bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms
Authors: Kuiper Irene , Lagendijk Ellen L. , Pickford Russell , Derrick Jeremy P. , Lamers Gerda E. M. , Thomas‐Oates Jane E. , Lugtenberg Ben J. J. , Bloemberg Guido V. 
Published: December 2004

Summary Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High‐performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface‐tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface‐active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N‐terminus of a 12‐amino‐acid peptide moiety, in which the C‐terminal carboxylic acid group forms an ester with the hydroxyl side‐chain of Ser9. The difference between the two structures is located in the second amino acid from the C‐terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio‐synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild‐type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.
Global regulation of virulence and the stress response by CsrA in the highly adapted human gastric pathogen Helicobacter pylori
Authors: Barnard Faye M. , Loughlin Michael F. , Fainberg Hernan P. , Messenger Michael P. , Ussery David W. , Williams Paul , Jenks Peter J. 
Published: December 2004

SummaryAlthough successful and persistent colonization of the gastric mucosa depends on the ability to respond to changing environmental conditions and co‐ordinate the expression of virulence factors during the course of infection, Helicobacter pylori possesses relatively few transcriptional regulators. We therefore investigated the contribution of the regulatory protein CsrA to global gene regulation in this important human pathogen. CsrA was necessary for full motility and survival of H. pylori under conditions of oxidative stress. Loss of csrA expression deregulated the oxidant‐induced transcriptional responses of napA and ahpC, the acid induction of napA, cagA, vacA, the urease operon, and fur, as well as the heat shock responses of napA, groESL and hspR. Although the level of napA transcript was higher in the csrA mutant, its stability was similar in the wild‐type and mutant strains, and less NapA protein was produced in the mutant strain. Finally, H. pylori strains deficient in the production of CsrA were significantly attenuated for virulence in a mouse model of infection. This work provides evidence that CsrA has a broad role in regulating the physiology of H. pylori in response to environmental stimuli, and may be important in facilitating adaptation to the different environments encountered during colonization of the gastric mucosa. Furthermore, CsrA appears to mediate its effects in H. pylori at the post‐transcriptional level by influencing the processing and translation of target transcripts, with minimal effect on the stability of the target mRNAs.
Is modification sufficient to protect a bacterial chromosome from a resident restriction endonuclease?
Authors: Makovets Svetlana , Powell Lynn M. , Titheradge Annette J. B. , Blakely Garry W. , Murray Noreen E. 
Published: December 2004

SummaryIt has been generally accepted that DNA modification protects the chromosome of a bacterium encoding a restriction and modification system. But, when target sequences within the chromosome of one such bacterium (Escherichia coli K‐12) are unmodified, the cell does not destroy its own DNA; instead, ClpXP inactivates the nuclease, and restriction is said to be alleviated. Thus, the resident chromosome is recognized as ‘self’ rather than ‘foreign’ even in the absence of modification. We now provide evidence that restriction alleviation may be a characteristic of Type I restriction–modification systems, and that it can be achieved by different mechanisms. Our experiments support disassembly of active endonuclease complexes as a potential mechanism. We identify amino acid substitutions in a restriction endonuclease, which impair restriction alleviation in response to treatment with a mutagen, and demonstrate that restriction alleviation serves to protect the chromosome even in the absence of mutagenic treatment. In the absence of efficient restriction alleviation, a Type I restriction enzyme cleaves host DNA and, under these conditions, homologous recombination maintains the integrity of the bacterial chromosome.
Aberrant cardiolipin metabolism in the yeast taz1 mutant: a model for Barth syndrome
Authors: Gu Zhiming , Valianpour Fredoen , Chen Shuliang , Vaz Frederic M. , Hakkaart Gertjan A. , Wanders Ronald J. A. , Greenberg Miriam L. 
Published: December 2004

SummaryIn eukaryotic cells, the acyl species of the phospholipid cardiolipin (CL) are more highly unsaturated than those of the other membrane phospholipids. Defective acylation of CL with unsaturated fatty acids and decreased total CL are associated with Barth syndrome, an X‐linked cardio‐ and skeletal myopathy attributed to a defect in the gene G4.5 (also known as tafazzin). We constructed a yeast mutant (taz1) containing a null mutation in the homologue of the human G4.5 gene. The yeast taz1Δ mutant was temperature sensitive for growth in ethanol as sole carbon source, but grew normally on glucose or glycerol plus ethanol. Total CL content was reduced in the taz1Δ mutant, and monolyso‐CL accumulated. The predominant CL acyl species found in wild‐type cells, C18:1 and C16:1, were markedly reduced in the mutant, whereas CL molecules containing saturated fatty acids were present. Interestingly, CL synthesis increased in the mutant, whereas expression of the CL structural genes CRD1 and PGS1 did not, suggesting that de novo biosynthetic enzyme activities are regulated by CL acylation. These results indicate that the taz1Δ mutant is an excellent genetic tool for the study of CL remodelling and may serve as a model system for the study of Barth syndrome.
EthR, a repressor of the TetR/CamR family implicated in ethionamide resistance in mycobacteria, octamerizes cooperatively on its operator
Authors: Engohang‐Ndong Jean , Baillat David , Aumercier Marc , Bellefontaine Flore , Besra Gurdyal S. , Locht Camille , Baulard Alain R. 
Published: December 2004

SummaryEthionamide (ETH) is an important second‐line antitubercular drug used for the treatment of patients infected with multidrug‐resistant Mycobacterium tuberculosis. Although ETH is a structural analogue of isoniazid, only little cross‐resistance to these two drugs is observed among clinical isolates. Both isoniazid and ETH are pro‐drugs that need to be activated by mycobacterial enzymes to exert their antimicrobial activity. We have recently identified two M. tuberculosis genes, Rv3854c (ethA) and Rv3855 (ethR), involved in resistance to ETH. ethA encodes a protein that belongs to the Flavin‐containing monooxygenase family catalysing the activation of ETH. We show here that ethR, which encodes a repressor belonging to the TetR/CamR family of transcriptional regulators, negatively regulates the expression of ethA. By the insertion of the ethA promoter region upstream of the lacZ reporter gene, overexpression of ethR in trans was found to cause a strong inhibition of ethA expression, independently of the presence of ETH in the culture media. Electrophoretic mobility shift assays indicated that EthR interacts directly with the ethA promoter region. This interaction was confirmed by DNA footprinting analysis, which, in addition, identified the EthR‐binding region. Unlike other TetR/CamR members, which typically bind 15 bp operators, EthR recognises an unusually long 55 bp region suggesting multimerization of the repressor on its operator. Identification by primer‐extension of the ethA transcriptional start site indicated that it is located within the EthR‐binding region. Taken together, bacterial two‐hybrid experiments and gel filtration assays suggested a dimerization of EthR in the absence of its operator. In contrast, surface plasmon resonance analyses showed that eight EthR molecules bind cooperatively to the 55 bp operator, which represents a novel repression mechanism for a TetR/CamR member.
The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage
Authors: Pullinger Gillian D. , Bevir Thomas , Lax Alistair J. 
Published: December 2004

SummaryToxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45–50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.
Restricting restriction
Authors: Bickle Thomas A. 
Published: December 2004

SummarySystems biology is a new, fashionable and well‐funded discipline, which to quote from a recent review aims to ‘examine the structure and dynamics of cellular and organismal function, rather than the characteristics of isolated parts of a cell or organism…’ (Kitano, H. (2002) Science 295:1662–1664). Systems biology will do this by profiting from the vast amounts of biological information that are available in the genomics era and make extensive use of computer modelling. But: ‘many breakthroughs in experimental devices, advanced software and analytical methods are required before the achievements of system biology can live up to their much‐touted potential’. This edition of Molecular Microbiology contains a paper that is the product of traditional experimental biology but which could serve as a test case for systems biology. The paper shows how bacteria integrate such disparate subsystems as DNA restriction, homologous recombination and regulated proteolysis to protect their chromosomes from degradation. When systems biology can predict this level of choreography, it will be a mature discipline.
Global impact of mature biofilm lifestyle on Escherichia coli K‐12 gene expression
Authors: Beloin Christophe , Valle Jaione , Latour‐Lambert Patricia , Faure Philippe , Kzreminski Mickaël , Balestrino Damien , Haagensen Janus A. J. , Molin Søren , Prensier Gérard , Arbeille Brigitte , Ghigo Jean‐Marc 
Published: December 2004

SummaryThe formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons. We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K‐12 biofilm. We show that, when biofilm is compared with the exponential growth phase, 1.9% of the genes showed a consistent up‐ or downregulation by a factor greater than two, and that 10% of the E. coli genome is significantly differentially expressed. The functions of the genes induced in these conditions correspond to stress response as well as energy production, envelope biogenesis and unknown functions. We provide evidence that the expression of stress envelope response genes, such as the psp operon or elements of the cpx and rpoE pathways, is a general feature of E. coli mature biofilms. We also compared biofilm with the stationary growth phase and showed that the biofilm lifestyle, although sharing similarities with the stationary growth phase, triggers the expression of specific sets of genes. Using gene disruption of 54 of the most biofilm‐induced genes followed by a detailed phenotypic study, we validated the biological relevance of our analysis and showed that 20 of these genes are required for the formation of mature biofilm. This group includes 11 genes of previously unknown function. These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E. coli biofilms and provide insights into its physiological signature.
Multiple stress signal integration in the regulation of the complex σS‐dependent csiD‐ygaF‐gabDTP operon in Escherichia coli
Authors: Metzner Martin , Germer Jens , Hengge Regine 
Published: December 2004

SummaryThe csiD‐ygaF‐gabDTP region in the Escherichia coli genome represents a cluster of σS‐controlled genes. Here, we investigated promoter structures, sigma factor dependencies, potential co‐regulation and environmental regulatory patterns for all of these genes. We find that this region constitutes a complex operon with expression being controlled by three differentially regulated promoters: (i) csiDp, which affects the expression of all five genes, is cAMP‐CRP/σS‐dependent and activated exclusively upon carbon starvation and stationary phase; (ii) gabDp1, which is σS‐dependent and exhibits multiple stress induction like σS itself; and (iii) gabDp2[previously suggested by Schneider, B.L., Ruback, S., Kiupakis, A.K., Kasbarian, H., Pybus, C., and Reitzer, L. (2002) J. Bacteriol. 184: 6976–6986], which appears to be Nac/σ70‐controlled and to respond to poor nitrogen sources. In addition, we identify a novel repressor, CsiR, which modulates csiDp activity in a temporal manner during early stationary phase. Finally, we propose a physiological  role  for σS‐controlled  GabT/D‐mediated γ‐aminobutyrate (GABA) catabolism and glutamate accumulation in general stress adaptation. This physiological role is reflected by the activation of the operon‐internal gabDp1 promoter under the different conditions that also induce σS, which include shifts to acidic pH or high osmolarity as well as starvation or stationary phase.
Application of AgaR repressor and dominant repressor variants for verification of a gene cluster involved in N‐acetylgalactosamine metabolism in Escherichia coli K‐12
Authors: Ray W. Keith , Larson Timothy J. 
Published: December 2004

SummaryThe agaZVWEFASYBCDI gene cluster encodes the phosphotransferase systems and enzymes responsible for the uptake and metabolism of N‐acetylgalactosamine and galactosamine in Escherichia coli. In some strains of E. coli, particularly the common K‐12 strain, a portion of this cluster is missing because of a site‐specific recombination event that occurred between sites in agaW and agaA. Strains that have undergone this recombination event have lost the ability to utilize either N‐acetylgalactosamine or galactosamine as sole sources of carbon. Divergently transcribed from this gene cluster is the gene agaR encoding a transcriptional repressor belonging to the DeoR/GlpR family of transcriptional regulators. Promoters upstream of agaR, agaZ and agaS were characterized. All three promoters had elevated activity in the presence of N‐acetylgalactosamine or galactosamine, were regulated in vivo by AgaR and possessed specific DNA‐binding sites for AgaR upstream from the start sites of transcription as determined by DNase I footprinting. In vivo analysis and DNase I footprinting indicated that the promoter specific for agaZ also requires activation by cAMP‐CRP. Previous work with GlpR and other members of the DeoR/GlpR family have identified highly conserved amino acid residues that function in DNA‐binding or response to inducer. These residues of AgaR were targeted for site‐directed mutagenesis and yielded variants of AgaR that were either negatively dominant or non‐inducible. The apparent ability to produce negatively dominant and non‐inducible variants of proteins of the DeoR/GlpR family of currently unknown function will likely facilitate screening for function.
The HCC‐domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction
Authors: Rummel Andreas , Mahrhold Stefan , Bigalke Hans , Binz Thomas 
Published: December 2004

SummaryTetanus and botulinum neurotoxins selectively invade neurons following binding to complex gangliosides. Recent biochemical experiments demonstrate that two ganglioside binding sites within the tetanus neurotoxin HC‐fragment, originally identified in crystallographic studies to bind lactose or sialic acid, are required for productive binding to target cells. Here, we determine by mass spectroscopy studies that the HC‐fragment of botulinum neurotoxins A and B bind only one molecule of ganglioside GT1b. Mutations made in the presumed ganglioside binding site of botulinum neurotoxin A and B abolished the formation  of  these  HC‐fragment/ganglioside  complexes, and drastically diminished binding to neuronal membranes and isolated GT1b. Furthermore, correspondingly mutated full‐length neurotoxins exhibit significantly reduced neurotoxicity, thus identifying a single ganglioside binding site within the carboxyl‐terminal half of the HC‐fragment of botulinum neurotoxins A and B. These binding cavities are defined by the conserved peptide motif H…SXWY…G. The roles of tyrosine and histidine in botulinum neurotoxins A and B in ganglioside binding differ from those in the analogous tetanus neurotoxin lactose site. Hence, these findings provide valuable information for the rational design of potent botulinum neurotoxin binding inhibitors.
MurAA, catalysing the first committed step in peptidoglycan biosynthesis, is a target of Clp‐dependent proteolysis in Bacillus subtilis
Authors: Kock Holger , Gerth Ulf , Hecker Michael 
Published: December 2004

SummaryThe carboxyvinyl transfer from phosphoenolpyruvate to UDP‐N‐acetylglucosamine is the first committed step in the pathway of peptidoglycan formation. This crucial reaction for bacterial cell growth is catalysed by the MurA enzymes. Gram‐negative bacteria carry one murA gene, whereas in a subgroup of Gram‐positive bacteria two separate paralogues, MurAA and MurAB, exist. This study provides evidence that in the Gram‐positive bacterium Bacillus subtilis, the MurAA protein is specifically degraded by the ClpCP protease. This Clp‐dependent degradation is especially enhanced upon entry into stationary phase, thus ensuring an immediate growth arrest due to stalled murein biosynthesis. The MurAA protein can therefore be addressed as a target of Clp‐dependent regulatory proteolysis such as the transcriptional regulators CtsR, ComK, Spx in B. subtilis, CtrA in Caulobacter crescentus or RpoS in Escherichia coli. Taking into account all other known regulatory targets of ATP‐dependent proteases, MurAA of B. subtilis represents the first example of a metabolic enzyme which is a unique regulatory substrate of Clp‐dependent proteolysis. Its function as a regulatory metabolic checkpoint resembles that of homoserine trans‐succinylase (MetA) in E. coli which is similarly ATP‐dependently degraded.
Structural studies of Streptococcus pneumoniae EPSP synthase in unliganded state, tetrahedral intermediate‐bound state and S3P‐GLP‐bound state
Authors: Park HaJeung , Hilsenbeck Jacqueline L. , Kim Hak Jun , Shuttleworth Wendy A. , Park Yong Ho , Evans Jeremy N. , Kang ChulHee 
Published: December 2004

SummaryThe shikimate pathway synthesizes aromatic amino acids and other essential metabolites that are necessary for bacteria, plants and fungi to survive. This pathway is not present in vertebrates and therefore represents an attractive target for antibacterial agents. We have successfully crystallized and solved the structure of unliganded, inhibitor‐liganded and tetrahedral intermediate (TI)‐liganded forms of Streptococcus pneumoniae EPSP synthase. The overall topology of the S. pneumoniae EPSP synthase is similar to that of the Escherichia coli EPSP synthase. In addition, the majority of residues responsible for ligand binding were conserved between the two proteins. TI‐liganded structure provides absolute configuration of the C‐2 atom from the F‐PEP moiety of the enzyme‐bound intermediate and also defines key residues responsible for the enzyme reaction. Comparison of the unliganded state and substrate‐bound state of the enzyme provides insights into the structural mechanisms involved in dynamic events of ligand binding, domain movement and closure. This structural study of the pathogenic bacteria S. pneumoniae EPSP synthase with inhibitor and TI will provide invaluable information for the design of new‐generation antibiotics.
The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing
Authors: Schuster Martin , Hawkins Andrew C. , Harwood Caroline S. , Greenberg E. P. 
Published: December 2004

SummaryIn Escherichia coli and some other γ‐Proteobacteria, the alternative σ factor RpoS functions as a regulator of the general stress response. The role of RpoS in Pseudomonas aeruginosa is not clear. Although P. aeruginosa RpoS contributes to the resistance to several environmental stresses, its role appears to be less pivotal than in E. coli. In P. aeruginosa, RpoS also regulates the production of several virulence factors and influences the expression of individual genes that are controlled by quorum sensing. Some quorum‐controlled genes are induced by RpoS, whereas others are repressed. To gain insights about RpoS function in P. aeruginosa and to understand better the regulation of quorum‐controlled genes, we used transcript profiling to define an RpoS regulon. We identified 772 genes regulated by RpoS in stationary but not in logarithmic growth phase (504 were induced and 268 were repressed), and we identified putative RpoS promoter sequence elements with similarity to the E. coli RpoS consensus in several of these genes. Many genes in the regulon, for example a set of chemotaxis genes, have assigned functions that are distinct from those in E. coli and are not obviously related to a stress response. Furthermore, RpoS affects the expression of more than 40% of all quorum‐controlled genes identified in our previous transcriptome analysis. This highlights the significance of RpoS as a global factor that controls quorum‐sensing gene expression at the onset of stationary phase. The transcription profiling results have allowed us to build a model that accommodates previous seemingly conflicting reports.