bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tn5‐mediated bleomycin resistance in Escherichia coli requires the expression of host genes
Authors: Blot Michel , Heitman Joseph , Arber Werner 
Published: October 2006

SummaryThe transposon Tn5 expresses a gene, ble, whose product increases the viability of Escherichia coli and also confers resistance to the DNA‐cleaving antibiotic bleomycin and the DNA‐alkylating agent ethyl‐methanesulphonate. We find that the Ble protein induces expression of an alkylation inducible gene, aidC, and that both the AidC gene product and DNA polymerase I are required for Ble to confer bleomycin resistance. These findings support models in which Ble enhances DNA repair and suggest that Tn5 confers a fitness advantage to the host bacterium by increasing the repair of spontaneous DNA lesions. Such co‐operation between a transposon and its host suggests that Tn5 is a symbiotic rather than a selfish DNA element.
Physical organization of lipids in the cell wall of Mycobacterium chelonae
Authors: Nikaido Hiroshi , Kim Sung‐Hou , Rosenberg Emiko Y. 
Published: October 2006

SummaryMycobacterial cell wall functions as an effective permeability barrier, making these bacteria resistant to most antibacterial agents. It has been assumed that this low permeability was due to the presence of a large amount of unusual lipids in the cell wall, but it was not known how these lipids are able to produce such an exceptional barrier. We report here the first experimental evidence on the physical arrangement of these lipids based on X‐ray diffraction studies of purified Mycobacterium chelonae cell wall, a result suggesting that the hydrocarbon chains of the cell‐wall lipids are arranged predominantly in a direction perpendicular to the cell wall surface, probably producing an asymmetric bilayer structure.
Molecular genetic analysis of the moa operon of Escherichia coli K‐12 required for molybdenum cofactor biosynthesis
Authors: Rivers S. L. , McNairn E. , Blasco F. , Giordano G. , Boxer D. H. 
Published: October 2006

SummaryA 3.2 kb chromosomal DNA fragment which complements the defects in a series of twelve moa::Mucts insertion mutants has been sequenced. Five open reading frames (ORFs) were identified and these are arranged in a manner consistent with their forming an operon. The encoded proteins (MoaA‐MoaE) have predicted molecular weights of 37346, 18665, 17234, 8843 and 16981 respectively. Examination of subclones of the whole locus in an expression system demonstrated the predicted products. N‐terminal amino acid sequences for the moa A, B, C and E products confirmed the translational starts. Genetic analysis distinguished four classes of moa mutants corresponding to genes moaA, C, D and E. Potential promoter sequences upstream of moaA and a possible transcription termination signal have been identified. Genetic analysis of the chlA1 and chlM mutants, which have been biochemically characterized as defective in molybdopterin biosynthesis, indicates that these carry lesions in moaA and moaD respectively. The moa locus is orientated clockwise at 17.7 minutes in the chromosome.
The obligate intracellular bacterium Chlamydia trachomatis is auxotrophic for three of the four ribonucleoside triphosphates
Authors: Tipples Graham , McClarty Grant 
Published: October 2006

SummaryUsing we11‐characterized mutant host cell lines, deficient in specific enzymes of energy and nucleotide metabolism, we addressed numerous questions regarding nucleotide metabolism in the obligate intracellular bacterium Chlamydia trachomatis. The results presented indicate that C. trachomatis: (i) does not absolutely depend on mitochondrial generated ATP for survival; (ii) does have a significant draw on host‐cell NTP pools but does not have a detrimental effect on the ability of the host cell to maintain its energy charge; (iii) lacks the ability to synthesize purine and pyrimidine nucleotides de novo; (iv) is not capable of interconverting purine nucleotides; and (v) possesses the pyrimidine metabolic‐pathway enzymes CTP synthetase and deoxycytidine nucleotide deaminase. In total our results indicate that C. trachomatis is auxotrophic for host‐cell ATP, GTP and UTP. In contrast, CTP can be obtained from the host cell or it can be synthesized from UTP by the parasite.
ATPase activity and ATP/ADP‐induced conformational change in the soluble domain of the bacterial protein translocator HlyB
Authors: Koronakis Vassilis , Hughes Colin , Koronakis Eva 
Published: October 2006

SummaryThe haemolysin exporter HlyB and its homologues are central to the unconventional signal‐peptide‐independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP‐binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug‐resistance P‐glycoprotein, combining both integral membrane and cytosolic domains. Overproduction of the HlyB cytopiasmic domain as a C ‐terminal peptide fused to glutathione S‐transferase allowed the direct affinity purification and concentration of 30‐50 mg ml−1 of soluble protein (GST‐Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST‐Bctp bound to ADP‐agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full‐length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro. The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein. Mg2+ ‐dependent ATPase activity of GST‐Bctp Vmax 17mu;mol min−1 mg−1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P‐glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F‐ and V‐type ATPases, non‐hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P‐type) ATPases, and the acidic phospholipid phosphatidyl glycerol.
The role of mRNA degradation in the regulated expression of bacterial photosynthesis genes
Authors: Klug Gabriele 
Published: October 2006

SummaryRegulation of gene expression in bacteria, as in eukaryotic cells, is often achieved by variation of mRNA levels. Since the steady state levels of mRNA depend on both the rate of synthesis and the rate of decay, both mechanisms are important for gene regulation. After considerable effort undertaken over many years to understand the regulation of transcription, mRNA degradation has recently gained Increasing attention as an important step in the regulation of some bacterial genes, and many investigations have addressed the mechanisms involved in mRNA decay. The puf mRNA of Rhodobacter capsulatus encoding pigment binding proteins has become a model system to study decay of a polycistronic mRNA and the role of mRNA degradation in gene expression. Individual segments of the polycistronic puf mRNA display extremely different half‐lives. These differences in stability of mRNA segments are involved in the differential expression of puf encoded genes and consequently contribute to the stoichiometry of light‐harvesting I and reaction centre complexes that results in optimal growth. In addition, control of mRNA stability is involved in the oxygen‐dependent regulation of photosynthesis genes. High oxygen tension results in decreased stability of the reaction‐centre specific puf mRNA segment, most likely by affecting the rate of endonucleolytic cleavage within the reaction centre coding region. The results obtained from studying puf mRNA degradation in R. capsulatus and Escherichia coli suggest that a specific distribution of decay promoting and decay impeding mRNA elements along the polycistronic mRNA is responsible for the different half‐lives of individual puf segments.
Adaptation of Escherichia coli to redox environments by gene expression
Authors: Iuchi S. , Lin E. C. C. 
Published: October 2006

Summary Escherichia coli is adroit in exploiting environmental energy sources to its greatest profit. A key strategy is to channel electron transport from donors to a terminal acceptor(s) so that the voltage drop is maximal. At the level of transcription, the goal is achieved by the interaction of three global regulatory systems, Fnr, NarL/NarX and ArcB/ArcA. In addition, the regulator FhlA is involved in a cascade‐controlled pathway for the formate branch of the pyruvate fermentation pathway.
Molecular cloning and characterization of a sym plasmid locus that regulates cultivar‐specific nodulation of soybean by Rhizobium fredii USDA257
Authors: Meinhardt L. W. , Krishnan H. B. , Balatti P. A. , Pueppke S. G. 
Published: October 2006

Summary Rhizobium fredii strain USDA257 produces nitrogen‐fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposonmutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain U5DA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolWand nolX are of the opposite polarity. The initiation codon for nolW lies 155bp upstream from that of nolB, and it is separated from nolXby 281 bp. The predicted NolT and NolW proteins have putative membrane‐spanning regions. The N‐terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site‐directed mutagenesis with mudll1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU anti nolX is induced as much as 30‐fold by flavonoid signal molecules, even though these genes lack nod‐box promoters. Histochemical staining of McCall roots inoculated with nolB–, nolU–, or nolX–lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU–ORF4–nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad‐host‐range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.
S‐layer protein from Thermus thermophilus HB8 assembles into porin‐like structures
Authors: Castón José R. , Berenguer José , de Pedro Miguel A. , Carrascosa José L. 
Published: October 2006

SummaryThe cells of the extreme thermophile Thermus thermophilus are surounded by a regular layer (S‐layer) built up by a protein with an apparent molecular mass of 100 kDa (P100). From purified membrane fractions, three different class of two‐dimensional crystals can be obtained by following alternative extractive procedures. One of these crystals, with p6 symmetry, clearly represents the native S‐layer detected by freeze etching on whole cells, while the other two, showing p2 and p3 symmetries respectively, closely resemble aggregates of bacterial porins. We demonstrate here by limited protreolysis and Western blotting the surprising fact that the protein component of the three crystals is the P100 protein. Our biochemical data also show how this protein forms Ca2+‐stabilized trimers in each crystal, which support the structural analysis that points to p3 units as the common structural block in all of them, and again resembles the situation found in bacterial porins.
Cloning and nucleotide sequence of frpC, a second gene from Neisseria meningitidis encoding a protein similar to RTX cytotoxins
Authors: Thompson Stuart A. , Wang Lisa L. , Sparling P. Frederick 
Published: October 2006

Summary Neisseria meningitidis FAM20 has recently been shown to produce two Fe‐regulated proteins (FrpA and FrpC) related to the RTX family of cytotoxins. Here we report the cloning and DNA sequence of the locus containing the gene encoding the larger meningococcal RTX protein FrpC. FrpC was highly similar to FrpA throughout much of the predicted protein, with two main differences. Whereas the FrpA protein had 13 copies of the nine‐amino‐acid repeat units typical of RTX proteins, FrpC had 43 copies. The additional copies in FrpC apparently arose from a threefold tandem amplification of a 600bp DNA fragment encoding the repeats. In addition, the frpC gene lacked good promoter consensus sequences. An open reading frame (0RF1) of unknown function was found immediately upstream of frpC, suggesting the possibility that frpC was cotranscribed with ORF1. A probable promoter was found 300 bp upstream of ORF1, and it contained a Fur protein‐binding sequence found in the promoters of Fe‐regulated Escherichia coli genes. DNA upstream of the ORF 1/frpC promoter was homologous to IStO76‐like elements surrounding capsulation loci of strains of Haemophilus influenzae. A FrpC‐like protein (reactive in immunoblots with monoclonal antibody 9D4; multiple reactive bands of about 200 to 120kDa) was found in five out of eight meningococcal strains but only in one out of 14 other Neisseria, suggesting that FrpC may participate in the pathogenesis of meningococcal disease.
Characterization of the cspB gene encoding PS2, an ordered surface‐layer protein in Corynebacterium glutamicum
Authors: Peyret J. L. , Bayan N. , Joliff G. , Gulik‐Krzywicki T. , Mathieu L. , Shechter E. , Leblon G. 
Published: October 2006

SummaryPS2 is one of two major proteins detected in the culture media of various Corynebacterium glutamicum strains. The coding and promoter regions of the cspB gene encoding PS2 were cloned in lambda gt11 using polyclonal antibodies raised against PS2 for screening. Expression of the cspB gene in Escherichia coli led to the production of a major anti‐PS2 labelled peptide of 63 000 Da, corresponding presumably to the mature form of PS2. It was detected in the cytoplasm, periplasm and surrounding medium of E. coli. Three other slower migrating bands of 65000, 68 000 and 72 000 Da were detected. The largest one probably corresponds to the precursor form of PS2 in E. coli. Analysis of the nucleotide sequence revealed an open reading frame (ORF) of 1533 nucleotides. The deduced 510‐amino‐acid polypeptide had a calculated molecular mass of 55 426 Da. According to the predicted amino acid sequence, PS2 is synthesized with a W‐terminal segment of 30‐amino‐acid residues reminiscent of eukaryotic and prokaryotic signal pep‐tides, and a hydrophobic domain of 21 residues near the C‐terminus. Although no significant homologies were found with other proteins, it appears that some characteristics and the amino acid composition of PS2 share several common features with surface‐layer proteins. The cspB gene was then disrupted in C. glutamicum by gene replacement. Freeze‐etching electron microscopy performed on the wild‐type strain indicated that the cell wall of C. glutamicum is covered with an ordered surface of proteins (surface layer, S‐layer) which is in very close contact with other cell‐wall components. These structures are absent from the cspB‐disrupted strain but are present after reintroduction of the cspB gene on a plasmid into this mutant. Thus we demonstrate that the Slayer protein is the product of the cspB gene.
Complementation of an Enterococcus hirae (Streptococcus faecalis) mutant in the alpha subunit of the H+‐ATPase by cloned genes from the same and different species
Authors: Suzuki Tomoko , Shibata Chiyoko , Yamaguchi Akie , Igarashi Kazuei , Kobayashi Hiroshi 
Published: October 2006

SummaryWe isolated an Enterococcus hirae (formerly Streptococcus faecalis) mutant, designated MS117, in which ‘G’ at position 301 of the alpha‐subunit gene of the F1F0 type of H+‐ATPase was deleted. MS117 had low H+‐ATPase activity, was deficient in the regulatory system of cytoplasmic pH, and was unable to grow at pH6.0. When the alpha‐subunit gene of E. hirae H+‐ATPase was ligated with the shuttle vector pHY300PLK at the downstream region of the tet gene of the vector, it was expressed without its own promoter in MS117, and the mutation of MS117 was complemented; the mutant harbouring the plasmid had the ability to maintain a neutral cytoplasm and grew at pH6.0. We next transformed MS117 with pHY300PLK containing the alpha‐subunit gene of Bacillus megaterium F1F0‐ATPase constructed in the same way. The transformant grew at pH 6.0, and the ATP hydrolysis activity was recovered. These results suggested that an active hybrid H+‐ATPase containing the B. megaterium alpha subunit was produced, and that the hybrid enzyme regulated the enterococcal cytoplasmic pH, although the function of the B. megaterium enzyme did not include pH regulation. Thus, our present results support the previous proposal that the enterococcal cytoplasmic pH is regulated by the F1F0 type of H+‐ATPase.
Conservation of motifs within the unusually variable polypeptide sequences of type I restriction and modification enzymes
Authors: Murray Noreen E. , Daniel Anne S. , Cowan Gill M. , Sharp Paul M. 
Published: October 2006

SummaryType I restriction enzymes comprise three subunits encoded by genes designated hsdR, hsdM, and hsdS; S confers sequence specificity. Three families of enzymes are known and within families, but not between, hsdM and hsdR are conserved. Consequently, interfamily comparisons of M and R sequences focus on regions of putative functional significance, while both inter‐ and intrafamily comparisons address the origin, nature and role of diversity of type I restriction systems. We have determined the sequence of the hsdR gene for EcoA, thus making available sequences of all three hsd genes of one representative from each family. The predicted R polypeptide sequences share conserved regions with one superfamily of putative helicases, so‐called ‘DEAD box’ proteins; these conserved sequences may be associated with the ATP‐dependent translocation of DNA that precedes restriction. We also present hsdM and hsdR sequences for EcoE, a member of the same family as EcoA. The sequences of the M and R genes of EcoA and EcoE are at least as divergent as typical genes from Escherichia coli and Salmonella, perhaps as the result of selection favouring diversity of restriction specificities combined with lateral transfer among different species.
Regulation of the ansB gene of Salmonella enterica
Authors: Jennings Michael P. , Scott Shaun P. , Beacham Ifor R. 
Published: October 2006

SummaryThe expression of l‐asparaginase II (encoded by ansB) in Salmonella enterica was found to be positively regulated by the cAMP receptor protein (CRP) and anaerobiosis. The anaerobic regulation of the S. enterica ansB gene is not mediated by the anaerobic transcriptional activator FNR. This is unlike the situation of the ansB gene of Escherichia coli, which is dependent on both CRP and FNR. To investigate this fundamental difference in the regulation of l‐asparaginase II expression in S. enterica, the ansB gene was cloned and the nucleotide sequence of the promoter region determined. Sequence analysis and transcript mapping of the 5′ promoter region revealed a single transcriptional start point (tsp) and two regulatory sites with substantial homology with those found in E. coli. One site, centred –90.5 bp from the tsp, is homologous to a hybrid CRP/FNR (‘CF’) site which is the site of CRP regulation in the E. coli promoter. The other site, centred 40.5 bp upstream of the tsp, is homologous to the FNR binding site of the E. coli promoter. Significantly, however, a single base‐pair difference exists in this site, at a position of the related CRP and FNR DNA‐binding site consensus sequences known to be involved in CRP versus FNR specificity. Site‐directed mutagenesis indicates that this single difference, relative to the homologous E. coli site, results in a CRP binding site and the observed FNR‐independent ansB expression in S. enterica. Thus, not only may CRP and FNR sites be interconverted experimentally, with few nucleotide changes, but this has apparently contributed to the evolutionary divergence of ansB regulation in E. coli and S. enterica.
Analysis of diphtheria toxin repressor‐operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals
Authors: Schmitt Michael P. , Holmes Randall K. 
Published: October 2006

SummaryThe diphtheria toxin repressor (DtxR) is an Fe2+‐activated protein with sequence‐specific DNA‐binding activity for the diphtheria toxin (tox) operator. Under high‐iron conditions in Corynebacterium diphtheriae, DtxR represses toxin and siderophore biosynthesis as well as iron uptake. DtxR and a mutant repressor with His–47 substituted for Arg–47, designated DtxR‐R47H, were purified and compared. Six different divalent cations (Cd2+, Co2+, Fe2+, Mn2+, Ni2+, and Zn2+) activated the sequence‐specific DNA‐binding activity of DtxR and enabled it to protect the fox operator from DNase I digestion, but Cu2+ failed to activate DtxR. Hydroxyl radical footprinting experiments indicated that DtxR binds symmetrically about the dyad axis of the tox operator. Methylation protection experiments demonstrated that DtxR binding alters the susceptibility to methylation of three G residues within the AT‐rich tox operator. These findings suggest that two or more monomers of DtxR are involved in binding to the tox operator, with symmetrical DNA‐protein interactions occurring at each end of the palindromic operator. In this regard, DtxR resembles several other well‐characterized prokaryotic repressor proteins but differs dramatically from the Fe2+‐activated ferric uptake repressor protein (Fur) of Escherichia coli. The concentration of Co2+ required to activate DtxR‐R47H was at least 10‐foid greater than that needed to activate DtxR, but the sequence‐specific DNA binding of activated DtxR‐R47H was indistinguishable from that of wild‐type DtxR. The markedly deficient repressor activity of DtxR‐R47H is consistent with a significant decrease in its binding activity for divalent cations.
Structure–function correlation for the EcoRV restriction enzyme: from non‐specific binding to specific DNA cleavage
Authors: Vipond I. Barry , Halford Stephen E. 
Published: October 2006

SummaryThe EcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence. The only cofactor it requires for activity is Mg2+: but in binding to DNA in the absence of Mg2+, the EcoRV enzyme shows no specificity for its recognition site. Instead, the reason why EcoRV cuts one DNA sequence faster than any other is that the rate of cleavage is controlled by the binding of Mg2+ to EcoRV‐DNA complexes: the complex at the recognition site has a high affinity for Mg2+, while the complexes at other DNA sequences have low affinities for Mg2+. The structures of the EcoRV endonuclease, and of its complexes with either 8pecific or non‐specific DNA, have been solved by X‐ray crystallography. In the specific complex, the protein interacts with the bases in the recognition sequence and the DNA takes up a highly distorted structure. In the non‐specific complex with an unrelated DNA sequence, there are virtually no interactions with the bases and the DNA retains a B‐like structure. Since the free energy changes for the formation of specific and non‐specific complexes are the same, the energy from the specific interactions balances that required for the distortion of the DNA. The distortion inserts the phosphate at the scissile bond into the active site of the enzyme, where it forms part of the binding site for Mg2+. Without this distortion, the EcoRV–DNA complex would be unable to bind Mg2+ and thus unable to cleave DNA. The specificity of the EcoRV restriction enzyme is therefore governed, not by DNA binding as such, but by its ability to organize the structure of the DNA to which it is bound.