bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular characterization of pmbA, an Escherichia coli chromosomal gene required for the production of the antibiotic peptide MccB17
Authors: Rodríguez‐Sáinz M. C. , Hernández‐Chico C. , Moreno F. 
Published: October 2006

SummaryMicrocin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains harbouring plasmid pMccB17. We have isolated two mutations that strongly reduce the production of MccB17. These mutations, which map at 96 min on the E. coli chromosome, define a new gene that we have caiied pmbA. A chromosomal DNA fragment of about 13kb, including the wild‐type pmbA allele, was cloned into a mini‐Mu piasmid vector. pmbA was located within the cloned DNA fragment by insertional mutagenesis and deletion analysis. The nucleotide sequence of a 1.7 kb DNA region containing the gene was determined. pmbA encodes a hydrophilic protein of 450‐amino‐acid residues with a predicted molecular size of 48375 D, which was visualized in polyacrylamide gels. Protein profiles of cellular envelope and soluble fractions from cells with plasmids overproducing PmbA indicated that it is cytoplasmic. Physiological experiments suggested that pmbA mutants synthesize a molecule (pro‐MccB17) able to inhibit DNA replication but unable to the released from cells. We propose that PmbA facilitates the secretion of the antibiotic by completing its maturation.
The Escherichia coli unc transcription terminator enhances expression of uncC, encoding the e subunit of F1‐ATPase, from plasmids by stabilizing the transcript
Authors: Patel A.M. , Dallmann H.G. , Skakoon E.N. , Kapala T.D. , Dunn S.D. 
Published: October 2006

SummaryThe effect of the unc transcription terminator on expression of uncC, encoding the e subunit of Escherichia coli F1‐ATPase, from plasmids was studied. The cloned sequence in pTK1 included the uncC ribosome binding site, the uncC structural gene, and unc transcription terminator. The cloned region in pSD37 was similar, but lacked the unc transcription terminator. Transformants carrying pTK1 produced the e subunit of F1‐ATPase, encoded by uncC, in 10‐fold greater abundance than transformants carrying pSD37. Northern blots revealed similar differences in the steady‐state uncC mRNA levels. The half‐life of the message transcribed from pTK1 was 90–100 seconds, while that from pSD37 was 25–30 seconds. These studies indicate the importance of message stabilization through features at the 3 end of the transcript in ensuring adequate production of. We have exploited this stabilization to develop a simple, efficient, and gentle method of purifying the overproduced subunit.
Molecular evolution of class A β‐lactamases: phylogeny and patterns of sequence conservation
Authors: Pastor N. , Piñero D. , Valdés A. M. , Soberón X. 
Published: October 2006

SummaryWe present a multiple alignment of the amino acid sequences of eight class A β‐lactamases and utilized it to propose a phylogeny, based on the nucleotide sequences of their corresponding genes. We have also used the alignment, together with the α‐carbon co‐ordinates of the Staphylococcus aureus protein, to search systematically for neighbouring residues that share the same pattern of conservation among the different members of the protein family. The distribution of invariant residues and of groups of residues with co‐ordinate changes map, predominantly, at the region of the active site and at interfaces between structural elements, respectively. We have also contrasted the distribution of conserved residues with the positions which are known to differ in mutants and variants of class A β‐lactamases.
Larvicidal activity of chimeric Bacillus thuringiensis protoxins
Authors: Raymond K. C. , John T. R. , Bulla L. A. 
Published: October 2006

Summary Bacillus thuringiensis subspecies kurstaki (Btk) and subspecies berliner (Btb) both produce lepidopteran‐specific larvicidal protoxins with different activities against the same insect species. Toxic activity resides in the amino‐terminal half of both protoxins, whereas the carboxy‐terminal half of the molecules is not required for toxicity. The protoxins are 90% homologous, with a major cluster of differences in the amino‐terminal half, and a 26 consecutive amino‐acid insertion within the carboxy‐terminal half of the Btk protoxin. Protoxin chimeras composed of the amino‐terminal half of one subspecies and the carboxy‐terminal half of the other were generated. Wild‐type and chimeric protoxins were compared in bioassays against tobacco horn worm larvae. The amino‐terminal half, the toxin itself, dictates specific larvicidal activity.
Identification and characterization of a gene and protein required for glycosylation in the yeast Golgi
Authors: Devlin C. , Ballou C. E. 
Published: October 2006

SummaryThe MNN2 gene of Saccharomyces cerevisiae has been cloned by complementation of the mnn2 mutant phenotype scored by a change in cell surface carbohydrate structure resulting from a lack of α1→2‐man‐nose branching in the outer chain. The gene was subcloned as a 3 kb DNA fragment that integrated at the MNN2 locus, and a gene disruption yielded the mnn2 phenotype. A lacZ–MNN2 gene fusion protein, produced in Escherichia coli, was used to raise a specific antiserum that recognized a 65kD wild‐type yeast protein. This MMN2 gene product lacks N‐linked carbohydrate but appears to be an integral membrane protein. Overproduction of MNN2p does not enhance the α1→2‐mannosyltransferase activity of yeast cells. The results suggest that MNN2p is a Golgi‐associated protein that is involved in mannoprotein sorting rather than glycosylation.
Corrected sequence of the mannitol (mtl) operon in Escherichia coli
Authors: Jiang W. , Wu L.‐F. , Tomich J. , Saier M.H. , Niehaus W.G. 
Published: October 2006

SummaryThe previously published sequences of the operator‐promoter region of the mannitol operon of Escherichla coli and of the mtlD gene have been found to contain a number of errors. The major conclusions reported previously were correct, but additionally it is now clear that a C‐terminal portion of mannitol‐1‐phosphate dehydrogenase (the mtlD gene product) exhibits significant sequence identity with an amino‐terminal region of human liver fructose‐6‐phoshate‐2‐kinase; fructose‐2,6‐bishosphatase.
Triple post‐transcriptional control
Authors: Bechhofer D. H. 
Published: October 2006

SummaryThe ermC gene confers resistance to MLS antibiotics in a Bacillus subtilis host. Synthesis of the ermC gene product, a ribosomal RNA methylase, is inducible by the addition of subinhibitory concentrations of erythromycin. Regulation of ermC gene expression occurs at the post‐transcriptional level in three ways: translational attenuation, translational autoregulation, and messenger RNA stabilization.
Escherichia coli molecular genetic map (1000Kbp):update
Authors: Medigue C. , Hénaut A. , Danchin A. 
Published: October 2006

SummeryThe sequenced genes from Escherichia coli that are available in the EMBL library (release 21) have been localized on an updated and corrected version of the restriction map of the chromosome generated by kohara et al. (1987). One thousand kbp of sequenced DNA are incorporated in this update; this is equivalent to 23% of the total genome. The accuracy of the map is assessed, and it is corrected and updated where appropriate, A significant number of sites were missing from the original map, mainly involving two of the eight enzymes used by Kohara et al. (1987), ie. Pvull and EcoRV, The nucleotide environment of such missing sites was examined and, using an Artificial intelligence approach, it appears that the site for these enzymes is sensitive to context effects. Several genes of known position on the E. coli chromosome could not be placed on the restriction map; this suggests that additional gaps are likely to exists on the restriction map, in addition to the original seven identified by Kohara et al. We have also obtained information about the probable direction of transcription of chromosomal genes with respect to the map. Most genes are transcribed in the same direction as the replication forks, particularly around oriC at 84 min
Evidence that conserved residues Cys‐62 and Cys‐154 within the Azotobacter vinelandii nitrogenase MoFe protein α‐subunit are essential for nitrogenase activity but conserved residues His‐83 and Cys‐88 are not
Authors: Dean D. R. , Setterquist R. A. , Brigle K. E. , Scott D. J. , Laird N. F. , Newton W. E. 
Published: October 2006

SummaryMetallocluster extrusion requirements, interspecies MoFe‐protein primary sequence comparisons and comparison of the primary sequences of the MoFe‐protein subunits with each other have been used to assign potential P‐cluster (Fe‐S cluster) domains within the MoFe protein. In each β unit of the MoFe protein, subunit domains, which include potential Fe‐S cluster ligands Cys‐62, His‐83, Cys‐88 and Cys‐154, and β‐subunit domains, which include potential Fe‐S cluster ligands Cys‐70, His‐90, Cys‐95 and Cys‐153, are proposed to comprise nearly equivalent P‐cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the α‐ subunit Cys‐62, His‐83, Cys‐88 and Cys‐154 positions by site‐directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates. MoFe—protein acetylene‐reduction activities, and whole‐cell S 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe‐protein α‐submit residues, Cys‐62 and Cys‐154, are probably essential for MoFe‐protein activity but that His‐83 and Cys‐88 residues are not. These results indicate either that His‐83 and Cys‐88 do not provide essential P‐cluster ligand or that a new cluster‐ligand arrangement is formed in their absence.
Cysteine‐rich outer membrane proteins of Chlamydia trachomatis display compensatory sequence changes between biovariants
Authors: Allen J. E. , Cerrone M. C. , Beatty P. R. , Stephens R. S. 
Published: October 2006

SummaryTwo cysteine‐rich proteins of Chlamydia trachomatis are essential structural components of the unique outer membrane of the infectious elementary body. These 58000 (outer membrane protein 2; OMP2) and 15000 (OMP3) proteins also differ structurally and chemically between biovariants that differ in invasive capability. We have identified the gene for OMP3 and sequenced both trachoma and lymphogranuloma venereum (LGV) omp3 genes. We have previously sequenced omp2 from the LGV biovar and now describe the omp2 sequence for a trachoma biovariant. Amino acid sequence differences between biovariants were few but, significantly, these changes have altered the charge of both OMP2 and OMP3 such that the net charge of each protein differs between biovariants. These compensatory charge alterations have implications for the outer membrane organization of these proteins. In addition, examination of the OMP3 sequence suggests that OMP3 may be a lipoprotein.
Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family
Authors: McAdam R. A. , Hermans P. W. M. , van Soolingen D. , Zainuddin Z. F. , Catty D. , van Embden J. D. A. , Dale J. W. 
Published: October 2006

SummaryA repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment‐length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30‐bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).
Elimination of the disulphide bridge in fragment B of diphtheria toxin: effect on membrane insertion, channel formation, and ATP binding
Authors: Stenmark H. , Olsnes S. , Madshus I. H. 
Published: October 2006

SummaryActive diphtheria toxin consists of two disulphide‐linked fragments, termed A and B. Fragment B, which contains an internal disulphide bridge, facilitates translocation of the enzymatically active fragment A to the cytosol of eukaryotic cells. In this process cation‐selective channels are formed. An in vitro translated full‐length mutant lacking the internal disulphide bridge (A‐58**) was functionally indistinguishable from its disulphide‐containing counterpart (A‐58) with respect to trypsin sensitivity, receptor binding, A‐fragment translocation, and channel formation. In contrast, the B fragment of A‐58** (B‐36**) was slightly less trypsin resistant than the S‐S‐Containing B fragment, B‐36, and was 300‐fold less efficient than B‐36 in permeabilizing cells. When first dialysed and then reconstituted with A fragment, B fragment without disulphide bridge yielded a less‐active toxin than did wild‐type B fragment. We conclude that the disulphide bridge in fragment B is not necessary for toxicity, as earlier believed, and that channel formation may play a role in membrane translocation.
IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola
Authors: Romantschuk M. , Richter G. Y. , Mukhopadhyay P. , Mills D. 
Published: October 2006

SummaryA transposable element, designated IS801, was isolated from strain LR781 of Pseudomonas syringae pathovar phaseolicola in two independent events using the entrapment plasmid, pUCD800. IS801 is 1517 base pairs in length and contains open reading frames that potentially encode proteins of 311 and 172 amino acids, as well as smaller proteins. Unlike most other prokaryotic transposable elements, IS801 lacks terminal repeats. Sequence analysis revealed two target pentamers for IS801 insertion that differ by one base pair. One copy of IS801 generated a perfect duplication of its target, TGAAC. The second copy of IS801 was flanked by the target, TGGAC, at one end, and TGAAC at the other end. A third copy of IS801 was cloned from pMMC7105, an indigenous plasmid of strain LR781, and it was flanked by copies of the pentamer TGAAC.
nolC, a Rhizobium fredii gene involved in cultivar‐specific nodulation of soybean, shares homology with a heat‐shock gene
Authors: Krishnan H. B. , Pueppke S. G. 
Published: October 2006

Summary Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall. Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene‐reduction activity is absent. We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176 bp open reading frame that we designate nolC. nolC encodes a protein of deduced molecular weight 43564. Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea. nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat‐shock protein. Both R. fredii USDA257 and mutant 257DH5 produce heat‐shock proteins of 78, 70, 22, and 16kDa. A 4.3kb EcoRI–HindIII subclone containing nolC expresses a single 43 kDa polypeptide in mini‐cells. A longer, 9.4kb Eco RI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat‐shock protein. Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat‐shock gene, it does not directly function in the heat‐shock response.
A family of clostridial and streptococcal ligand‐binding proteins with conserved C‐terminal repeat sequences
Authors: Wren B. W. 
Published: October 2006

SummaryAnalysis of the derived amino acid sequences of toxins A and B from Clostridium difficile has identified an extraordinarily large number of repeat amino acid units in the C‐terminal regions of the proteins. Nearly one third of each of the proteins consist of repeating units which appear, at least in the case of toxin A, to be responsible for carbohydrate binding. Similar repeat units are also found in the C‐terminal region of four glucosyltransferases from Streptococcus mutans and Streptococcus downel, and in four lytic enzymes from Streptococcus pneumoniae and its bacteriophages (HB‐3, Cp‐1 and Cp‐9). In each case the repeats constitute the ligand‐binding portion of the respective enzymes. A glucan‐binding protein from S. mutans, which lacks enzymatic activity, has similar repeats spanning almost the entire molecule. This family of ligand‐binding proteins appears to be of modular design, with one module consisting of a repetitive ligand‐binding domain located in the C‐terminal region and the other module(s) providing enzymatic functions.
Translational coupling varying in efficiency between different pairs of genes in the central region of the atp operon of Escherichia coli
Authors: Hellmuth K. , Rex G. , Surin B. , Zinck R. , McCarthy J. E. G. 
Published: October 2006

SummaryA series of afp::lacZ fusions has been constructed for use in a study of translational coupling in the central region of the Escherichia coli atp operon. Five genes, atpE, atpF, atpH, atp A and atpG, were shown to be translationally coupled to various degrees of tightness. A new lac promoter vector, compatible with the afp::lacZ fusion vectors, was used to express individual atp genes in the same hosts as the fusion genes. The H+‐ATPase subunits thus synthesized exercised no significant trans‐regulation on the expression of the atp::lacZ fusions, indicating that the coupling is primarily cis. The mechanism of this coupling was investigated using in vitro mutagenesis. At least in the case of the pair atpHA, coupling seems to involve facilitated binding of fresh ribosomes to the atpA translational initiation regions.
Isolation and properties of the RepA1 protein of the IncFII replicon, RepFIC
Authors: Maas R. , Oppenheim J. , Saadi S. , Fuchs T. , Maas W. K. 
Published: October 2006

SummaryThe initiator protein RepA1 of the IncFII replicon RepFIC derived from the enterotoxin plasmid EntP307 has been cloned under the control of the λ Pl promoter. This has enabled us to overproduce this protein and study its properties. Here we show that RepA1 is a soluble basic protein with an experimentally determined molecular weight of 40000. Deletion analysis indicates that the overproduced protein originates from the open reading frame which we previously designated as coding for RepA1. We have also shown that the replication function of the replicon RepFIC depends on the intact RepA1 coding frame.
A sugar‐specific porin, ScrY, is involved in sucrose uptake in enteric bacteria
Authors: Schmid K. , Ebner R. , Jahreis K. , Lengeler J. W. , Titgemeyer F. 
Published: October 2006

SummaryDuring the molecular analysis of a plasmid‐coded sucrose metabolic pathway of enteric bacteria, a gene, scrY, was found whose product, ScrY, had all the properties of a bacterial porin (Schmid et al, 1988). Loss of this protein (Mr 58kDa), localized in the outer membrane, led, as shown here, to an increase in the apparent Km for sucrose transport in whole cells from 10 μM in wild‐type cells to 300 μM in mutant cells. This contrasts with the Km for sucrose phosphorylation as measured in membrane vesicles from mutant and wild‐type cells, which remained unchanged at about 10 μM, and reflects the activity of the sucrose‐specific Enzymell of the phosphoenolpyruvate‐dependent carbohydrate:phosphotransferase system (PTS) responsible for uptake through the inner membrane. Furthermore, the presence of ScrY restored growth on maltodextrins in cells devoid of LamB, thus complementing the lack of this maltoporin. The amino acid sequence deduced from the DNA sequence was determined for the plasmid‐coded and the ScrY porin coded in the chromosome of Klebsiella pneumoniae. Both show high identity (86%) to each other, and to the channel domain of LamB, further corroborating the conclusion that they constitute porins.
Pleiotropic changes resulting from depletion of Era, an essential GTP‐binding protein in Escherichia coli
Authors: Lerner C. G. , Inouye M. 
Published: October 2006

SummaryPhenotypic analysis of a temperature‐sensitive era mutant strain indicates that Escherichia coli cells depleted of Era undergo many physiological changes. At 43° C., a completely non‐permissive temperature, growth is arrested because of loss of the gene and depletion of the Era protein. Depletion of Era at 43° C results in depressed synthesis of heat‐shock proteins DnaK, GroEL/ES, D33.4 and C62.5, lack of thermal induction of ppGpp pool levels, and increased capacity for carbon source metabolism through the citric acid cycle. Thus, in addition to inhibition of cell growth and viability, ioss of Era function results in pleiotropic changes including abnormal adaptation to thermal stress.
The surface‐located YopN protein is involved in calcium signal transduction in Yersinia pseudotuberculosis
Authors: Forsberg Å. , Viitanen A.‐M. , Skurnik M. , Wolf‐Watz H. 
Published: October 2006

SummaryThe low‐calcium response (Icr) is strongly conserved among the pathogenic Yersinia species and is observed when the pathogen is grown at 37°C in Ca2+‐depleted medium. This response is characterized by a general metabolic downshift and by a specific induction of virulence‐plasmid‐encoded yop genes. Regulation of yop expression is exerted at transcriptional level by a temperature‐regulated activator and by Ca2+‐regulated negative elements. The yopN gene was shown to encode a protein (formerly also designated Yop4b) which is surface‐located when Yersmia is grown at 37°C. yopN was found to be part of an operon that is induced during the low‐calcium response. Insertional inactivation of the yopN gene resulted in derepressed transcription of yop genes. A hybrid plasmid containing the yopN gene under the control of the tac promoter fully restored the wild‐type phenotype of the yopN mutant. Thus the surface‐located YopN somehow senses the calcium concentration and transmits a signal to shut off yop transcription when the calcium concentration is high.
The RecA protein as a recombinational repair system
Authors: Cox M. M. 
Published: October 2006

SummaryThe Escherichia coli RecA protein plays a central role in homologous genetic recombination, recombinational repair, and several other processes in bacteria. In vitro, an extended filament involving thousands of RecA monomers promotes a reaction in which individual ONA strands switch pairing partners (DNA strand exchange). This reaction has been extensively studied as a paradigm for the central steps in recombination. Because the strand‐exchange reaction is relatively simple and Isoenergetic, the complexity of the RecA system that carries it out has led to controversy about the functional significance of many fundamental properties of RecA. Filamentous protein structures involving thousands of RecA monomers, which hydrolyse 100 ATPs per base pair of heteroduplex DNA formed, are hard to rationalize in the context of recombination between two homologous DNAs. The thermodynamic barriers to strand exchange are much too small. These molecular features of the system can be easily rationalized, however, by shifting the focus to DNA repair.