bluuebunny/crossref_metadata_2025_split huggingface dataset:
IS 10 mRNA stability and steady state levels in Escherichia coli: indirect effects of translation and role of rne function
Authors: Jain Chaitanya , Kleckner Nancy 
Published: October 2006

SummaryTranslation of the IS 10 transposase gene is known to be very infrequent. We have identified mutations whose genetic properties suggest that they act directly to increase or decrease the intrinsic level of translation initiation. Also, we have analysed in detail the effects of these mutations on IS 10 mRNA using one particular IS 10 derivative. In this case, increases or decreases in translation are accompanied by increases or decreases in both the steady state level and the half‐life of transposase mRNA; effects on steady state levels are much more dramatic than effects on message half‐life. At wild‐type levels of translation initiation, the rate‐limiting step in physical decay of full length IS 10 message for a particular IS 10 derivative is shown to be rne‐dependent endonucleolytic cleavage; 3′ exonucleases appear to play a secondary role, degrading primary cleavage products. Analysis of interplay between translation mutations and rne function, together with the above observations, suggests that translation stabilizes messages in a general way against rne‐dependent endonucleolytic cleavage, and that significant protection may be conferred by one or a few ribosomes. However, dramatic effects of translation on steady state message levels are still observed in an rne mutant and involve the 3′ end of the transcript; we propose that these additional effects reflect translation‐mediated stimulation of transcript release.
Preferential cis action of IS 10 transposase depends upon its mode of synthesis
Authors: Jain Chaitanya , Kleckner Nancy 
Published: October 2006

SummaryA number of bacterial DNA‐binding proteins, including IS element transposases, act preferentially in cis. We show below that the degree of preferential cis action by IS 10 transposase depends upon its mode of synthesis at steps subsequent to transcription initiation. Cis preference is increased several fold by mutations that decrease translation initiation, by the presence of IS 10‐specific antisense RNA and by plasmids that increase the level of cellular RNases. Conversely, cis preference is decreased by mutations that increase translation initiation; in some cases, cis preference is nearly abolished. Mutations that alter the rate of transcription initiation have no effect. In light of other observations, we suggest that cis preference is strongly dependent upon the rate at which transcripts are released from their templates and/or the half‐life of the transposase message. These observations provide further evidence that inefficient translation plays multiple roles in the biology of IS 10.
The int genes of bacteriophages P22 and λ are regulated by different mechanisms
Authors: Wulff Daniel L. , Ho Yen Sen , Powers Susan , Rosenberg Martin 
Published: October 2006

SummaryBacteriophage P22 and λ are related bacteriophages with similar gene organizations. In λ the cII‐dependent PI promoter is responsible for λint gene expression. The only apparent counterpart to PI in P22 is oriented in the opposite direction, and cannot transcribe the P22 int gene. We show that this promoter, called Pal, is active both in vivo and in vitro, and is dependent upon the P22 cII‐like gene, called c1. We have also determined the DNA sequence of a 3.3 kb segment that closes the gap between previously reported sequences to give a continuous sequence between the P22 pL promoter and the int gene. The newly determined sequence is densely packed with genes from the pL direction, and the proteins predicted by the sequence show excellent correlation with the proteins mapped by Youderian and Susskind in 1980. However, the sequence contains no apparent genes in the opposite (pal) direction, and no additional binding motifs for the P22 c1 protein. We conclude that int gene expression in P22 is regulated by a different mechanism than in λ.
Hc1‐mediated effects on DNA structure: a potential regulator of chlamydial development
Authors: Barry Clifton E. , Brickman Timothy J. , Hackstadt Ted 
Published: October 2006

SummaryChlamydiae are obligate intracellular bacteria which undergo a unique developmental cycle, alternating between non‐replicative elementary bodies (EBs) and replicatlve reticulate bodies (RBs). The transition from RB to EB is characterized by condensation of the chromosome into a dense nucleoid structure. The chlamydial histone homologue Hc1 is sufficient to induce formation of a similar structure in Escherichia coli. High‐level Hc1 expression in E. coli is self‐limiting and down‐regulates transcription, translation, and replication at concentrations similar to those observed in chlamydial elementary bodies. Expression of Hc1 at sub‐structural levels may have specific regulatory functions through its interaction with chromosomal DNA, In E. coli this is reflected in a dramatic shift in the pattern of gene expression. The differential expression of the outer membrane porin proteins OmpC and OmpF and analysis of lacZ fusions with promoter regions sensitive to supercoiling suggests that low‐level Hc1 expression results in a net relaxation of chromosomal DNA. Topological analysis of plasm id DNA from both E. coli and Chlamydia trachomatis supports a decrease in superhelicity preceding nucleoid formation. In vitro analysis of purified Hc1–DNA interactions supports preferential binding based upon DNA conformation. These results suggest a dual role in which Hc1‐mediated changes in gene expression may precede metabolic inactivity.
Pleiotropic effects of the rpoC10 mutation affecting the RNA polymerase β′ subunit of Escherichia coli on factor‐dependent transcription termination and antitermination
Authors: Ito Koichi , Nakamura Yoshikazu 
Published: October 2006

Summary Escherichia coli RNA polymerase is composed of four different subunits, 2α, β, β′ and σ;. Among these subunits, the role of β′ is poorly understood. The rpoC10 mutation affecting β′ has been isolated as a suppressor mutation of the temperature‐sensitive nusA11 mutant. DNA sequence analysis revealed that the rpoC10 mutant is a substitution of Lys for Glu‐402. This increased positive charge appears to compensate for the increased negative charge present in the nusA11 protein (Asp for Gly‐181). In vivo measurements of reporter gene expression have revealed that rpoC10 restores ρ‐dependent termination but fails to restore ρ‐independent termination in nusA.11 Moreover, the rpoC10 mutation, in combination with any nusA mutation, inhibited λ Q‐mediated antitermination without affecting N antitermination and severely restricted λ phage development. The inhibition of Q function and λ growth could be compensated for by overproducing Q. These results suggest that the RNA polymerase β′ subunit plays a crucial rote in factor‐dependent transcription termination and antitermination.
Processing and methylation of PulG, a pilin‐like component of the general secretory pathway of Klebsiella oxytoca
Authors: Pugsley Anthony P. 
Published: October 2006

SummaryThe signal sequence of the Klebsiella oxytoca pulG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me‐Phe) pilins. The significance of this similarity is confirmed by the observation that the pulO gene product processes prePulG at the consensus type IV prepilin peptidase cleavage site at the amino‐terminal end of the PulG signal sequence. Like most type IV pilins, processed PuiG was found to have a methylated amino‐terminal phenylaianine residue. Site‐directed mutagenesis was used to replace amino acids in prePulG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino‐terminal side of the prePulG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11 (+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently IV‐methylated. Cleavage of prePulG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the car‐boxy‐terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePulG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.
Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP‐L‐rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems
Authors: Zhang Lijuan , Al‐Hendy Ayman , Toivanen Paavo , Skumik Mikael 
Published: October 2006

SummaryThe Yersinia enterocolitica O:3 lipopotysaccharide O‐antigen is a homopotymer of 6‐deoxy‐L‐altrose. The cloned rfb region was sequenced, and 10 open reading frames were identified. Transposon mutagenesis, deletion analysis and transcomplementatton experiments showed that eight of the genes, organized into two operons, rfbABC and rfbDEFGH, are essential for 0‐antigen synthesis. Functional tandem promoters were identified upstream of both operons. Of the deduced polypeptides RfbA, RfbF and RfbG were similar to Salmonella proteins involved in the dTDP‐l‐rhamnose biosynthesis. Rhamnose and 6‐deoxy‐l‐altrose are C3‐epimers suggesting that analogous pathways function in their biosynthesis. RfbD and RfbE were similar to capsular polysaccharide export proteins, e.g. KpsM and KpsT of Escherichia coli. This and transposon mutagenesis showed that RfbD and RfbE function as O‐antigen exporters.
Cloning, expression and sequencing of Helicobacter felis urease genes
Authors: Ferrero Richard L. , Labigne Agnès 
Published: October 2006

SummaryUrease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A‐digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen‐limiting medium. Subcloning of DNA from an urease‐positive cosmid cione led to the construction of plLL205 (9.5 kb) which conferred a urease activity of 1.2±0.5 μmole urea min‐1 mg‐1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen‐limiting medium. Random mutagenesis using a MiniTn3‐Km transposable element permitted the identification of three DNA regions on plLL205 which were necessary for the expression of an urease‐positive phenotype in E. coii clones. To localize the putative structural genes of H. felis on plLL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti‐H. felis rabbit serum. One mutant cione did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had caicuiated moiecuiar masses of 26 074 and 61 663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.
A pleiotropic reduced virulence (Rvi−) mutant of Erwinia carotovora subspecies atroseptica is defective in flagella assembly proteins that are conserved in plant and animal bacterial pathogens
Authors: Mulholland Vincent , Hinton Jay C. D. , Sidebotham Julie , Toth Ian K. , Hyman Lisabeth J. , Perombelon Michel C. M. , Reeves Philip J. , Salmond George P. C. 
Published: October 2006

Summary Erwinia carotovora subsp. atroseptica was mutage‐nized and assayed for virulence in planta. Those mutants which exhibited reduced virulence (Rvi‐) were assayed for growth rate, auxotrophy and extracellular enzyme secretion and seven mutants were found to be wild type for all of these phenotypes. When screened for other phenotypes, two were found to be non‐motile. One mutant was complemented for motility by a heterologous gene library. A 2.7kb XmaIII‐Clal complementing fragment was sequenced and the gene products were found to have similarity to flagella biosynthesis gene products from several bacteria. Further similarity was found to a pathogenicity protein from the plant pathogen Xanthomonas campestris pv. glycines and to the Spa pathogenicity proteins of the human pathogen Shigella fiexneri, which are involved in the surface presentation of antigens. These studies highlight the emergence of common themes in the molecular strategies employed by both plant and animal bacterial pathogens for the targeting of proteins involved in the elaboration of disease.
Generation of isogenic K54 capsule‐deficient Escherichia coli strains through TnphoA‐mediated gene disruption
Authors: Russo Thomas A. , Guenther Jane E. , Wenderoth Suzanne , Frank Michael M. 
Published: October 2006

SummaryTransposon muta genesis, using IS50L::phoA(Tn‐phoA), was performed in a K54/O4/H5 blood isolate of Escherichia coli (CP9), to generate a library of random mutants. Five hundred and twenty‐six independent CP9 TnphoA mutants were isolated with active gene fusions to alkaline phosphatase. From this mutant library, eight capsule‐deficient strains were detected and were found to have a single copy of TnphoA. Sixteen additional capsule deficient mutants with TnphoA inserts were subsequently obtained that did not possess active PhoA fusions. In conjunction with the initial eight capsule‐deficient isolates we have defined genes on three different XbaI fragments as being involved in capsule production. Generalized transduction with the bacteriophage T4 established that these insertions were responsible for the loss of capsule and that they are linked. These capsule‐deficient strains can be used to assess the pathogenic role of the K54 capsular polysaccharide.
Sequence and properties of mecA, a negative regulator of genetic competence in Bacillus subtilis
Authors: Kong Liyun , Siranosian Kathryn Jaacks , Grossman Alan D. , Dubnau David 
Published: October 2006

SummaryThe development of competence in Bacillus subtilis is regulated by growth conditions and several regulatory genes. In complex media competence development is poor, and there is little or no expression of late competence genes, mec mutations permit competence development and late competence gene expression in complex media, and bypass the requirements for many of the competence regulatory genes. In this paper we describe the cloning and characterization of mecA. The mecA gene product acts negatively in the development of competence. Null mutations in mecA allowed expression of a late competence gene comG, under conditions where it is not normally expressed, including in complex media and in cells mutant for several competence regulatory genes. Overexpression of MecA from a multicopy plasmid resulted in inhibition of comG transcription. The DNA sequence of mecA was determined and the predicted gene product showed no significant similarity to any protein in the database. Expression of a mecA–lacZ translational fusion was constitutive during growth and did not vary significantly in the different media tested. The rote of mec A in competence development and other stationary phase phenomena is discussed.
Isolation and characterization of hydroxylamine‐induced mutations in the Erwinia herbicola ice nucleation gene that selectively reduce warm temperature ice nucleation activity
Authors: Gurian‐Sherman Douglas , Lindow Steven E. , Panopoulos Nickolas J. 
Published: October 2006

SummaryCells of ice nucleation active bacterial species catalyse ice formation over the temperature range of ‐2 to ‐12°C. Current models of ice nucleus structure associate the size of ice nucleation protein aggregates with the temperature at which they catalyse ice formation. To better define the structural features of ice nucleation proteins responsible for the functional heterogeneity of ice nuclei within a genetically homogeneous collection of cells we used in vitro chemical mutagenesis to isolate mutants with reduced ability to nucleate ice at warm assay temperatures but which retain normal or near normal nucleation activity at cold temperatures (WIND, i.e. warm ice nucleus‐deficient mutants). Nearly half of the mutants obtained after hydroxylamine mutagenesis of the iceE gene from Erwinia herbicola had this phenotype. The phenotypes and location of lesions on the genetic map of iceE were determined for a number of mutants. All WIND mutations were restricted to the portion of iceE encoding the repetitive region of the poty peptide. DNA sequencing of two WIND mutants revealed single nucleotide substitutions changing a conserved serine or glycine residue to phenylalanine and serine, respectively. The implications of these findings in structure/function models for the ice nucleation protein are discussed.
Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1
Authors: Klena J. D. , Schnaitman C. A. 
Published: October 2006

SummaryA plasmid that included both an 8.9 kb chromosomal DNA insert containing genes from the rfb cluster of Shigella dysenteriae 1 and a smaller insert containing the rfp gene from a S. dysenteriae 1 multicopy plasmid resulted in efficient expression of O antigen in an rfb‐deleted strain of Escherichia coli K‐12. Eight genes were identified in the rfb fragment: the rfbB‐CAD cluster which encodes dTDP‐rhamnose synthesis, rfbX which encodes a hydrophobic protein involved in assembly of the O antigen, rfc which encodes the O antigen polymerase, and two sugar transferase genes. The production of an O antigen also required the E. coli K‐12 rfe gene, which is known to encode a transferase which adds N‐acetyl‐glucosamine phosphate to the carrier lipid unde‐caprenol phosphate. Thus Rfe protein appears to function as an analogue of the Salmonella RfbP protein to provide the first sugar of the O unit. Functional analysis of the other genes was facilitated by the fact that partial O units of one, two or three sugars were efficiently transferred to the lipopolysaccharide core. This analysis indicated that the plasmid‐encoded Rfp protein is the transferase that adds the second sugar of the O unit while the two rfb transferases add the distal sugars to make an O antigen whose structure is (Rha–Rha–Gal–GlcNAc)n. The use of the rfe gene product as the transferase that adds the first sugar of an O unit is a novel mechanism which may be used for the synthesis of other enteric O antigens.
Nitrate regulation of anaerobic respiratory gene expression in Escherichia coli
Authors: Stewart Valley 
Published: October 2006

SummarySynthesis of most anaerobic respiratory pathways is subject to dual regulation by anaerobiosis and nitrate. Anaerobic induction is mediated by the FNR protein. Dual interacting two‐component regulatory systems mediate nitrate induction and repression. The response regulator protein NARL binds DNA to control nitrate induction and repression of genes encoding nitrate respiration enzymes and alternate anaerobic respiratory enzymes, respectively. The homologous protein NARP controls nitrite induction of at least two operons. Nitrate and nitrite signalling are both mediated by the homologous sensor proteins NARX and NARQ. Recent mutational analyses have defined a heptamer sequence necessary for specific DNA binding by the NARL protein. These heptamers are located at different positions in the control regions of different operons. The NARL protein‐binding sites in the narG (nitrate reductase) and narK (nitrate‐nitrite antiporter) operon control regions are located approximately 200 bp upstream of the transcription initiation site. The integration host factor (IHF) greatly stimulates nitrate induction of these operons, indicating that a specific DNA loop brings NARL protein, bound at the upstream region, into the proximity of the promoter for transcription activation. Other NARL protein‐dependent operons do not require IHF for nitrate induction, and the arrangement of NARL heptamer sequences in these control regions is quite different. This complexity of signal transduction pathways, coupled with the diversity of control region architecture, combine to provide many interesting areas for future investigation. An additional challenge is to determine how or if the FNR and NARL proteins interact to mediate dual positive control of transcription initiation.
The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration
Authors: Wang Xunde , Lutkenhaus Joe 
Published: October 2006

SummaryThe ftsZ gene is essential for cell division in both Escherichia coli and Bacillus subtilis. In E. coli FtsZ forms a cytokinetic ring at the division site whose formation is under cell‐cycle control. In addition, the FtsZ from E. coli has a GTPase activity that shows an unusual lag in vitro. In this study we show that FtsZ in Bacillus subtilis forms a ring that is at the tip of the invaginating septum. The FtsZ ring is dynamic since it is formed as division is initiated, changes diameter during septation, and disperses upon completion of septation. In vitro the purified FtsZ from B. subtilis exhibits a GTPase activity without a demonstrable lag, but the GTPase activity is markedly dependent upon the FtsZ concentration, suggesting that the FtsZ protein must oligomerize to express the GTPase activity.
The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen in Escherichia coli
Authors: van Heeswijk Wally C. , Rabenberg Maarten , Westerhoff Hans V. , Kahn Daniel 
Published: October 2006

SummaryRegulation of glutamine‐synthetase (GS) activity in enteric bacteria involves a complex cascade of events. In response to nitrogen limitation, a transferase catalyses the uridylylation of the PII protein, which in turn stimulates deadenylylation of GS. Deadenylylated GS is the more active form of the enzyme. Here we characterize in detail the genes from Escherichia coli encoding uridylyl‐transferase (gInD), the PII protein (glnB), and adenylyl‐transferase (glnE). glnD is transcribed from its own promoter, glnE is cotranscribed with another gene, orfXE, whereas glnB is partly cotranscribed with a gene encoding a homologue of the transcription activator NtrC. All three gln regulatory genes were constitutively expressed at a tow level, i.e. their expression was independent of the nitrogen status and the RNA polymerase sigma factor σ;54. We conclude that the functioning of the GS adenylylation cascade is regulated by modulation of the activities of uridylyl‐transferase and adenylyl‐transferase, rather than by changes in the expression of their genes.