bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bacterial subcellular architecture: recent advances and future prospects
Authors: Lewis Peter J. 
Published: November 2004

SummaryTraditional textbook representations of the prokaryotic cytoplasm show an amorphous, unstructured amalgamation of proteins and small molecules in which a randomly arranged chromosome resides. The development and application of a swathe of microscopic techniques over the last 10 years in particular, has shown this image of the microbial cell to be incorrect: the cytoplasm is highly structured with many proteins carrying out their assigned functions at specific subcellular locations; bacteria contain cytoskeletal elements including microtubule, actin and intermediate filament homologues; the chromosome is not randomly folded and is organized in such a way as to facilitate efficient segregation upon cell division. This review will concentrate on recent advances in our understanding of subcellular architecture and the techniques that have led to these discoveries.
The related effector proteins SopD and SopD2 from Salmonella enterica serovar Typhimurium contribute to virulence during systemic infection of mice
Authors: Jiang Xiuju , Rossanese Olivia W. , Brown Nat F. , Kujat‐Choy Sonya , Galán Jorge E. , Finlay B. Brett , Brumell John H. 
Published: November 2004

Summary Salmonella resides within host cells in a vacuole that it modifies through the action of virulence proteins called effectors. Here we examined the role of two related effectors, SopD and SopD2, in Salmonella pathogenesis. Salmonella enterica serovar Typhimurium (S. Typhimurium) mutants lacking either sopD or sopD2 were attenuated for replication in the spleens of infected mice when competed against wild‐type bacteria in mixed infection experiments. A double mutant lacking both effector genes did not display an additive attenuation of virulence in these experiments. The double mutant also competed equally with both of the single mutants. Deletion of either effector impaired bacterial replication in mouse macrophages but not human epithelial cells. Deletion of sopD2 impaired Salmonella’s ability to form tubular membrane filaments [Salmonella‐induced filaments (Sifs)] in infected cells; the number of Sifs decreased, whereas the number of pseudo‐Sifs (thought to be a precursor of Sifs) was increased. Transfection of HeLa cells with the effector SifA induced the formation of Sif‐like tubules and these were observed in greater size and number after co‐transfection of SifA with SopD2. In infected cells, SifA and SopD2 were localized both to Sifs and to pseudo‐Sifs. In contrast, deletion of sopD had no effect on Sif formation. Our results indicate that both SopD and SopD2 contribute to virulence in mice and suggest a functional relationship between these two proteins during systemic infection of the host.
Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion
Authors: Atmakuri Krishnamohan , Cascales Eric , Christie Peter J. 
Published: November 2004

SummaryBacteria use type IV secretion systems (T4SS) to translocate DNA (T‐DNA) and protein substrates across the cell envelope. By transfer DNA immunoprecipitation (TrIP), we recently showed that T‐DNA translocates through the Agrobacterium tumefaciens VirB/D4 T4SS by forming close contacts sequentially with the VirD4 receptor, VirB11 ATPase, the inner membrane subunits VirB6 and VirB8 and, finally, VirB2 pilin and VirB9. Here, by TrIP, we show that nucleoside triphosphate binding site (Walker A motif) mutations do not disrupt VirD4 substrate binding or transfer to VirB11, suggesting that these early reactions proceed independently of ATP binding or hydrolysis. In contrast, VirD4, VirB11 and VirB4 Walker A mutations each arrest substrate transfer to VirB6 and VirB8, suggesting that these subunits energize this transfer reaction by an ATP‐dependent mechanism. By co‐immunoprecipitation, we supply evidence for VirD4 interactions with VirB4 and VirB11 independently of other T4SS subunits or intact Walker A motifs, and with the bitopic inner membrane subunit VirB10. We reconstituted substrate transfer from VirD4 to VirB11 and to VirB6 and VirB8 by co‐synthesis of previously identified ‘core’ components of the VirB/D4 T4SS. Our findings define genetic requirements for DNA substrate binding and the early transfer reactions of a bacterial type IV translocation pathway.
Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin
Authors: Schlünzen Frank , Pyetan Erez , Fucini Paola , Yonath Ada , Harms Jörg M. 
Published: November 2004

SummaryTiamulin, a prominent member of the pleuromutilin class of antibiotics, is a potent inhibitor of protein synthesis in bacteria. Up to now the effect of pleuromutilins on the ribosome has not been determined on a molecular level. The 3.5 Å structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin provides for the first time a detailed picture of its interactions with the 23S rRNA, thus explaining the molecular mechanism of the antimicrobial activity of the pleuromutilin class of antibiotics. Our results show that tiamulin is located within the peptidyl transferase center (PTC) of the 50S ribosomal subunit with its tricyclic mutilin core positioned in a tight pocket at the A‐tRNA binding site. Also, the extension, which protrudes from its mutilin core, partially overlaps with the P‐tRNA binding site. Thereby, tiamulin directly inhibits peptide bond formation. Comparison of the tiamulin binding site with other PTC targeting drugs, like chloramphenicol, clindamycin and streptogramins, may facilitate the design of modified or hybridized drugs that extend the applicability of this class of antibiotics.
Transcriptional profiling in Candida albicans reveals new adaptive responses to extracellular pH and functions for Rim101p
Authors: Bensen Eric S. , Martin Samuel J. , Li Mingchun , Berman Judith , Davis Dana A. 
Published: November 2004

SummaryThe human pathogen Candida albicans grows and colonizes sites that can vary markedly in pH. The pH response in C. albicans is governed in part  by the Rim101p pathway. In Saccharomyces cerevisiae, Rim101p promotes alkaline responses by repressing expression of NRG1, itself a transcriptional repressor. Our studies reveal that in C. albicans, Rim101p‐mediated alkaline adaptation is not through repression of CaNRG1. Furthermore, our studies suggest that Rim101p and Nrg1p act in parallel pathways to regulate hyphal morphogenesis, an important contributor to virulence. To determine the wild‐type C. albicans transcriptional response to acidic and alkaline pH, we utilized microarrays and identified 514 pH‐responsive genes. Of these, several genes involved in iron acquisition were upregulated at pH 8, suggesting that alkaline pH induces iron starvation. Microarray analysis of rim101–/– cells indicated that Rim101p does not govern transcriptional responses at acidic pH, but does regulate a subset of transcriptional responses at alkaline pH, including the iron acquisition genes. We found that rim101–/– cells are sensitive to iron starvation, which suggests that one important aspect of the Rim101p‐dependent alkaline pH response is to adapt to iron starvation conditions.
A conservative amino acid change alters the function of BosR, the redox regulator of Borrelia burgdorferi
Authors: Seshu J. , Boylan Julie A. , Hyde Jenny A. , Swingle Kristen L. , Gherardini Frank C. , Skare Jonathan T. 
Published: November 2004

Summary Borrelia burgdorferi, the aetiologic agent of Lyme disease, modulates gene expression in response to changes imposed by its arthropod vector and mammalian hosts. As reactive oxygen species (ROS) are known to vary in these environments, we asked how B. burgdorferi responds to oxidative stress. The B. burgdorferi genome encodes a PerR homologue (recently designated BosR) that represses the oxidative stress response in other bacteria, suggesting a similar function in B. burgdorferi. When we tested the sensitivity of B. burgdorferi to ROS, one clonal non‐infectious B. burgdorferi isolate exhibited hypersensitivity to t‐butyl hydroperoxide when compared with infectious B. burgdorferi and other non‐infectious isolates. Sequence analysis indicated that the hypersensitive non‐infectious isolates bosR allele contained a single nucleotide substitution, converting an arginine to a lysine (bosRR39K). Mutants in bosRR39K exhibited an increase in resistance to oxidative stressors when compared with the parental non‐infectious strain, suggesting that BosRR39K functioned as a repressor. Complementation with bosRR39K and bosR resulted in differential sensitivity to t‐butyl hydroperoxide, indicating that these alleles are functionally distinct. In contrast to BosR, BosRR39K did not activate transcription of a napA promoter–lacZ reporter in Escherichia coli nor bind the napA promoter/operator domain. However, we found that both BosR and BosRR39K bound to the putative promoter/operator region of superoxide dismutase (sodA). In addition, we determined that cells lacking BosRR39K  synthesized  fourfold  greater  levels  of the decorin binding adhesin DbpA suggesting that BosRR39K regulates genes unrelated to oxidative stress. Based on these data, we propose that the single amino acid substitution, R39K, dramatically alters the activity of BosR by altering its ability to bind DNA at target regulatory sequences.
The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels
Authors: Datta Kaustuv , Skidmore Jennifer M. , Pu Kun , Maddock Janine R. 
Published: November 2004

SummaryThe Obg subfamily of bacterial GTP‐binding proteins are biochemically distinct from Ras‐like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene (cgtAC) which, in Ras‐like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP‐binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtAC, we generated a temperature‐sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtAC is important for 50S assembly and/or stability. Surprisingly, at the non‐permissive temperature, G80E  cells  rapidly  lose  viability  and  yet  do not display an additional ribosome defect. Thus, the essential nature of the cgtAC gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1‐S arrest. Therefore, CgtAC is necessary for DNA replication and progression through the cell cycle.
Genetic recombination and the cell cycle: what we have learned from chromosome dimers
Authors: Lesterlin Christian , Barre François‐Xavier , Cornet François 
Published: November 2004

SummaryGenetic recombination is central to DNA metabolism. It promotes sequence diversity and maintains genome integrity in all organisms. However, it can have perverse effects and profoundly influence the cell cycle. In bacteria harbouring circular chromosomes, recombination frequently has an unwanted outcome, the formation of chromosome dimers. Dimers form by homologous recombination between sister chromosomes and are eventually resolved by the action of two site‐specific recombinases, XerC and XerD, at their target site, dif, located in the replication terminus of the chromosome. Studies of the Xer system and of the modalities of dimer formation and resolution have yielded important knowledge on how both homologous and site‐specific recombination are controlled and integrated in the cell cycle. Here, we briefly review these advances and highlight the important questions they raise.
The C‐terminal domains of the gingipain K polyprotein are necessary for assembly of the active enzyme and expression of associated activities
Authors: Sztukowska Maryta , Sroka Aneta , Bugno Marcin , Banbula Agnieszka , Takahashi Yusuke , Pike Robert N. , Genco Caroline A. , Travis James , Potempa Jan 
Published: November 2004

SummaryThe Porphyromonas gingivalis lysine‐specific cysteine protease (gingipain K, Kgp) is expressed as a large precursor protein consisting of a leader sequence, a pro‐fragment, a catalytic domain with a C‐terminal IgG‐like subdomain (IgSF) and a large haemagglutinin/adhesion (HA) domain. In order to directly study the role of these non‐catalytic domains in pro‐Kgp processing and maturation in P. gingivalis, the wild‐type form of the gene was replaced with deletion variants encoding C‐terminally truncated proteins, including KgpΔHA3/4 (Δ1292–1732 aa), KgpΔHA2‐4 (Δ1157–1732 aa), KgpΔHA1‐4 (Δ738–1732 aa), KgpΔC‐term/HA (Δ681–1732 aa) and KgpΔIg/C‐term/HA (602–1732 aa). Northern blot and reverse transcription polymerase chain reaction (RT‐PCR) analysis revealed that all truncated variants of the kgp gene were transcribed in P. gingivalis. Despite high levels of kgpΔC‐term/HA and kgpΔIg/C‐term/HA transcripts, no Kgp‐specific antigen was detected in cultures of these mutants as determined by Western blot analysis with monoclonal antibodies specific for the Kgp catalytic domain. Furthermore, only barely measurable amounts of Kgp‐specific activity were detected in these two mutants. The remaining mutants expressed significant Kgp activity, however, at lower levels when compared with the parental strain. The decreased activity most probably resulted from altered folding and/or hindered secretion of the protein. The kgp gene truncation was also demonstrated to alter the distribution of the gingipain protein between membrane‐associated and ‐secreted forms. While both gingipain K activity and the protein were cell membrane‐associated in the parental strain, the mutants released significant amounts of both protein and activity into the media. Taken together, these results suggest that the C‐terminal HA domains of Kgp are not only essential for full expression of gingipain activity, but also for proper processing of the multiprotein complex assembly on the P. gingivalis outer membrane. Moreover, our results indicate that the immunoglobulin‐like subdomain is indispensable for proper folding and expression of the gingipains.
Two discrete elements are required for the replication of a repABC plasmid: an antisense RNA and a stem–loop structure
Authors: Venkova‐Canova Tatiana , Soberón Nora E. , Ramírez‐Romero Miguel A. , Cevallos Miguel A. 
Published: November 2004

SummaryThe repABC replicons contain an operon encoding the initiator protein (RepC) and partitioning proteins (RepA and RepB). The latter two proteins negatively regulate the transcription of the operon. In this article we have identified two novel regulatory elements, located within the conserved repB–repC intergenic sequence, which negatively modulate the expression of repC, in plasmid p42d of Rhizobium etli. One of them is a small antisense RNA and the other is a stem–loop structure in the repABC mRNA that occludes the Shine‐Dalgarno sequence of repC. According to in vivo and in vitro analyses, the small antisense RNA (57–59 nt) resembles canonical negative regulators of replication because: (i) it is transcribed from a strong constitutive promoter (P2), (ii) the transcript overlaps untranslated region upstream of the RepC coding sequences, (iii) the RNA forms one secondary structure acting as a rho‐independent terminator, (iv) the antisense RNA is a strong trans‐incompatibility factor and (v) its presence reduces the level of repC expression. Surprisingly, both of these seemingly negative regulators are required for efficient plasmid replication.
The Campylobacter jejuni dccRS two‐component system is required for optimal in vivo colonization but is dispensable for in vitro growth
Authors: MacKichan Joanna K. , Gaynor Erin C. , Chang Christopher , Cawthraw Shaun , Newell Diane G. , Miller Jeff F. , Falkow Stanley 
Published: November 2004

SummaryA Campylobacter jejuni two‐component signal transduction system (TCSTS), designated dccR‐dccS (diminished capacity to colonize; Cj1223c‐Cj1222c), has been found to be important for in vivo colonization but dispensable for in vitro growth. A ΔdccR response regulator mutant generated using the virulent strain 81–176 background exhibited significantly reduced colonization of immunocompetent limited flora (I‐LF) mice, severe combined immunodeficient limited flora (SCID‐LF) mice, and 1‐day‐old chicks. A ΔdccS sensor kinase mutant was likewise defective for colonization in the I‐LF mouse model. ΔdccR‐infected SCID‐LF mice also exhibited dramatically reduced inflammation relative to wild type‐infected SCID‐LF mice. Despite this diminished colonization capacity, the ΔdccRS mutants were indistinguishable from wild type for growth under numerous in vitro conditions as well as for various phenotypes. Microarray analysis identified several genes encoding putative periplasmic and membrane proteins as being regulated by this two‐component system; binding of purified His‐tagged DccR to the promoter region of two of these genes supports a direct protein–DNA interaction. A conserved repeat sequence was identified in the promoter regions of these genes and in three other promoter regions in the genome, including that of an operon encoding a putative type I secretion system. Two of the regulated target genes were found to be essential for optimal colonization. Both the two‐component system and the putative regulated genes have uncharacterized homologues in other Campylobacter and Helicobacter spp., suggesting that they may perform an important function in colonization among a variety of related pathogenic species.
Application of liquid chromatography–mass spectrometry to monitoring plasma cyclophosphamide levels in phase I trial cancer patients*
Authors: Liu Johnson J , Kestell Philip , Findlay Michael , Riley Greta , Ackland Stephen , Simpson Andrew , Isaacs Richard , McKeage Mark J 
Published: November 2004

Summary1. A specific and efficient liquid chromatography–mass spectrometry (LC‐MS) method was established for monitoring patient plasma cyclophosphamide levels in a phase I trial of an oral cyclophosphamide‐based combination chemotherapy regimen.2. An Agilent 1100 Series LC‐MSD system (Agilent Technologies, Avondale, PA, USA), with a single quadrupole mass detector using a positive atmospheric pressure chemical ionization (APCI) interface and single ion monitoring at m/z 261, was used. Chromatography was performed using a LUNA C8 5 µm 30 × 4.6 mm stainless steel column (Phenomenex, Torrance, CA, USA) and a mobile phase of aqueous acetonitrile pumped at a flow rate of 0.7 mL/min. High‐throughput solid‐phase sample extraction was performed using a Gilson ASPEC XL4 system (Gilson Medical, Middleton, WI, USA) controlled by prestored programs.3. The standard curve for cyclophosphamide was linear over the concentration range 0.026–1.08 µg/mL (r2 > 0.994). Intra‐ and interassay accuracy and precision were 97–107 and 3–10%, respectively. The limit of detection was determined to be 0.01 µg/mL. Single ion monitoring at m/z 261 provided a high degree of specificity without interference from the matrix or other chemotherapy drugs.4. Automated sample processing allowed the analysis of a large number of plasma samples from a clinical trial of repeated daily oral dosing of cyclophosphamide. One hour after dosing, cyclophosphamide was detected in 98 of 106 plasma specimens at concentrations ranging between 0.03 and 4.88 µg/mL. Twenty‐four hours after dosing, cyclophosphamide was detected in 72 of 77 plasma specimens at concentrations ranging between 0.06 and 3.13 µg/mL. There were no time‐dependent changes in cyclophosphamide concentration during the 43 day period of repeated daily oral dosing. There was no correlation between cyclophosphamide dose and plasma concentration, despite the wide range of doses given in the clinical trial (50–125 mg/m2).5. We conclude that a solid‐phase extraction LC‐MS technique was validated for determining cyclophosphamide in human plasma. Interoccasion variability in the rate of oral absorption and in the clearance of systemically available drug may have contributed to the wide range of cyclophosphamide concentrations found at 1 and 24 h after tablet ingestion.
Effects of antidigoxin antiserum on endoxin levels, apoptosis and the expression of Bax and Bcl‐2 protein in ischaemia–reperfusion myocardium
Authors: Ke Yong‐Sheng , Liu Zhao‐Fang , Wang De‐Guo , Wang He‐Gui 
Published: November 2004

Summary1. The aim of the present study was to investigate the effects of antidigoxin antiserum (ADA), an endoxin special antagonist, on endoxin levels, apoptosis and the expression of the apoptosis‐related protein bcl‐2 and bax in myocardial ischaemia–reperfusion (MIR).2. The left anterior descending coronary artery was subjected to 30 min ischaemia followed by 45 min reperfusion in open‐chest anaesthetized rats. The rats were divided randomly into seven groups: a sham‐operated group, an MIR group, a vehicle control (normal saline) group, and groups receiving verapamil (5 mg/kg) or ADA (9, 18 and 36 mg/kg). The drugs were injected into rats via the femoral vein before reperfusion was commenced.3. Myocardial endoxin levels were measured by radioimmunoassay. Apoptotic cells was detected using the terminal deoxyribonucleotidyl transferase‐mediated dUTP–digoxigenin nick end‐labeling method. The expression of the apoptosis‐related proteins bcl‐2 and bax was detected by immunohistochemistry and their semiquantification scores were recorded by a computer image analysis system.4. Myocardial endoxin levels, the number of apoptotic cells and bax protein expression were increased in the MIR group compared with the sham group.5. Although bcl‐2 protein expression was elevated in the MIR group, there was no significant difference between the MIR and sham groups. However, the ratio of bcl‐2/bax was significantly decreased in the MIR group.6. In the group receiving 36 mg/kg ADA, myocardial endoxin levels, the number of apoptotic cells and bax protein expression were significantly decreased; bcl‐2 protein expression was enhanced. The bcl‐2/bax ratio was increased.7. The results suggest that ADA inhibited myocardial apoptosis induced by MIR in rats. The mechanisms involved require further investigation, but the present study may suggest that ADA prevents bax upregulation and enhances bcl‐2 upregulation by antagonizing the effects of endoxin.