bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of CRP‐dependent transcription at the Escherichia coli acsP2 promoter by nucleoprotein complexes: anti‐activation by the nucleoid proteins FIS and IHF
Authors: Browning Douglas F. , Beatty Christine M. , Sanstad Erik A. , Gunn Kathryn E. , Busby Stephen J. W. , Wolfe Alan J. 
Published: December 2004

Summary acs encodes acetyl‐coenzyme A synthetase, a high‐affinity enzyme that allows cells to scavenge for acetate during carbon starvation. CRP activates acs transcription by binding tandem DNA sites located upstream of the major promoter, acsP2. Here, we used electrophoretic mobility shift assays and DNase I footprint analyses to demonstrate that the nucleoid proteins FIS and IHF each bind multiple sites within the acs regulatory region, that FIS competes successfully with CRP for binding to their overlapping and neighbouring sites and that IHF binds independently of either FIS or CRP. Using in vitro transcription assays, we demonstrated that FIS and IHF independently reduce CRP‐dependent acs transcription. Using in vivo reporter assays, we showed that disruption of DNA sites for FIS or deletion of DNA sites for IHF increases acs transcription. We propose that FIS and IHF each function directly as anti‐activators of CRP, each working independently at different times during growth to set the levels of CRP‐dependent acs transcription.
Regulatory proteins with a sense of direction: cell cycle signalling network in Caulobacter
Authors: Jacobs‐Wagner Christine 
Published: December 2004

SummaryLocalization of kinases and other signalling molecules at discrete cellular locations is often an essential component of signal transduction in eukaryotes. Caulobacter crescentus is a small, single‐celled bacterium that presumably lacks intracellular organelles. Yet in Caulobacter, the subcellular distribution of several two‐component signal transduction proteins involved in the control of polar morphogenesis and cell cycle progression changes from a fairly dispersed distribution to a tight accumulation at one or both poles in a spatial and temporal pattern that is reproduced during each cell cycle. This cell cycle‐dependent choreography suggests that similarly to what happens in eukaryotes, protein localization provides a means of modulating signal transduction in bacteria. Recent studies have provided important insights into the biological role and the mechanisms for the differential localization of these bacterial signalling proteins during the Caulobacter cell cycle.
Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation
Authors: Burke Mary E. , Arnold Patricia H. , He Jiuya , Wenwieser Sandra V. C. T. , Rowland Sally‐J. , Boocock Martin R. , Stark W. Marshall 
Published: December 2004

SummaryCatalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent ‘accessory’ binding sites II and III. ‘Hyperactive’ resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res × site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I × site I plasmid. Site I × site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2–3′ interface that abolish activity of wild‐type resolvase. Activating mutations may allow regulatory mechanisms of the wild‐type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.
Characterization of cis‐acting prgQ mutants: evidence for two distinct repression mechanisms by Qa RNA and PrgX protein in pheromone‐inducible enterococcal plasmid pCF10
Authors: Bae Taeok , Kozlowicz Briana K. , Dunny Gary M. 
Published: December 2004

SummaryThe pCF10‐encoded negative regulators PrgX and Qa (prgQ antisense) RNA inhibit pCF10 transfer by blocking prgQ transcription extension past a potential transcription terminator sequence IRS1. To identify potential target sites for negative regulation, we isolated and analysed 13 cis‐acting mutations in the prgXQ region. Determination of the 3′ end of Qa RNA showed that eight mutations mapped in the region encoding Qa RNA. Four mutations were in the Qa promoter region and one was in IRS1. Three mutations in Qa greatly reduced the intracellular level of this RNA but did not affect that of PrgX. However, both Qa RNA and PrgX protein were reduced in three Qa promoter region mutants and the expression of prgQ transcripts extending 3′ from IRS1 became constitutive. Qa RNA could mediate its negative regulatory activity in the absence of PrgX, and this activity was not abolished by cCF10, the peptide pheromone that induces pCF10 transfer. RNA analysis showed that Qa RNA abolished transcription readthrough. Based on the experimental data as well as computer analysis of predicted secondary structures of prgQ mRNA in the presence or absence of Qa, we concluded that Qa RNA is a pheromone‐insensitive effector of prgQ mRNA termination or degradation at IRS1. In cells lacking a Qa target sequence, expression of PrgX repressed transcription from the prgQ promoter, and this repression was relieved by addition of exogenous cCF10. Thus, even though the synthesis of these negative regulators is coupled, they each act independently on separate targets to regulate expression of conjugation functions.
Structure‐function analysis of the self‐recognizing Antigen 43 autotransporter protein from Escherichia coli
Authors: Klemm Per , Hjerrild Louise , Gjermansen Morten , Schembri Mark A. 
Published: December 2004

SummaryAntigen 43 (Ag43) is a self‐recognizing surface adhesin found in most Escherichia coli strains. Expression of Ag43 confers aggregation and fluffing of cells, promotes biofilm formation and is associated with enhanced resistance to antimicrobial agents. Ag43 is an autotransporter protein and consists of two moieties: a transporter, the β‐module, and a passenger domain, the α‐module. Here we have employed various molecular approaches to probe structure/function aspects of Ag43. An entire family of Ag43 variants was identified. The gene encoding Ag43 (flu) was cloned from a diverse range of E. coli subtypes and found to encode variant proteins with different properties. Several novel variants were identified and characterized that were unable to promote cell–cell aggregation. By employing a combination of linker insertion mutagenesis and domain swapping between clumping and non‐clumping variants, we have pinpointed the region of the protein responsible for autoaggregation to be located within the N‐terminal one‐third of the passenger domain. Our data suggest that ionic interactions between charged residues residing in interacting pairs of Ag43α domains may be important for the self‐recognition process. Based on its similarity to other related proteins, we predict the passenger, Ag43α, domain primarily to consist of an extended β‐helix structure in which numerous repeats or rungs are stacked in parallel orientation in an extended cylindrical formation. Finally, we found that in spite of their different aggregative pattern all Ag43 variants promoted biofilm formation to abiotic surfaces.
Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization – RING‐FISH
Authors: Zwirglmaier K. , Ludwig W. , Schleifer K. ‐H. 
Published: December 2004

SummaryFluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 104−105 copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (101−103 copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method – a halo‐like, ring‐shaped concentration of fluorescence in the cell periphery – we coined the term RING‐FISH (recognition of individual genes) to differentiate it from conventional FISH.
Site‐directed mutagenesis of a LuxR‐type quorum‐sensing transcription factor: alteration of autoinducer specificity
Authors: Chai Yunrong , Winans Stephen C. 
Published: December 2004

SummaryTraR is a quorum‐sensing transcription factor from Agrobacterium tumefaciens that regulates replication and conjugation genes of the tumour‐inducing (Ti) plasmid. TraR activity requires the autoinducer pheromone N‐3‐oxooctanoyl‐l‐homoserine lactone (OOHL). Structural studies of TraR–OOHL–DNA complexes showed that one molecule of OOHL is completely engulfed within the N‐terminal domain of each TraR subunit. TraR is thought to bind OOHL via four hydrogen bonds, three of them direct and one water mediated, and by numerous hydrophobic interactions. Here, we show that all residues predicted to hydrogen bond with OOHL are essential for wild‐type protein function. Mutants that failed to detect OOHL in vivo invariably failed to sequester exogenous OOHL. We showed previously that TraR is protected from cellular proteases by OOHL, and now show that mutants that failed to detect OOHL were also not protected from proteolysis by OOHL. We also describe several mutants with altered autoinducer specificity. Three mutants (T129V, T129A and T115I) detected 3‐oxo‐AHLs and 3‐unsubstituted AHLs with equal sensitivity, indicating that these mutations perturb the water‐mediated hydrogen bond to the 3‐oxo moiety of OOHL. Three other mutants (A49I, A49M and Q58L) preferentially detected AHLs containing six or seven carbon atoms rather than eight. The bulkier residues in these mutations appear to have occupied a portion of the OOHL binding site, interfering with binding of the acyl chain of AHLs.
A novel biotin protein required for reductive carboxylation of 2‐oxoglutarate by isocitrate dehydrogenase in Hydrogenobacter thermophilus TK‐6
Authors: Aoshima Miho , Ishii Masaharu , Igarashi Yasuo 
Published: December 2004

SummaryIsocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2‐oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP‐Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N‐terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase. Isocitrate dehydrogenase, the novel biotin protein, ATP‐Mg and NADH were essential for the reductive carboxylation of 2‐oxoglutarate. These observations indicate that the novel biotin protein is an ATP‐dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase.
The tetraspanin BcPls1 is required for appressorium‐mediated penetration ofBotrytis cinereainto host plant leaves
Authors: Gourgues M. , Brunet‐Simon A. , Lebrun M‐H. , Levis C. 
Published: December 2004

SummaryAnimal tetraspanins are membrane proteins controlling cell adhesion, morphology and motility. In fungi, the tetraspanin MgPls1 controls an appressorial function required for the penetration ofMagnaporthe griseainto host plants. An orthologue ofMgPLS1,BcPLS1, was identified in the necrotrophic fungal plant pathogenBotrytis cinerea. We constructed aBcpls1::barnull mutant by targeted gene replacement.Bcpls1::baris not pathogenic on intact plant tissues of bean, tomato or rose, but it infects wounded plant tissues. Both wild type andBcpls1::bardifferentiate appressoria on plant and artificial surfaces, a process involving an arrest of polarized growth, apex swelling and its cell wall reinforcement. Although wild‐type appressoria allowed the penetration of the fungus into the host plant within 6–12 h, no successful penetration events were observed withBcpls1::bar, suggesting that its appressoria are not functional. AneGFPtranscriptional fusion showed thatBcPLS1was specifically expressed in conidia, germ tubes and appressoria during host penetration. Our results indicate thatBcPLS1is required for the penetration ofB. cinereainto intact host plants. The defect in pathogenicity ofBcpls1::baralso demonstrates that functionalB. cinereaappressoria are required for a successful penetration process. AsBcpls1::barandMgpls1Δ::hphpenetration defects are similar, fungal tetraspanins are likely to be required for an essential appressorial function widespread among fungi.
Initiation of intracellular offspring in Epulopiscium
Authors: Angert Esther R. , Clements Kendall D. 
Published: December 2004

Summary Epulopiscium spp. are the largest heterotrophic bacteria yet described. A distinguishing feature of the Epulopiscium group is their viviparous production of multiple, internal offspring as a means of cellular reproduction. Based on their phylogenetic position, among low G + C Gram‐positive endospore‐forming bacteria, and the remarkable morphological similarity between developing endospores and Epulopiscium offspring, we hypothesized that intracellular offspring production in Epulopiscium evolved from endospore formation. These observations also raise the possibility that a cell with the capacity to form multiple intracellular offspring was the ancestor of all contemporary endospore‐forming bacteria. In an effort to characterize mechanisms common to both processes, we describe the earliest stages of offspring formation in Epulopiscium. First, in anticipation of polar division, some of the mother cell DNA coalesces at the cell poles. FtsZ then localizes in a bipolar pattern and the cell divides. A portion of the pole‐associated DNA is trapped within the small cells formed by division at both poles. As development progresses, more pole‐associated DNA is apparently packaged into the offspring primordia. These results illustrate three mechanisms, the reorganization of cellular DNA, asymmetric division and DNA packaging, that are common to both endospore formation in Bacillus subtilis and the production of active, intracellular offspring in Epulopiscium. Unlike most endospore formers, Epulopiscium partitions only a small proportion of mother cell DNA into the developing offspring.
Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK‐DnaJ‐GrpE system in the management of protein misfolding under severe stress conditions
Authors: Mujacic Mirna , Bader Martin W. , Baneyx François 
Published: December 2004

Summary Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48°C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50°C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB‐DnaK‐DnaJ‐GrpE system to clear thermally aggregated proteins at 30°C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, ΔclpB or ΔibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK‐DnaJ‐GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding.
Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms
Authors: Friedman Lisa , Kolter Roberto 
Published: December 2004

Summary Pseudomonas aeruginosa forms diverse matrix‐enclosed surface‐associated multicellular assemblages (biofilms) that aid in its survival in a variety of environments. One such biofilm is the pellicle that forms at the air–liquid interface in standing cultures. We screened for transposon insertion mutants of P. aeruginosa PA14 that were unable to form pellicles. Analysis of these mutants led to the identification of seven adjacent genes, named pel genes, the products of which appear to be involved in the formation of the pellicle's extracellular matrix. In addition to being required for pellicle formation, the pel genes are also required for the formation of solid surface‐associated biofilms. Sequence analyses predicted that three pel genes encode transmembrane proteins and that five pel genes have functional homologues involved in carbohydrate processing. Microscopic and macroscopic observations revealed that wild‐type P. aeruginosa PA14 produces a cellulase‐sensitive extracellular matrix able to bind Congo red; no extracellular matrix was produced by the pel mutants. A comparison of the carbohydrates produced by the wild‐type strain and pel mutants suggested that glucose was a principal component of the matrix material. Together, these results suggest that the pel genes are responsible for the production of a glucose‐rich matrix material required for the formation of biofilms by P. aeruginosa PA14.
A bacterial cell–cell communication signal with cross‐kingdom structural analogues
Authors: Wang Lian‐Hui , He Yawen , Gao Yunfeng , Wu Ji En , Dong Yi‐Hu , He Chaozu , Wang Su Xing , Weng Li‐Xing , Xu Jin‐Ling , Tay Leng , Fang Rong Xiang , Zhang Lian‐Hui 
Published: December 2004

SummaryExtracellular signals are the key components of microbial cell–cell communication systems. This report identified a diffusible signal factor (DSF), which regulates virulence in Xanthomonas campestris pv. campestris, as cis‐11‐methyl‐2‐dodecenoic acid, an α,β unsaturated fatty acid. Analysis of DSF derivatives established the double bond at the α,β positions as the most important structural feature for DSF biological activity. A range of bacterial pathogens, including several Mycobacterium species, also displayed DSF‐like activity. Furthermore, DSF is structurally and functionally related to farnesoic acid (FA), which regulates morphological transition and virulence by Candida albicans, a fungal pathogen. Similar to FA, which is also an α,β unsaturated fatty acid, DSF inhibits the dimorphic transition of C. albicans at a physiologically relevant concentration. We conclude that α,β unsaturated fatty acids represent a new class of extracellular signals for bacterial and fungal cell–cell communications. As prokaryote–eukaryote interactions are ubiquitous, such cross‐kingdom conservation in cell–cell communication systems might have significant ecological and economic importance.
Overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance
Authors: de Pinto B. , Lippolis R. , Castaldo R. , Altamura N. 
Published: December 2004

SummaryIn yeast the UPF1, UPF2 and UPF3 genes encode three interacting factors involved in translation termination and nonsense‐mediated mRNA decay (NMD). UPF1 plays a central role in both processes. In addition, UPF1 was originally isolated as a multicopy suppressor of mitochondrial splicing deficiency, and its deletion leads to an impairment in respiratory growth. Here, we provide evidence that inactivation of UPF2 or  UPF3,  like  that  of  UPF1,  leads  to  an  impairment in respiratory competence, suggesting that their products, Upf1p, Upf2p and Upf3p, are equivalently involved in mitochondrial biogenesis. In addition, however, we show that only Upf1p acts as a multicopy suppressor of mitochondrial splicing deficiency, and its activity does not require either Upf2p or Upf3p. Mutations in the conserved cysteine‐ and histidine‐rich regions and ATPase and helicase motifs of Upf1p separate the ability of Upf1p to complement the respiratory impairment of a Δupf1 strain from its ability to act as a multicopy suppressor of mitochondrial splicing deficiency, indicating that distinct pathways express these phenotypes. In addition, we show that, when overexpressed, Upf1p is not detected within mitochondria, suggesting that its role as multicopy suppressor of mitochondrial splicing deficiency is indirect. Furthermore, we provide evidence that cells overexpressing certain upf1 alleles accumulate a phosphorylated isoform of Upf1p. Altogether, these results indicate that overexpression of Upf1p compensates for mitochondrial splicing deficiency independently of its role in mRNA surveillance, which relies on Upf1p–Upf2p–Upf3p functional interplay.
Dynamics and processivity of 40S ribosome scanning on mRNA in yeast
Authors: Berthelot Karine , Muldoon Mark , Rajkowitsch Lukas , Hughes John , McCarthy John E. G. 
Published: December 2004

SummaryThe eukaryotic 40S ribosomal subunit locates the translation initiation codon on an mRNA via the so‐called scanning process that follows 40S binding to the capped 5′ end. This key step in translation is required for the expression of almost all eukaryotic genes, yet the mechanism and dynamics of scanning are unknown. We have performed quantitative studies in vivo and in vitro of the movement of yeast 40S ribosomes along 5′ untranslated regions (UTRs) of different lengths. 40S subunits perform cap‐dependent scanning with high processivity for more than 1700 nucleotides in cells of Saccharomyces cerevisiae. Moreover, the observed rates of expression indicate that scanning is performed by an untethered 40S subunit that has been released from the 5′ cap complex. Unexpectedly, the capability to maintain scanning competence on a long 5′ UTR is more dependent on the Ded1/Dbp1 type of helicase than on eIF4A or eIF4B. In a yeast cell‐free extract, scanning shows reduced processivity, with an estimated net 5′→3′ rate of approximately 10 nucleotides per second at 26°C. We have developed a biased bidirectional walking model of ribosomal scanning that provides a framework for understanding the above observations as well as other known quantitative and qualitative features of this process.