bluuebunny/crossref_metadata_2025_split huggingface dataset:
The molar ratio of σ73 to core polymerase in the obligate intracellular bacterium, Rickettsia prowazekii
Authors: Ding Han‐Fei , Winkler Herbert H. 
Published: October 2006

SummaryIn the obligate Intracellular parasitic bacterium, Rickettsia prowazekii, the molar ratio of σ73 to core RNA polymerase, that is, the degree of saturation of the core polymerase by the catalytically active sigma factor, was very low. This ratio was determined from the radioactivity in rickettsial RNA polymerase immuno‐precipitated from crude extracts of infected L929 cells in which the parasite was exponentially growing. If we assume that, as Is true for the σ subunit, in R. prowazekii and Escherichia coli the β’and β subunits of the RNA polymerase have similar methionine and cysteine contents (the radiolabelled amino acids), the molar ratio of σ73 to core polymerase in R prowazekii would be 0.1. This is in striking contrast to E. Coli where the ratio is typically 0.4. it remains to be established whether this low sigma saturation results In a limitation of active RNA polymerase in R. prowazekii and contributes to its slow growth.
A louse involved in the regulation of replication in plasmid pSC 101
Authors: Manen Danielle , Xia Guixian , Caro Lucien 
Published: October 2006

SummaryThe origin of replication of plasmid pSC101 contains three directly repeated sequences RS1, RS2, and RS3 separated by 22 bp from two palindromic sequences, IR1 and IR2, which are partially homologous to the direct repeats. These inverted repeat (IR) sequences overlap the promoter of the repA gene which encodes a protein essential for plasmid replication. We have shown that RepA binds to the RS sites as a monomer and to the IR sites as a dimer. The influence of the IR1 site, and of the DNA segment that separates it from RS3, on plasmid copy number control has been studied in detail. We show that the integrity of IR1 is essential for efficient replication and plasmid stability, the critical site extending to the left of IR1 proper. We also show that the presence of IR1 modifies profoundly the binding properties of purified RepA protein to a segment of DNA containing the RS sequences. IR1 is separated from its homologous site on RS3 by approximately four turns of the DNA helix. Replication is abolished if this distance is increased by half a turn of the helix but it is restored if the distance is increased by a whole turn. These results suggest a DNA looping interaction, in the initiation of replication, between the RepA dimer that binds iR1 and the RepA monomers that bind the RS sequences.
Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis
Authors: Gold S. E. , Kronstad J. W. 
Published: October 2006

SummaryThe phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non‐pathogenic, yeast‐like cells mate to form a pathogenic, filamentous dikaryon. Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U. maydis are expressed at similar levels in yeast‐like cells and in cells undergoing the mating reaction leading to the filamentous cell type. A mutation was constructed in each of the chitin synthase genes by targeted gene disruption. Each mutant showed a reduction in the level of trypsin‐activated enzyme activity, compared with a wild‐type strain, but retained the wild‐type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type.
Escherichia coli cad operon functions as a supplier of carbon dioxide
Authors: Takayama Miho , Ohyama Takao , Igarashi Kazuel , Kobayashi Hiroshi 
Published: October 2006

SummaryWe examined the gene expression of the Escherichia coli cad operon, which consisted of the genes cadB and cadA (lysine decarboxylase), using cells possessing cadB–lacZ fusion gene. The cad operon was expressed when O2 was limited, and the expression was optimal at pH6.3. The β‐galactosidase activity was lowered by the addition of sodium carbonate to the medium. The expression of the cad operon was reduced in cells containing the plasmid‐encoding ornithine decarboxylase, which produced carbon dioxide, indicating that the gene expression of the cad operon was regulated by carbon dioxide (or its derivatives). It is known that the Krebs cycle is a major pathway for producing carbon dioxide, and that its activity is repressed when O2 is limited. Thus, our present results suggested that the physiological role of the cad operon is to supply carbon dioxide when its internal level is lowered under O2‐limiting conditions at a low pH.
Coupling between mRNA synthesis and mRNA stability in Escherichia coli
Authors: Chow Janet , Dennis Patrick P. 
Published: October 2006

SummaryTransiently stable products derived from the endonuclease cleavage of transcripts from the secEnusG and rplKAJLrpoBC operons have been identified. Cleavage sites for RNase III occur in the leader of the secEnusG transcript and in the L12‐β intercistronic space of the rplKAJLrpoBC transcript. A single RNase E cleavage site was located in the L1‐L10 intergenic space. Inactivation of RNase III and RNase E results respectively in a one‐ to twofold and a greater than 10‐fold stabilization of five mRNA sequences from within the secE, nusG, L11‐L1, L10 and β encoding cistrons. The relative amounts of each of these five mRNA sequences were found to be nearly constant when measured either in the presence or absence of cleavage by RNase III or RNase E. This clearly implies that any increases in the stability of these mRNA sequences resulting from the inactivation of processing by RNase III or RNase E are counterbalanced by changes in the mRNA synthesis rates. The mechanism that links mRNA synthesis to mRNA decay is not known.
Repression of tonB transcription during anaerobic growth requires Fur binding at the promoter and a second factor binding upstream
Authors: Young Glenn M. , Postle Kathleen 
Published: October 2006

SummaryAlthough iron is an essential nutrient, its toxicity at high levels necessitates regulated transport. In Gram‐negative bacteria a central target for regulation is the TonB protein, an energy transducer that couples the cytoplasmic membrane proton motive force to active transport of (FeIII)‐siderophore complexes across the outer membrane. We have previously demonstrated the threefold repression of tonB transcription by excess iron in the presence of Fur repressor protein under aerobic conditions. In this report, we examine tonB regulation under anaerobic conditions where the solubility of iron is not a limiting factor and, presumably, siderophore‐mediated transport is not required. Under these conditions, tonB transcription is repressed at least 10‐foid by excess iron in the presence of Fur, but can be fully derepressed in the absence of Fur. Based on several lines of evidence, this anaerobic repression is not due to increased negative supercoiling as previously postulated. Our results rule out both supercoiling mediated decreased promoter function and increased Fur binding as mediators of anaerobic repression. Under iron‐limiting anaerobic conditions tonB expression is as high or higher than under iron‐limiting aerobic conditions, suggesting that promoter function has not decreased anaerobically. Furthermore, under anaerobic conditions in tonB+ strains, tonB promoter function is insensitive to the gyrase inhibitor novobiocin and to changes in medium osmolarity and temperature, three conditions known to change levels of supercoiling. We also rule out effects of mutations in arcA or fnr as mediators of anaerobic repression. Results from in vivo dimethyl sulphate protection foot‐printing indicate that Fur binds to an operator site between the ‐10 and ‐35 regions of the promoter but not to a less homologous operator site centered at +26. The binding is, if anything, weaker under anaerobic conditions, indicating that anaerobic repression is not mediated through Fur. Additional changes in the in vivo footprint upstream from the promoter implicate a second factor in tonB anaerobic repression. Together, these results suggest that the mechanism responsible for this regulation (and, by analogy, that of other anaerobically repressed, iron‐regulated genes such as cir, exbB, and fhuA) is a novel one.
Isolation and characterization of hypophosphite‐resistant mutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter
Authors: Suppmann Bernhard , Sawers Gary 
Published: October 2006

SummaryHypophosphite was used as a toxic analogue to identify genes whose products have a putative function in the transport of formate. Two Tn10‐derived insertion mutants were identified that exhibited increased resistance to high concentrations of hypophosphite in the culture medium. The transposon was located in the identical position in the focA (formate channel; previously termed orf) gene of the pfl operon in both mutants. A defined chromosomal focA nonsense mutant, which showed minimal polarity effects on pfl gene expression, had the same phenotype as the insertion mutants. Results obtained using a hycA‐lacZ fusion to monitor changes in the intracellular formate concentration in a focA mutant indicated that the level of formate inside the cell was elevated compared with the wild type. Moreover, it could be shown that there was a corresponding reduction of approximately 50% in the amount of formate excreted by a focA mutant into the culture medium. Taken together, these results indicate that formate accumulates in anaerobic ceils which do not have a functional focA gene product and that one function of FocA may be to export formate from the cell. A further significant result was that hypophosphite could substitute for formate in activating hycA gene expression. This hypophosphite‐dependent activation of hycA gene expression was reduced 10‐fold in a focA null mutant, suggesting that hypophosphite must first enter the cell before it can act as a signal to activate hycA expression. By analogy, these data suggest that FocA may also be functional in the import of formate into anaerobic Escherichia coli cells.Site‐specific mutagenesis identified the translation initiation codon of focA as a GUG. Therefore, the FocA polypeptide has a molecular weight of 30958. FocA shows significant similarity at both the primary and secondary structural levels with the NirC protein of E. coli and the FdhC protein of Methanobacterium formicicum. All three proteins are predicted to be integral membrane proteins. A detailed in vivo TnphoA mutagenesis study predicted that FocA has six membrane‐spanning segments.
Rho and RNA: models for recognition and response
Authors: Platt Terry 
Published: October 2006

Summary Escherichia coli Rho factor Is required for termination of transcription at certain sites by RNA polymerase. Binding to unstructured cytosine‐containing RNA target sites, subsequent RNA‐dependent ATP hydrolysis, and an RNA‐DNA helicase activity that presumably facilitates termination, are considered essential for Rho function. Yet the RNA recognition elements have remained elusive, the parameters relating RNA binding to ATPase activation have been obscure, and the mechanistic steps that integrate Rho's characteristics with its termination function in vitro and in vivo have been largely undefined. Recent work offers new insights into these interactions with results that are both surprising and satisfying in the context of Rho's emerging structure. These include the requirements for binding and ATPase activation by a variety of RNA substrates, dynamic analyses of Rho tracking, helicase and termination activity, and the participation of a new factor (NusG) that interacts with Rho. Models for Rho function are considered in the light of these recent revelations.
Regulation of the Bacillus subtilis trp operon by an RNA‐binding protein
Authors: Gollnick Paul 
Published: October 2006

SummaryThe Bacillus subtilis tryptophan (trpEDCFBA) operon is regulated by transcription attenuation. Transcription is controlled by two alternative RNA secondary structures, which form in the leader transcript. In the presence of L‐tryptophan, a transcription terminator forms and the operon is not expressed, whereas in the absence of tryptophan, an antiterminator structure forms allowing transcription of the operon. The mechanism of selection between these alternative structures involves a trans‐acting RNA‐binding regulatory protein. This protein is the product of the mtrB gene and is called TRAP for trp attenuation p/rotein. TRAP has been shown to bind specifically to trp leader RNA, and to cause transcription of the trp operon to terminate in the leader region. The model for regulation suggests that in the presence of tryptophan, TRAP binds to the leader RNA and induces formation of the transcription terminator structure, whereas in the absence of tryptophan, the protein does not bind and the antiterminator is formed.
Structural dynamics of translating ribosomes: 16S ribosomai RNA bases that may move twice during translocation
Authors: Laughrea Michael 
Published: October 2006

SummaryRecent footprinting, sedimentation and neutron‐scattering results obtained In vivo or on pre‐translocation and post‐translocation ribosomal complexes are integrated with cross‐linking and immunoelectron microscopy information, it is proposed that the 30S subunit pulses during translocation and that its preand post‐translocation structures are not necessarily identical. Accordingly, transiocation is characterized by three consecutive conformational states of the 30S and 50S subunits. State 1 (the pre‐translocation state) lasts until the elongation factor EF‐G·GTP complex binds to the ribosome or adopts the GTPase conformation. State 2 (the translocation state, or the peak or plateau of the pulse) follows and lasts until EF‐G adopts a subsequent conformation or is released from the ribosome. State 3 (the post‐translocation state) ensues and lasts until A (aminoacyl) site binding of aminoacyl‐tRNA. In state 2, 16S RNA hairpins 26 and 33‐33A, located in the platform and the head of the 30S subunit, respectively, become kinked or twisted, and residue A1503, near the decoding site, becomes exposed. A platform twist is associated with P (peptide) to E (exit) site tRNA movements and a head twist with pivoting of the peptidyl‐tRNA elbow from the A to the P site, around a (retractable?) S19 domain. These twists result in an unlocking of the platform and the head from the 50S subunit. Exposure of A1503 is tentatively associated with movements of mRNA or tRNA anticodon stem‐loops. These twisted or otherwise‐exposed residues readopt their previous setting upon completion of translocation, i.e. states 1 and 3 of 16S RNA differ more from state 2 than from each other. Yet the ribosome is never fully locked or unlocked at any time during elongation. It is unlocked in one or another respect in the pre‐ and post‐translocation states, and unlocked to the largest extent in state 2.‘Le plus grand Phénomène de la Nature, le plus Merveilleux, est le Mouvement’ (The greatest, the most wonderful Phenomenon in Nature is Motion).Pierre Moreau de Maupertuis
Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by the rpoS(katF) gene
Authors: Chang Ying‐Ying , Wang Ai‐Yu , Cronan John E. 
Published: October 2006

SummaryThe activity of Escherichia coli pyruvate oxidase (PoxB) was shown to be growth‐phase dependent; the enzyme activity reaches a maximum at early stationary phase. We report that PoxB activity is dependent on a functional rpoS(katF) gene which encodes a σ factor required to transcribe a number of stationary‐phase‐induced genes. PoxB activity as well as the β‐galactosidase encoded by a poxB::lacZ protein fusion was completely abolished in a strain containing a defective rpoS gene. Northern and primer extension analyses showed that poxB expression was regulated at the transcriptional level and was transcribed from a single promoter; the 5′ end of the mRNA being located 27 bp upstream of the translational initiation codon of poxB. The poxB gene was expressed at decreased levels under anaerobiosis; however, the anaerobic regulatory genes arcA, arcB or fnr were not involved in anaerobic poxB gene expression. Expression of the rpoS(katF) gene has been reported to be affected by acetate, the product of PoxB reaction. However, we found that poxB null mutations had no effect on rpoS(katF) expression. Inactivation of two genes involved In acetate metabolism, ackA and pta, had no effect on either poxB or rpoS(katF) expression.
Starvation‐induced expression of SspA and SspB: the effects of a null mutation in sspA on Escherichia coli protein synthesis and survival during growth and prolonged starvation
Authors: Williams Mark D. , Ouyang Tracy X. , Flickinger Michael C. 
Published: October 2006

SummaryMaxicell labelling and two‐dimensional gel electro‐phoresis (2‐D PAGE) have identified the proteins encoded by sspA and sspB (SspA, SspB) as proteins D27.1 and A25.8, respectively, in the Escherichia coli gene‐protein database. SspA expression increases with decreasing growth rate and is induced by glucose, nitrogen, phosphate or amino acid starvation. The promoter, Pssp, is similar to gearbox promoters. Inactivation of SspA (sspA::neo) blocks sspB expression. [35S]‐methionine‐labelled proteins synthesized during growth and during stationary phase are different in δsspA strains compared to sspA strains. This difference is enhanced during extended stationary phase (24–72 h). Long‐term (10 d) viability of arginine‐starved isogenic strains shows that sspA cultures remain viable significantly longer than δsspA mutants. 2‐D PAGE of proteins expressed during exponential growth shows that expression of at least 11 proteins is altered in δsspA strains. A functional relA gene is required for sspA to affect protein synthesis.
Characterization of the carbon starvation‐inducible and stationary phase‐inducible gene slp encoding an outer membrane lipoprotein in Escherichia coli
Authors: Alexander Denise M. , St John Ann C. 
Published: October 2006

Summary Escherichia coli induces the expression of more than 50 proteins in response to starvation for a carbon source. Strains MC7 (csi7::phoA) and MC19 (csi19::phoA) contain fusions of a signal peptide‐deficient phoA reporter sequence to a csi (carbon starvation‐inducible) gene. PhoA expression increased when these strains were deprived of a carbon source or entered stationary phase but did not when the cells were deprived of a nitrogen source or subjected to osmotic, oxidative or thermal stress. Mapping and sequence analysis of the cloned phoA fusions in strains MC7 and MC19 indicated that they had occurred in different locations within the same previously unidentified gene. The wild‐type allele of this gene was cloned and the encoded protein was found to be a new lipoprotein. Therefore we propose to call this locus slp (starvation lipoprotein). The 22 kDa Slp protein is associated with the outer membrane fraction. The slp gene was located at 78.6 centisomes on the E. coli genetic map. The ‐10 and ‐35 regions upstream of the mRNA start site were characteristic of a σ70 promoter. The major transcript from this promoter was sufficiently large to contain slp sequences but not the downstream open reading frame. Induction of β‐galactosidase activity from a slp::lacZ translational fusion during carbon starvation or stationary phase was independent of cAMP, RpoS (KatF) and DnaK, all of which are known to affect the expression of certain starvation‐inducible or stationary phase‐inducible proteins.
Conjugative transposition of Tn916: preferred targets and evidence for conjugative transfer of a single strand and for a double‐stranded circular intermediate
Authors: Scott June R. , Bringel Françoise , Marra Diana , Van Alstine Gaylene , Rudy Christine K. 
Published: October 2006

SummaryTransposition of conjugative transposons proceeds by excision and formation of a covalently closed circular Intermediate that includes at its joint the six flanking bases from its previous host (coupling sequences). To elucidate the role of the coupling sequences in this process and to determine the sequence of targets used by Tn916, we studied its insertion into a plasmid following conjugation. The results differ from those previously observed when Tn916 was introduced by transformation. They suggest that only one specific strand of the transposon molecule is transferred during the conjugation event and that complementary strand synthesis produces a double‐stranded transposon circle with no mismatches which serves as the reaction intermediate. Tn916 inserts preferentially at specific sites and the same targets are used when Tn916 comes from donors with different coupling sequences. An analysis of the sequences of preferred targets is presented.
A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein
Authors: Mhammedi‐Alaoul Amine , Pato Martin , Gama Marie‐Jo , Toussaint Ariane 
Published: October 2006

SummaryWe have shown previously that some particular mutations in bacteriophage Mu repressor, the frameshift vir mutations, made the protein very sensitive to the Escherichia coli ATP‐dependent Clp protease. This enzyme is formed by the association between a protease subunit (ClpP) and an ATPase subunit. ClpA, the best characterized of these ATPases, is not required for the degradation of the mutant Mu repressors. Recently, a new potential ClpP associated ATPase, ClpX, has been described. We show here that this new subunit is required for Mu vir repressor degradation. Moreover, ClpX (but not ClpP) was found to be required for normal Mu replication. Thus ClpX has activities that do not require its association with ClpP. In the pathway of Mu replicative transposition, the block resides beyond the strand transfer reaction, i.e. after the transposition reaction per se is completed, suggesting that ClpX is required for the transition to the formation of the active replication complex at one Mu end. This is a new clear‐cut case of the versatile activity of polypeptides that form multi‐component ATP‐dependent proteases.
Transcriptional regulation of prfA and PrfA‐regulated virulence genes in Listeria monocytogenes
Authors: Bohne Jutta , Sokolovic Zeljka , Goebel Werner 
Published: October 2006

SummaryThe ActA protein, the lecithinase PlcB and listeriolysin are the major PrfA‐dependant proteins synthesized when brain‐heart infusion (BHI)‐cultured Listeria monocytogenes is shifted to minimum essential medium (MEM) In the presence of the transcriptional inhibitor rifampicin. Enhanced synthesis of all three proteins under these conditions depends, however, on a short incubation (about 5 min) of the bacteria in MEM without rifampicin, suggesting that induction of these proteins in MEM requires de novo transcription. The enhanced synthesis of these three proteins is observed in the L. monocytogenes wild‐type strains EGD and NCTC 7973, both of which belong to the serotype 1/2a. A significant induction of the bicistronic mRNA for ActA and PlcB is observed in both strains shortly after shifting the bacteria from BHI to MEM. This mRNA as well as the monocistronic listeriolysin (hly)‐specific mRNA is highly stable in L. monocytogenes NCTC 7973 shifted to MEM. In contrast to the actA‐plcB mRNA, no enhanced transcription in MEM is observed for the regulatory prfA gene or for the PrfA‐controlled virulence genes hlyA and plcA in strain NCTC 7973. However, transcription of these genes is induced in strain EGD. Transcriptional induction of the mpl gene is observed in neither strain NCTC 7973 nor in strain EGD. The life‐time of the prfA, plcA, and mpl transcripts is short. ActA was also found to be the most abundant newly synthesized surface protein when the two wild‐type strains of L. monocytogenes replicated within the phagocytic cell line J774. ActA synthesis seemed to be induced in the cytoplasm since the non‐haemolytic mutant M3 did not induce ActA when taken up by J774 cells.