong , Stewart Valley , Palmer Tracy , Berks Ben C. 
Published: March 2003

SummaryThe Escherichia coli Tat system mediates Sec‐independent export of protein
precursors bearing twin arginine signal peptides. Formate dehydrogenase‐N is a three‐subunit
membrane‐bound enzyme, in which localization of the FdnG subunit to the membrane
is Tat dependent. FdnG was found in the periplasmic fraction of a mutant lacking
the membrane anchor subunit FdnI, confirming that FdnG is located at the periplasmic
face of the cytoplasmic membrane. However, the phenotypes of gene fusions between
fdnG and the subcellular reporter genes phoA (encoding alkaline phosphatase)
or lacZ (encoding β‐galactosidase) were the opposite of those expected for analogous fusions targeted to the Sec translocase. PhoA fusion experiments have previously been used to argue that the peripheral membrane DmsAB subunits of the Tat‐dependent enzyme dimethyl sulphoxide reductase are located at the cytoplasmic face of the inner membrane. Biochemical data are presented that instead show DmsAB to be at the periplasmic side of the membrane. The behaviour of reporter proteins targeted to the Tat system was analysed in more detail. These data suggest that the Tat and Sec pathways differ in their ability to transport heterologous passenger proteins. They also suggest that caution should be observed when using subcellular reporter fusions to determine the topological organization of Tat‐dependent membrane protein complexes.
Antitermination by GlpP, catabolite repression via CcpA and inducer exclusion triggered by P~GlpK dephosphorylation control Bacillus subtilis glpFK expression
Authors: Darbon Emmanuelle , Servant Pascale , Poncet Sandrine , Deutscher Josef 
Published: March 2003

SummaryThe Bacillus subtilis glpFK operon encoding the glycerol transport facilitator (GlpF) and glycerol kinase (GlpK) is induced by glycerol‐3‐P and repressed by rapidly metabolizable sugars. Carbon catabolite repression (CCR) of glpFK is partly mediated via a catabolite response element cre preceding glpFK. This operator site is recognized by the catabolite control protein A (CcpA) in complex with one of its co‐repressors, P‐Ser‐HPr or P‐Ser‐Crh. HPr is a component of the phosphoenolpyruvate:sugar phos‐photransferase system (PTS), and Crh is an HPr homologue. The hprK‐encoded HPr kinase phosphorylates HPr and Crh at Ser‐46. But in neither ccpA nor hprK mutants was expression of a glpF′–lacZ fusion relieved from CCR, as a second, CcpA‐independent CCR mechanism implying the terminator tglpFK, whose formation is prevented by the glycerol‐3‐P‐activated antiterminator GlpP, is operative. Deletion of tglpFK led to elevated expression of the glpF′–lacZ fusion and to partial relief from CCR. CCR completely disappeared in ΔtglpFK mutants carrying a disruption of ccpA or hprK. The tglpFK‐requiring CCR mechanism seems to be based on insufficient synthesis of glycerol‐3‐P, as CCR of glpFK was absent in ccpA mutants growing on glycerol‐3‐P or synthesizing H230R mutant GlpK. In cells growing on glycerol, glucose prevents the phosphorylation of GlpK by P~His‐HPr. P~GlpK is much more active than GlpK, and the absence of P~GlpK formation in ΔptsHI strains prevents glycerol metabolism. As a consequence, only small amounts of glycerol‐3‐P will be formed in glycerol and glucose‐exposed cells (inducer exclusion). The uptake of glycerol‐3‐P via GlpT provides high concentrations of this metabolite in the ccpA mutant and allows the expression of the glpF′–lacZ fusion even when glucose is present. Similarly, despite the presence of glucose, large amounts of glycerol‐3‐P are formed in a glycerol‐exposed strain synthesizing GlpKH230R, as this mutant GlpK is as active as P~GlpK.
Truncation of merozoite surface protein 3 disrupts its trafficking and that of acidic–basic repeat protein to the surface of Plasmodium falciparum merozoites
Authors: Mills Kerry E. , Pearce J. Andrew , Crabb Brendan S. , Cowman Alan F. 
Published: March 2003

SummaryMerozoite surface protein 3 (MSP3), an important vaccine candidate, is a soluble polymorphic antigen associated with the surface of Plasmodium falciparum merozoites. The MSP3 sequence contains three blocks of heptad repeats that are consistent with the formation of an intramolecular coiled‐coil. MSP3 also contains a glutamic acid‐rich region and a putative leucine zipper sequence at the C‐terminus. We have disrupted the msp3 gene by homologous recombination, resulting in the expression of a truncated form of MSP3 that lacks the putative leucine zipper sequence but retains the glutamic acid‐rich region and the heptad repeats. Here, we show that truncated MSP3, lacking the putative leucine zipper region, does not localize to the parasitophorous vacuole or interact with the merozoite surface. Furthermore, the acidic–basic repeat antigen (ABRA), which is present on the merozoite surface, also was not localized to the merozoite surface in parasites expressing the truncated form of MSP3. The P. falciparum merozoites lacking MSP3 and ABRA on the surface show reduced invasion into erythrocytes. These results suggest that MSP3 is not absolutely essential for blood stage growth and that the putative leucine zipper region is required for the trafficking of both MSP3 and ABRA to the parasitophorous vacuole.
Selective expression of the β‐subunit of nucleoid‐associated protein HU during cold shock in Escherichia coli
Authors: Giangrossi Mara , Giuliodori Anna Maria , Gualerzi Claudio O. , Pon Cynthia L. 
Published: March 2003

SummaryExpression of Escherichia coli hupA and hupB, the structural genes

encoding the most abundant nucleoid‐associated proteins HUα and HUβ

has been studied during cold shock. This article demonstrates that: (i) transcriptional

expression of hupA is blocked following a sudden temperature downshift (from

37°C to 10°C), whereas transcription of hupB from the

P2 and P3 promoters is maintained at a constitutive level and is activated de

novo from the P4 promoter; (ii) all three hupB mRNAs (transcribed from

the three natural promoters P2, P3 and P4) become much more stable than the single

hupA transcript; and (iii) the hupB transcripts, unlike that of hupA,

are efficiently translated in vivo during cold acclimation and can be actively

translated in vitro at low temperature. Taken together, the results indicate

that during cold shock the expression of the HUβ subunit is preferentially

stimulated and that of HUα repressed, suggesting that an altered HUα

to HUβ expression ratio resulting in an increase of HUα/HUβ

heterodimers and/or (HUβ)2 homodimers may play an important role during cold adaptation.
Yeast Rio1p is the founding member of a novel subfamily of protein serine kinases involved in the control of cell cycle progression
Authors: Angermayr Michaela , Roidl Andreas , Bandlow Wolfhard 
Published: March 2003

SummaryRio1p was identified as a protein serine kinase founding a novel subfamily. It is highly conserved from Archaea to man and only distantly related to previously established protein kinase families. Nevertheless, analysis of multiple protein sequence alignments shows that those amino acid residues that are important for either structure or catalytic activity in conventional protein kinases are also conserved in members of the Rio1p family at the respective positions (corresponding to domains I–XI of protein kinases). Recombinant Rio1p from Escherichia coli and tagged Rio1p from yeast has kinase activity in vitro, and mutation of amino acid residues that are conserved and indispensable for catalytic activity (i.e. ATP‐binding motif, catalytic centre) abrogates activity. RIO1 is essential in yeast and plays a role in cell cycle progression. After sporulation of RIO1/rio1 diploids, RIO1‐disrupted progeny cease growth after one to three cell divisions and arrest as either large unbudded or large‐budded cells. Cells deprived of Rio1p are enlarged and arrest either in G1 or in mitosis mainly with the DNA at the bud neck and short spindles (a phenotype also seen in cells carrying a weak allele), suggesting that Rio1p activity is required for at least at two steps during the cell division cycle: for entrance into S phase and for exit from mitosis. The weak RIO1 allele leads to increased plasmid loss.
HF2: a double‐stranded DNA tailed haloarchaeal virus with a mosaic genome
Authors: Tang Sen‐Lin , Nuttall Stewart , Ngui Katrina , Fisher Clare , Lopez Philippe , Dyall‐Smith Mike 
Published: March 2003

SummaryHF2 is a haloarchaeal virus infecting two Halorubrum species (Family Halobacteriaceae).
It is lytic, has a head‐and‐tail morphology and belongs to the Myoviridae (contractile
tails). The linear double‐stranded DNA genome was sequenced and found to be 77 670
bp in length, with a mol% G+C of 55.8. A total of 121 likely open reading frames (ORFs) were identified, of which 37 overlapped at start and stop codons. The predicted proteins were usually acidic (average pI of 4.8), and less than about 12% of them had homologues in the sequence databases. Four complete tRNA‐like sequences (tRNA‐Arg, ‐Asx, ‐Pro and ‐Tyr) and an incomplete tRNA‐Thr were detected. A transcription map showed that most of the genome was transcribed and that the synthesis of transcripts occurred in a highly organized and reproducible pattern over a 5 h infection cycle. Transcripts often spanned multiple ORFs, suggesting that viral genes were organized into operons. The predicted ORF and observed transcript directions matched well and showed that transcription is mainly directed inwards from the genome termini, meeting at about 45–48 kb, and this was also a turning point in a cumulative GC‐skew plot. The low point in cumulative GC‐skew, near the left end, was a region rich in short repeats and lacking ORFs, which is likely to be an origin of replication. The HF2 genome is a mosaic of components from widely different sources, demonstrating clearly that viruses of haloarchaea, like their bacteriophage counterparts, are vectors for the exchange and transmission of genetic material between wide taxonomic distances, even across domains.