bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipopolysaccharides of polymyxin B‐resistant mutants of Escherichia coii are extensively substituted by 2‐aminoethyl pyrophosphate and contain aminoarabinose in lipid A
Authors: Nummila Kim , Kilpeläinen IIkka , Zähringer Ulrich , Vaara Martti , Helander IIkka M. 
Published: October 2006

SummaryLipopolysaccharides (LPS) of two polymyxin‐resistant (pmr) mutants and the corresponding parent strain of Escherichia Coli were chemically analysed for composition and subjected to 31P‐NMR (nuclear magnetic resonance) for assessment of phosphate substitution. Whereas the saccharide portions, fatty acids, and phosphate contents were similar in wild‐type and pmr LPS, the latter contained two‐ to threefold higher amounts of 2‐aminoethanol. The pmr LPS also contained 4‐amino‐4‐deoxy‐l‐arabinopyranose (l‐Arap4N), which is normally not a component of E. coli LPS. This aminopentose has been assigned to be linked to the 4′‐phosphate of lipid A. Comparative 31P‐NMR analysis of the de‐O‐acylated LPS of the wild‐type and pmr strains revealed that phosphate groups of the pmr LPS were mainly (71‐79%) diphosphate diesters, which accounted for only 20% in the wild‐type LPS. Diphosphate monoesters were virtually nonexistent in the pmr LPS, whereas they accounted for 42% of all phosphates in wild‐type LPS. In the lipid A of the pmr strains, the 4′‐phosphate was to a significant degree (35%) substituted by l‐Arap4N, whereas in the wild‐type LPS the l‐ArapN was absent. In the pmr lipid A1 2‐aminoethanol was completely substituting the glycosidic pyrophosphate but not the glycosidic monophosphate, forming a diphosphate diester linkage at this position in 40% of lipid A molecules. In the wild‐type LPS the glycosidic position of lipid A carried mostly unsubstituted monophosphate and pyrophosphate. Thus the polymyxin resistance was shown to be associated, along with the esterification of the lipid A 4′‐monophosphate by aminoarabinose, with extensive esterification of diphosphates in LPS by 2‐aminoethanol.
Enhanced secretion through the Shigella flexneri Mxi‐Spa translocon leads to assembly of extracellular proteins into macromolecular structures
Authors: Parsot Claude , Ménard Robert , Gounon Pierre , Sansonetti Philippe J. 
Published: October 2006

SummaryGenes required for entry of Shigella flexneri into epithelial cells in vitro are clustered in two adjacent loci, one of which encodes secretory proteins, the IpaA–D proteins, and the other their dedicated secretion apparatus, the Mxi–Spa translocon. Ipa secretion, which is induced upon contact of bacteria with epithelial cells, is prevented during growth in vitro. Here, we show that ipaB and ipaD mutations lead to enhanced secretion of a set of about 15 proteins. These extracellular proteins and some Ipas associate in organized structures consisting of extended sheets. Growth of the wild‐type strain in the presence of Congo red is shown to induce protein secretion through the Mxi–Spa translocon. Cultures grown to stationary phase in the presence of Congo red contain extracellular filaments whose composition and morphology are similar to those produced by the hyper‐secreting ipaB and ipaD mutants.
Isolation and characterization of genes that promote the expression of inositol transporter gene ITR1 in Saccharomyces cerevisiae
Authors: Nikawa J. , Hosaka K. 
Published: October 2006

SummaryThe expression of many genes of Saccharomyces cerevisiae, such as ITR1, is regulated by inositol and choline. In this work, a yeast strain has been constructed in which HIS3 expression is controlled by the ITR1 promoter. Using this strain, three genes were isolated which, when Introduced as multicopies, abolish the repression caused by inositol via the ITR1 promoter. Northern blot analysis revealed that two of these three genes, designated as DIE1 and DIE2, clearly increased the expression of ITR1. DIE2 is more effective for ITR1 expression than DIE1. Gene‐disruption experiments revealed that DIE1 was essential for the expression of ITR1 but that DIE2 was not. The sequence of the DIE1 gene was shown to be identical to that of INO2 (also called SCS1), which encodes a protein required for the expression of INO1. DIE2 is a new gene and is capable of encoding 525 amino acid residues with a calculated molecular weight of 61 789. Experiments involving lacZfusion genes showed that multicopy DIE2 resulted in an increase in the expression of both ITR1 and INO1. These results strongly suggest that the DIE1 and DIE2 gene products have an important regulatory function for gene expression of not only ITR1 but also INO1.
The Escherichia coli genes sspA and rnk can functionally replace the Pseudomonas aeruginosa alginate regulatory gene algR2
Authors: Schlictman David , Shankar Sandeep , Chakrabarty A. M. 
Published: October 2006

SummaryThe algR2 (also known as algQ) gene of Pseudomonas aeruginosa has previously been identified as being necessary for alginate production at 37°C. We have cloned two genes, from a cosmid library of Escherichia coli, which can restore mucoidy to an algR2 mutant of P. aeruginosa. The complementing regions of both cosmids were localized by subcloning restriction fragments. One of the E. coli genes identified here has not previously been described; we have named this gene rnk (regulator of nucleoside diphosphate kinase). It encodes a 14.9 kDa protein with no homo‐logy to any other protein. The other gene, sspA, is a regulator involved in stationary‐phase regulation in E. coli. Either gene will restore mucoidy to an algR2‐deficient strain of P. aeruginosa. AlgR2 has been shown to regulate at least two enzymes, succinyl‐CoA synthetase (Scs) and nucleoside diphosphate kinase (Ndk), which form a complex in P. aeruginosa. When we examined the ability of the E. coli analogues to regulate Ndk, we found that rnk but not sspA was able to restore Ndk activity to the P. aeruginosa algR2 mutant. Furthermore, rnk was able to restore growth of the algR2 mutant in the presence of Tween 20, which inhibits other Ndk‐like activities.
Genetic and molecular analysis of a regulatory region of the herbicide 2,4‐dichlorophenoxyacetate catabolic plasmid pJP4
Authors: You I.‐S. , Ghosal D. 
Published: October 2006

SummaryIn Alcaligenes eutrophus JMP134, pJP4 carries the genes coding for 2,4‐dichlorophenoxyacetate (2,4‐D) and 3‐chlorobenzoate (3‐Cba) degradation plus mercury resistance. The plasmid genes specifying 2,4‐D and 3‐Cba catabolism are organized in three operons: tfdA, tfdB, and tfdCDEF. Regulation of these operons by two unlinked genes, tfdR and tfdS, has been proposed. Physical and DNA sequence analyses revealed that the tfdR and tfdS genes were identical and were located within a longer inverted repeat of 1592bp. Similar stem‐loop structures were observed among other 2,4‐D plasmids. The tfdR gene is 888 bp long and capable of encoding a polypeptide of 32kDa. The deduced amino acid sequence of tfdR indicates that it is a member of the LysR‐type activators. Investigation of the regulation of the catabolic gene clusters through the construction of a pJP4 defined deletion mutant, pYG1010, which lacks a 4.2 kilobase Xbal fragment containing the inverted repeat region carrying the tfdR and tfdS regulatory genes, showed that Pseudomonas cepacia strains containing pYG1010 became 2,4‐D negative, but 3‐Cba positive. In vivo recombinants of pYG1010 and a cloned tfdS gene rescued the 2,4‐D phenotype, indicating that TfdS is a positive regulator of tfdA expression, but not for tfdCDEF expression.
The tagGH operon of Bacillus subtilis 168 encodes a two‐component ABC transporter involved in the metabolism of two wall teichoic acids
Authors: Lazarevic Vladimir , Karamata Dimitri 
Published: October 2006

SummaryWe report the nucleotide sequence and the characterization of the Bacillus subtilis tagGH operon. The latter is controlled by a σA‐dependent promoter and situated in the 308° chromosomal region which contains genes involved in teichoic acid biosynthesis. TagG is a hydrophobic 32.2 kDa protein which resembles integral membrane proteins belonging to polymerexport systems of Gram‐negative bacteria. Gene tagH encodes a 59.9 kDa protein whose N‐moiety contains the ATP‐binding motif and shares extensive homology with a number of ATP‐binding proteins, particularly with those associated with the transport of capsular polysaccharides and O‐antigens. That the tagGH operon is essential for cell growth was established by the failure to inactivate tagG and the 5′ ‐moiety of tagH by insertional mutagenesis. During limited tagGH expression, cells exhibited a cocoid morphology while their walls contained reduced amounts of phosphate as well as galactosamine. These observations, revealing impaired metabolism of both wall teichoic acids of B. subtilis 168, i.e. poly(glycerol phosphate), and poly(glucose galactosamine phosphate), combined with sequence homologies, suggest that TagG and TagH are involved in the translocation through the cytoplasmic membrane of the latter teichoic acids or their precursors.
Saturation of penicillin‐binding protein 1 by β‐lactam antibiotics in growing cells of Bacillus licheniformis
Authors: Lepage Sophie , Lakaye Bernard , Galleni Moreno , Thamm Iris , Crine Michel , Groslambert Sylvie , Frère Jean‐Marie 
Published: October 2006

SummaryWith the help of a new highly sensitive method allowing the quantification of free penicillin‐binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various β‐lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.
Repair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteins
Authors: Visick Jonathan E. , Clarke Steven 
Published: October 2006

SummaryProteins, like DNA, are subject to various forms of damage that can render them non‐functional. Conformational changes and covalent chemical alterations occur spontaneously, and the rates of these reactions can be increased by environmental stresses such as heat, oxidative agents, or changes in pH or osmotic conditions. Although affected proteins can be replaced by de novo biosynthesis, cells – especially those subjected to stress or nutrient limitation – have developed mechanisms which can either restore damaged polypeptides to an active state or remove them. Such mechanisms can spare the biosynthetic capacity of the cell and ensure that the presence of non‐functional molecules does not disrupt cell physiology. Three major mechanisms, which operate in bacteria as well as eukaryotic organisms, have been described. First, chaperones not only assist in proper de novo folding of proteins but also provide an important means of restoring activity to conformationally damaged proteins. Second, enzymatic‘repair’systems exist to directly reverse certain forms of protein damage, including proline isomerization, methionine oxidation and the formation of isoaspartyl residues. Finally, proteolysis provides a‘last‐resort’means of dealing with abnormal proteins which cannot be repaired. Protein maintenance and repair may be of special importance for bacteria preparing to survive extended periods in stationary phase: both constitutive and induced mechanisms are utilized to permit survival despite greatly reduced protein synthesis.
Cloning, expression and functional analyses of the catabolite control protein CcpA from Bacillus megaterium
Authors: Hueck Christoph J. , Kraus Alexandra , Schmiedel Dagmar , Hillen Wolfgang 
Published: October 2006

SummaryA mutant of Bacillus megaterium relieved from catabolite repression has been used to clone ccpA from B. megaterium by complementation. ccpA is the first gene of a presumed operon, in which it is followed by the motA homologue ORF1 and the motB homologue ORF2. The mutation maps in the 3′‐terminal region of ccpA, where an in‐frame duplication of 84 nucleotides located between two 9 bp direct repeats leads to an insertion of 28 amino acids near the C‐terminus of CcpA. An in‐frame deletion of 501 bp in ccpA exhibits the same phenotype as the 84 bp duplication. Deletion of ORF1 and ORF2 does not yield an apparent phenotype. A single‐copy ccpA::lacZ transcriptional fusion is constitutively expressed, independent of whether the growth medium triggers catabolite repression or not. The ccpA mutation leads to relief of catabolite repression exerted by glucose, fructose, mannitol, glucitol and glycerol, whereas only smaller effects were found with ribose, citrate and glutamate. The respective growth rates on these carbon sources are uniformly reduced to a generation time of about 90 min in the ccpA mutant. Catabolite repression of a plasmid‐encoded xylA::ccpA fusion is less efficient than that of a xylA::lacZ fusion in the same vector. Furthermore, overproduction of CcpA decreases catabolite repression of a singlecopy xylA::lacZ fusion approximately twofold. Thus, overexpression of CcpA may be counterproductive for catabolite repression, supporting the hypothesis that CcpA by itself may not bind sufficiently strongly to the cis‐active catabolite‐responsive element to exert catabolite repression.
Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae
Authors: Winter Nathalie , Triccas James A. , Rivoire Becky , Pessolani Maria Cristina V. , Eiglmeier Karin , Lim Eng‐Mong , Hunter Shirley W. , Brennan Patrick J. , Britton Warwick J. 
Published: October 2006

SummaryAnalysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N‐terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein‐encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an‘up‐regulated’promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein‐encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The avaibility of the recombinant 35 kDa protein will permit dissection of both antibody‐ and T‐cell‐mediated immune responses in leprosy patients.
Novel organization of the site‐specific integration and excision recombination functions of the Staphylococcus aureus serotype F virulence‐converting phages φ13 and φ42
Authors: Carroll David , Kehoe Michael A. , Cavanagh David , Coleman David C. 
Published: October 2006

SummaryFunctions required for site‐specific integration and excision of the Staphylococcus aureus serotype F virulence‐converting phages φ13 and φ42 were localized and characterized. Like other temperate phages, integration of φ13 and φ42 sequences was found to require the product of an int gene located close to the phage attP site. Both int genes are almost identical, express proteins possessing characteristic features of the Int (integrase) family of recombinases, but share very little homology with previously described int genes, including those of the serotype B S. aureus phages L54a and φ11. Nevertheless, all four S. aureus phages share an almost identical short sequence located immediately 5′ to these distinct int genes, suggesting a common mechanism of int gene regulation. Upstream from these common sequences, the sequences of φ13 and φ42 are quite distinct from each other, and from the corresponding regions of φ11 and L54a which encode the Xis proteins that are required with Int to mediate site‐specific excision of the latter phages. Surprisingly, φ13 and φ42 sequences encompassing the attP sites and int genes, but lacking either an adjacent or more distant phage excision protein gene, were sufficient to mediate site‐specific excision of integrated phage DNA sequences.
Identification of the ligand‐binding domain of the surface‐located fibrinogen receptor (clumping factor) of Staphylococcus aureus
Authors: McDevitt D. , Francois P , Vaudaux P. , Foster T. J. 
Published: October 2006

SummaryThe ability of Staphylococcus aureus to bind to fibrinogen and fibrin is believed to be an important factor in the initiation of foreign‐body and wound Infections. Recently, we reported the cloning and sequencing of the gene clfA encoding the fibrinogen receptor (clumping factor, ClfA) of S. aureus strain Newman and showed that the gene product was responsible for the clumping of bacteria in soluble fibrinogen and for the adherence of bacteria to solid‐phase fibrinogen. This was confirmed here by showing that antibodies raised against purified Region A inhibited both of these properties. Also, immunofluorescent microscopic analysis of wild‐type Newman and a clfA::Tn917 mutant of Newman with anti‐ClfA Region A sera confirmed that Region A is exposed on the bacterial cell surface. Furthermore, polystyrene beads coated with the Region A protein formed clumps in soluble fibrinogen showing that the ClfA protein alone is sufficient for the clumping phenotype. Western immunoblotting with anti‐ClfA Region A antibodies identified the native ClfA receptor as a 185 kDa protein that was released from the cell wall of S. aureus by lysostaphin treatment. A single extensive ligand‐binding site was located within Region A of the ClfA protein. Truncated ClfA proteins were expressed in Escherichia coli. Lysates of E. coli and proteins that had been purified by affinity chromatography were tested for (i) their ability to bind fibrinogen in Western ligand blotting experiments, (ii) for their ability to inhibit clumping of bacteria in fibrinogen solution and adherence of bacteria to solid‐phase fibrinogen, and (iii) for their ability to neutralize the blocking activity of anti‐ClfA Region A antibody. These tests allowed the ligand‐binding domain to be localized to a 218‐residue segment (residues 332‐550) within Region A.
The Mycobacterium leprae genome: systematic sequence analysis identifies key catabolic enzymes, ATP‐dependent transport systems and a novel polA locus associated with genomic variability
Authors: Fsihi Hafida , Cole Stewart T. 
Published: October 2006

SummaryIn the framework of the mycobacterial genome sequencing project, a continuous 37049bp sequence from the Mycobacterium leprae chromosome has been determined. Computer analysis revealed 10 complete open reading frames, and nine of their products show similarity to known proteins. Seven of these were identified as the enzyme isocitrate lyase, two P‐type ATPase cation transporters, two AMP‐binding proteins, the ribosomal protein S1, and DNA polymerase I. Interestingly, the polA gene, encoding DNA polymerase, is flanked by two inverted copies of a new class of the M. leprae specific repetitive sequence, RLEP, and this structure resembles a transposable element. A second copy of this element was found at another locus in the genome, but the two copies were not present in equal amounts and could not be found in all isolates of M. leprae. This is the first evidence for genomic variability in the leprosy bacillus and might ultimately be useful for developing a molecular test capable of distinguishing between strains of M. leprae.
Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae
Authors: Wieles Brigitte , van Soolingen Dick , Holmgren Arne , Offringa Rienk , Ottenhoff Tom , Thole Jelle 
Published: October 2006

SummaryThe thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox‐active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells. Thioredoxin is also a general protein disulphide reductase involved in redox regulation. In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB). In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene. Sequence analysis of the M. leprae gene demonstrated that the N‐terminal part of the protein corresponds to TR and the C‐terminal part to Trx. A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M. leprae. These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M. leprae.
Membrane topology and site‐specific mutagenesis of Pseudomonas aeruginosa porin OprD
Authors: Huang Honglin , Jeanteur Denis , Pattus Franc , Hancock Robert E. W. 
Published: October 2006

Summary Pseudomonas aeruginosa OprD is a 420‐amino‐acid protein that facilitates the uptake of basic amino acids, imipenem and gluconate across the outer membrane. OprD was the first specific porin that could be aligned with members of the non‐specific porin super‐family. Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD‐topology model was proposed. Sixteen β‐strands were predicted, connected by short loops at the periplasmic side. The eight external loops were of variable length but tended to be much longer than the periplasmic ones. Polymerase chain reaction (PCR)‐based site‐specific mutagenesis was performed to delete separately short stretches (4‐8 amino acid residues) from each of the predicted external loops. The mutants with deletions in the predicted external loops L1, L2, L5, L6, L7 and L8 were tolerated in both Escherichia coli and P. aeruginosa. The expressed mutant proteins maintained substantial resistance to trypsin treatment in the context of isolated outer membranes. Proteins with deletions in loops L1, L5, L6, L7 and L8 reconstituted similar imipenem supersusceptibility in a P. aeruginosa OprD::Ω background. The L2‐deletion mutant only partially reconstituted supersusceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.