bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing the Risk of Introducing Exotic Species via the Live Marine Species Trade
Authors: WEIGLE SHANNON M. , SMITH L. DAVID , CARLTON JAMES T. , PEDERSON JUDITH
Published: January 2005

Abstract:  Although the shipping industry has received considerable attention as a dispersal mechanism for aquatic nuisance species, many invasions have been linked to other mechanisms of transfer. The threat posed to coastal ecosystems by these alternative mechanisms, however, remains largely unquantified. We assessed the potential risks of introducing marine and estuarine species associated with seven mechanisms of transfer: seafood companies, aquaculture operations, bait shops, stores that sell marine ornamental species, research and educational organizations, public aquariums, and coastal restoration projects. For each, we compiled a comprehensive database of organizations in coastal Massachusetts. We then designed and administered a survey to a subset of organizations that inquired about (1) their proximity to saltwater and methods of handling live imports; (2) the type and quantity of marine species being imported; and (3) the organization's familiarity with marine invasions. Respondents in five of the seven categories acknowledged importing nonlocal live marine species to the area. Seafood companies handled the majority of individuals but relatively few taxa. This mechanism of transfer also had the most complex trade patterns and the greatest number of operations located near saltwater. In contrast, the other transfer mechanisms each had simpler trade pathways and fewer operations but varied in the quantity and taxonomic diversity of their imports. Significantly, no single mechanism of transfer stood out as presenting a primary risk. Rather, each had characteristics or used handling practices at different points in the importation process that could facilitate introductions. To prevent future marine invasions, better reporting requirements for live species imports are needed, and best‐management practices and outreach strategies specific to the transfer mechanism should be developed and implemented.
Environmental Education as a Component of Multidisciplinary Conservation Programs: Lessons from Conservation Initiatives for Critically Endangered Fruit Bats in the Western Indian Ocean
Authors: TREWHELLA W. J. , RODRIGUEZ‐CLARK K. M. , CORP N. , ENTWISTLE A. , GARRETT S. R. T. , GRANEK E. , LENGEL K. L. , RABOUDE M. J. , REASON P. F. , SEWALL B. J.
Published: January 2005

Abstract:  Pteropus livingstonii, P. voeltzkowi,and P. rodricensis are three critically endangered fruit bats from western Indian Ocean islands for which multidisciplinary conservation programs have been established that include environmental education programs (EEPs). We describe these EEPs in terms of the strategies used to achieve them and evaluate the educational and conservation outcomes and impacts of the programs. Educational outputs (including posters, stickers, videos, lesson plans, and workshops), primarily linking human needs to the ecosystem services provided by bats, were delivered to schools and community groups, and local environmental educators were trained to further develop the EEPs. Outcomes included increased local awareness about the bats and their conservation, training of environmental educators, inclusion of bat conservation and environmental issues in the school curricula, and establishment of community‐based environmental nongovernmental organizations (NGOs). Extensive prior planning, presentations in local languages, distribution of outputs through existing networks of educators, training of local educators, establishment of local environmental NGOs, and local capacity‐building were all associated with these EEPs achieving their goals in the under‐resourced island locations where these bats are found. The EEPs were also important in the development of other components of their respective conservation programs, such as population monitoring programs. Although long‐term conservation impacts, particularly tackling habitat loss, are slow to materialize and social and economic issues need to be addressed, these EEPs have already had important outcomes and have established the foundation for future conservation actions.
Beetle Responses to Habitat Fragmentation Depend on Ecological Traits, Habitat Condition, and Remnant Size
Authors: DRISCOLL DON A. , WEIR TOM
Published: January 2005

Abstract:  Under extreme (>80%) levels of habitat loss and fragmentation, exponentially increasing risks of extinction have been predicted; however, the proportion of species that will decline is uncertain. Factors influencing species declines include patch characteristics, such as size and condition, and species' ecological traits, such as dispersal ability. In central New South Wales, Australia, 90% of the mallee woodland has been cleared for agriculture. At each of three 100‐km2 locations, we sampled beetles in 10 sites and asked: (1) How is the impact of habitat loss and fragmentation mediated by remnant condition and size? (2) What proportion of the beetle fauna is declining? (3) Are ecological traits based on body size, trophic group, flight, and position relative to the ground correlated with beetle responses to habitat loss and fragmentation? Seven of 34 beetle species (21%) occurred in fragmented and isolated populations in the agricultural landscape, implying that they may be at risk of local extinction. Most declining species depended on large remnants, but two species were confined to disturbed linear remnants, emphasizing the importance of diverse management regimes for invertebrate conservation. In contrast, approximately one‐quarter of the beetle fauna survived in paddocks and so was not at risk of decline. Greatest species richness was observed in narrow linear remnants, not square reserves, because of the influx of species from the matrix and the presence of strip‐specialist species. Flying ability and position explained most of the variation in species responses to fragmentation. Flightless species or species living underground were most vulnerable to decline in agricultural landscapes. Combinations of traits were also implicated in beetle responses, however, suggesting that causal mechanisms involve more than just the effects of flight or position. To identify species vulnerable to decline in fragmented landscapes, then, many traits need to be considered simultaneously.
Cutaneous Tumors as the Initial Presentation of Non‐T, Non‐B, Nonmyeloid CD4+ CD56+ Hematolymphoid Malignancy in an Adolescent Boy
Authors: Fass Jonathan , Tichy Elizabeth H. , Kraus Eric W. , Herrick Jennifer L. , Ehsan Aamir , Peterson Joe , Grimwood Ronald E.
Published: January 2005

Abstract:  We describe a 14‐year‐old Hispanic boy who presented with a 2‐month history of enlarging plum‐colored cutaneous tumors on his face, trunk, and proximal extremities. Histopathologic examination showed nodular infiltrates of malignant mononuclear cells extending from the superficial dermis to the deep subcutis. Immunohistochemical staining of the biopsy specimen and flow cytometry studies on a bone marrow aspirate revealed a CD4+, CD56+ hematolymphoid tumor that was negative for all other myeloid and lymphoid markers. Based on this information, the patient was diagnosed with the recently described, rare non‐T, non‐B, nonmyeloid CD4+ CD56+ hematolymphoid malignancy. To our knowledge, this is the youngest patient reported in the literature.
Is Disseminated Juvenile Xanthogranulomatosis Benign Cephalic Histiocytosis?
Authors: Sidwell R. U. , Francis N. , Slater D. N. , Mayou S. C.
Published: January 2005

Abstract:  We describe an infant with a congenital form of non‐Langerhans cell histiocytosis with clinical and pathologic features of both disseminated juvenile xanthogranulomatosis and benign cephalic histiocytosis. The findings in this case support the concept of these non‐Langerhans cell histiocytoses forming part of a spectrum of disease rather than being separate pathologic entities.
Atopic Dermatitis Exclusively Localized on Nipples and Areolas
Authors: Laura Amato , Samantha Berti , Caterina Chiarini , Paolo Fabbri
Published: January 2005

Abstract:  We present an 11‐year‐old girl, with celiac disease, and a 9‐ month history of itchy and erythemato‐edematous lesions with vesicles and exudation on her nipples and areolas. No other lesions or signs of scratching were present on her face or folds. She had no specific lesions of atopic dermatitis in typical sites nor in other body surface during her life. Patch tests showed a positive reaction to nickel and thimerosal that was not significantly related to the clinical appearance.

This presentation documents the clinical relevance of atopic dermatitis minor diagnostic criteria. We discuss the importance of nipple eczema in AD and its differential diagnosis.
Treatment of Multiple Trichoepitheliomas with Topical Imiquimod and Tretinoin
Authors: Urquhart Jean Liu , Weston William L.
Published: January 2005

Abstract:  An 11‐year‐old white girl presented multiple flesh‐colored dome‐shaped papules on the forehead, nose, scalp, and posterior neck. The lesions had been present for 2 years and were asymptomatic. Family history was negative for skin diseases. A skin biopsy specimen revealed histopathology consistent with trichoepitheliomas. Treatment of multiple trichoepitheliomas is usually difficult. Methods reported in the literature include laser treatment, surgery, cryotherapy, electrodessication, and radiation. The parents were concerned about the risk of scarring and wanted to pursue a nonscarring treatment. The patient was initially treated with topical imiquimod three times a week. She progressively increased the frequency of application to twice daily and added topical tretinoin gel once daily to her regimen for more resistant lesions. After 3 years of treatment, the patient experienced ≈80% clearing of lesions without scarring. The advantage of using this nonsurgical treatment is no scarring, painless, and no need for other invasive procedures such as injection of local anesthetic.
Characterization of complement receptor 1 domains for prevention of complement‐mediated red cell destruction
Authors: Mqadmi Amina , Abdullah Yazan , Yazdanbakhsh Karina
Published: January 2005

BACKGROUND:  Complement activation resulting in intravascular hemolysis can cause transfusion‐associated mortality. We recently showed that a recombinant soluble form of complement receptor 1 (CR1) effectively reduces complement‐mediated red blood cell (RBC) destruction in vitro and more importantly prolongs the survival of transfused human RBCs in mice. To determine CR1‐active sites that prevent RBC destruction, structure‐function analysis of its extracellular 1930‐amino‐acid domain has been performed.

STUDY DESIGN AND METHODS:  Several CR1‐truncated soluble proteins were prepared and tested for their ability to prevent complement‐mediated RBC destruction in vitro and in mice.

RESULTS:  A 250‐amino‐acid region in CR1 that possesses antihemolytic activity and is effective in prolonging survival of transfused RBCs in vivo was identified. Mutation of two critical residues (D109N and E116K) in this 250‐amino‐acid domain, previously shown to improve complement‐inhibitory functions of CR1 derivatives, resulted in a more potent inhibition of complement activation in vitro. In vivo, however, the activity of mutant proteins was comparable to the wild‐type molecules.

CONCLUSION:  Our structure‐function studies have characterized smaller CR1‐based complement inhibitors for future development of structure‐derived transfusion therapeutics. Our studies underscore the importance of testing CR1 inhibitors in vivo.
Large‐volume leukapheresis yields more viable CD34+ cells and colony‐forming units than normal‐volume leukapheresis, especially in patients who mobilize low numbers of CD34+ cells
Authors: Abrahamsen J.F. , Stamnesfet S. , Liseth K. , Hervig T. , Bruserud O.
Published: January 2005

BACKGROUND:  Large‐volume leukapheresis (LVL) differs from normal‐volume leukapheresis (NVL) by increased blood flow and altered anticoagulation regimen. LVL is now regarded as a safe procedure for collection of peripheral blood progenitor cells (PBPCs), but it is not known whether the procedure will alter CD34+ cell quality or will be useful for patients who mobilize few CD34+ cells into peripheral blood.

STUDY DESIGN AND METHODS:  The results from 82 LVL and 125 NVL (4.0‐5.3 and 2.7‐3.5 times the patients’ blood volumes processed, respectively) were retrospec‐tively analyzed in altogether 112 consecutive patients with malignant diseases.

RESULTS:  The LVL yielded significantly more CD34+ cells (4.2 × 106 vs. 3.1 × 106/kg, p = 0.006, all patients; and 1.8 × 106 vs. 1.3 × 106/kg, p = 0.004, bad mobilizers) and significantly higher colony‐forming units (77 × 104 vs. 33 × 104/kg; all patients and 33 × 104 vs. 20 × 104/kg, p < 0.001, both groups). Significantly fewer leukapheresis procedures were required to obtain 2 × 106 CD34+ cells per kg (one vs. two, p = 0.001, all patients; and two vs. three, p = 0.009, bad mobilizers). No significant differences in CD34+ cell viability and time to hematologic recovery were observed between the patients who received PBPCs harvested by NVL and LVL.

CONCLUSION:  Although a median platelet loss of 36 percent can be expected, LVL can be recommended as the standard apheresis method for PBPC collections in patients with malignant diseases. LVL is particularly useful in patients who mobilize a low number of CD34+ cells into the peripheral blood.
Preparation and storage of white blood cell–reduced split apheresis platelet concentrates for pediatric use
Authors: Van Der Meer Pieter F. , Vrielink Hans , Pietersz Ruby N.I.
Published: January 2005

BACKGROUND: White blood cell (WBC) reduction and bacterial screening induce unacceptable product loss when platelet (PLT) concentrates (PCs) for pediatric transfusion are prepared from whole blood. The aim was to investigate PCs, WBC reduced and bacterially screened, from single‐donor apheresis procedures, divided in 3 or 4 pediatric units and stored up to 5 days.

STUDY DESIGN AND METHODS: PCs were collected with an apheresis machine and WBC reduced by in‐process filtration. The PCs were sampled for bacterial screening and subsequently divided in 70‐mL products. Initially, storage characteristics of split units in 400‐mL polyvinylchloride (PVC) bags with 17 split PCs originating from five apheresis donations were studied. When a 600‐mL container made of the more gas‐permeable polyolefin became available, a paired comparison was performed with 9 split PCs from nine donations and with a higher‐yield PLT collection procedure.

RESULTS: Split PCs contained 69 × 109 ± 14 × 109PLTs in 69 ± 1 mL of plasma, and storage in the PVC containers gave a pH value of 6.86 ± 0.10 on Day 6 (mean ± SD, n = 17). When comparing the containers, the PVC bag contained 98 × 109 ± 15 × 109PLTs in 72 ± 4 mL versus 102 × 109 ± 18 × 109PLTs in 74 ± 8 mL for the polyolefin bag (n = 9, not significant). This gave pH values on Day 6 of 6.12 ± 0.50 in the PVC container, whereas pH remained acceptable in the polyolefin container: 6.85 ± 0.10 on Day 6 (p < 0.01).

CONCLUSION: PCs for pediatric use from split single‐donor apheresis concentrates, WBC reduced and bacterially screened, can be stored for up to 5 days in a 600‐mL polyolefin container with maintenance of good in vitro storage variables.
Evaluation of the Scansystem method for detection of bacterially contaminated platelets
Authors: Jacobs Michael R. , Bajaksouzian Saralee , Windau Anne , Palavecino Elizabeth L. , Yomtovian Roslyn
Published: January 2005

BACKGROUND:  Platelet (PLT) bacterial contamination occurs in approximately 1 in 2000 PLT units. The College of American Pathologists recommends and AABB requires procedures to detect PLT bacterial contamination. Although two methods, BacT/ALERT (bioMérieux) and Pall BDS (Pall Corporation), have FDA approval for quality control testing, additional methods are in development. One such method was evaluated, the Scansystem (Hemosystem), which has been developed for use on leukoreduced PLT components between 30 and 72 hours after collection.

STUDY DESIGN AND METHODS:  Leukoreduced, single‐donor apheresis PLT units (LR‐SDPs) were inoculated with 10 bacterial species (low and high inocula) associated with PLT contamination. Bacterial detection was compared with the Scansystem and BacT/ALERT. Testing was initiated (10 replicates performed) when LR‐SDPs were experimentally inoculated with bacteria. The Scansystem was evaluated 30 hours later, the shortest manufacturer recommended time after PLT collection.

RESULTS:  All replicates were positive with the Scansystem at 30 hours and with the BacT/ALERT, at 9.3 to 24.0 hours after inoculation. The Scansystem detected bacteria in 83 of 200 replicates (42%) at the time of inoculation indicating a potential for earlier application.

CONCLUSIONS:  The Scansystem, used to test LR‐SDPs 30 hours after bacterial inoculation, detected all 20 replicates with a sensitivity equal to the BacT/ALERT system. Based on use of Scansystem with LR‐SDPs 30 hours after collection and the BacT/ALERT being inoculated 24 hours after collection and incubated for an additional 24 hours before being determined to be negative, the Scansystem will potentially provide results at an earlier time point (32 hr) than provided by the BacT/ALERT system (48 hr).
Induction of Rapid Detachment in
Shewanella oneidensis
MR-1 Biofilms
Authors: Thormann Kai M. , Saville Renée M. , Shukla Soni , Spormann Alfred M.
Published: January 2005

Active detachment of cells from microbial biofilms is a critical yet poorly understood step in biofilm development. We discovered that detachment of cells from biofilms of
Shewanella oneidensis
MR-1 can be induced by arresting the medium flow in a hydrodynamic biofilm system. Induction of detachment was rapid, and substantial biofilm dispersal started as soon as 5 min after the stop of flow. We developed a confocal laser scanning microscopy-based assay to quantify detachment. The extent of biomass loss was found to be dependent on the time interval of flow stop and on the thickness of the biofilm. Up to 80% of the biomass of 16-h-old biofilms could be induced to detach. High-resolution microscopy studies revealed that detachment was associated with an overall loosening of the biofilm structure and a release of individual cells or small cell clusters. Swimming motility was not required for detachment. Although the loosening of cells from the biofilm structure was observed evenly throughout thin biofilms, the most pronounced detachment in thicker biofilms occurred in regions exposed to the flow of medium, suggesting a metabolic control of detachability. Deconvolution of the factors associated with the stop of medium flow revealed that a sudden decrease in oxygen tension is the predominant trigger for initiating detachment of individual cells. In contrast, carbon limitation did not trigger any substantial detachment, suggesting a physiological link between oxygen sensing or metabolism and detachment. In-frame deletions were introduced into genes encoding the known and putative global transcriptional regulators ArcA, CRP, and EtrA (FNR), which respond to changes in oxygen tension in
S. oneidensis
MR-1. Biofilms of null mutants in
arcA
and
crp
were severely impacted in the stop-of-flow-induced detachment response, suggesting a role for these genes in regulation of detachment. In contrast, an Δ
etrA
mutant displayed a variable detachment phenotype. From this genetic evidence we conclude that detachment is a biologically controlled process and that a rapid change in oxygen concentration is a critical factor in detachment and, consequently, in dispersal of
S. oneidensis
cells from biofilms. Similar mechanisms might also operate in other bacteria.
Characterization of the Pheromone Response of theEnterococcus faecalisConjugative Plasmid pCF10: Complete Sequence and Comparative Analysis of the Transcriptional and Phenotypic Responses of pCF10-Containing Cells to Pheromone Induction
Authors: Hirt Helmut , Manias Dawn A. , Bryan Edward M. , Klein Joanna R. , Marklund Jesper K. , Staddon Jack H. , Paustian Michael L. , Kapur Vivek , Dunny Gary M.
Published: January 2005

The sex pheromone plasmids inEnterococcus faecalisare one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10−1transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by theprgBgene) was present on the cell surface for a long period of time after pheromone-induced transcription ofprgBand plasmid transfer ability had ceased. This observation could have relevance for the virulence ofE. faecalis.
Regulation of Virulence by a Two-Component System in Group B
Streptococcus
Authors: Jiang Sheng-Mei , Cieslewicz Michael J. , Kasper Dennis L. , Wessels Michael R.
Published: January 2005

Group B
Streptococcus
(GBS) is frequently carried in the gastrointestinal or genitourinary tract as a commensal organism, yet it has the potential to cause life-threatening infection in newborn infants, pregnant women, and individuals with chronic illness. Regulation of virulence factor expression may affect whether GBS behaves as an asymptomatic colonizer or an invasive pathogen, but little is known about how such factors are controlled in GBS. We now report the characterization of a GBS locus that encodes a two-component regulatory system similar to CsrRS (or CovRS) in
Streptococcus pyogenes
. Inactivation of
csrR
, encoding the putative response regulator, in two unrelated wild-type strains of GBS resulted in a marked increase in production of beta-hemolysin/cytolysin and a striking decrease in production of CAMP factor, an unrelated cytolytic toxin. Quantitative RNA hybridization experiments revealed that these two phenotypes were associated with a marked increase and decrease in expression of the corresponding genes,
cylE
and
cfb
, respectively. The CsrR mutant strains also displayed increased expression of
scpB
encoding C5a peptidase. Similar, but less marked, changes in gene expression were observed in CsrS (putative sensor component) mutants, evidence that CsrR and CsrS constitute a functional two-component system. Experimental infection studies in mice demonstrated reduced virulence of both CsrR and CsrS mutant strains relative to the wild type. Together, these results indicate that CsrRS regulates expression of multiple GBS virulence determinants and is likely to play an important role in GBS pathogenesis.
Genome-Wide Transcriptional Response of Chemostat-CulturedEscherichia colito Zinc
Authors: Lee Lucy J. , Barrett Jason A. , Poole Robert K.
Published: January 2005

Zinc is an essential trace metal ion for growth, but an excess of Zn is toxic and microorganisms express diverse resistance mechanisms. To understand global bacterial responses to excess Zn, we conducted transcriptome profiling experiments comparingEscherichia coliMG1655 grown under control conditions and cells grown with a toxic, sublethal ZnSO4concentration (0.2 mM). Cultures were grown in a defined medium lacking inorganic phosphate, permitting maximum Zn bioavailability, and in glycerol-limited chemostats at a constant growth rate and pH. Sixty-four genes were significantly up-regulated by Zn stress, including genes known to be involved in Zn tolerance, particularlyzntA,zraP, andhydG. Microarray transcriptome profiling was confirmed by real-time PCR determinations ofcusF(involved in Ag and Cu efflux),ais(an Al-inducible gene),asr(encoding an acid shock-inducible periplasmic protein),cpxP(a periplasmic chaperone gene), andbasR. Five up-regulated genes,basRandbasS[encoding a sensor-regulator implicated inSalmonellain Fe(III) sensing and antibiotic resistance],fliM(flagellar synthesis), andycdMandyibD(both with unknown functions), are important for growth resistance to zinc, since mutants with mutations in these genes exhibited zinc sensitivity in liquid media and on metal gradient plates. Fifty-eight genes were significantly down-regulated by Zn stress; notably, several of these genes were involved in protection against acid stress. Since themdtoperon (encoding a multidrug resistance pump) was also up-regulated, these findings have important implications for understanding not only Zn homeostasis but also how bacterial antibiotic resistance is modulated by metal ions.
NopB, a Type III Secreted Protein of
Rhizobium
sp. Strain NGR234, Is Associated with Pilus-Like Surface Appendages
Authors: Saad Maged M. , Kobayashi Hajime , Marie Corinne , Brown Ian R. , Mansfield John W. , Broughton William J. , Deakin William J.
Published: January 2005

Rhizobium
sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGRΩ
rhcN
). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with
Pachyrhizus tuberosus
and augments nodulation of
Tephrosia vogelii
. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.
Specificity and Polymorphism of the PlcR-PapR Quorum-Sensing System in theBacillus cereusGroup
Authors: Slamti Leyla , Lereclus Didier
Published: January 2005

The expression of extracellular virulence factors in various species of theBacillus cereusgroup is controlled by theplcRandpapRgenes, which encode a transcriptional regulator and a cell-cell signaling peptide, respectively. A processed form of PapR, presumably a pentapeptide, specifically interacts with PlcR to facilitate its binding to its DNA targets. This activating mechanism is strain specific, with this specificity being determined by the first residue of the pentapeptide. We carried out in vivo complementation assays and compared the PlcR-PapR sequences of 29 strains from theB. cereusgroup. Our findings suggested that the fifth amino acid of the pentapeptide is also involved in the specificity of activation. We identified four classes of PlcR-PapR pairs, defining four distinct pherotypes in theB. cereusgroup. We used these findings to look at the evolution of the PlcR-PapR quorum-sensing system with regard to the phylogeny of the species forming theB. cereusgroup.
Characterization of Phospholipase Activity of the
Pseudomonas aeruginosa
Type III Cytotoxin, ExoU
Authors: Sato Hiromi , Feix Jimmy B. , Hillard Cecilia J. , Frank Dara W.
Published: January 2005

Recombinant ExoU (rExoU) and yeast extract were used to optimize an in vitro phospholipase assay as a basis for identifying the mechanism for enzyme activation and substrate specificity. Our results support a model in which a eukaryotic protein cofactor or complex facilitates the interaction of rExoU with phospholipid substrates.
Acetic Acid Induces Expression of the
Staphylococcus aureus cidABC
and
lrgAB
Murein Hydrolase Regulator Operons
Authors: Rice Kelly C. , Nelson Jeremy B. , Patton Toni G. , Yang Soo-Jin , Bayles Kenneth W.
Published: January 2005

The
Staphylococcus aureus lrg
and
cid
operons encode homologous proteins that regulate extracellular murein hydrolase activity and penicillin tolerance in a diametrically opposing manner. Although their specific regulatory functions remain unknown, it has been postulated that the functions of CidA and LrgA are analogous to those of bacteriophage holins and antiholins, respectively, and that these proteins serve as molecular control elements of bacterial programmed cell death. Although these studies demonstrated that
cidBC
transcription is abundant in σ
B
-proficient strains,
cidABC
transcription was only minimally expressed under standard growth conditions. In this study, we demonstrate that
cidABC
and
lrgAB
transcription in the clinical isolate UAMS-1 is induced by growth in the presence of 35 mM glucose and that this enhances murein hydrolase activity and decreases tolerance to vancomycin and rifampin. The effect of glucose on murein hydrolase activity was not observed in the
cidA
mutant, indicating that the induction of this activity was dependent on enhanced
cidABC
expression. Furthermore, we demonstrate that the effects of glucose on
cidABC
and
lrgAB
transcription are mediated by the generation of acetic acid produced by the metabolism of this and other carbon sources. These results shed new light on the control of the
S. aureus cidABC
and
lrgAB
genes and demonstrate that these operons, as well as murein hydrolase activity and antibiotic tolerance, are responsive to carbohydrate metabolism.
Expression and Functional Analysis of the Subtilin Immunity Genes
spaIFEG
in the Subtilin-Sensitive Host
Bacillus subtilis
MO1099
Authors: Stein Torsten , Heinzmann Stefan , Düsterhus Stefanie , Borchert Stefan , Entian Karl-Dieter
Published: January 2005

Bacillus subtilis
ATCC 6633 produces the cationic pore-forming lantibiotic subtilin, which preferentially acts on gram-positive microorganisms; self protection of the producer cells is mediated by the four genes
spaIFEG
. To elucidate the mechanism of subtilin autoimmunity, we transferred different combinations of subtilin immunity genes under the control of an inducible promoter into the genome of subtilin-sensitive host strain
B. subtilis
MO1099. Recipient cells acquired subtilin tolerance through expression of either
spaI
or
spaFEG
, which shows that subtilin immunity is based on two independently acting systems. Cells coordinately expressing all four immunity genes acquired the strongest subtilin protection level. Quantitative in vivo peptide release assays demonstrated that SpaFEG diminished the quantity of cell-associated subtilin, suggesting that SpaFEG transports subtilin molecules from the membrane into the extracellular space. Homology and secondary structure analyses define SpaFEG as a prototype of lantibiotic immunity transporters that fall into the ABC-2 subfamily of multidrug resistance proteins. Membrane localization of the lipoprotein SpaI and specific interaction of SpaI with the cognate lantibiotic subtilin suggest a function of SpaI as a subtilin-intercepting protein. This interpretation was supported by hexahistidine-mediated 0-Å cross-linking between hexahistidine-tagged SpaI and subtilin.
Disparate Subcellular Localization Patterns of
Pseudomonas aeruginosa
Type IV Pilus ATPases Involved in Twitching Motility
Authors: Chiang Poney , Habash Marc , Burrows Lori L.
Published: January 2005

The opportunistic pathogen
Pseudomonas aeruginosa
expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a
pilC
mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.
Mutants with Temperature-Sensitive Defects in the
Escherichia coli
Mismatch Repair System: Sensitivity to Mispairs Generated In Vivo
Authors: Hong Esther S. , Yeung Annie , Funchain Pauline , Slupska Malgorzata M. , Miller Jeffrey H.
Published: January 2005

We have used direct selections to generate large numbers of mutants of
Escherichia coli
defective in the mismatch repair system and have screened these to identify mutants with temperature-sensitive defects. We detected and sequenced mutations that give rise to temperature-sensitive MutS, MutL, and MutH proteins. One mutation,
mutS60
, results in almost normal levels of spontaneous mutations at 37°C but above this temperature gives rise to higher and higher levels of mutations, reaching the level of null mutations in
mutS
at 43°C. However, at 37°C the MutS60 protein can be much more easily titrated by mispairs than the wild-type MutS, as evidenced by the impaired ability to block homeologous recombination in interspecies crosses and the increased levels of mutations from weak mutator alleles of
mutD
(
dnaQ
),
mutC
, and
ndk
. Strains with
mutS60
can detect mispairs generated during replication that lead to mutation with much greater sensitivity than wild-type strains. The findings with
ndk
, lacking nucleotide diphosphate kinase, are striking. An
ndk mutS60
strain yields four to five times the level of mutations seen in a full knockout of
mutS
. These results pose the question of whether similar altered Msh2 proteins result from presumed polymorphisms detected in tumor lines. The role of allele interactions in human disease susceptibility is discussed.
Identification and Characterization of Genes Involved in the Downstream Degradation Pathway of γ-Hexachlorocyclohexane in
Sphingomonas paucimobilis
UT26
Authors: Endo Ryo , Kamakura Mayuko , Miyauchi Keisuke , Fukuda Masao , Ohtsubo Yoshiyuki , Tsuda Masataka , Nagata Yuji
Published: January 2005

Sphingomonas paucimobilis
UT26 utilizes γ-hexachlorocyclohexane (γ-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of γ-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated
linF
, that is essential for the utilization of γ-HCH in UT26. A gene named
linEb
, whose deduced product showed significant identity to LinE (53%), was located close to
linF
. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in
Escherichia coli
transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for γ-HCH utilization.
Pyruvate:Quinone Oxidoreductase from
Corynebacterium glutamicum
: Purification and Biochemical Characterization
Authors: Schreiner Mark E. , Eikmanns Bernhard J.
Published: January 2005

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO
2
with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in
Escherichia coli
. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer
Corynebacterium glutamicum
. The activity was highest (0.055 ± 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type
C. glutamicum
, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (
k
cat
= 37.8 ± 3 s
−1
;

K
m

= 30 ± 3 mM) and 2-oxobutyrate (
k
cat
= 33.2 ± 3 s
−1
;

K
m

= 90 ± 8 mM). Thiamine pyrophosphate (

K
m

= 1 μM) and certain divalent metal ions such as Mg
2+
(

K
m

= 29 μM), Mn
2+
(

K
m

= 2 μM), and Co
2+
(

K
m

= 11 μM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol,
p
-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (

K
m

= 106 μM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in
C. glutamicum
.
Heat Shock Proteome Analysis of Wild-Type
Corynebacterium glutamicum
ATCC 13032 and a Spontaneous Mutant Lacking GroEL1, a Dispensable Chaperone
Authors: Barreiro Carlos , González-Lavado Eva , Brand Sven , Tauch Andreas , Martín Juan F.
Published: January 2005

Proteome analysis of
Corynebacterium glutamicum
ATCC 13032 showed that levels of several proteins increased drastically in response to heat shock. These proteins were identified as DnaK, GroEL1, GroEL2, ClpB, GrpE, and PoxB, and their heat response was in agreement with previous transcriptomic results. A major heat-induced protein was absent in the proteome of strain 13032B of
C. glutamicum
, used for genome sequencing in Germany, compared with the wild-type ATCC 13032 strain. The missing protein was identified as GroEL1 by matrix-assisted laser desorption ionization-time of flight peptide mass fingerprinting, and the mutation was found to be due to an insertion sequence, Is
Cg1
, that was integrated at position 327 downstream of the translation start codon of the
groEL1
gene, resulting in a truncated transcript of this gene, as shown by Northern analysis. The GroEL1 chaperone is, therefore, dispensable in
C. glutamicum
. On the other hand, GroEL2 appears to be essential for growth. Based on these results, the role of the duplicate
groEL1
and
groEL2
genes is analyzed.
Trimeric Structure of Major Outer Membrane Proteins Homologous to OmpA inPorphyromonas gingivalis
Authors: Nagano Keiji , Read Erik K. , Murakami Yukitaka , Masuda Takashi , Noguchi Toshihide , Yoshimura Fuminobu
Published: January 2005

The major outer membrane proteins Pgm6 (41 kDa) and Pgm7 (40 kDa) ofPorphyromonas gingivalisATCC 33277 are encoded by open reading framespg0695andpg0694, respectively, which form a single operon. Pgm6 and Pgm7 (Pgm6/7) have a high degree of similarity toEscherichia coliOmpA in the C-terminal region and are predicted to form eight-stranded β-barrels in the N-terminal region. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pgm6/7 appear as bands with apparent molecular masses of 40 and 120 kDa, with and without a reducing agent, suggesting a monomer and trimer, respectively. To verify the predicted trimeric structure and function of Pgm6/7, we constructed three mutants withpg0695,pg0694, or both deleted. The double mutant produced no Pgm6/7. The single-deletion mutants appeared to contain less Pgm7 and Pgm6 and to form homotrimers that migrated slightly faster (115 kDa) and slower (130 kDa), respectively, than wild-type Pgm6/7 under nonreducing conditions. N-terminal amino acid sequencing and mass spectrometry analysis of partially digested Pgm6/7 detected only fragments from Pgm6 and Pgm7. Two-dimensional, diagonal electrophoresis and chemical cross-linking experiments with or without a reducing agent clearly showed that Pgm6/7 mainly form stable heterotrimers via intermolecular disulfide bonds. Furthermore, growth retardation and arrest of the three mutants and increased permeability of their outer membranes indicated that Pgm6/7 play an important role in outer membrane integrity. Based on results of liposome swelling experiments, these proteins are likely to function as a stabilizer of the cell wall rather than as a major porin in this organism.
Transcriptional Regulation ofsitABCDofSalmonella entericaSerovar Typhimurium by MntR and Fur
Authors: Ikeda Jack S. , Janakiraman Anuradha , Kehres David G. , Maguire Michael E. , Slauch James M.
Published: January 2005

Salmonella entericaserovar Typhimurium has two manganese transport systems, MntH and SitABCD. MntH is a bacterial homolog of the eukaryotic natural resistance-associated macrophage protein 1 (Nramp1), and SitABCD is an ABC-type transporter. Previously we showed thatmntHis negatively controlled at the transcriptional level by thetrans-acting regulatory factors, MntR and Fur. In this study, we examined the transcriptional regulation ofsitABCDand compared it to the transcriptional regulation ofmntHby constructinglacZfusions to the promoter regions with and without mutations in putative MntR and/or Fur binding sites. The presence of Mn caused transcriptional repression of thesitABCDandmntHpromoters primarily via MntR, but Fur was also capable of some repression in response to Mn. Likewise, Fe in the medium repressed transcription of bothsitandmntHprimarily via Fur, although MntR was also involved in this response. Transcriptional control by MntR and Fur was disrupted by site-specific mutations in the putative MntR and Fur binding sites, respectively. Transcription of thesitoperon was also affected by the oxygen level and growth phase, but the increased expression observed under high oxygen conditions and higher cell densities is consistent with decreased availability of metals required for repression by the metalloregulatory proteins.
Markerless Mutagenesis inMethanococcus maripaludisDemonstrates Roles for Alanine Dehydrogenase, Alanine Racemase, and Alanine Permease
Authors: Moore Brian C. , Leigh John A.
Published: January 2005

Among the archaea,Methanococcus maripaludishas the unusual ability to usel- ord-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools forM.maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of theM.maripaludis hptgene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of theuptgene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow ond-alanine. The wild-type gene for alanine dehydrogenase, incorporated into theuptsite, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for thelisomer, and the racemase converts thedisomer to thelisomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria.
Functional Interactions between the Carbon and Iron Utilization Regulators, Crp and Fur, in
Escherichia coli
Authors: Zhang Zhongge , Gosset Guillermo , Barabote Ravi , Gonzalez Claudio S. , Cuevas William A. , Saier Milton H.
Published: January 2005

In
Escherichia coli
, the ferric uptake regulator (Fur) controls expression of the iron regulon in response to iron availability while the cyclic AMP receptor protein (Crp) regulates expression of the carbon regulon in response to carbon availability. We here identify genes subject to significant changes in expression level in response to the loss of both Fur and Crp. Many iron transport genes and several carbon metabolic genes are subject to dual control, being repressed by the loss of Crp and activated by the loss of Fur. However, the
sodB
gene, encoding superoxide dismutase, and the
aceBAK
operon, encoding the glyoxalate shunt enzymes, show the opposite responses, being activated by the loss of Crp and repressed by the loss of Fur. Several other genes including the
sdhA-D
,
sucA-D
, and
fumA
genes, encoding key constituents of the Krebs cycle, proved to be repressed by the loss of both transcription factors. Finally, the loss of both Crp and Fur activated a heterogeneous group of genes under σ
S
control encoding, for example, the cyclopropane fatty acid synthase, Cfa, the glycogen synthesis protein, GlgS, the 30S ribosomal protein, S22, and the mechanosensitive channel protein, YggB. Many genes appeared to be regulated by the two transcription factors in an apparently additive fashion, but apparent positive or negative cooperativity characterized several putative Crp/Fur interactions. Relevant published data were evaluated, putative Crp and Fur binding sites were identified, and representative results were confirmed by real-time PCR. Molecular explanations for some, but not all, of these effects are provided.
Multiple Regulators Control Expression of the Entner-Doudoroff Aldolase (Eda) of
Escherichia coli
Authors: Murray Elizabeth L. , Conway Tyrrell
Published: January 2005

The
Escherichia coli eda
gene, which encodes the Entner-Doudoroff aldolase, is central to the catabolism of several sugar acids. Here, we show that Eda synthesis is induced by growth on gluconate, glucuronate, or methyl-β-
d
-glucuronide; phosphate limitation; and carbon starvation. Transcription of
eda
initiates from three promoters, designated P1, P2, and P4, each of which is responsible for induction under different growth conditions. P1 controls
eda
induction on gluconate and is regulated by GntR. P2 controls
eda
induction on glucuronate and galacturonate and is regulated by KdgR. P4 is active under conditions of phosphate starvation and is directly controlled by PhoB. In addition, CsrA activates Eda synthesis, apparently by an indirect mechanism that may be involved in the modest changes in expression level that are associated with carbon starvation. The complex regulation of
eda
is discussed with respect to its several physiological roles, which apparently accommodate not only sugar acid catabolism but also detoxification of metabolites that could accumulate during starvation-induced stress.