bluuebunny/crossref_metadata_2025_split huggingface dataset:
Significance of plasmolysis spaces as markers periseptal annuli and adhesion sites
Authors: Woldringh Conrad L. 
Published: October 2006

SummaryDuring hyperosmotic shock, the protoplast and stretched‐out peptidoglycan layer first shrink together until the turgor pressure in the cell is relieved. Being non‐compressible, the outer and inner membranes must fold their superfluous surfaces. While the protoplast contracts further, the inner membrane rearranges into plasmolysis spaces visible by phase contrast microscopy. Two opposing theories predict a similar positioning of spaces in dividing cells and filaments: the ‘periseptal annulus model, based on adhesion zones, involved in the predetermination of the division site; and a ‘restricted, random model’, based on physical properties of the protoplast. Strong osmotic shock causes retraction of the inner membrane over almost the entire surface forming the so‐called ‘Bayer bridges’. These tubular adhesion sites are preserved by chemical fixation, and can be destroyed by cryofixation and freeze‐substitution of unfixed ceils. Both the regular positioning of the plasmolysis spaces and the occurrence of tubular adhesion sites can be explained on the basis of physical properties of the membrane which necessitate rearrangements by membrane flow during shrinkage of the protoplast.
Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae: cloning, identification and characterization of the α1,5 heptosyltransferase I gene (rfaC)
Authors: Zhou Daoguo , Lee Na‐Gyong , Apicella Michael A. 
Published: October 2006

SummaryThe identical partial deep‐core structure of Hepα1–3Hepα1–5KDO In Salmonella typhimurium LT2 LPS and Neisseria gonorrhoeae LOS enabled us to isolate a DNA fragment from N. gonorrhoeae that was able to complement the α1,5 LOS heptosyltransferase defect in the S. typhimurium rfaC630 (SA1377) mutant. SDS‐PAGE analysis confirmed the production of wild‐type LPS in the transformant. Subcloning revealed that complementation was due to a 1.2 kb fragment. Sequence analysis revealed a complete open reading frame capable of encoding a 36–37 kDa peptide. In vitro transcription‐translation analysis of the 1.2 kb clone confirmed that a 37 kDa protein was encoded by this DNA fragment. The DNA sequence‐deduced protein had 36% identity and 58% similarity to S. typhimurium heptosyltransferase I (RfaC). Primer extension analysis indicated that transcription of the cloned gene in N. gonorrhoeae strain 1291 begins 144bp upstream of the start codon at a G nucleotide. An isogenic mutant of N. gonorrhoeae strain 1291 with an m‐Tn3 insertion inside the coding sequence expressed a single truncated LOS with a similar molecular mass to S. typhimurium rfaC LPS. We conclude that the 1.2 kb fragment encodes the α1,5 LOS heptosyltransferase 1 (RfaC) in N. gonorrhoeae. Our studies also provide further evidence that the third KDO residue in S. typhimurium LPS is added after the core synthesis is completed.
Purification of a MaIE‐SoxS fusion protein and identification of the control sites of Escherichia coli superoxide‐inducible genes
Authors: Fawcett William P. , Wolf Richard E. 
Published: October 2006

SummaryIn Escherichia coli, the soxRS genes effect the cell's defence against superoxide by activating the transcription of more than 14 genes, including zwf, sodA, nfo, micF and fumC. Previous work from other laboratories has Indicated that SoxR is the sensor of oxidative stress and induces synthesis of SoxS, which in turn activates transcription of the regulon's target genes. Although SoxS appears to be a DNA‐binding protein, its ability to bind to the promoter regions of target genes has not been demonstrated. To facilitate purification and characterization of SoxS, we constructed a fusion of soxS to MalE, which encodes maltose‐binding protein, and demonstrated that the in vivo expression of the MaIE‐SoxS fusion protein can provide SoxS function to a soxRS deletion mutant. We purified the fusion protein by affinity chromatography on an amylose column. The purified fusion protein stimulated m vitro expression of zwf in a coupled transcription‐translation system and formed specific complexes with DNA fragments carrying target gene promoters. Moreover, MalE–SoxS protected from DNase I attack 22–27 bp segments immediately adjacent to or overlapping the −35 hexamers of the zwf, sodA, nfo, micF. and fumC promoters. The protected regions revealed a consensus ‘soxbox’ sequence.
Characterization and occurrence of two repeated palindromic DNA elements of Brucella spp.: Bru‐RS1 and Bru‐RS2
Authors: Halling Shirley M. , Bricker Betsy J. 
Published: October 2006

SummaryTwo repeated DNA elements of 103 bp and 105 bp were discovered in brucellae and designated Bru‐RS1 and Bru‐RS2, respectively. The two elements are palindromic, are 65% similar in sequence, form two families of elements that are slightly divergent in sequence, appear to be intergenic, and are found, collectively, in more than 35 copies in brucellae. These elements are bounded by perfect or nearly perfect inverted repeats. A third copy of the terminal repeat is found within the elements and is the terminus for several truncated copies of the Bru‐RS1 family. Hybridization patterns for the elements among brucellae were unique. The elements are dispersed, highly conserved among brucellae, and hot‐spots for insertion by IS711.
Non‐motile mutants of Helicobacter pylori and Helicobacter mustelae defective in flagellar hook production
Authors: O'Toole Paul W. , Kostrzynska Magdalena , Trust Trevor J. 
Published: October 2006

SummaryFlagellar hooks were purified from Helicobacter pylori and Helicobacter mustelae. The 70 × 16nm H. pylori hook was composed of FIgE subunits of 78kDa, while the 72 × 16nm H. mustelae hook was composed of 87kDa subunits. N‐terminal sequence was obtained for the FIgH proteins of both species, and for an internal H. mustelae FlgE peptide. Degenerate oligonucleotide primers allowed amplification of a 1.2 kb fragment from the H. mustelae chromosome, which carried part of the flgE gene. The corresponding H. pylori gene was cloned by immunoscreening of a genomic library constructed in λZAP Express, The translated H. pylori flgE sequence indicated a protein with limited homology with the hook proteins from Salmonella typhimurium and Treponema phagedenis. Mutants of H. pylori and H. mustelae defective in hook production generated by allele replacement were non‐motile and devoid of flagellar filaments but produced both flagellin subunits, which were localized in the soluble fraction of the cell. The level of flagellin production was unchanged in the mutants, indicating that the regulation of flagellin expression in Helicobacter differs from that in the Enterobacteriaceae.
The ccoNOQP gene cluster codes for a cb‐type cytochrome oxidase that functions in aerobic respiration of Rhodobacter capsulatus
Authors: Thöny‐Meyer Linda , Beck Christoph , Preisig Oliver , Hennecke Hauke 
Published: October 2006

SummaryThe genes for a new type of a haem‐copper cytochrome oxidase were cloned from Rhodobacter capsulatus strain 37b4, using the Bradyrhizobium japonicum fixNOQP gene region as a hybridizing probe. Four genes, probably organized in an operon (ccoNOQP), were identified; their products share extensive amino acid sequence similarity with the FixN, O, Q and P proteins that have recently been shown to be the subunits of a cb‐type oxidase. CcoN is a c‐type cytochrome, CcoO and CcoP are membrane‐bound mono‐ and dihaem c‐type cytochromes and CcoQ is a small membrane protein of unknown function. Genes for a similar oxidase are also present in other non‐rhizobial bacterial species such as Azoto‐bacter vinelandii, Agrobacterium tumefaciens and Pseudomonas aeruginosa, as revealed by polymerase chain reaction analysis. A ccoN mutant was constructed whose phenotype, in combination with the structural information on the gene products, provides evidence that the CcoNOQP oxidase is a cytochrome c oxidase of the cb type, which supports aerobic respiration in R. capsulatus and which is probably identical to the cbb3‐type oxidase that was recently purified from a different strain of the same species. Mutant analysis also showed that this oxidase has no influence on photosynthetic growth and nitrogen‐fixation activity.
mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem‐loop structures
Authors: Causton Helen , Py Béatrice , McLaren Robert S. , Higgins Christopher F. 
Published: October 2006

SummaryStem‐loop structures can protect upstream mRNA from degradation by impeding the processive activities of 3′–5′ exoribonucleases. The ability of such structures to impede exonuclease activity in vitro is insufficient to account for the stability they can confer on mRNA in vivo. In this study we identify a factor from Escherichia coli which specifically impedes the processive activity of the 3′–5′ exonuclease PNPase at stem‐loop structures in vitro. This factor can, potentialiy, reconcile the apparent discrepancy between the ability of 3′ stem‐loop structures to stabilize upstream mRNA in vitro and in vivo. Its mechanism of action, and possible role in regulating mRNA degradation, is discussed.
Transmembrane signalling by the chimeric chemosensory receptors of Escherichia coli Tsr and Tar with heterologous membrane‐spanning regions
Authors: Tatsuno Ichiro , Lee Lan , Kawagishi Ikuro , Homma Michio , Imae Yasuo 
Published: October 2006

SummaryThe serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane‐spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N‐terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C‐terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TW2 regions of Tsr and Tar are interchangeable, suggesting that sequence‐specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane.The cells expressing either of the chimeric receptors, however, showed ‘smooth’, biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase‐defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming.
SeqA limits DnaA activity in replication from oriC in Escherichia coli
Authors: Ulrik von Freiesleben  , Rasmussen Knud V. , Schaechter Moselio 
Published: October 2006

SummaryA mutant Escherichia coli that transforms minichromosomes with high efficiency in the absence of Dam methylation has been Isolated and the mutation mapped to 16.25 min on the E. coli map. The mutant strain containing seqA2 is defective for growth in rich medium but not in minimal medium. A similar mutation In this gene, named seqA1, has also been isolated.Here we show that the product of the seqA gene, SeqA, normally acts as an inhibitor of chromosomal initiation. In the seqA2‐containing mutant, the frequency of initiation increases by a factor of three. Introduction of the wild‐type seqA gene on a low‐copy plasmid suppresses the cold sensitivity of a dnaAcos mutant known to overinitiate at temperatures below 39°C. In addition, the seqA2 mutation is a suppressor of several dnaA (Ts) alleles.The seqA2 mutant overinitiates replication from oriC and displays the asynchronous initiation phenotype. Also the seqA2 mutant has an elevated level of DnaA protein (twofold).The introduction of minichromosomes or a low‐copy‐number plasmid carrying five DnaA‐boxes from the oriC region increases the growth rate of the seqA2 mutant in rich medium to the wild‐type level, reduces overinitiation but does not restore synchrony. We propose that the role of SeqA is to limit the activity level of the E. coli regulator of chromosome initiation, DnaA.
Multiple roles for DNA polymerase I in establishment and replication of the promiscuous plasmid pLS1
Authors: Díaz Asunción , Lacks Sanford A. , López Paloma 
Published: October 2006

SummaryThe polymerase activity of DNA polymerase I is important for the establishment of the pLS1 replicon by reconstitutive assembly in Streptococcus pneumoniae after uptake of exogenous pLS1 plasmid DNA. In polA mutants lacking the polymerase domain, such establishment was reduced at least 10‐fold in frequency. Chromosomally facilitated establishment of pLS1‐based plasmids carrying DNA homologous to the host chromosome was not so affected. However, both types of plasmid transfer gave mostly small colonies on initial selection, which was indicative of a defect in replication and filling of the plasmid pool. Once established, the pLS1‐based plasmids replicated in polA mutants, but they showed segregational instability. This defect was not observed in strains with the wild‐type enzyme or in an S. pneumoniae strain that encodes the polymerase and exonuclease domains of the enzyme on separate fragments. The role of DNA polymerase I in stably maintaining the plasmids depends on its polymerizing function in three separate steps of rolling‐circle replication, as indicated by the accumulation of different replication intermediate forms in polA mutants. Furthermore, examination of the segregational stability of the pLS1 replicon in an Escherichia coli mutant system indicated that both the polymerase and the 5′‐to‐3′ exonuclease activities of DNA polymerase I function in plasmid replication.
Genes required for both gliding motility and development in Myxococcus xanthus
Authors: MacNeil Spencer D. , Mouzeyan Aram , Hartzell Patricia L 
Published: October 2006

Summary Myxococous xanthus cells can glide both as individual cells, dependent on Adventurous motility (A motility), and as groups of cells, dependent upon Social motility (S motility), Tn5‐lac mutagenesis was used to generate 16 new A‐ and nine new S‐ mutations. In contrast with previous results, we find that subsets of A‐ mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S‐ mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S‐ mutants produce a wild‐type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5–lac insertions result in surprising phenotypes. Colonies of two S‐ mutants glide on ‘swim’ (0.35% agar) plates to form fractal patterns. These S‐ mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild‐type strain with one S‐ insertion resembles the frz‐ sglA1‐ mutants upon development, suggesting that this S‐ gene defines a new chemotaxis component in M. xanthus.
Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC7120
Authors: Frías José E. , Flores Enrique , Herrero Antonia 
Published: October 2006

SummaryThe cyanobacterial ntcA gene encodes a DNA‐binding protein that belongs to the Crp family of bacterial transcriptional regulators. In this work, we describe the isolation of an ntcA insertional mutant of the dinitrogen‐fixing, heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120. The Anabaena ntcA mutant was able to use ammonium as a source of nitrogen for growth, but was unable to assimilate atmospheric nitrogen (dinitrogen) or nitrate. Nitrogenase and enzymes of the nitrate reduction system were not synthesized in the ntcA mutant under derepressing conditions, and glutamine synthetase levels were lower in the mutant than in the wild‐type strain. In the ntcA mutant, in response to removal of ammonium, accumulation of mRNA of the genes encoding nitrogenase (nifHDK), nitrite reductase (nir, the first gene of the nitrate assimilation operon), and glutamine synthetase (glnA) was not observed. A transcription start point of the Anabaena glnA gene (corresponding to RNA1), that has been shown to be used preferentially after nitrogen step‐down, was not used in the ntcA insertional mutant. Heterocyst development (which is necessary for the aerobic fixation of dinitrogen) and induction of hetR (a regulatory gene that is required for heterocyst development) were also impaired in the ntcA mutant. These results showed that the ntcA gene product, NtcA, is required in Anabaena sp. PCC 7120 for the expression of genes encoding proteins involved in the assimilation of nitrogen sources alternative to ammonium including dinitrogen and nitrate, and that the process of heterocyst development is also controlled by NtcA.
Transcriptional attenuation control of the tylosin‐resistance gene tlrA in Streptomyces fradiae
Authors: Kelemen Gabriella H. , Zalacain Magdalena , Culebras Esther , Seno Eugene T. , Cundliffe Eric 
Published: October 2006

SummaryThe tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase‐coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome‐mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD.
Iron piracy: acquisition of transferrin‐bound iron by bacterial pathogens
Authors: Cornelissen Cynthia Nau , Sparling P. Frederick 
Published: October 2006

SummaryThe mechanism of iron utilization from transferrin has been most extensively characterized in the pathogenic Neisseria species and Haemophilus species. Two transferrin‐binding proteins, Tbp1 and Tbp2, have been identified in these pathogens and are thought to be components of the transferrin receptor. Tbp1 appears to be an integral, TonB‐dependent outer membrane protein while Tbp2, a lipoprotein, may be peripherally associated with the outer membrane. The relative contribution of each of these proteins to transferrin binding and utilization is discussed and a model of iron uptake from transferrin is presented. Sequence comparisons of the genes encoding neisserial transferrin‐binding proteins suggest that they are probably under positive selection for variation and may have resulted from inter‐species genetic exchange.
Specificity domain localization of Bacillus thuringiensis insecticidal toxins is highly dependent on the bioassay system
Authors: Masson Luke , Mazza Alberto , Gringorten Larry , Baines Danica , Aneliunas Victoria , Brousseau Roland 
Published: October 2006

SummaryThe Bacillus thuringiensis cryIA(a) and cryIA(c) gene specificity regions were probed by creating and testing hybrid toxins both in vivo and in vitro against cultured insect cells or dissociated midgut epithelial cells. Toxin threshold dose determinations revealed that CryIA(c) is highly active against cultured Choristoneure fumiterana cells (CF‐1) whereas CryIA(a) is nontoxic. In live insect bioassays, a reversed order of toxicity was observed. Hybrid analysis reversed that the CryIA(c) toxicity‐determining region is located between codons 258 and 510. Two smaller subsections of this region (residues 258–358 and 450–510) were able to confer toxicity, although at lower levels, and one region (358–450) was present where progressive substitutions of CryIA(a) with cryIA(c) sequences had no effect. Exchanging the non‐homologous N‐terminal regions of CryIA(c) with CryIE suggested that the W‐terminus does not play a role in specificity. One hybrid clone, MP80, displays a 99.3% homology to CryIA(b) but shows an 800‐fold increase in toxicity to CF–1 cells relative to that shown by CryIA(b). Direct comparison between live Bombyx mori bioassays and a newly developed in vitro lawn assay using dissociated midgut epithelial cells from the same insect revealed striking differences in toxicity. The toxicity‐determining region for B. mori larvae was determined to be between codons 283 and 450, although the 450–620 codon region may exert an influence on toxicity. In general, native or hybrid toxins showing little or no insect intoxication were very active against the epithelial cells, suggesting that factors other than toxin amino acid sequence play an important role in determining toxin specificity.
Successive action of Escherichia coli chaperones in vivo
Authors: Gaitanaris George A. , Vysokanov Alexander , Hung Siu‐Chun , Gottesman Max E , Gragerov Alexander 
Published: October 2006

Summary Escherichia coli DnaK, DnaJ and GrpE are required for renaturation of heat‐inactivated λ CI857 repressor (Gaitanaris et al., 1990). Here we demonstrate that in addition to the above three proteins, GroEL and GroES are necessary for the CI857 repressor to acquire full activity at the permissive temperature. Although full‐length soluble repressor is present at normal amounts, the protein has reduced specific activity and migrates abnormally on native gels. To determine where the different chaperones act in protein folding, we identified their cellular locations. DnaK and DnaJ are associated with nascent polypeptide chains in translating ribosomes. In contrast, GroEL, although it is transiently associated with newly synthesized proteins, is absent from the ribosomes. This suggests that DnaK and DnaJ ptay an early role in protein maturation, whereas GroEL acts at a later stage.
Topological and mutational analysis of KpsM, the hydrophobic component of the ABC‐transporter involved in the export of polysialic acid in Escherichia coli K1
Authors: Pigeon Ronald P. , Silver Richard P. 
Published: October 2006

SummaryThe 17 kb kps gene cluster of Escherichia coli K1, which encodes the information required for synthesis, assembly and translocation of the polysialic acid capsule of E. coli K1, is divided into three functional regions. Region 3 contains two genes, kpsM and kpsT, essential for the transport of capsule polymer across the cytoplasmic membrane. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein while KpsT contains a consensus ATP‐binding site. KpsM and KpsT belong to the ATP‐binding cassette (ABC) superfamily of membrane transporters. In this study, we investigate the topology of KpsM within the cytoplasmic membrane using β‐lactamase fusions and alkaline phosphatase sandwich fusions. Our analysis provides evidence for a model of KpsM having six membrane‐spanning regions, with the N‐ and C‐terminal domains facing the cytoplasm, and a short domain within the third periplasmic loop, which we refer to as the SV–SVI linker localizing in the membrane. Protease digestion studies are consistent with regions of KpsM exposed to the periplasmic space. In vivo cross‐linking studies provide support for dimerization of KpsM within the cytoplasmic membrane. Linker‐insertion and site‐directed mutagenesis define the N‐terminus, the first cytoplasmic loop, and the SV‐SVI linker as regions that are important for the function of KpsM in K1 polymer transport.