bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Candida albicans Cdr2p ATP‐binding cassette (ABC) transporter confers resistance to caspofungin
Authors: Schuetzer‐Muehlbauer Manuela , Willinger Birgit , Krapf Gerd , Enzinger Sabine , Presterl Elisabeth , Kuchler Karl 
Published: December 2004

SummaryMultidrug resistance may pose a serious problem to antifungal therapy. The Candida albicans Cdr2p is one of two ATP‐binding cassette (ABC) transporters mediating antifungal resistance in vivo through increased drug efflux. Echinocandins such as caspofungin represent the newest class of antifungals that target cell wall synthesis. We show here by agar plate resistance assays that cross‐resistant clinical isolates of C. albicans display high minimal inhibitory concentrations (MICs) to caspofungin when compared with a sensitive ATCC reference strain. Northern analysis and immunoblotting indicate that these isolates also show high levels of CDR1 and CDR2 expression. To determine a possible contribution of Cdr1p or Cdr2p to caspofungin resistance, we have functionally expressed Cdr1p and Cdr2p in appropriate recipient strains of the yeast Saccharomyces cerevisiae. Yeast cells expressing Cdr1p or Cdr2p exhibit cross‐resistance to established antifungal drugs such as azoles and terbinafine. However, Cdr2p and, to a much lesser extent, Cdr1p confer caspofungin hyper‐resistance when expressed in yeast. Likewise, Cdr2p confers caspofungin resistance when constitutively overexpressed in a drug‐sensitive C. albicans strain. We therefore propose that Cdr2p may contribute to clinical candin resistance. Finally, our data suggest that cross‐resistance phenotypes of clinical isolates are the consequence of distinct mechanisms that may operate simultaneously.
The Haemophilus influenzae HMW1 adhesin is glycosylated in a process that requires HMW1C and phosphoglucomutase, an enzyme involved in lipooligosaccharide biosynthesis
Authors: Grass Susan , Buscher Amy Z. , Swords W. Edward , Apicella Michael A. , Barenkamp Stephen J. , Ozchlewski Neil , St Geme Joseph W. 
Published: December 2004

SummaryNon‐typeable Haemophilus influenzae is a common respiratory pathogen and an important cause of morbidity in humans. The non‐typeable H. influenzae HMW1 and HMW2 adhesins are related proteins that mediate attachment to human epithelial cells, an essential step in the pathogenesis of disease. Secretion of these adhesins requires accessory proteins called HMW1B/HMW2B and HMW1C/HMW2C. In the present study, we investigated the specific function of HMW1C. Examination of mutant constructs demonstrated that HMW1C influences both the size and the secretion of HMW1. Co‐immunoprecipitation and yeast two‐hybrid assays revealed that HMW1C interacts with HMW1 and forms a complex in the cytoplasm. Additional experiments and homology analysis established that HMW1C is required for glycosylation of HMW1 and may have glycotransferase activity. The glycan structure contains galactose, glucose and mannose and appears to be generated in part by phosphoglucomutase, an enzyme important for lipooligosaccharide biosynthesis. In the absence of glycosylation, HMW1 is partially degraded and is efficiently released from the surface of the organism, resulting in reduced adherence. Based on these results, we conclude that glycosylation is a prerequisite for HMW1 stability. In addition, glycosylation appears to be essential for optimal HMW1 tethering to the bacterial surface, which in turn is required for HMW1‐mediated adherence, thus revealing a novel mechanism by which glycosylation influences cell–cell interactions.
Chlamydia trachomatis type III secretion: evidence for a functional apparatus during early‐cycle development
Authors: Fields K. A. , Mead D. J. , Dooley C. A. , Hackstadt T. 
Published: December 2004

SummaryThe obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole termed an inclusion. During its intracellular developmental cycle, C. trachomatis maintains this intracellular niche, presumably by expressing a type III secretion system, which deploys a set of host cell‐interactive proteins including inclusion membrane‐localized proteins termed Incs. Some Incs are expressed and secreted by 2 h (early cycle) after infection, whereas the expression of type III‐specific genes is not detectable until 6–12 h (mid‐cycle). To resolve this paradox, we investigated the presence of a type III apparatus on elementary bodies (EBs) that might function early in infection. We demonstrate the existence of the type III secretory apparatus by matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) and immunoblot analyses of purified EB extracts. Immunoblots using polyclonal antibodies specific for the core apparatus component CdsJ identified this protein in both EB and reticulate body (RB) extracts. Furthermore, CdsJ‐specific signals were detected by immunoblot of whole infected‐culture extracts and by indirect immunofluorescence of infected monolayers at times before the detection of cdsJ‐specific message. Finally, expression of IncC, expressed by 2 h after infection during C. trachomatis infections, in Yersinia pseudotuberculosis resulted in its secretion via the Yersinia type III apparatus. Based on these data, we propose a model in which type III secretion pores are present on EBs and mediate secretion of early Incs and possible additional effectors. Mid‐cycle expression of type III genes would then replenish secretion apparatus on vegetative RBs and serve as a source of secretion pores for subsequently formed EBs.
Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium
Authors: Mundy Rosanna , Pickard Derek , Wilson Rebecca K. , Simmons Cameron P. , Dougan Gordon , Frankel Gad 
Published: December 2004

Summary Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. In order to identify genes required for the colonization of A/E‐forming pathogens, a library of signature‐tagged transposon mutants of C. rodentium was constructed and screened in mice. Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon. Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis. Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide‐binding proteins. The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C. rodentium (named colonization factor Citrobacter, CFC). The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E. coli (ETEC), the toxin co‐regulated pilus of Vibrio cholerae and the bundle‐forming pilus of EPEC. A non‐polar mutation in cfcI, complementation of this strain with wild‐type cfcI and complementation of strain P5H2 with wild‐type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C. rodentium. Thus, CFC provides a convenient model to study type IV pilus‐mediated pathogen–host interactions under physiological conditions in the natural colonic environment.
Signalling early developmental events in two highly diverged Streptomyces species
Authors: Chater Keith F. , Horinouchi Sueharu 
Published: December 2004

SummaryWe review three main aspects of extracellular signalling in the initiation of aerial mycelium formation in two phylogenetically distant streptomycetes, S. coelicolor A3(2) and S. griseus: (1) γ ‐butyrolactones; (2) a complex cascade of mostly undefined signals; and (3) progress towards defining an integrating endpoint of all this signalling. Although apparent orthologues of many of the genes involved are found in both species, some of the connectivities are different. Moreover, some of the genes involved in signalling have diverged more rapidly than known housekeeping genes. We propose that that this may be an important aspect of speciation, and that the differences in gene interactions may reflect the diverse soil microecologies to which different streptomycetes are adapted.
RNA‐modifying machines in archaea
Authors: Omer Arina D. , Ziesche Sonia , Decatur Wayne A. , Fournier Maurille J. , Dennis Patrick P. 
Published: December 2004

SummaryIt has been known for nearly half a century that coding and non‐coding RNAs (mRNA, and tRNAs and rRNAs respectively) play critical roles in the process of information transfer from DNA to protein. What is both surprising and exciting, are the discoveries in the last decade that cells, particularly eukaryotic cells, contain a plethora of non‐coding RNAs and that these RNAs can either possess catalytic activity or can function as integral components of dynamic ribonucleoprotein machines. These machines appear to mediate diverse, complex and essential processes such as intron excision, RNA modification and editing, protein targeting, DNA packaging, etc. Archaea have been shown to possess RNP complexes; some of these are authentic homologues of the eukaryotic complexes that function as machines in the processing, modification and assembly of rRNA into ribosomal subunits. Deciphering how these RNA‐containing machines function will require a dissection and analysis of the component parts, an understanding of how the parts fit together and an ability to reassemble the parts into complexes that can function in vitro. This article summarizes our current knowledge about small‐non‐coding RNAs in Archaea, their roles in ribosome biogenesis and their relationships to the complexes that have been identified in eukaryotic cells.
Bordetella bronchiseptica PagP is a Bvg‐regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract
Authors: Preston Andrew , Maxim Elizabeth , Toland Elinor , Pishko E. Jane , Harvill Eric T. , Caroff Martine , Maskell Duncan J. 
Published: December 2004

Summary Bordetella bronchiseptica lipopolysaccharide (LPS) expression varies depending on growth conditions, regulated by the Bvg system. A B. bronchiseptica pagP homologue was identified that is required for Bvg‐mediated modification of the lipid A core region of LPS that occurs on switching from the Bvg– to the Bvg+ phase. Structural analysis demonstrated that the lipid A of a B. bronchiseptica pagP mutant differed from wild‐type lipid A by the absence of a palmitate group in secondary acylation at the C3′ position. The putative pagP promoter drove the expression of a green fluorescent protein (GFP) reporter gene in a Bvg‐regulated fashion. These data suggest that B. bronchiseptica pagP encodes a Bvg‐regulated lipid A palmitoyl transferase that mediates modification of the lipid A as part of the overall Bvg‐mediated adaptation of this organism to changing environmental conditions. We also show that pagP is not required for the initial colonization of the mouse respiratory tract by B. bronchiseptica, but is required for persistence of the organism within this organ.
3D structure of EspA filaments from enteropathogenic Escherichia coli
Authors: Daniell Sarah J. , Kocsis Eva , Morris Edward , Knutton Stuart , Booy Frank P. , Frankel Gad 
Published: December 2004

SummaryThe type III secretion system (TTSS) is a modular apparatus assembled by many pathogenic Gram‐negative bacteria and is designed to translocate proteins through the bacterial cell wall into the eukaryotic host cell. The conserved components of the TTSS comprise stacks of rings spanning the inner and outer bacterial membrane and a narrow, needle‐like structure projecting outwards. The TTSS of enteropathogenic E. coli is unique in that one of the translocator proteins, EspA, polymerizes to form an extension to the needle complex which interacts with the host cell. In this study we present the 3D structure of EspA filaments to c. 26 Å resolution determined from electron micrographs of negatively stained preparations by image processing. The structure comprises a helical tube with a diameter of 120 Å enclosing a central channel of 25 Å diameter through which effector proteins may be transported. The subunit arrangement corresponds to a one‐start helix with 28 subunits present in five turns of the helix and an axial rise of 4.6 Å per subunit. This is the first report of a 3D structure of a filamentous extension to the TTSS.
The RepA and RepB autorepressors and TraR play opposing roles in the regulation of a Ti plasmid repABC operon
Authors: Pappas Katherine M. , Winans Stephen C. 
Published: December 2004

SummaryThe replicator regions of the Ti plasmids of Agrobacterium tumefaciens belong to the repABC family of replication and partitioning systems, members of which are widely distributed among alpha proteobacteria. In the region upstream of the octopine‐type Ti plasmid repABC operon, three promoters were recently shown to be activated by the LuxR‐type regulator TraR. Activation of these promoters by TraR led to enhanced rep gene expression and increased Ti plasmid copy number. Here we describe a fourth promoter, designated P4. This promoter lies directly upstream of repA and is not regulated by TraR. The promoter was localized by subcloning and demonstrated to be strongly autorepressed. RepA is the major cis‐acting autorepressor of this promoter, though RepB enhanced repression and was essential for RepA‐mediated repression in trans. Purified RepA bound to an approximately 70‐nucleotide operator site overlapping the P4 promoter and extending well downstream. Binding affinity was increased by adenosine di‐ and tri‐phosphates and also by purified RepB. Activation of P1, P2, and P3 enhanced the activity of P4, suggesting that P4 somehow communicates with the upstream promoters. These findings demonstrate that both autoinduction and autorepression play critical and opposing roles in regulating repABC expression and hence in the replication, stability and copy number of the Ti plasmid.
A lytic transglycosylase homologue, PleA, is required for the assembly of pili and the flagellum at the Caulobacter crescentus cell pole
Authors: Viollier Patrick H. , Shapiro Lucy 
Published: December 2004

SummaryTwo distinct protein complexes, the flagellum and the pilus biogenesis machinery, are asymmetrically assembled at one pole of the Caulobacter predivisional cell. Cell division yields dissimilar daughter cells: a stalked cell and a swarmer cell that assembles several pili at the flagellated cell pole. Strains bearing mutations in the pleA gene are pililess and non‐flagellated. The PleA protein contains a region that is similar to a peptidoglycan‐hydrolytic active site, and a point mutation at this site in PleA results in the loss of flagellum and pili biogenesis. PleA was found to be required for the insertion of the outer membrane pilus secretion channel at the cell pole and for the accumulation of the PilA pilin subunit. PleA is also required for the assembly of substructures of the flagellar basal body hook complex that are located in or traverse the peptidoglycan layer. These results argue that PleA facilitates the assembly of envelope‐spanning structures at the cell pole. In support of this, PleA was found to be present only during a short interval in the cell cycle that coincides with the assembly of the flagellum and the pilus secretion apparatus.
Prophages and bacterial genomics: what have we learned so far?
Authors: Casjens Sherwood 
Published: December 2004

Epigraph
There is something fascinating about science. One gets such wholesale returns of conjecture out of such a trifling investment of fact.Mark Twain 1883

Life on the Mississippi
Summary Bacterial genome nucleotide sequences are being completed at a rapid and increasing rate. Integrated virus genomes (prophages) are common in such genomes. Fifty‐one of the 82 such genomes published to date carry prophages, and these contain 230 recognizable putative prophages. Prophages can constitute as much as 10–20% of a bacterium's genome and are major contributors to differences between individuals within species. Many of these prophages appear to be defective and are in a state of mutational decay. Prophages, including defective ones, can contribute important biological properties to their bacterial hosts. Therefore, if we are to comprehend bacterial genomes fully, it is essential that we are able to recognize accurately and understand their prophages from nucleotide sequence analysis. Analysis of the evolution of prophages can shed light on the evolution of both bacteriophages and their hosts. Comparison of the Rac prophages in the sequenced genomes of three Escherichia coli strains and the Pnm prophages in two Neisseria meningitidis strains suggests that some prophages can lie in residence for very long times, perhaps millions of years, and that recombination events have occurred between related prophages that reside at different locations in a bacterium's genome. In addition, many genes in defective prophages remain functional, so a significant portion of the temperate bacteriophage gene pool resides in prophages.
The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l‐ornithine N 5‐monooxygenase (sidA) and a non‐ribosomal peptide synthetase (sidC)
Authors: Eisendle Martin , Oberegger Harald , Zadra Ivo , Haas Hubertus 
Published: December 2004

SummaryThe filamentous ascomycete A. nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin intracellularly. In this study we report the characterization of two siderophore biosynthetic genes, sidA encoding l‐ornithine N5‐monooxygenase and sidC encoding a non‐ribosomal peptide synthetase respectively. Disruption of sidC eliminated synthesis of ferricrocin and deletion of sidA completely blocked siderophore biosynthesis. Siderophore‐deficient strains were unable to grow, unless the growth medium was supplemented with siderophores, suggesting that the siderophore system is the major iron assimilatory system of A. nidulans during both iron depleted and iron‐replete conditions. Partial restoration of the growth of siderophore‐deficient mutants by high concentrations of Fe2+ (but not Fe3+) indicates the presence of an additional ferrous transport system and the absence of an efficient reductive iron assmilatory system. Uptake studies demonstrated that TAFC‐bound iron is transferred to cellular ferricrocin whereas ferricrocin is stored after uptake. The siderophore‐deficient mutant was able to synthesize ferricrocin from triacetylfusarinine C. Ferricrocin‐deficiency caused an increased intracellular labile iron pool, upregulation of antioxidative enzymes and elevated sensitivity to the redox cycler paraquat. This indicates that the lack of this cellular iron storage compound causes oxidative stress. Moreover, ferricrocin biosynthesis was found to be crucial for efficient conidiation.
The Pseudomonas syringae type III‐secreted protein HopPtoD2 possesses protein tyrosine phosphatase activity and suppresses programmed cell death in plants
Authors: Espinosa Avelina , Guo Ming , Tam Vincent C. , Fu Zheng Qing , Alfano James R. 
Published: December 2004

SummaryThe bacterial plant pathogen Pseudomonas syringae possesses a type III protein secretion system that delivers many virulence proteins into plant cells. A subset of these proteins (called Avr proteins) is recognized by the plant's innate immune system and triggers defences. One defence‐associated response is the hypersensitive response (HR), a programmed cell death (PCD) of plant tissue. We have previously identified HopPtoD2 as a type III secreted protein from P. s. pv. tomato DC3000. Sequence analysis revealed that an N‐terminal domain shared homology with AvrPphD and a C‐terminal domain was similar to protein tyrosine phosphatases (PTPs). We demonstrated that purified HopPtoD2 possessed PTP activity and this activity required a conserved catalytic Cys residue (Cys378). Interestingly, HopPtoD2 was capable of suppressing the HR elicited by an avirulent P. syringae strain on Nicotiana benthamiana. HopPtoD2 derivatives that lacked Cys378 no longer suppressed the HR indicating that HR suppression required PTP activity. A constitutively active MAPK kinase, called NtMEK2DD, is capable of eliciting an HR‐like cell death when transiently expressed in tobacco. When NtMEK2DD and HopPtoD2 were co‐delivered into plant cells, the HR was suppressed indicating that HopPtoD2 acts downstream of NtMEK2DD. DC3000 hopPtoD2 mutants were slightly reduced in their ability to multiply in planta and displayed an enhanced ability to elicit an HR. The identification of HopPtoD2 as a PTP and a PCD suppressor suggests that the inactivation of MAPK pathways is a virulence strategy utilized by bacterial plant pathogens.
A translocated protein tyrosine phosphatase of Pseudomonas syringae pv. tomato DC3000 modulates plant defence response to infection
Authors: Bretz James R. , Mock Norton M. , Charity James C. , Zeyad Syed , Baker C. Jacyn , Hutcheson Steven W. 
Published: December 2004

Summary Pseudomonas syringae strains translocate effector proteins into host cells via the hrp‐encoded type III protein secretion system (TTSS) to facilitate pathogenesis in susceptible plants. However, the mechanisms by which pathogenesis is favoured by these effectors are not well understood. Individual strains express multiple effectors with apparently distinct activities that are co‐ordinately regulated by the alternative sigma factor HrpL. Genes for several effectors were identified in the P. syringae pv. tomato DC3000 genome using a promoter trap assay to identify HrpL‐dependent promoters. In addition to orthologues of avrPphE and hrpW, an unusual allele of avrPphD was detected that carried an IS52 insertion. Using this avrPphD::IS52 allele as a probe, a wild‐type allele of avrPphD, hopPtoD1, and a chimeric homologue were identified in the DC3000 genome. This chimeric homologue, identified as HopPtoD2 in the annotated DC3000 genome, consisted of an amino terminal secretion domain similar to that of AvrPphD fused to a potential protein tyrosine phosphatase domain. Culture filtrates of strains expressing HopPtoD2 were able to dephosphorylate pNPP and two phosphotyrosine peptides. HopPtoD2 was shown to be translocated into Arabidopsis thaliana cells via the hrp‐encoded TTSS. A ΔhopPtoD2 mutant of DC3000 exhibited strongly reduced virulence in Arabidopsis thaliana. Ectopic expression of hopPtoD2 in P. syringae Psy61 that lacks a native hopPtoD2 orthologue delayed the development of several defence‐associated responses including programmed cell death, active oxygen production and transcription of the pathogenesis‐related gene PR1. The results indicate that HopPtoD2 is a translocated effector with protein tyrosine phosphatase activity that modulates plant defence responses.