bluuebunny/crossref_metadata_2025_split huggingface dataset:
Beta‐lactamases and bacterial resistance to antibiotics
Authors: Frère Jean‐Marie 
Published: October 2006

SummaryThe efficiency of β‐lactam antibiotics, which are among our most useful chemotherapeutic weapons, is continuously challenged by the emergence of resistant bacterial strains. This is most often due to the production of β‐lactamases by the resistant cells. These enzymes inactivate the antibiotics by hydrolysing the β‐lactam amide bond. The elucidation of the structures of some β‐lactamases by X‐ray crystallography has provided precious insights into their catalytic mechanisms and revealed unsuspected similarities with the DD‐transpeptidases, the bacterial enzymes which constitute the lethal targets of β‐lactams. Despite numerous kinetic, structural and site‐directed mutagenesis studies, we have not completely succeeded in explaining the diversity of the specificity profiles of β‐lactamases and their surprising catalytic power. The solutions to these problems represent the cornerstones on which better antibiotics can be designed, hopefully on a rational basis.
The role of anti‐sigma factors in gene regulation
Authors: Brown Kit L. , Hughes Kelly T. 
Published: October 2006

SummaryDespite the isolation of an anti‐sigma factor over 20 years ago, it is only recently that the concept of an anti‐sigma factor emerged as a general mechanism of transcriptional regulation in prokaryotic systems. Anti‐sigma factors bind to sigma factors and inhibit their transcriptional activity. Studies on the mechanism of action of anti‐sigma factors has shed new light on the regulation of gene expression in bacteria, as the anti‐sigma factors add another layer to transcriptional control via negative regulation. Their cellular roles are as diverse as FlgM of Salmonella typhimurium, which can be exported to sense the structural state of the flagellar organelle, to SpollAB of Bacillus subtilis participating in the switch from one cell type to another during the process of sporulation. Additionally, the bacteriophage T4 uses an anti‐sigma factor to sabotage the Escherichia coli E·σ70 RNA polymerase in order to direct exclusive transcription of its own genes. Cross‐linking., co‐immuno‐precipitations, and co‐purification indicate that the anti‐sigma factors directly interact with their corresponding sigma factor to negatively regulate transcription. in B. subtilis, anti anti‐sigma factors regulate anti‐sigma factors by preventing an anti‐sigma factor from interacting with its cognate sigma factor, thereby allowing transcription to occur.
IHF‐ and RpoN‐dependent regulation c hydrogenase expression in Bradyrhizobium japonicum
Authors: Black Lori K. , Maier Robert J. 
Published: October 2006

SummarySequence analysis of the Bradyrhizobium japonicum hydrogenase promoter regulatory region indicated the presence of a –24/–12 type promoter, which is recognized by RpoN, and a potential integration host factor (IHF)‐binding site. B. japonicum rpoN−/rpoN2 double mutants were deficient in hydrogen‐uptake activity. Using plasmid‐borne hup‐lacZ fusions, it was shown that the rpoN mutants were also deficient in nickel‐dependent transcriptional regulation of hydrogenase. Gel‐shift assays of the hydrogenase promoter regulatory region showed that purified IHF from Escherichia coli binds to a 210bp fragment. DNase footprint analysis revealed a protected region of 31 bp between bases –44 and –75 from the transcription start site. Western analysis with B. japonicum soluble extract and antibodies against E. coli IHF gave two bands equivalent to molecular masses of 12 and 14kDa approximately. When the IHF‐binding area is mutated on a plasmid‐borne hup‐lacZ fusion, nickel‐dependent transcriptional regulation of hydrogenase is still observed, but the transcriptional rates are clearly less than in the parent hup‐lacZ fusion plasmid. Like the results with nickel, regulation of hydrogenase by other transcriptional regulators (hydrogen and oxygen) still occurs, but at a diminished level in the IHF‐binding‐area‐mutated construct.
Organization of tcp, acf, and toxT genes within a ToxT‐dependent operon
Authors: Brown R. Clark , Taylor Ronald K. 
Published: October 2006

SummaryThe toxin coregulated pilus (TCP) is required for Vibrio cholerae to colonize the human intestine. The expression of the pilin gene, tcpA, is dependent upon ToxR and upon ToxT. The toxT gene was recently mapped within the TCP biogenesis gene cluster and shown to be capable of activating a tcpA::TnphoA fusion when cloned in Escherichia coli. In this study, we determined that ToxR/ToxT activation occurs at the level of tcpA transcription. ToxT expressed in E. coli could activate a tcp operon fusion, while ToxR, ToxR with ToxS, or a ToxR‐PhoA fusion failed to activate the tcp operon fusion and we could not demonstrate binding of a ToxR extract to the tcpA promoter region in DNA mobility‐shift assays. The start site for the regulated promoter was shown by primer extension to lie 75 bp upstream of the first codon of tcpA. An 800‐base tcpA message was identified, by Northern analysis, that correlates by size to the distance between the transcriptional start and a hairpin‐loop sequence between tcpA and tcpB. The more‐sensitive assay of RNase protection analysis demonstrated that a regulated transcript probably extends through the rest of the downstream tcp genes, including ToxT and the adjacent accessory colonization factor (acf) genes. An in‐frame tcpA deletion, but not a polar tcpA::TnphoA fusion, could be complemented for pilus surface expression by providing tcpA in trans. This evidence suggests that the tcp genes, including toxT, are organized in an operon directly activated by ToxT in a ToxR‐dependent manner. Most of the ToxT expression under induced conditions requires transcription of the tcpA promoter. Further investigation of how tcp::TnphoA insertions that are polar on ToxT expression retain regulation showed that a low basal level of toxT expression is present in toxR and tcp::TnphoA strains. Overall, these observations support the ToxR/ToxT cascade of regulation for tcp. Once induced, toxT expression becomes autoregulatory via the tcp promoter, linking tcp expression to that of additional colonization factors, exotoxin production, and genes of unknown function in cholera pathogenesis.
Plasmid pT181 replication is decreased at high levels of RepC per plasmid copy
Authors: Lordanescu Serban 
Published: October 2006

SummaryThe replication of staphylococcal plasmid pT181 is indirectly controlled at the level of the synthesis of its replication initiator, RepC. As a result, high levels of RepC synthesis per plasmid copy were expected to lead to autocatalytic plasmid replication, which secondarily would affect host physiology. Surprisingly, RepC overexpression was found to lead to a rapid decrease in pT181 copy number and replication rate. These effects depended on the ratio of RepC lo the PT181 replication origin rather than on the absolute amount of RepC in the cell. In a wild‐type host, the increase in RepC/plasmid copy also inhibited chromosome replication and cell division. The changes in host physiology did not play any role in the decrease in pT181 replication caused by RepC overexpression since pT181 replication responded in the same way in a host mutant insensitive to the effects of RepC induction. These results suggest that pT181, the prototype of an entire class of plasmids from Gram‐positive bacteria, responds to overexpression of its replication initiator by a decrease in plasmid replication.
Characterization of the pilF—pilD pilus‐assembly locus of Neisseria gonorrhoeae
Authors: Freitag Nancy E. , Seifert H. Steven , Koomey Michael 
Published: October 2006

SummaryExpression of Type IV pili by the bacterial pathogen Neisseria gonorrhoeae appears to be essential for colonization of the human host. Several N. gonorrhoeae gene products have been recently identified which bear homology to proteins involved in pilus assembly and protein export in other bacterial systems. We report here the isolation and characterization of transposon insertion mutants in N. gonorrhoeae whose phenotypes indicate that the N. gonorrhoeae pilF and pilD gene products are required for gonoccocal pilus biogenesis. Mutants lacking the pilD gene product, a pre‐pilin peptidase, were unable to process the pre‐pilin subunit into pilin and thus were non‐piliated. pilF mutants processed pilin but did not assemble the mature subunit. Both classes of mutants released S‐pilin, a soluble, truncated form of the pilin subunit previously correlated with defects in pilus assembly. In addition, mutants containing transposon insertions in pilD or in a downstream gene, orfX, exhibited a severely restricted growth phenotype. Deletion analysis of pilD indicated that the poor growth phenotype observed for the pilD transposon mutants was a result of polar effects of the insertions on orfX expression. orfX encodes a predicted polypeptide of 23 kDa which contains a consensus nucleotide‐binding domain and has apparent homologues in Pseudomonas aeruginosa, Pseudomonas putida, Thermus thermophilus, and the eukaryote Caenorhabditis elegans. Although expression of orfX and pilD appears to be transcriptionally coupled, mutants containing transposon insertions in orfX expressed pili. Unlike either pilF or pilD mutants, orfX mutants were also competent for DNA transformation.
Resistance to mecillinam produced by the co‐operative action of mutations affecting iipopolysaccharide, spoT, and cya or crp genes of Salmonella typhimurium
Authors: Antón Dora N. 
Published: October 2006

SummaryLipopolysaccharide (LPS), spoT, and cya or crp mutations individually do not affect the minimum inhibitory concentration of mecillinam on Salmonella typhimurium. However, when mutations of two of these types were combined in the same strain, high‐level resistance appeared, and increased even further when all three types of mutations were present. Most mutations affecting LPS (rfa, rfb, rfc) showed this behaviour, although to different degrees. The highest resistance to mecillinam was caused by galE and rfc mutations whereas almost no effect was noticed with rfaB or rfaK mutations. This phenomenon appears to be specific for mecillinam since none of several other antibiotics elicited it. Reduction of guanosine tetraphosphate (ppGpp) levels by introduction of a relA mutation did not significantly affect the MIC of mecillinam on strains carrying different combinations of SpoT, galE, and cya or crp mutations. All the strains produced spherical cells in medium with a low concentration (0.05 μg ml−1) of the antibiotic. These results suggest that the antibacterial action of mecillinam on S. typhimurium is somehow dependent on the interaction of LPS, cyclic AMP/cyclic AMP receptor protein (cAMP/CRP), and SpoT. The reported resistance to mecillinam of cya and crp mutants of Escherichia coli K‐12 is probably due to the natural LPS defectiveness of this strain.
The chaperone‐like protein YerA of Yersinia pseudotuberculosis stabilizes YopE in the cytoplasm but is dispensible for targeting to the secretion loci
Authors: Frithz‐Lindsten Elisabet , Rosqvist Roland , Johansson Lenore , Forsberg Åke 
Published: October 2006

SummaryThe virulence plasmid‐encoded YopE cytotoxin of Yersinia pseudotuberculosis is secreted across the bacterial membranes and subsequently translocated into the eukaryotic cell. Translocation of YopE into target cells was recently shown to be polarized and only occurred at the zone of contact between the pathogen and the eukaryotic cell.Immunogold electron microscopy on cryosectioned Y. pseudotuberculosis revealed that YopE is secreted and deposited on the bacterial cell surface when the bacteria are grown in Ca2+‐depleted media at 37°C. No YopE was detected in the cytoplasm or in the membranes. In yerA mutants which are downregulated for VopE at a post‐transcriptional level, the cytotoxin could only be detected in the cytoplasm. The overall recovery of YopE from the yerA mutant strain was, however, considerably lower than from the wild‐type strain. yerA had no major effect on the translation of YopE, but was found to stabilize YopE in the cytoplasm. YerA was shown to specifically interact with YopE in the cytoplasm in vivo and this binding also correlated with YopE secretion. Targeting of YopE to the secretion loci as well as translocation of YopE into HeLa cells occurred also in the absence of YerA. Based on our findings, we suggest that YerA by binding to YopE stabilizes and maintains the cytotoxin in a secretion‐competent conformation.
Promoter selectivity control of Escherichia coli RNA polymerase by ionic strength: differential recognition of osmoregulated promoters by EσD and EσS holoenzymes
Authors: Ding Qingquan , Kusano Shuichi , Villarejo Merna , Ishihama Akira 
Published: October 2006

SummaryTranscription in vitro of two osmoregulated promoters, for the Escherichia coli osmB and osmY genes, was analysed using two species of RNA polymerase holoenzyme reconstituted from purified core enzyme and either σD (σ70, the major σ in exponentially growing cells) or σS (σ38, the principal σ at stationary growth phase). Under conditions of low ionic strength, the osmB and osmY promoters were transcribed by both EσD and EσS. Addition of up to 400 mM potassium glutamate (K glutamate), mimicking the intracellular ionic conditions under hyper‐osmotic stress, specifically enhanced transcription at these promoters by EσS but inhibited that by EσD. At similar high concentrations of potassium chloride (KCI), however, initiation at both these promoters was virtually undetectable. These data suggest that the RNA polymerase, EσS, itself can sense osmotic stress by responding to changes in intracellular K glutamate concentrations and altering its promoter selectivity in order to recognize certain osmoregulated promoters.
The bgl1 gene of Trichoderma reesei QM 9414 encodes an extracellular, cellulose‐inducible β‐glucosidase involved in cellulase induction by sophorose
Authors: Mach Robert L. , Seiboth Bernhard , Myasnikov Andrey , Gonzalez Ramon , Strauss Josef , Harkki Anu M. , Kubicek Christian P. 
Published: October 2006

SummaryWe have investigated the effect of disruption of the bgl1‐(β‐glucosidase l‐encoding) gene of Trichoderma reesei on the formation of other β‐glucosidase activities and on the induction of cellulases. To this end the bgl1 locus was disrupted by insertion of the Aspergillus nidulans amdS (acetamidase‐encoding) gene. The bgl1‐disrupted strain did not produce the 75kDa extracellular β‐glucosidase on cellulose or lactose, but still formed β‐glucosidase activity on glucose, cellobiose, xylan or β‐1,3‐glucan, suggesting that the enzyme(s) exhibiting this β‐glucosidase activity is (are) not encoded by bgl1. The cellulose‐inducer sophorose induced the bgl1‐encoded β‐glucosidase, whereas the remaining β‐glucosidase activity was induced by methyl‐β‐D‐glucoside. The bgl1‐gene product was mainly secreted into the medium, whereas the other β‐glucosidase activity was mainly associated with the cells. A bgl1‐multicopy strain formed higher amounts of cellulases than the parent strain. Nonsaturating concentrations of sophorose efficiently induced cellobiohydrolase I formation in the bgl1‐multicopy strain, but less efficiently in the bgl1‐disrupted strain. The multicopy strain and the parent strain were comparably efficient at saturating sophorose concentrations. The β‐glucosidase inhibitor nojirimycin strongly inhibited induction in all strains. These data suggest that the bgl1‐encoded β‐glucosidase is not identical to the plasma‐membrane‐bound, constitutive, methyl‐β‐glucoside inducible β‐glucosidase, but represents an extracellular cellulose‐induced enzyme. Both enzymes contribute to rapid induction of cellulases by modifying the inducer sophorose.
Mutational analysis of the putative nucleic acid‐binding surface of the cold‐shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of single‐stranded DNA containing the Y‐box motif
Authors: Schröder Katja , Graumann Peter , Schnuchel Arndt , Holak Tad A. , Marahiel Mohamed A. 
Published: October 2006

SummaryThe major cold‐shock protein of Bacillus subtilis, CspB, is a member of a protein family widespread among prokaryotes and eukaryotes that share the highly conserved cold‐shock domain (CSD). The CSD domain is involved in transcriptional and translational regulation and was shown to bind the Y‐box motif, a cis‐element that contains the core sequence ATTGG, with high affinity. The three‐dimensional structure of CspB, a prototype of this protein family, revealed that this hydrophilic CSD domain creates a surface rich in aromatic and basic amino acids that may act as the nucleic acid‐binding site. We have analysed the potential role of conserved aromatic and basic residues in nucleic acid binding by site‐directed mutagenesis. In gel retardation and ultraviolet cross‐linking experiments, the ability of CspB mutants to bind single‐stranded oligonucleotides (ssDNA) that contain the Y‐box motif was investigated. Single substitutions of three highly conserved phenylalanine residues (Phe‐15, Phe‐17, Phe‐27) by alanine and substitution of one histidine (His‐29) by glutamine, all located within the putative RNA‐binding sites RNP‐1 and RNP‐2, abolished the nucleic acid‐binding activity of CspB. Conservative substitutions of Phe‐15 to tyrosine (F15Y) showed a small increase in binding affinity, whereas separate replacement of Phe‐17 and Phe‐27 by tyrosine caused a reduction in binding activity. These and other substitutions including the conserved basic residues Lys‐7, Lys‐13 and Arg‐56 as well as the aromatic residues Trp‐8 and Phe‐30 strongly suggest that CspB uses the sidechains of these amino acids for specific interaction with nucleic acids. Ultraviolet cross‐linking experiments for CspB mutants with ssDNA supported the idea of specific CspB/nucleic acid interaction and indicated an essential role for the aromatic and basic residues in this binding. In addition, two‐dimensional nuclear magnetic resonance studies with F17A, K13Q, F15Y and F27Y revealed that the mutants have the same overall structure as the wild‐type CspB protein.
Differential effect of dsbA and dsbC mutations on extracellular enzyme secretion in Erwinia chrysanthemi
Authors: Shevchik Vladimir E. , Bortoli‐Gernnan Isabelle , Robert‐Baudouy Janine , Robinet Sandrine , Barras Frederic , Condemine Guy 
Published: October 2006

SummaryAn Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation has been cloned and sequenced. This gene codes for a periplasmic protein with disulphide isomerase activity that has 69% identity and 94% similarity with the E. coli DsbA protein. An E. chrysanthemi dsbA‐uidA fusion mutant has been constructed. dsbA expression seems to be constitutive. This mutant has multiple phenotypes resulting from the absence of disulphide bond formation in periplasmic and secreted proteins. Pectate lyases and the cellulase EGZ are rapidly degraded in the periplasm of the dsbA mutant. E. chrysanthemi synthesizes another periplasmic protein with disulphide isomerase activity, namely DsbC. The dsbC gene introduced on a multicopy plasmid in a dsbA mutant was only partially able to restore EGZ secretion, indicating that even if DsbA and DsbC possess disulphide oxydoreductase activity, they are not completely interchangeable. Moreover, pectate lyases expressed in an E. coli dsbA mutant were very instable but their stability was unaffected in a dsbC mutant. These results indicate that DsbA and DsbC could have different substrate specificities.
Restriction alleviation and modification enhancement by the Rac prophage of Escherichia coli K‐12
Authors: King Gareth , Murray Noreen E. 
Published: October 2006

SummaryBacteriophage λ. encodes an antirestriction function, Ral, which is able to modulate the activity of the Escherichia coli K‐12 restriction and modification system, EcoKl. Here we report the characterization of an analogous function, Lar, expressed by E. coli sbcA mutants and the hybrid phage λreverse. E. coli sbcA mutants and λ.reverse both express genes of the Rac prophage, and we have located the lar gene immediateiy downstream of recT in this element. The lar gene has been cloned in an expression plasmid, and a combination of site‐directed mutagenesis and labelling of plasmid‐encoded proteins has enabled us to identify a number of translational products of lar, the smallest of which is sufficient for restriction alleviation. Lar, like Ral, is able both to alleviate restriction and to enhance modification by Eco Kl. Lar, therefore, is functionally similar to Ral and the nucleotide sequences of their genes share 47% identity, indicating a common origin. A comparison of the predicted amino acid sequences of Lar and Ral shows only a 25% identity, but a few short regions do align and may indicate residues important for structure and/or function.
Molecular analysis of asmA, a locus identified as the suppressor of OmpF assembly mutants of Escherichia coli K‐12
Authors: Misra Rajeev , Miao Yun 
Published: October 2006

SummaryWe present the molecular characterization of the asmA gene, whose product is involved in the assembly of outer membrane proteins in Escherichia coli K‐12. The asmA locus was initially identified as a site for suppressor mutations of an assembly defective OmpF315. Our data suggest that these suppressor mutations either completely abolish or reduce asmA expression and can be complemented in trans by piasmid clones carrying asmA sequences. The recessive nature of asmA suppressor mutations suggests that the functional AsmA protein participates in Inhibiting the assembly of OmpF315 and other mutant OmpFs. As the assembly of wild‐type and parental OmpF proteins was not affected by asmA mutations, AsmA must provide an environment refractory only to the assembly of mutant OmpF proteins. However, we cannot completely rule out the possibility that AsmA plays a minor role in the assembly of wild‐type and parental OmpF in wild‐type cells. The presence of a putative signal sequence within the amino‐terminal sequence of AsmA suggests that it is either a periplasmic or an outer membrane protein. This predicted location of AsmA is compatible with its role in the assembly of outer membrane proteins.