bluuebunny/crossref_metadata_2025_split huggingface dataset:
Common features of the NAD‐binding and catalytic site of ADP‐ribosylating toxins
Authors: Domenighini Mario , Magagnoli Claudia , Pizza Mariagrazia , Rappuoli Rino 
Published: October 2006

SummaryComputer analysis of the three‐dimensional structure of ADP‐ribosylating toxins showed that in all toxins the NAD‐binding site is located in a cavity. This cavity consists of 16 contiguous amino acids that form an a‐helix bent over β‐strand. The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence. Catalysis is supported by two spatially conserved amino acids, each flanking the NAD‐binding site. These are: a glutamic acid that is conserved in all toxins, and a nucleophillc residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat‐labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus. The latter group of toxins presents an additional histidine that appears important for catalysis. This structure suggests a general mechanism of ADP‐ribosylation evolved to work on different target proteins.
Probing the structure‐activity relationship of Escherichia coli LT‐A by site‐directed mutagenesis
Authors: Pizza Mariagrazia , Domenighini Mario , Hol Wim , Giannelli Valentine , Fontana Maria Rita , Giuliani Marzia M. , Magagnoli Claudia , Peppoloni Samuele , Manetti Roberto , Rappuoli Rino 
Published: October 2006

SummaryComputer analysis of the crystallographic structure of the A subunit of Escherichia coil heat‐labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site‐directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non‐toxic new molecules containing mutations of single amino acids such as Val‐53 → Glu or Asp, Ser‐63 → Lys, Val‐97 → Lys, Tyr‐104 → Lys or Asp, and Ser‐14 → Lys or Glu. This group also included mutations in amino acids such as Arg‐7, Glu‐110 and Glu‐112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu‐41 → Phe, Ala‐45 → Tyr or Glu, Val‐53 → Tyr, Val‐60 → Gly, Ser‐68 → Pro, His‐70 → Pro, Val‐97 → Tyr and Ser‐114 → Tyr. The third group contained those molecules that maintained a wild‐type level of toxicity in spite of the mutations introduced: Arg‐54 → Lys or Ala, Tyr‐59 → Met, Ser‐68 → Lys, Ala‐72 → Arg, His or Asp and Arg‐192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non‐toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.
New clusters of genes required for gliding motility in Myxococcus xanthus
Authors: MacNeil Spencer D. , Calara Federico , Hartzell Patricia L. 
Published: October 2006

SummaryGilding is the directed movement of cells across surfaces which occurs in the absence of external organelles such as flagella. Gliding of the complex prokaryote, Myxococcus xanthus, results from the action of two independent sets of genes known as the A (adventurous motility) and S (social motility) genes. Strains with mutations in both systems (A−S−) do not spread on agar surfaces because both individual and group movement is abolished. To generate regulated, transcriptional fusions with operons including A and S genes, we introduced TN5‐lac into A− and S− strains to obtain non‐motile A−S::Tn5‐lac and A::Tn5‐lac S− double mutants. These insertions identify five separate clusters of A genes and three separate clusters of S genes on the M. xanthus genome. Some Tn5‐lac insertions map near two of the five previously identified motility gene clusters, but at least five new clusters were identified in this search. Single mutations at only one locus, mgIA, block motility; the mgIA locus is epistatic to A and S motility genes. A‐and S‐Tn5‐lac insertions were transduced into mgl+ and δmgl strains. The levels of β‐galactosidase activity produced from each A‐or S‐Tn5‐lac insertion are similar in otherwise isogenic mgl+ and δmgl strains, showing that MgIA does not regulate the transcription of many A and S genes.
The FeSII protein of Azotobacter vinelandii is not essential for aerobic nitrogen fixation, but confers significant protection to oxygen‐mediated inactivation of nitrogenase in vitro and in vivo
Authors: Moshiri Farhad , Kim John W. , Fu Changlin , Maier Robert J. 
Published: October 2006

SummarySummary The FeSII protein of Azotobacter vinelandii has been proposed to mediate the ‘conformational protection’ of the molybdenum‐dependent nitrogenase components against oxygen inactivation. We have cloned and characterized the structural gene for the FeSII protein (the fesII Iocus). Hybridization studies did not reveal the presence of fesII‐like genes in a number of diverse species of well‐studied nitrogen‐fixing bacteria, with the exception of Azotobacter chroococcum. The fesll locus is transcriptionally expressed during both nitrogen fixing and non‐nitrogen fixing conditions, although the level of its message is up‐regulated by approximately 2.5‐fold during nitrogen fixation. The promoter region was identified by primer extension analysis, and is similar to other σ70‐type promoters. Mutants devoid of the FeSII protein were constructed. These mutants possessed growth characteristics on a variety of carbon substrates during non‐diazotrophic as well as diazotrophic growth that were essentially indistinguishable from the wild‐type strain. Nevertheless, the nitrogenase activity in cell‐free extracts is significantly more sensitive to irreversible oxygen inactivation in the mutants as compared with the wild type. When treated with 250 mM NaCI (a condition known to dissociate FeSII from nitrogenase components), the wild‐type and mutant extracts were equally hypersensitive to oxygen Inactivation. Upon energy starvation, conditions in which ‘respiratory protection’ is inoperable, the MoFe and Fe proteins of nitrogenase are degraded much more rapidly in vivo in the deletion mutants, compared to the wild type. Strains relying on either the vanadium or the ‘iron‐only’ alternative nitrogenases exhibited similar growth rates irrespective of the presence of absence of the FeSII protein, and the in vitro inactivation of the vanadium nitrogenase components was not affected by the lack of the FeSII protein. All in all, these results are consistent with a model whereby ‘respiratory protection’ is the major physiological mechanism responsible for the protection of all three nitrogenases during energy supplemented growth. Upon energy starvation, however, ‘conformational protection’ mediated by the FeSII protein is capable of temporarily protecting the conventional molybdenum nitrogenase components from inactivation and subsequent degradation.
Molecular characterization of catalase from Bordetella pertussis: identification of the katA promoter in an upstream insertion sequence
Authors: DeShazer David , Wood Gwendolyn E. , Friedman Richard L. 
Published: October 2006

SummaryIn this report we evaluate the role of catalase in the survival of Bordetella pertussis within human polymorphonuclear leukocytes (PMNs). Crude extracts of B. pertussis exhibited a single catalase activity when subjected to non‐denaturing polyacrylamide gel electrophoresis and assayed for catalase activity. A plasmid containing B. pertussis katA was identified by complementation of UM255, a catalase‐deficient strain of Escherichia coli. The nucleotide sequence of katA predicts a 55kDa protein that shares homology with a class of haem‐containing catalases found in both eubacteria and eukaryotes. Analysis of the nucleotide sequence upstream of katA revealed the presence of a copy of IS481, a B. pertussis‐specific insertion sequence. The start site of transcription of katA was mapped to a T residue in IS481 by primer extension analysis performed with B. pertussis RNA and a katA‐specific primer. A catalase‐deficient strain of B. pertussis, DD900, was constructed by gene replacement. DD900 was more sensitive to killing by 1 and 5 mM H2O2 than the parental strain, BP339. However, there was no difference in the ability of DD900 and BP339 to survive for 2h in human PMNs. This suggests that catalase plays no significant role in the survival of B. pertussis within PMNs
Molecular analysis of the biosynthesis pathway of the α‐2,8 polysialic acid capsule by Neisseria meningitidls serogroup B
Authors: Edwards Ulrike , Müller Astrid , Hammerschmidt Sven , Gerardy‐Schahn Rita , Frosch Matthias 
Published: October 2006

SummaryThe genes encoding all enzymes necessary for capsular polysaccharide biosynthesis in Neisseria meningitidis B are located on a 5 kb DNA fragment within the chromosomal cps gene cluster. Nucleotide sequence analysis revealed four open reading frames (ORFs), which can encode proteins with molecular masses of 41.4 kDa, 24.9kDa, 38.3 kDa, and 54.4 kDa, respectively. These ORFs constitute a transcriptional unit as demonstrated by Northern blots. Primer extension analysis revealed that the transcriptional start site is preceded by a nucleotide sequence with homologies to the σ70consensus promoter sequence of Escherichia coli. Functional analysis of the proteins encoded by the ORFs indicated that ORF2 encodes the CMP‐NeuNAc synthetase, ORF3 encodes the NeuNAc condensing enzyme, and ORF4 encodes the α‐2,8 polysialyltransferase. ORF1 encodes an enzyme, which provides a precursor molecule for synthesis of monomeric NeuNAc. In E. coli the subcloned ORFs 2–4 were able to synthesize a high‐molecular‐weight α‐2,8 polysialic acid. In contrast, inactivation of ORF1 in the meningococcal genome resulted in a complete loss of capsule production. A regulatory enzyme, the CMP‐NeuNAc hydrolase, which cleaves CMP‐NeuNAc to CMP and NeuNAc, was not found as a part of the capsular polysaccharide biosynthesis gene operon or within the cps gene cluster.
Distinct mechanisms of interactions of Opc‐expressing meningococci at apical and basolateral surfaces of human endothelial cells; the role of integrins in apical interactions
Authors: Virji Mumtaz , Makepeace Katherine , Moxon E. Richard 
Published: October 2006

SummaryInteractions of Opc‐expressing Neisseria meningitidis with polarized and non‐polarized human umbilical vein endothelial cells (Huvecs) were investigated. Metabolic inhibitors and cytochalasin D treatment showed that host cellular and cytoskeletal functions were important for Opc‐expressing bacterial association with Huvecs at the apical surface. In addition, this interaction required the presence of serum in the incubation medium whilst association with nonpolarized cells did not require serum. Pre‐exposure of Opc‐expressing bacteria to serum was sufficient to increase the number of bacterial interactions at the apical surface; B306, a monoclonal antibody (mAb) against Opc, inhibited these interactions, suggesting that Ope binds to serum factor(s) and this in turn increases adherence to Huvecs. The receptors involved in this ‘sandwich’ adherence belong to the integrin family since the interaction was inhibited by peptides containing the amino acid sequence arginine‐glycine‐aspartic acid (RGD) and the tetrapeptide RGDS (but not the peptide RGES) was inhibitory. Non‐polarized cells appeared to expose receptors/sites that bound to Opc‐expressing bacteria directly, did not require serum factors and were not inhibited by RGD‐containing peptides. Serum‐dependent interactions of Opc‐expressing bacteria to apical surface was inhibited significantly by severai mAbs against avβ3 integrins. Some mAbs against α5 and β1 caused partial inhibition; antibodies that did not block the function of β1 integrins or the mAbs against α2 integrins were not inhibitory to bacterial interactions with Huvecs. Purified vitronectin supported adherence of Opc‐expressing bacteria to Huvecs but not of Opc‐bacteria. These interactions were inhibited by mAb B306 against Opc, by RGDS peptides as well as by blocking antibodies directed against αvβ‐3 but not antibodies against other integrins. These data suggest that a sequence of molecular events resulting in trimolecular complexes at the endothelial surface may drive neisserial invasion of Huvesc. The expression of Opc appears to enable bacteria to utilize the normal signal‐transduction mechanism of host cells via ligands in sera that adhere to endothelial cell integrins.
A Haemophilus influenzae IgA protease‐like protein promotes intimate interaction with human epithelial cells
Authors: St. Geme Joseph W. , Maria L. de la Morena  , Falkow Stanley 
Published: October 2006

Summary Haemophilus influenzae represents a common cause of human disease and an important source of morbidity and mortality. Disease caused by this organism begins with colonization of the upper respiratory tract. Several studies indicate that H. influenzae is capable of binding to and entering cultured human cells, properties which are potentially of relevance to the process of colonization. In the present study, we isolated an H. influenzae gene designated hap, which is associated with the capacity for In vitro attachment and entry. Analysis of the derived amino acid sequence of hap demonstrated significant homology with the serine‐type lgA1 proteases expressed by H. influenzae and Neisseria gonorrhoeae. It is notable that the hap product shares the catalytic domain of the lgA1 proteases and appears to be processed and secreted in an analogous manner. We speculate that the hap gene product is an important determinant of colonization, perhaps enabling the organism to evade the local immune response and thereby persist within the respiratory tract.
Growth and viability of Streptomyces coelicolor mutant for the cell division gene ftsZ
Authors: McCormick Joseph R. , Su Edwin P. , Driks Adam , Losick Richard 
Published: October 2006

SummaryA homologue of the bacterial cell division gene ftsZ was cloned from the filamentous bacterium Streptomyces coelicolor. The gene was located on the physical map of the chromosome at about ‘11 o'clock’ (in the vicinity of glkA, hisA and trpB). Surprisingly, a null mutant in which the 399‐codon ftsZ open reading frame was largely deleted was viable, even though the mutant was blocked in septum formation. This indicates that cell division may not be essential for the growth and viability of S. coelicolor. The ftsZ mutant was able to produce aerial hyphae but was unable to produce spores, a finding consistent with the idea that ftsZ is required in order for aerial hyphae to undergo septation into the uninucleoid cells that differentiate into spores.
Observations on the formation of deletions on monomeric and dimeric plasmids in Escherichia coli
Authors: Ravn Peter , Givskov Michael , Eegholm Karen‐Magrethe , Madsen Søren M. , Boe Lars 
Published: October 2006

SummaryWe have studied the formation of spontaneous mutations on plasmids present In the monomeric and dimeric states in a recF strain of Escherichia coli. Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (Oc) mutations on the pBR322‐derived plasmid pPY97. The rate of Oc mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the Oc phenotype was caused by small deletions in the operator sequence. No apparent mutational hot‐spot was found. The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids. Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322. A mechanism to account for this is suggested. Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans‐acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid. Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.
Permeability of the cell wall of Mycobacterium smegmatis
Authors: Trias Joaquim , Benz Roland 
Published: October 2006

SummaryThe cell wail of Mycobacterium smegmatis me2155 was shown to be an effective permeability barrier to hydrophilic compounds. Permeability coefficients to β‐lactams ranged from 10 × 10 −7 to 0.5 × 10 −7 cm s−1. Cell wall proteins were solubilized with EDTA and Genapol and were tested for channel‐forming activity by reconstitution into lipid bilayers. Proteins were able to induce a voltage‐gated cation‐selective channel. The mycobacterial porin channel appeared to be water‐filled since the single‐channel conductance followed the mobility sequence of hydrated ions in the aqueous phase. On the basis of the Renkin equation and the single‐channel conductance, the channel diameter was estimated to be around 3 nm. Model calculations showed that cation selectivity may be caused by four negative point‐charges at the channel mouth. The permeability properties of the cell wall of intact cells were in good agreement with those of the reconstituted channel. Negatively charged cephalosporins, cefamandole and cephalothin, diffused at a 10‐ to 20‐fold lower rate than the zwitterionic cephaloridine. The mycobacterial porin represents a major hydrophilic pathway of the cell wall of M. smegmatis.
Overexpression of fIbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brIA and premature initiation of development
Authors: Lee Bee Na , Adams Thomas H. 
Published: October 2006

Summary Aspergillus nidulans reproduces asexually by forming thousands of mitotically derived spores atop highly specialized multicellular organs termed conidiophores. We have identified a gene called flbA (for fluffy low brlA expression) that is required for initiation of A. nidulans conidiophore development. flbA mutants form abnormal colonies that have a distinct fluffy phenotype characterized by tightly interwoven aerial hyphae that autolyse as the colony matures. The requirement for fIbA in conidiophore development precedes activation of brlA, a primary regulator of conidiophore development. The wild‐type flbA gene was isolated and found to encode a 3.0 kb mRNA that is expressed throughout the A. nidulans asexual life cycle. Overexpression of fIbA using an Inducible promoter resulted in misscheduled expression of brlA in vegetative ceils and caused hyphal tips to differentiate into spore‐producing structures. Sequence analysis of a nearly full‐length fIbA cDNA clone showed that fibA is predicted to encode a 717‐amino‐acid polypeptide with 30% identity to the Saccharomyces cerevisiae SST2 protein. SST2 is required by yeast cells for resuming growth following prolonged exposure to yeast mating pheromone and for mating partner discrimination. We propose that fIbA plays a related role in a signalling pathway for Aspergillus conidiophore development.
Multiple protein‐DNA and protein‐protein interactions are involved in transcriptional activation by MaIT
Authors: Danot Olivier , Raibaud Olivier 
Published: October 2006

SummaryThe promoters of the Escherichia coli Maltose regulon are positively regulated by the MaIT protein, which recognizes a short asymmetric nucleotide sequence that is present as several copies in each promoter of the regulon. We report a detailed biochemical characterization of the interaction of MaIT with the promoter of the malPQ operon. The MaIT sites in malPp were precisely located and their occupation as a function of MaiT concentration was quantified using DNase I and dimethyl sulphate footprinting experiments. The contribution of each site to malPp activity was assessed by Introducing mutations that destroy them and measuring the residual in vivo and in vitro activity. Two main results were obtained. First, although the proximal MaIT site is centred at ‐37.5, RNA polymerase is likely to establish a contact required for matPp activity with at least one base pair of the promoter ‐35 region; this close proximity between RNA polymerase and MaIT bound to site 1 suggests that the two proteins interact. Second, even if the interaction of MaIT with the three functional sites in maIPp is a co‐operative process, the MaIT molecules bound to the two distal sites play a more subtle rote than simply increasing the occupancy of the proximal site and may also contact RNA polymerase. We suggest that, in the nucleoprotein structure responsible for the initiation of transcription, MaIT, RNA polymerase and malPp are held together by several weak interactions.
Klebsiella pneumoniae genes for citrate lyase and citrate lyase ligase: localization, sequencing, and expression
Authors: Bott Michael , Dimroth Peter 
Published: October 2006

SummaryIn the course of studies on anaerobic citrate metabolism in Klebsiella pneumoniae, the DNA region upstream of the gene for the sodium‐dependent citrate carrier (dtS) was investigated. Nucleotide sequence analysis revealed a cluster of five new genes that were oriented inversely to citS and probaby form an operon. The genes were named citCDEFG. Based on known protein sequence data, the gene products derived from citD, citE and citF could be identified as the λ‐, β‐, and α‐subunits of citrate lyase, respectively. This enzyme catalyses the cleavage of citrate to oxaloacetate and acetate. The gene product derived from citC (calculated Mr 36476) exhibited no obvious similarity to other proteins. In the presence of acetate and ATP, cell extracts from a citC‐expressing Escherichia coli strain were able to reactivate purified citrate lyase from K. pneumoniae that had been inactivated by chemical deacetylation of the prosthetic group. This represents 5‐phosphoribosyi‐dephospho‐acetyl‐coenzyme A which is covalently bound to serine‐14 of the acyl carrier protein (λ‐subunit). CitC was thus identified as acetate:SH‐citrate lyase ligase. The function of the gene product derived from citG (Mr 32 645) has not yet been identified. Expression of the CitCDEFG gene cluster in E. coli led to the formation of citrate lyase which was active only in the presence of acetyl‐coenzyme A, a compound known to substitute for the prosthetic group. These and other data strongly indicated that the enzyme synthesized in E. coli lacked its prosthetic group. Thus, additional genes besides citCDEFG appear to be required for the formation of holo‐citrate lyase.
Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages
Authors: Russel Marjorie 
Published: October 2006

SummaryThe filamentous phage protein pIV is required for assembly and secretion of the virus and possesses regions homologous to those found in a number of Gram‐negative bacterial proteins that are essential components of a widely distributed extracellular protein‐export system. These proteins form multimers that may constitute an outer membrane channel that allows phage/protein egress. Three sets of f1 gene IV mutants were isolated at positions that are absolutely (G355 and P375) or largely (F381) conserved amongst the 16 currently known family members. The G355 mutants were non‐functional, interfered with assembly of plV+ phage, and made Escherichia coli highly sensitive to deoxycholate. The P375 mutants were non‐functional and defective in multimerization. Many of the F381 mutants retained substantial function, and even those in which charged residues had been introduced supported some phage assembly. Some inferences about the roles of these conserved amino acids are made from the mutant phenotypes.