bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning, characterization and expression of the Zymononas mobilis eda gene that encodes 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase of the Entner‐Doudoroff pathway
Authors: Conway T. , Fliege R. , Jones‐Kilpatrick D. , Liu J. , Barnell W. O. , Egan S. E. 
Published: October 2006

SummaryThe eda gene that encodes 2‐keto‐3‐deoxy‐6‐phos‐phogluconate aldolase of the Entner‐Doudoroff pathway was cloned from Zymomonas mobilis by genetic complementation of an Escherichia coli mutant. The gene is present in a single copy on the Z mobilis genome and is not tightly linked to the edd gene. Nucleotide sequence analysis of the eda region revealed that the structural gene is 627 bp long and capable of encoding a protein of 208 amino acids with a deduced molecular weight of 21 505. The eda gene is monocistronic and is transcribed from a single promoter. The transcriptional initiation site was determined and an improved consensus promoter sequence for Z. mobilis was derived. High‐level expression of the eda gene can be attributed to very efficient translational initiation caused by the high quality of the ribosome‐binding site and stability of the mRNA, which has a decay rate of 7.6 min. A comparison of highly expressed Z mobilis genes indicated that the relative quality of the ribosome‐binding sites of these genes might play an important role in determining the level of enzyme synthesis. This possibility is discussed with regard to the role of gene expression in coordinating the enzyme levels of the Entner‐Doudoroff glycolytic pathway.
Genetic diversity among the T‐protein genes of group A streptococci
Authors: Jones K. F. , Schneewind O. , Koomey J. M. , Fischetti V. A. 
Published: October 2006

SummaryT protein is a trypsin‐ and pepsin‐resistant molecule on the surface of group A streptococci used as a serological tool to differentiate streptococci of this group. The purpose of this study was to determine the relatedness among the T protein genes of the 25 known T serotypes. DNA probes were constructed which represented various regions of the structural gene for the T6 protein, tee6. The probes were assayed for their ability to hybridize Hin dlll digests of chromosomal DNA from the 25 different T serotypes. Probe pTEE6.3, coding for the entire T6 protein, and pTEE61–299, coding for the amino‐terminal half of T6, displayed the highest amount of homology, each binding to 10 of 25 T serotypes. Probes coding for sequences in the carboxy‐terminal half of T6 showed considerably less homology among T serotypes with one probe hybridizing with only three out of 25. A synthetic oligonucleotide coding for the carboxy‐terminal hydrophobic domain of T6, an area conserved to some degree among several bacterial surface proteins, showed homology with only seven out of 25 T serotypes. Hybridization with sequences outside the tee6 coding area provided additional information on the relatedness of certain sets of T serotypes according to restriction‐fragment size heterogeneity. Clearly, there is considerable diversity among T‐serotype genes. The data suggest that two or more families of structurally variant T proteins exist, which share only the property of proteolytic resistance and/or, perhaps, some biological function.
A protein kinase C‐like activity in Escherichia coli
Authors: Norris V. , Baldwin T. J. , Sweeney S. T. , Williams P. H. , Leach K. L. 
Published: October 2006

SummaryThe protein kinase C (PKC) family comprises calcium‐ and phospholipid‐dependent kinases whose activity is stimulated by diacylglycerol and tumour‐promoting phorbol esters such as 12‐tetradecanoyl phorbol‐13‐acetate (TPA). In the Gram‐negative bacterium Escherichia coli, functional similarity to PKC was demonstrated in crude extracts by calcium and phospholipid‐dependent, TPA‐stimulated phosphorylation of a small number of endogenous substrates. Activity was reduced by sphingosine, a known inhibitor of eukaryotic PKC. Structural similarity to PKC was demonstrated in crude and partially purified bacterial extracts by cross‐reactivity with several monoclonal antibodies. This revealed isozyme‐specific homology between a protein(s) of relative molecular mass 80–85000 in E. coli and the α‐and γ‐isozymes, but probably not the β‐isozyme, of eukaryotic PKC.
Molecular action of tricholin, a ribosome‐inactivating protein isolated from Trichoderma viride
Authors: Lin A. , Chen C.‐K. , Chen Y.‐J. 
Published: October 2006

SummaryAn extracellular protein was isolated from a species of soil‐borne fungi (Trichoderma viride) and its amino acid composition has been determined. The protein is acidic with a molecular mass of 14 200 daltons and is given the trivial name tricholin. Tricholin is a potent inhibitor of cell‐free protein synthesis. When rabbit reticulocyte lysate was incubated with tricholin at a concentration of 6.3 × 10−7 M, it completely abolished the capacity of the lysate to support protein synthesis. The inhibition appears to be due to its reaction to ribosomes, since it generates a specific cleavage product, an α‐sarcin RNA fragment, from reticulocyte ribosomal RNA. This reaction to ribosomes mimics that of α‐sarcin. The antibody of α‐sarcin strongly cross‐reacts with tricholin, while the antibody of tricholin shows a weak reaction with α‐sarcin.
A cytosine‐ over guanosine‐rich sequence in RNA activates rho‐dependent transcription termination
Authors: Rivellini F. , Alifano P. , Piscitelli C. , Blasi V. , Brunt C. B. , Carlomagno M. S. 
Published: October 2006

SummaryWe have constructed an expression vector carrying the Escherichia coli his operon control region to study the ability of defined segments of DNA to cause rho tactor‐mediated transcription termination both in vivo and in vitro. We have previously identified a consensus motif consisting of a region of high cytosine over guanosine content common to several cryptic intracistronic transcription termination elements unmasked by polar mutations. We show that a DNA fragment possessing features similar to the ones previously identified is capable of causing rho‐mediated release of transcripts in vivo and in vitro. The efficiency of termination depends on the length and relative cytosine over guanosine ratio of the element.
The tol gene products and the import of macronmolecules into Escherichia coli
Authors: Webster R. E. 
Published: October 2006

SummaryGenetic studies have identified a number of genes whose products appear to be required for the transport of the group A colicins and the single‐stranded DNA of certain filamentous bacteriophages into Escherichia coli. Mutations in these genes allow normal binding of the colicins to their outer‐membrane receptors and of the bacteriophage to the tip of specific conjugative pili, but do not allow translocation of the macromolecules to their target. These mutations have been designed‘tolerant’(tol) mutations and the protein products specified by these genes appear to comprise part of a transport system known as the Tol import system. Some of these genes have been isolated, sequenced and their protein products localized to the membranes or periplasm of E. coli. Information is also available regarding the domains of the colicins or phage proteins which interact with the Tol proteins. A preliminary model of the location and possible interactions of the Tol proteins is presented.
Structure‐function analysis of the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli
Authors: Pedersen P. A. 
Published: October 2006

SummarySeveral in‐frame linker insertions have been made in various positions in the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The effects of the linker insertions have been investigated with regard to the ability of the mutated fimbrial subunits to be exported to the surface of the bacterial cell and assembled into a fimbrial structure. The structure/function relationship of the subunit protein is discussed in the light of the phenotypes of the mutations constructed, secondary structure predictions, and hydrophilicity plots.
Gonococcal penicillin‐binding protein 3 and the surface‐exposed 44kDa peptidoglycan‐binding protein appear to be the same molecule
Authors: Shafer W. M. , Judd R. C. 
Published: October 2006

SummaryThe outer membrane of Neisseria gonorrhoeae contains a 44000 datton (44kDa) surface‐exposed protein which has the reported ability to form covalent interactions with peptidoglycan (PG). This PG‐binding outer‐membrane protein (OMP) appears to be highly conserved since it has been detected in all isolates examined. It also appears to be invariant since its primary structure among strains gives evidence of being identical (Judd et al., 1991). While studying the interaction of gonococcal penicillin‐binding proteins (PBPs) with human lysosomal cathepsin G, we noticed that the 44kDa PG‐binding OMP exhibited certain properties similar to PBP3. In this study we sought to obtain biochemical evidence to ascertain whether these proteins were the same. We found that both proteins fractionated with other sarkosyl‐insoluble OMPs and that they exhibited similar susceptibility to cleavage in situ by enzymatically active cathepsin G. Moreover, a purified preparation of the 44kDa OMP was found to covalently bind radiolabelled benzylpenicillin in vitro. Thus, the data presented herein suggest that the 44kDa PG‐binding OMP and PBP3 are the same OMP.
Molecular cloning of a gene involved in lipooligosaccharide biosynthesis and virulence expression by Haemophilus influenzae type B
Authors: Cope L D. , Yogev R. , Mertsola J. , Latimer J. L. , Hanson M. S. , McCracken G. H. , Hansen E. J. 
Published: October 2006

SummaryA wild‐type Haemophilus influenzae type b (Hib) genomic DNA library was constructed in the plasmid shuttle vector pGJB103. A virulence‐deficient lipooligosaccharide (LOS) mutant of Hib was used as a recipient for genetic transformation to screen this Hib genomic DNA library for genes involved in LOS expression. A recombinant plasmid containing a 7.8 kb Pstl fragment of Hib DNA was shown to transform this LOS mutant to reactivity with a monoclonal antibody (mAb) specific for a wild‐type LOS epitope. Transformation of two different virulence‐deficient LOS mutants with a 4.4kb BglII fragment of this recombinant plasmid yielded transformants which expressed LOS that bound the wild‐type LOS‐specific mAb and yielded profiles in sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis different from those of the original LOS mutants. These transformants with structurally altered LOS molecules also exhibited increased virulence in an animal model for invasive Hib disease. The virulence‐transforming ability was further localized to a 1.8kb BglII‐AlwNI fragment of the Hib DNA insert. Nucleotide sequence analysis indicated the presence of a single large open reading frame within this fragment. This open reading frame contained 19 consecutive repeats of the tetramer CAAT near the 5’end. Linker insertion mutagenesis was used to demonstrate directly the involvement of this open reading frame in both LOS biosynthesis and virulence expression by Hib.
Characterization of legiolysin (lly), responsible for haemolytic activity, colour production and fluorescence of Legionella pneumophila
Authors: Wintermeyer E. , Rdest U. , Ludwig B. , Debes A. , Hacker J. 
Published: October 2006

SummaryA genomic library of Legionella pneumophila, the causative agent of Legionnaires’disease in humans was constructed in Escherichia coli K12 and the recombinant clones were tested for haemolysis and other phenotypic properties. Seven clones were identified which were able to confer haemolysis of human, sheep, and canine erythrocytes but which were unable to mediate proteolytic activities or cyto‐toxic effects on CHO‐ or Vero cells. Clones that exhibited this haemolytic property were also able to produce a brown colour and a yellow‐green fluorescence activity detected on M9 plates containing tyrosine. The genetic determinant encoding these properties, termed legiolysin (lly) was mapped by Tn 1000 mutagenesis and by subcloning experiments. Southern hybridization with an lly‐specific gene probe showed that this determinant is part of the genome of L. pneumophila but is not identical to a protease gene of L. pneumophila which also mediates haemolysis. Minicell analysis of lly‐specific plasmids exhibited a protein of 39kDa. Polyclonal antibodies generated against a LacZ‐Lly hybrid protein also recognized a 39kDa protein produced either by the recombinant legiolysin‐positive E. coli K12 clones or by L. pneumophila wild‐type strains.
Chromosome strand segregation during sporulation in Bacillus subtilis
Authors: Errington J. , Wake R. G. 
Published: October 2006

SummaryAfter the initiation of spore formation In Bacillus subtilis, the products of the final round of DNA replication segregate into two cells, i.e. the prespore and the mother cell. The prespore, which is known to contain a single completed chromosome, develops into a mature endospore which can be readily separated from mother cells and non‐sporulating cells on the basis of its resistance properties. We have used a procedure originally developed to label the terminus region of the B. subtilis chromosome to specifically label the newly synthesized strands of DNA during the final round of DNA replication before sporulation. We have purified prespore DNA and used strand‐specific probes to measure the radioactivity incorporated. The results show that the sister chromosomes segregate at random into the prespore. This result has implications for the segregation of chromosomes during vegetative growth and for the generation of cellular asymmetry during sporulation.
Selenocysteyl‐tRNA occurs in the diatom Thalassiosira and in the ciliate Tetrahymena
Authors: Hatfield D. L. , Lee B. J. , Price N. M. , Stadtman T. C. 
Published: October 2006

SummarySelenocysteyl‐tRNAs that decode UGA were identified previously in animal and bacterial cells and the genes for these tRNAs have been shown to be widespread in animals and eubacteria. In the present study, we identify a selenocysteyl‐tRNA that codes for UGA in Thalassiosira pseudonana, which is a diatom, and in Tetrahymena borealis, which is a ciliate. The fact that these very diverse unicellular organisms also contain a selenocysteyl‐tRNA suggests that selenocysteine‐containing proteins and the use of UGA as a codon for selenocysteine are widespread, if not ubiquitous, in nature.
A cloned ompR‐like gene of Streptomyces lividans 66 suppresses defective melC1, a putative copper‐transfer gene
Authors: Tseng H.‐C. , Chen C. W. 
Published: October 2006

SummaryExpression of tyrosinase in Streptomyces requires functional MelC1 protein, which is postulated to transfer copper to apotyrosinase. We have previously isolated a mutant of Streptomyces lividans, HT32, that phenotypically suppressed mutations in cloned melC1 (H.‐C. Tseng and C. W. Chen, in preparation). Plasmid pLUS132, containing an ATG to ATA transition at the initiation codon of melC1, was used for cloning the suppressor gene from HT32. A 1687bp suppressor DNA was isolated that contained two characteristic Streptomyces coding sequences: a 217‐amino‐acid open reading frame (cutR) and a truncated open reading frame (cutR) downstream. Subcloning analysis attributed the phenotypic suppression activity to the putative cutR gene from HT32. The putative CutR exhibited similarity to the response regulator OmpR of the osmoregulatory signal‐transduction system in Escherichia coli. The truncated CutS resembled, to a lesser degree, the N‐terminus of EnvZ, the histidine protein kinase counterpart of OmpR. DNA hybridizing to the cloned cutR‐cutS sequence was detected in 16 other Streptomyces species. We postulate that the putative cutR‐cutS operon regulates copper metabolism in Streptomyces.
The glucose permease of the phosphotransferase system of Bacillus subtilis: evidence for IIGlc and IIIGlc domains
Authors: Gonzy‐Tréboul G. , de Waard J. H. , Zagorec M. , Postma P. W. 
Published: October 2006

SummaryGlucose is taken up in Bacillus subtilis via the phosphoenolpyruvate:glucose phosphotransferase system (glucose PTS). Two genes, orfG and ptsX, have been implied in the glucose‐specific part of this PTS, encoding an Enzyme IIGlc and an Enzyme IIIGlc, respectively. We now show that the glucose permease consists of a single, membrane‐bound, polypeptide with an apparent molecular weight of 80000, encoded by a single gene which will be designated ptsG. The glucose permease contains domains that are 40‐50% identical to the IIGlc and IIIGlc proteins of Escherichia coli. The B. subtilis IIIGlc domain can replace IIIGlc in E. coli crr mutants in supporting growth on glucose and transport of methyl α‐glucoside.Mutations in the IIGlc and IIIGlc domains of the B. subtilis ptsG gene impaired growth on glucose and in some cases on sucrose. ptsG mutants lost all methyl α‐glucoside transport but retained part of the glucose‐transport capacity. Residual growth on glucose and transport of glucose in these ptsG mutants suggested that yet another uptake system for glucose existed, which is either another PT system or regulated by the PTS.The glucose PTS did not seem to be involved in the regulation of the uptake or metabolism of non‐PTS compounds like glycerol. In contrast to ptsl mutants in members of the Enterobacteriaceae, the defective growth of B. subtilis ptsl mutants on glycerol was not restored by an insertion in the ptsG gene which eliminated IIGlc. Growth of B. subtilis ptsG mutants, lacking IIGlc, was not impaired on glycerol. From this we concluded that neither non‐phosphorylated nor phosphorylated IIGlc was acting as an inhibitor or an activator, respectively, of glycerol uptake and metabolism.
Regulation of methionine synthesis in Escherichia coli
Authors: Weissbach H. , Brot N. 
Published: October 2006

SummaryThe biosynthesis of methionine in Escherichia coli is under complex regulation. The repression of the biosynthetic pathway by methionine is mediated by a repressor protein (MetJ protein) and S‐adenosyl‐methionine which functions as a corepressor for the MetJ protein. Recently, a new regulatory locus, metR has been identified. The MetR protein is required for both metE and metH gene expression, and functions as a transactivator of transcription of these genes. MetR is a unique prokaryotic transcription activator in that it possesses a leucine zipper motif, first described for eukaryotic DNA‐binding proteins. The transcriptional activity of MetR is modulated by homocysteine, the metabolic precursor of methionine. Finally, it is known that vitamin B12 can repress expression of the metE gene. This effect is mediated by the MetH holoenzyme, which contains a cobamide prosthetic group.
Regulation of capsular polysaccharide synthesis in Escherichia coli K12
Authors: Gottesman S. , Stout V. 
Published: October 2006

SummarySynthesis of the capsular polysaccharide colanic acid in Escherichia coli K12 Is regulated by a complex network of regulatory proteins. This regulation is expressed at the level of transcription of the cps (capsular polysaccharide synthesis) genes. Two positive regulators, RcsA and RcsB, are necessary for maximal capsule expression. The availability of RcsA is normally limited because the RcsA protein is rapidly degraded by the Lon ATP‐dependent protease. Therefore Lon acts, indirectly, as a negative regulator of capsule synthesis. The sequence predicted for RcsB suggests that it is the effector component of a two‐component system; a protein with homology to sensors, RcsC, also plays a role in capsule regulation. We propose a model for capsule synthesis in which RcsA interacts with RcsB to stimulate transcription of the cps genes. The mechanism of regulation of colanic acid synthesis in E. coli may apply to other capsules in a variety of Gram‐negative bacteria.
Thirty‐three nucleotides of 5′ flanking sequence including the ‘TATA’ box are necessary and sufficient for efficient U2 snRNA transcription in Schizosaccharomyces pombe
Authors: Dandekar T. , Tollervey D. 
Published: October 2006

SummaryWe have sequenced the 5′ flanking region of the U2 gene and compared this with the 5′ flanking sequences of other snRNA genes from Schizosac‐charomyces pombe. This revealed no regions of clear homology 5′ to a region surrounding the ‘TATA’ box at −32 to −29. Deletion analysis shows that a 5′ flanking region extending to only −33 is sufficient for accurate and efficient transcription of U2 in Schizosaccharomyces pombe.
A gene in the archaebacterium Sulfolobus solfataricus that codes for a protein equivalent to the alpha subunits of the signal recognition particle receptor in eukaryotes
Authors: Ramirez C. , Matheson A. T. 
Published: October 2006

SummaryWe have sequenced a gene in the archaebacterium Sulfolobus solfataricus that codes for a protein that shows sequence similarity to the alpha subunit of the signal recognition particle receptor or docking protein In eukaryotes and the product of the ftsY gene in Escherichia coli. Comparison of the Sulfolobus‘docking protein’ with its eukaryotic and eubacterial counterparts showed that the region of highest sequence similarity corresponds to a GTP‐binding site. The presence of this gene in archaebacteria suggests that some of the components involved in protein transport have been conserved in the three kingdoms.
Both genes for EF‐1α in Candida albicans are translated
Authors: Sundstrom P. , Irwin M. , Smith D. , Sypherd P. S. 
Published: October 2006

SummaryIn previous work, we showed that Candida albicans has two genes, TEF‐1 and TEF‐2, which encode identical polypeptides for the highly conserved, essential, protein synthesis factor EF‐1α (Breviario et al., 1988). This result prompted questions as to whether C. albicans preferentially uses one of the genes over the other and whether both genes are actually translated into protein. Gene‐specific sequence differences in the untranslated portion of each gene made it possible to prepare gene‐specific oligonucleotide hybridization probes. Results with the probes showed that the relative steady‐state mRNA levels of the two genes were equivalent and that the mRNA for each gene was present in active translation complexes.
Absence of acetohydroxy acid synthase III in Salmonella typhimurium is due to early termination of translation within the ilvl gene
Authors: Ricca E. , Lago C. T. , Sacco M. , De Fellce M. 
Published: October 2006

SummaryThe cryptic ilvlH locus of Salmonella typhimurium has genetic information for two distinct subunits of acetohydroxy acid synthase III. We show that the ilvH‐encoded subunit is normally translated and the lack of activity is due to early termination of translation within the promoter‐proximal ilvl gene. Analysis of the 5’region of the operon led to identification of the promoter and the amino‐terminal part of ilvl. Expression of this gene in a mutant producing acetohydroxy acid synthase is due to a transversion which creates a UUA (leucine) codon in the place of a UGA (stop) codon present in position 12 of the wild‐type coding region.