bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and molecular characterization of a transcriptional regulator from Pseudomonas aeruginosa PAO1 exhibiting structural and functional similarity to the FNR protein of Escherichia coli
Authors: Sawers R. G. 
Published: October 2006

SummaryA gene library of chromosomal DNA from Pseudomonas aeruginosa contained a DNA fragment which was able to restore anaerobic growth to an Escherichia coli fnr deletion mutant on glycerol/nitrate medium. The cloned gene (termed anr) was sequenced and shown to encode a protein of 244 amino acids with a calculated molecular weight of 27 129. The deduced amino acid sequence of the anr gene product showed considerable similarity to the FNR protein from E. coli. Expression of the anr gene in a T7 promoter/polymerase system identified ANR as a 31 kDa protein. Transcriptional analysis of the anr gene showed that it is monocistronic but apparently lacks the equivalent sites for negative autoregulation which have been shown to be present in the promoter region of the E. coli fnr gene. The ANR protein was shown to activate transcription of the pfl gene in E. coli in response to anaerobiosis, as well as being able to restore the activity of three anaerobicaliy inducible enzymes. A P. aeruginosa mutant incapable of growing anaerobically with nitrate or on arginine was fully complemented by the anr gene, indicating that it probably has a function in controlling anaerobic gene expression in Pseudomonas. Further corroboration for this assumption was provided by S1 nuclease analysis of transcription of the multiple promoters of the E. coli pfl operon in P. aeruginosa. Transcription was induced by oxygen limitation and was completely ANR‐dependent in both aerobic and anaerobic cells. Removal of the upstream regulatory sequence of the pfl operon, which includes the sequences required for FNR‐dependent regulation in E. coli, removed ANR‐dependent transcriptional control of the remaining pfl promoters, irrespective of the cellular oxygen status. These results imply that the mechanisms by which ANR and FNR regulate transcription are fundamentally similar.
Mutations in the aphA‐2 gene of transposon Tn5 mapping within the regions highly conserved in aminoglycoside‐phosphotransferases strongly reduce aminoglycoside resistance
Authors: Blázquez J. , Davies J. , Moreno F. 
Published: October 2006

SummaryAminoglycoside‐phosphotransferases contain several conserved amino acid sequence motifs. Using hydroxylamine we have obtained five independent missense mutations within the aphA‐2 gene of transposon Tn5. Four of the mutations dramatically reduced antibiotic resistance. Two were identical and included the replacement of His‐188 with Tyr. One other resulted from the replacement of Gly‐189 with Asp. These three mutations map within the first of the conserved motifs. The replacement of Asp‐261 with Asn maps to the third of these structural motifs. A mutation diminishing but not eliminating aminoglycoside resistance resulted from replacement of the conserved Val‐36 with Met. By site‐directed mutagenesis three additional mutants were obtained: His‐188 was replaced with Leu and Ser, and Arg‐211 within the second conserved motif was substituted by Gly. All three showed reduced levels of resistance to kanamycin. Our results show that these conserved motifs are essential for the biological activity of aminoglycoside phosphotransferases.
The Haemophilus influenzae capsulation gene cluster: a compound transposon
Authors: Kroll J. S. , Loynds B. M. , Moxon E. R. 
Published: October 2006

SummaryThe population of capsulate Haemophilus influenzae is divided into two phylogenetic divisions. Here we show that in division I strains the capsulation (cap) gene cluster lies between direct repeats of a novel insertion sequence (IS)‐like element, IS 1016. cap has apparently been mobilized in the chromosome as a compound transposon by IS 1016, and the repeats have provided a molecular substrate for reversible cap gene amplification, with augmentation of capsule production, through unequal homologous recombination. Such amplification has occurred in serotype b strains, but in these a large direct repeat of cap genes has become fixed in the population. We have found a 1.2kb deletion at one end of this duplicated cap b locus, removing most of one copy of the polysaccharide export gene bexA. We have shown that this makes capsulation dependent on preservation of the direct repeat structure in order to avoid recombination‐mediated loss of the other copy of bexA. Type b strains with this cap configuration are disseminated worldwide and currently cause nearly all invasive Haemophilus infections, leading us to speculate that the 1.2 kb deletion occurred in an ancestral type b strain and conferred significant biological advantage.
Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host‐range Gram‐negative cosmid vector pJRD215
Authors: Hermans J. , Martin C. , Huijberts G. N. M. , Goosen T. , de Bont J. A. M. 
Published: October 2006

SummaryThe transformation of Mycobacterium aurum and Mycobacterium smegmatis with the Gram‐negative RSF1010‐derived cosmid pJRD215 is described. The plasmid is stably maintained in both species and the antibiotic resistance determinants for kanamycin and streptomycin are expressed. Southern blot analysis shows that rearrangements take place both in M. aurum and in M. smegmatis. The use of pJRD215 in mycobacterial cloning systems is discussed.
Macronuclear and micronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1α in Stylonychia lemnae
Authors: Bierbaum P. , Dönhoff T. , Klein A. 
Published: October 2006

SummaryThe micronuclear and macronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1α in the hypotrich ciliate Stylonychia lemnae were compared. The two sequences are generally colinear. The coding sequence of the micronuclear gene is, however, interrupted by a 64 bp insert flanked by a 2 bp direct repeat in a gene region which is moderately conserved among EF 1α genes of different organisms. The insertion site is distinct from known intron positions in eukaryotic EF 1α genes. The insert sequence shows inverted repeats at its ends and thus exhibits typical features of an internal eliminated sequence (IES). Comparison with other such sequences in the related organism Oyxtricha nova shows that the IES falls into a new group of such elements. The macronuclear gene exhibits a strikingly limited codon usage, which cannot be simply explained by the overall base composition of the DNA but probably also relates to the very high copy number of the macronuclear gene and the putative high amount of the gene product.
The Entamoeba histolytica rDNA episome: nuclear localization, DNAase I sensitivity map, and specific DNA‐protein interactions
Authors: Zurita M. , Alagón A. , Vargas‐Villarreal J. , Lizardi P. M. 
Published: October 2006

SummaryStructural and functional features of the extrachromosomal DNA element that contains the ribosomal RNA genes of Entamoeba histolytica were studied using a variety of techniques. Using in situ hybridization, the element was found to be distributed along the inner phase of the nuclear membrane in the trophozoite stage; it appears to be part of the so‐called peripheral chromatin. DNAase I‐sensitive regions on the episome were mapped and found to correspond to the borders of the ribosomal RNA coding region. Other DNAase I‐sensitive regions were found to correspond to DNA containing a 145bp sequence that exists in the episome as tandem repeats. Electrophoretic shift assays and footprinting experiments demonstrate the existence of specific nuclear factors that bind specifically to the 145bp repeat. Preliminary analysis of the binding factors showed that a 28 kDa polypeptide is a likely candidate for a specific DNA:protein interaction involving the repetitive element. These results suggest that a protein‐binding domain within the 145bp repeat may have a specific function in the episome.
The spoIIIA operon of Bacillus subtilis defines a new temporal class of mother‐cell‐specific sporulation genes under the control of the σE form of RNA polymerase
Authors: Illing N. , Errington J. 
Published: October 2006

SummaryWe have cloned and characterized a 5 kbp region of the Bacillus subtilis chromosome and show that it contains the promoter‐proximal part of the spoIIIA locus. The locus consists of a polycistronic operon containing at least three genes. We show that the operon is regulated at the transcrlptional level, from a promoter that is first activated about 80 minutes after the induction of sporulation, immediately after septation. Expression of spoIIIA in different spo mutant backgrounds correlates with the ability of each strain to synthesize the sporulation‐specific sigma factor, σE. Moreover, synthesis of σE in vegetative cells by use of an inducible promoter causes expression of mother‐cell‐specific genes spoIID, spoIIIA, and spoIIID, but not the prespore‐specific genes, spoIIIG and spoVA. We suggest that σE may be the primary determinant of mother‐cell‐specific gene expression and that the SpoIIID protein exerts an additional level of regulation on spoIIIA, apparently by acting as a transcriptional repressor. Since the onset of spoIIID expression occurs about 10 minutes after that of spoIIIA, spoIIIA expression is transient. Thus spoIIIA defines a third temporal class of gene controlled by the σE form of RNA polymerase.
IS900‐promoted stable integration of a foreign gene into mycobacteria
Authors: England P. M. , Wall S. , McFadden J. 
Published: October 2006

SummaryAn artificial mycobacterial transposon was constructed by placing two copies of the insertion sequence IS900 flanking a kanamycin resistance gene into a non‐(mycobacterial) replicating vector. Constructs were introduced into mycobacteria by electroporation and transposition events conferring kanamycin resistance were selected. Integration of IS900 into several genomic sites was analysed by Southern blotting and shown to involve both simple insertions and cointegrate formation, suggesting that IS900 can transpose by a replicative mechanism. Kanamycin resistance of IS900‐integrated transformants was shown to be stable in the absence of selection.
Computer modelling of the NAD binding site of ADP‐ribosylating toxins: active‐site structure and mechanism of NAD binding
Authors: Domenighini M. , Montecucco C. , Ripka W. C. , Rappuoli R. 
Published: October 2006

SummarySummary Five ADP‐ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site‐directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three‐dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.
987P fimbrial gene identification and protein characterization by T7 RNA polymerase‐induced transcription and TnphoA mutagenesis
Authors: Schifferli D. M. , Beachey E. H. , Taylor R. K. 
Published: October 2006

SummaryThe 987P fimbrial gene cluster has been previously cloned as a 12 kb fragment from prototype strain 987. Gene products encoded by the whole clone were analysed by utilizing an in vivo system based on the induction of transcription by T7 RNA polymerase. The sensitivity of this technique permitted us to identify new proteins involved in 987P fimbriation. In total, eight proteins were detected, their genes (fasA to fasH) were mapped and their orientation of transcription determined. Several of the gene products demonstrated typical properties of exported proteins. Precursor and processed forms could be correlated after inhibiting protein transport with ethanol. The detection of enzymatically active fusion proteins after TnphoA (Tn5IS50L::phoA) mutagenesis supported and complemented these results. One protein encoded by the 12kb fragment was found not to be related to fimbriation but rather the product of the STla gene, identified as a component of a Tn1681‐like transposon.
Chlamydia trachomatis contains a protein similar to the Legionella pneumophila mip gene product
Authors: Lundemose A. G. , Birkelund S. , Fey S. J. , Larsen P. Mose , Christiansen G. 
Published: October 2006

SummaryA 27kDa Chlamydia trachomatis L2 protein was characterized by the use of monoclonal antibodies and by two‐dimensional gel electrophoresis. The protein was shown to be located in the membrane of reticulate bodies as well as elementary bodies. Its synthesis could be detected from 10 hours post‐infection. Cloning and sequence analysis of the distal part of the gene revealed an open reading frame of 175 amino acids. Comparison of the deduced amino acid sequence with the NBRF data base revealed significant homology between the 27kDa chlamydial membrane protein and the product of the macrophage infectivity potentiator (mip) gene of Legionella pneumophila.
Folding in vitro and transport in vivo of pre‐β‐lactamase are SecB independent
Authors: Laminet A. A. , Kumamoto C. A. , Plückthun A. 
Published: October 2006

SummaryThe rate of folding of the precursor of β‐lactamase is not influenced by the presence of SecB under conditions in which GroEL/ES retards the folding. Wild‐type β‐lactamase and several mutants in the signal or the mature protein, affecting either transport or enzyme kinetics and probably folding, were examined for total expression, total enzymatic activity, and transported β‐lactamase (in vivo resistance) in secB‐ and secB+ strains. We conclude that there is no indication of any relevant interaction between SecB and pre‐β‐lactamase in vitro, nor did the secB‐ mutation affect the transport of wild–type β‐lactamase or any of the mutants in vivo. Thus, putative Escherichia coli‘folding modulators’must be of limited specificity.
Control of pilus expression in Neisseria gonorrhoeae as an original system in the family of two‐component regulators
Authors: Taha M. K. , Dupuy B. , Saurin W. , So M. , Marchal C. 
Published: October 2006

SummaryWe have previously reported the identification of two genes, pilA and pilB, which act in trans to regulate pilus expression in Neisseria gonorrhoeae. Here we show that PilA and PilB have amino acid sequence similarities with members of the two component 'sensor‐regulator’family of proteins. PilB has homology with histidine kinase sensors. Alkaline phosphatase fusions to the predicted sensor and transmitter domains are described. Their PhoA activity and cellular location suggest that PilB is inserted in the cytoplasmic membrane and predict periplasmic and cytoplasmic locations for the sensor and the transmitter domains, respectively. PilA has homology with response regulators in its N‐terminal part, and with components of the eukaryotic protein secretory apparatus (SRP 54 and SRP receptor) as well as two Escherichia coli gene products in its C‐terminal part. In particular, it contains a putative GTP‐binding site. Mini‐transposon insertions into different regions of pilA were obtained. The phenotypes and genotypes of these mutants and preliminary biochemical studies of the gene products of two of these mutants lend further support to the hypothesis that PitA is a DNA‐binding response regulator and confirm that it participates in an essential function in the bacterium.
Sequences downstream from the transcriptional start site are essential for microaerobic, but not symbiotic, expression of the Rhizobium meliloti nifHDK promoter
Authors: Wang Y.‐P. , Birkenhead K. , Dobson A. , Boesten B. , O'Gara F. 
Published: October 2006

SummaryDeletion analysis studies have been carried out on the nifHDK promoter (p1) of R. meliloti in the expression of this promoter under both free‐living microaerobic and symbiotic conditions. Deletion of a region down‐stream (+17 to +61) from the promoter element resulted in low levels of expression under free‐living microaerobic conditions However, wild‐type levels of expression were obtained during symbiosis with Alfalfa plants. The sequences in this region were designated the‘downstream sequences'. The pattern of expression observed when the downstream sequences were deleted was similar to that observed when a previously identified upstream activator sequence (UAS) was deleted. Only when both the downstream sequences and the UAS were deleted, did activity form the P1 promoter become significantly decreased during symbiosis. Expression studies of the P1 promoter in a nifA mutant background indicate that nifA is required for symbiotic expression of P1 which is enhanced by the presence of the downstream sequences.
The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation
Authors: Perego M. , Higgins C. F. , Pearce S. R. , Gallagher M. P. , Hoch J. A. 
Published: October 2006

Summary Bacillus subtilis spo0K mutants are blocked at the first step in sporulation. The spo0K strain was found to contain two mutations: one was linked to the trpS locus, and the other was elsewhere on the chromosome. The mutation linked to trpS was responsible for the sporulation defect (spo‐). The unlinked mutation enhanced this sporulation deficiency but had no phenotype on its own. The spo‐ mutation was located in an operon of five genes highly homologous to the oligopeptide transport (Opp) system of Gram‐negative species. Studies with toxic peptide analogues showed that this operon does indeed encode a peptide‐transport system. However, unlike the Opp system of Salmonella typhimurium, one of the two ATP‐binding proteins, OppF, was not required for peptide transport or for sporulation. The OppA peptide‐binding protein, which is periplasmically located in Gram‐negative species, has a signal sequence characteristic of lipo‐proteins with an amino‐terminal lipo‐amino acid anchor. Cellular location studies revealed that OppA was associated with the cell during exponential growth, but was released into the medium in stationary phase. A major role of the Opp system in Gram‐negative bacteria is the recycling of cell‐wall peptides as they are released from the growing peptidoglycan. We postulate that the accumulation of such peptides may play a signaling role in the initiation of sporulation, and that the sporulation defect in opp mutants results from an inability to transport these peptides.
Dissecting the functional role of PriA protein‐catalysed primosome assembly in Escherichia coli DNA replication
Authors: Zavitz K. H. , Marians K. J. 
Published: October 2006

SummaryThe multi‐functional PriA protein of Escherichia coli (formerly replication factor Y or protein n′) serves to guide the ordered assembly of the primosome, a mobile multiprotein replication priming/helicase complex. Primosome assembly is essential for bacteriophage ØX174 complementary DNA strand synthesis and ColE1‐type plasmid replication reconstituted in vitro with purified proteins. The biochemical activities of the primosome suggest that it can fulfil the primase/helicase requirement on the lagging‐strand DNA template during cellular DNA replication. However, reconstruction in vitro of DNA replication of small plasmids containing the E. coli origin of DNA replication (oriC) does not require the complete complement of primosomal proteins. Thus, the extent to which PriA‐catalysed primosome assembly participates in chromosomal replication has remained unclear. The recent isolation of the genes encoding PriA, PriB (protein n), PriC (protein n″), and DnaT (protein i) has provided the necessary tools for addressing this issue. The phenotype of mutations in these genes, and other results described in this review, suggest that assembly of the primosome catalysed by PriA does in fact contribute at some stage to normal cellular DNA replication. A model for primososme‐catalysed reactivation of a dysfunctional replication fork is discussed.
Molecular analysis of two fructokinases involved in sucrose metabolism of enteric bacteria
Authors: Aulkemeyer P. , Ebner R. , Heilenmann G. , Jahreis K. , Schmid K. , Wrieden S. , Lengeler J. W. 
Published: October 2006

SummarySucrose‐positive derivatives of Escherichia coli K‐12, containing the plasmid pUR400, and of Klebsiella pneumoniae hydrolyse intracellular sucrose 6‐phosphate by means of an invertase into d‐glucose 6‐phosphate and free d‐fructose. The latter is phosphorylated by an ATP‐dependent fructokinase (gene scrK of an scr regulon) to d‐fructose 6‐phosphate. The lack of ScrK does not cause any visible phenotype in wild‐type strains of both organisms. Using genes and enzymes normally involved in d‐arabinitol metabolism from E. coli C and K. pneumoniae, derivatives of E. coli K‐12 were constructed which allowed the identification of scrK mutations on conventional indicator plates. Cloning and sequencing of scrK from sucrose plasmid pUR400 and from the chromosome of K. pneumoniae revealed an open reading frame of 924 bp in both cases — the equivalent of a peptide containing 307 amino acid residues (Mr, 39 and 34 kDa, respectively, on sodium dodecyl sulphate gels). The sequences showed overall identity among each other (69% identical residues) and to a kinase from Vibrio alginoiyticus (57%) also involved in sucrose metabolism, lower overall identity (39%) to a d‐ribose‐kinase from E. coli, and local similarity to prokaryotic, and eukaryotic phosphofructokinases at the putative ATP‐binding sites.
Molecular analysis of the Corynebacterium glutamicum gene involved in lysine uptake
Authors: Seep‐Feldhaus A. H. , Kalinowski J. , Pühler A. 
Published: October 2006

SummaryTwo Corynebacterium glutamicum mutants defective in lysine uptake were identified by analysing mutants resistant to S‐(2‐amtnoethyl)‐cysteine (AEC). A 5.6kb genomic DNA fragment restoring AEC sensitivity and lysine uptake was isolated. A 4.2kb subfragment was sequenced and three open reading frames were identified. Subcloning and gene disruption experiments showed that only the first open reading frame, termed lysl, is involved in lysine uptake. Lysl consists of 501 amino acids with a Mr of 53600. The hydrophobicity profile suggests that the lysl gene product is an integral membrane protein with 13 transmembrane segments. The amino acid sequence of lysl displays strong homology to that of the arcD gene product of Pseudomonas aeruginosa, which is proposed to act as an arginine–ornithine antiporter. Investigation of the influence of the lysl gene on lysine secretion suggests the existence of a separate lysine efflux system in C. glutamicum.