bluuebunny/crossref_metadata_2025 huggingface dataset:
Delineating and Quantifying Ground Water Discharge Zones Using Streambed Temperatures
Authors: Conant Brewster
Published: December 2005

Streambed temperature mapping, hydraulic testing using minipiezometers, and geochemical analyses of interstitial water of the streambed were used to delineate the pattern of ground water discharge in a sandy streambed and to develop a flux‐based conceptual model for ground water/surface water interactions. A new and simple empirical method was used to relate fluxes obtained from minipiezometer data to streambed temperatures. The relationship allowed flux to be calculated at locations where only streambed temperature measurements were made. Slug testing and potentiomanometer measurements at 34 piezometers indicated ground water discharge ranged from 0.03 to 446 L/m2/day (and possibly as high as 7060 L/m2/day) along a 60 m long by 11 to 14 m wide reach of river. Complex but similar plan‐view patterns of flux were calculated for both summer and winter using hundreds of streambed temperatures measured on a 1 by 2 m grid. The reach was dominated by ground water discharge and 5% to 7% of the area accounted for ∼20% to 24% of the total discharge. < 12% of the total area consisted of recharge zones or no‐discharge zones. A conceptual model for ground water/surface water interactions consisting of five different behaviors was developed based on the magnitude and direction of flux across the surface of the streambed. The behaviors include short‐circuit discharge (e.g., high‐flow springs), high discharge (e.g., preferential flowpaths), low to moderate discharge, no discharge (e.g., horizontal hyporheic or ground water flow), and recharge. Geological variations at depth played a key role in determining which type of flow behavior occurred in the streambed.
Validation of Numerical Ground Water Models Used to Guide Decision Making
Authors: Hassan Ahmed E.
Published: December 2005

Many sites of ground water contamination rely heavily on complex numerical models of flow and transport to develop closure plans. This complexity has created a need for tools and approaches that can build confidence in model predictions and provide evidence that these predictions are sufficient for decision making. Confidence building is a long‐term, iterative process and the author believes that this process should be termed model validation. Model validation is a process, not an end result. That is, the process of model validation cannot ensure acceptable prediction or quality of the model. Rather, it provides an important safeguard against faulty models or inadequately developed and tested models. If model results become the basis for decision making, then the validation process provides evidence that the model is valid for making decisions (not necessarily a true representation of reality). Validation, verification, and confirmation are concepts associated with ground water numerical models that not only do not represent established and generally accepted practices, but there is not even widespread agreement on the meaning of the terms as applied to models. This paper presents a review of model validation studies that pertain to ground water flow and transport modeling. Definitions, literature debates, previously proposed validation strategies, and conferences and symposia that focused on subsurface model validation are reviewed and discussed. The review is general and focuses on site‐specific, predictive ground water models used for making decisions regarding remediation activities and site closure. The aim is to provide a reasonable starting point for hydrogeologists facing model validation for ground water systems, thus saving a significant amount of time, effort, and cost. This review is also aimed at reviving the issue of model validation in the hydrogeologic community and stimulating the thinking of researchers and practitioners to develop practical and efficient tools for evaluating and refining ground water predictive models.
Conduit Properties and Karstification in the Unconfined Floridan Aquifer
Authors: Screaton Elizabeth , Martin Jonathan B. , Ginn Brian , Smith Lauren
Published: December 2005

Exchange of water between conduits and matrix is an important control on regional chemical compositions, karstification, and quality of ground water resources in karst aquifers. A sinking stream (Santa Fe River Sink) and its resurgence (River Rise) in the unconfined portion of the Floridan Aquifer provide the opportunity to monitor conduit inflow and outflow. The use of temperature as a tracer allows determination of residence times and velocities through the conduit system. Based on temperature records from two high water events, flow is reasonably represented as pipe flow with a cross‐sectional area of 380 m2, although this model may be complicated by losses of water from the conduit system at higher discharge rates. Over the course of the study year, the River Rise discharged a total of 1.9 × 107 m3 more water than entered the River Sink, reflecting net contribution of ground water from the matrix into the conduit system. However, as River Sink discharge rates peaked following three rainfall events during the study period, the conduit system lost water, presumably into the matrix. Surface water in high flow events is typically undersatu‐rated with respect to calcite and thus may lead to dissolution, depending on its residence time in the matrix. A calculation of local denudation is larger than other regional estimates, perhaps reflecting return of water to conduits before calcite equilibrium is reached. The exchange of matrix and conduit water is an important variable in karst hydrology that should be considered in management of these water resources.
Spatial Variability of In Situ Microbial Activity: Biotracer Tests
Authors: Sandrin Susannah K. , Brusseau Mark L. , Piatt Joseph J. , Bodour Adria A. , Blanford William J. , Nelson Nicole T.
Published: December 2005

Biotracer tests have been proposed as a means by which to characterize the in situ biodegradation potential for field‐scale systems. In this study, field experiments were conducted at two sites to evaluate the utility of the biotracer method for characterizing the spatial variability of microbial activity. The first site is a mixed waste‐contaminated sur‐ficial aquifer in Utah, and the second site is a chlorinated solvent‐contaminated regional aquifer in Tucson, Arizona. Mass recovery of the biotracer decreased approximately linearly with increasing residence time for the Tucson site. Similar behavior was observed at the Utah site, except in the region adjacent to the injection zone, where percent recoveries were much lower than those predicted using a correlation determined using data collected downgradient of the injection zone. First‐order biodegradation rate coefficients obtained from model calibration of the tracer data varied between 0.2 and 0.5/day for the Tucson site. For the Utah site, the values varied between 0.1 and 0.6/day downgradient of the injection wells, and between 0.7 and 2.6/day near the injection wells. Considering the large range over which biodegradation rate coefficients can vary, the rate coefficient exhibited relatively minimal spatial variability (factor of 2.5) for the Tucson site. Conversely, the spatial variability of the rate coefficient was an order of magnitude greater for the Utah site. These differences in variability are consistent with conditions associated with the respective sites. For example, the greater microbial activity observed in the vicinity of the injection wells for the Utah site is consistent with the biomass distribution determined from analysis of core samples, which shows larger bacterial cell densities for the region near the injection wells. These results illustrate the utility of biotracer tests for in situ characterization of microbial activity (e.g., biodegradation potential), including evaluation of potential spatial variability.
Estrogen in a Karstic Aquifer
Authors: Wicks Carol , Kelley Cheryl , Peterson Eric
Published: December 2005

Adverse impacts on the health of some fish populations, such as skewed sex distributions, have been noted in surface waters and in laboratory experiments with relatively low concentrations (above 25 ng/L) of natural estrogen (17 β‐estradiol—E2). Sources of E2 to surface and ground waters can include avian, human, and mammalian waste products. The Ozark Plateau Aquifer (OPA) is a karstic basin that receives a significant portion of its water through losing reaches of rivers. Thus, there is a direct connection between surface water and ground water. The OPA was targeted for an E2 study to assess the potential for adverse health effects to aquatic organisms living in the system. Eight springs, which drain the aquifer, were sampled quarterly. The concentrations of E2 in the OPA ranged from 13 to 80 ng/L. For any one sampling event, the concentrations of E2 at the spring waters were statistically similar; however, the concentrations of E2 at all springs varied throughout the year. At Maramec Spring, one of the larger springs, the E2 concentration, was correlated with discharge. Based on the correlation between discharge and E2 concentration, aquatic organisms living in the plateau or in its discharged waters, including the threatened southern cavefish T. subterraneus, are exposed to concentration of E2 above 25 ng/L ∼60% of the time. This implies that organisms living in karst basins throughout the OPA are likely exposed to E2 concentrations that may adversely impact their reproductive success for a significant portion of each year.
Post‐fire recovery of ant communities in Submediterranean Pinus nigra forests
Authors: Rodrigo Anselm , Retana Javier
Published: December 2005

This study analyzes the variations in the structure and composition of ant communities in burned Pinus nigra forests in central Catalonia (NE Spain). Pinus nigra forests do not recover after fire, changing to shrublands and oak coppices. For this reason, we suggest that ant communities of burned P. nigra forests will change after fire, because the post‐fire scenario, in particular with the increase of open areas, is different to the unburned one, and more favourable for some species than for others. In four locations previously occupied by P. nigra forests where different fires occurred 1, 5, 13 and 19 yr before the sampling, we sampled the structure and composition of ant communities with pitfall traps, tree traps and net sweeping in unburned plots and in plots affected by canopy and understory fire. The results obtained suggest that canopy and understory fire had little effect on the structure of ant communities. Thus, many variables concerning ant communities were not modified either by fire type (understory or canopy fire) or by time since fire. However, a number of particular species were affected, either positively or negatively, by canopy fire: three species characteristic of forest habitats decreased after fire, while eight species characteristic of open habitats increased in areas affected by canopy fire, especially in the first few years after fire. These differences in ant community composition between burned and unburned plots imply that the maximum richness is achieved when there is a mixture of unburned forests and areas burned with canopy fire. Moreover, as canopy cover in P. nigra forests burned with canopy fire is not completed in the period of time studied, the presence of the species that are characteristic of burned areas remains along the chronosequence studied, while the species that disappear after fire do not recover in the period of time considered. Overall, the results obtained indicate that there is a persistent replacement of ant species in burned P. nigra forests, as is also the case with vegetation.
Mutations in the Central Cavity and Periplasmic Domain Affect Efflux Activity of the Resistance-Nodulation-Division Pump EmhB from
Pseudomonas fluorescens
cLP6a
Authors: Hearn Elizabeth M. , Gray Murray R. , Foght Julia M.
Published: December 2005

The EmhABC efflux system in
Pseudomonas fluorescens
cLP6a is homologous to the multidrug and solvent efflux systems belonging to the resistance-nodulation-division (RND) family and is responsible for polycyclic aromatic hydrocarbon transport, antibiotic resistance, and toluene efflux. To gain a better understanding of substrate transport in RND efflux pumps, the EmhB pump was subjected to mutational analysis. Mutagenesis of amino acids within the central cavity of the predicted three-dimensional structure of EmhB showed selective activity towards antibiotic substrates. An A384P/A385Y double mutant showed increased susceptibility toward rhodamine 6G compared to the wild type, and F386A and N99A single mutants showed increased susceptibility to dequalinium compared to the wild type. As well, the carboxylic acid side chain of D101, located in the central cavity region, was found to be essential for polycyclic aromatic hydrocarbon transport and resistance to all antibiotic substrates of EmhB. Phenylalanine residues located within the periplasmic pore domain were also targeted for mutagenesis, and the F325A and F281A mutations significantly impaired efflux activity for all EmhB substrates. One mutation (A206S) in the outer membrane protein docking domain increased antibiotic resistance and toluene tolerance, demonstrating the important role of this domain in transport activity. These data demonstrate the roles of the central cavity and periplasmic domains in the function of the RND efflux pump EmhB.
Hfq Is a Regulator of F-Plasmid TraJ and TraM Synthesis inEscherichia coli
Authors: Will William R. , Frost Laura S.
Published: December 2005

The F plasmid ofEscherichia coliallows horizontal DNA transfer between an F+donor cell and an F−recipient. Expression of the transfer genes is tightly controlled by a number of factors, including the following plasmid-encoded regulatory proteins: TraJ, the primary activator of the 33-kbtraoperon, and the autoregulators TraM and TraY. Here, we demonstrate that the host RNA binding protein, Hfq, represses TraJ and TraM synthesis by destabilizing their respective mRNAs. Mating assays and immunoblot analyses for TraM and TraJ showed that transfer efficiency and protein levels increased in host cells containing a disruption inhfqcompared to wild-type cells in stationary phase. The stability of transcripts containing a putative Hfq binding site located in the intergenic untranslated region betweentraMandtraJwas increased inhfqmutant donor cells, suggesting that Hfq destabilizes these transcripts. Electrophoretic mobility shift assays demonstrated that Hfq specifically binds this region but not the antisense RNA, FinP, encoded on the opposite strand. Together, these findings indicate that Hfq regulatestraMandtraJtranscript stability by a mechanism separate from FinOP-mediated repression.
The
Pseudomonas aeruginosa
Ribbon-Helix-Helix DNA-Binding Protein AlgZ (AmrZ) Controls Twitching Motility and Biogenesis of Type IV Pili
Authors: Baynham Patricia J. , Ramsey Deborah M. , Gvozdyev Borys V. , Cordonnier Ellen M. , Wozniak Daniel J.
Published: December 2005

Pseudomonas aeruginosa
is an opportunistic pathogen that is commonly found in water and soil. In order to colonize surfaces with low water content,
P. aeruginosa
utilizes a flagellum-independent form of locomotion called twitching motility, which is dependent upon the extension and retraction of type IV pili. This study demonstrates that AlgZ, previously identified as a DNA-binding protein absolutely required for transcription of the alginate biosynthetic operon, is required for twitching motility. AlgZ may be required for the biogenesis or function of type IV pili in twitching motility. Transmission electron microscopy analysis of an
algZ
deletion in nonmucoid PAO1 failed to detect surface pili. To examine expression and localization of PilA (the major pilin subunit), whole-cell extracts and cell surface pilin preparations were analyzed by Western blotting. While the PilA levels present in whole-cell extracts were similar for wild-type
P. aeruginosa
and
P. aeruginosa
with the
algZ
deletion, the amount of PilA on the surface of the cells was drastically reduced in the
algZ
mutant. Analysis of
algZ
and
algD
mutants indicates that the DNA-binding activity of AlgZ is essential for the regulation of twitching motility and that this is independent of the role of AlgZ in alginate expression. These data show that AlgZ DNA-binding activity is required for twitching motility independently of its role in alginate production and that this involves the surface localization of type IV pili. Given this new role in twitching motility, we propose that
algZ
(PA3385) be designated
amrZ
(
a
lginate and
m
otility
r
egulator
Z
).
Role of
Salmonella enterica
Serovar Typhimurium Two-Component System PreA/PreB in Modulating PmrA-Regulated Gene Transcription
Authors: Merighi Massimo , Carroll-Portillo Amanda , Septer Alecia N. , Bhatiya Aditi , Gunn John S.
Published: December 2005

The
PmrA/PmrB two-component system encoded by the
pmrCAB
operon regulates the modification of
Salmonella
enterica
serovar Typhimurium lipopolysaccharide leading to
polymyxin B resistance. PmrA and PhoP are the only known activators of
pmrCAB
. A transposon mutagenesis screen for additional
regulators of a
pmrC
::MudJ fusion led to
the identification of a two-component system, termed PreA/PreB
(
pmrCAB
regulators A and B), that controls the transcription
of the
pmrCAB
operon in response to unknown signals. The
initial observations indicated that insertions in, or a deletion of,
the
preB
sensor, but not the
preA
response regulator,
caused upregulation of
pmrCAB
. Interestingly, the
expression of
pmrCAB
was not upregulated in a
preAB
mutant grown in LB broth, implicating PreA in the increased expression
of
pmrCAB
in the
preB
strain. This was confirmed by
overexpression of
preA

+

in
preAB
or
preB
backgrounds, which resulted in
significant upregulation or further upregulation of
pmrCAB
. No
such effect was observed in any tested
preB

+

backgrounds. Additionally,
an ectopic construct expressing a
preA
[D51A] allele also
failed to upregulate
pmrC
in any of the
pre
backgrounds tested, which implies that there is a need for
phosphorylation in the activation of the target genes. The observed
upregulation of
pmrCAB
occurred independently of the response
regulators PmrA and PhoP. Although a
preB
mutation led to
increased transcription of
pmrCAB
, this did not result in a
measurable effect on polymyxin B resistance. Our genetic data support a
model of regulation whereby, in response to unknown signals, the PreB
sensor activates PreA, which in turn indirectly upregulates
pmrCAB
transcription.
The
Mycobacterium tuberculosis
TrcR Response Regulator Represses Transcription of the Intracellularly Expressed Rv1057 Gene, Encoding a Seven-Bladed β-Propeller
Authors: Haydel Shelley E. , Clark-Curtiss Josephine E.
Published: December 2005

The
Mycobacterium tuberculosis
TrcR response regulator binds and regulates its own promoter via an AT-rich sequence. Sequences within this AT-rich region determined to be important for TrcR binding were used to search the
M. tuberculosis
H37Rv genome to identify additional related TrcR binding sites. A similar AT-rich sequence was identified within the intergenic region located upstream of the Rv1057 gene. In the present work, we demonstrate that TrcR binds to a 69-bp AT-rich sequence within the Rv1057 intergenic region and generates specific contacts on the same side of the DNA helix. An
M. tuberculosis trcRS
deletion mutant, designated STS10, was constructed and used to determine that TrcR functions as a repressor of Rv1057 expression. Additionally, identification of the Rv1057 transcriptional start site suggests that a SigE-regulated promoter also mediates control of Rv1057 expression. Using selective capture of transcribed sequences (SCOTS) analysis as an evaluation of intracellular expression, Rv1057 was shown to be expressed during early
M. tuberculosis
growth in human macrophages, and the Rv1057 expression profile correlated with a gene that would be repressed by TrcR. Based on structural predictions, motif analyses, and molecular modeling, Rv1057 consists of a series of antiparallel β-strands which adopt a β-propeller fold, and it was determined to be the only seven-bladed β-propeller encoded in the
M. tuberculosis
genome. These results provide evidence of TrcR response regulator repression of the Rv1057 β-propeller gene that is expressed during growth of
M. tuberculosis
within human macrophages.
Reconstruction and Regulation of the Central Catabolic Pathway in the Thermophilic Propionate-Oxidizing SyntrophPelotomaculum thermopropionicum
Authors: Kosaka Tomoyuki , Uchiyama Taku , Ishii Shun-ichi , Enoki Miho , Imachi Hiroyuki , Kamagata Yoichi , Ohashi Akiyoshi , Harada Hideki , Ikenaga Hiroshi , Watanabe Kazuya
Published: December 2005

Obligate anaerobic bacteria fermenting volatile fatty acids in syntrophic association with methanogenic archaea share the intermediate bottleneck step in organic-matter decomposition. These organisms (called syntrophs) are biologically significant in terms of their growth at the thermodynamic limit and are considered to be the ideal model to address bioenergetic concepts. We conducted genomic and proteomic analyses of the thermophilic propionate-oxidizing syntrophPelotomaculum thermopropionicumto obtain the genetic basis for its central catabolic pathway. Draft sequencing and subsequent targeted gap closing identified all genes necessary for reconstructing its propionate-oxidizing pathway (i.e., methylmalonyl coenzyme A pathway). Characteristics of this pathway include the following. (i) The initial two steps are linked to later steps via transferases. (ii) Each of the last three steps can be catalyzed by two different types of enzymes. It was also revealed that many genes for the propionate-oxidizing pathway, except for those for propionate coenzyme A transferase and succinate dehydrogenase, were present in an operon-like cluster and accompanied by multiple promoter sequences and a putative gene for a transcriptional regulator. Proteomic analysis showed that enzymes in this pathway were up-regulated when grown on propionate; of these enzymes, regulation of fumarase was the most stringent. We discuss this tendency of expression regulation based on the genetic organization of the open reading frame cluster. Results suggest that fumarase is the central metabolic switch controlling the metabolic flow and energy conservation in this syntroph.
Two Residues in the Anticodon Recognition Domain of the Aspartyl-tRNA Synthetase from
Pseudomonas aeruginosa
Are Individually Implicated in the Recognition of tRNA
Asn
Authors: Bernard Dominic , Akochy Pierre-Marie , Beaulieu David , Lapointe Jacques , Roy Paul H.
Published: December 2005

In many organisms, the formation of asparaginyl-tRNA is not done by direct aminoacylation of tRNA
Asn
but by specific tRNA-dependent transamidation of aspartyl-tRNA
Asn
. This transamidation pathway involves a nondiscriminating aspartyl-tRNA synthetase (AspRS) that charges both tRNA
Asp
and tRNA
Asn
with aspartic acid. Recently, it has been shown for the first time in an organism (
Pseudomonas aeruginosa
PAO1) that the transamidation pathway is the only route of synthesis of Asn-tRNA
Asn
but does not participate in Gln-tRNA
Gln
formation.
P. aeruginosa
PAO1 has a nondiscriminating AspRS. We report here the identification of two residues in the anticodon recognition domain (H31 and G83) which are implicated in the recognition of tRNA
Asn
. Sequence comparisons of putative discriminating and nondiscriminating AspRSs (based on the presence or absence of the AdT operon and of AsnRS) revealed that bacterial nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs possess a leucine at position 31 and a residue other than a glycine at position 83. Mutagenesis of these residues of
P. aeruginosa
AspRS from histidine to leucine and from glycine to lysine increased the specificity of tRNA
Asp
charging over that of tRNA
Asn
by 3.5-fold and 4.2-fold, respectively. Thus, we show these residues to be determinants of the relaxed specificity of this nondiscriminating AspRS. Using available crystallographic data, we found that the H31 residue could interact with the central bases of the anticodons of the tRNA
Asp
and tRNA
Asn
. Therefore, these two determinants of specificity of
P. aeruginosa
AspRS could be important for all bacterial AspRSs.
Polymorphisms in the Nine Polymorphic Membrane Proteins ofChlamydia trachomatisacross All Serovars: Evidence for Serovar Da Recombination and Correlation with Tissue Tropism
Authors: Gomes João P. , Nunes Alexandra , Bruno William J. , Borrego Maria J. , Florindo Carlos , Dean Deborah
Published: December 2005

Chlamydia trachomatisis an intracellular bacterium responsible for ocular, respiratory, and sexually transmitted diseases. The genome contains a nine-member polymorphic membrane protein (Pmp) family unique to members of the orderChlamydiales. Genomic and molecular analyses were performed for the entirepmpgene family for the 18 reference serological variants (serovars) and genovariant Ja to identify specific gene and protein regions that differentiate chlamydial disease groups. The mean genetic distance among all serovars varied from 0.1% forpmpAto 7.0% forpmpF. Lymphogranuloma venereum (LGV) serovars were the most closely related for thepmpgenes and were also the most divergent, compared to ocular and non-LGV urogenital disease groups. Phylogenetic reconstructions showed that for six of ninepmpgenes (notpmpA,pmpD, orpmpE), the serovars clustered based on tissue tropism. The most globally successful serovars, E and F, clustered distantly from the urogenital group for fivepmpgenes. Thesepmpgenes may confer a biologic advantage that may facilitate infection and transmission for E and F. Surprisingly, serovar Da clustered with the ocular group frompmpEtopmpI, which are located together in the chromosome, providing statistically significant evidence for intergenomic recombination and acquisition of a genetic composition that could hypothetically expand the host cell range of serovar Da. We also identified distinct domains forpmpE,pmpF, andpmpHwhere substitutions were concentrated and associated with a specific disease group. Thus, our data suggest a possible structural or functional role that may vary amongpmpgenes in promoting antigenic polymorphisms and/or diverse adhesions-receptors that may be involved in immune evasion and differential tissue tropism.
Cooperativity Between Different Nutrient Receptors in Germination of Spores of
Bacillus subtilis
and Reduction of This Cooperativity by Alterations in the GerB Receptor
Authors: Atluri Swaroopa , Ragkousi Katerina , Cortezzo Donna E. , Setlow Peter
Published: December 2005

The GerA nutrient receptor alone triggers germination of
Bacillus subtilis
spores with
l
-alanine or
l
-valine, and these germinations were stimulated by glucose and K
+
plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K
+
(GFK) (termed cogerminants) plus GerK for triggering of germination with a number of
l
-amino acids. GerB and GerA also triggered spore germination cooperatively with
l
-asparagine, fructose, and K
+
and either
l
-alanine or
l
-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to
d
-alanine do not respond to
d
-alanine but respond to the same
l
-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these
l
-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to
l
-alanine germination via GerA, spore germination via
l
-alanine and GerB or GerB* was not inhibited by
d
-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K
+
somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to
l
-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to
l
-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to
l
-amino acids, and GerA interacts only with
l
-valine,
l
-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to
l
-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination.
Disruption of
sucA
, Which Encodes the E1 Subunit of α-Ketoglutarate Dehydrogenase, Affects the Survival of
Nitrosomonas europaea
in Stationary Phase
Authors: Hommes Norman G. , Kurth Elizabeth G. , Sayavedra-Soto Luis A. , Arp Daniel J.
Published: December 2005

Although
Nitrosomonas europaea
lacks measurable α-ketoglutarate dehydrogenase activity, the recent completion of the genome sequence revealed the presence of the genes encoding the enzyme. A knockout mutation was created in the
sucA
gene encoding the E1 subunit. Compared to wild-type cells, the mutant strain showed an accelerated loss of ammonia monooxygenase and hydroxylamine oxidoreductase activities upon entering stationary phase. In addition, unlike wild-type cells, the mutant strain showed a marked lag in the ability to resume growth in response to pH adjustments in late stationary phase.
Role of the Novel OprD Family of Porins in Nutrient Uptake in
Pseudomonas aeruginosa
Authors: Tamber Sandeep , Ochs Martina M. , Hancock Robert E. W.
Published: December 2005

To circumvent the permeability barrier of its outer membrane,
Pseudomonas aeruginosa
has evolved a series of specific porins. These channels have binding sites for related classes of molecules that facilitate uptake under nutrient-limited conditions. Here, we report on the identification of a 19-member family of porins similar to the basic-amino-acid-specific porin OprD. The members of this family fell into one of two phylogenetically distinct clusters, one bearing high similarity to OprD and the other bearing most similarity to the putative phenylacetic acid uptake porin PhaK of
Pseudomonas putida
. Analysis of the genome context, operon arrangement, and regulation of the PhaK-like porin OpdK indicated that it might be involved in vanillate uptake. This result was confirmed by demonstrating that an
opdK
mutant had a deficiency in the ability to grow on vanillate as a carbon source. To extrapolate these data to other paralogues within this family, the substrate specificities of 6 of the 17 remaining OprD homologues were inferred using an approach similar to that used with
opdK
. The specificities determined were as follows: OpdP, glycine-glutamate; OpdC, histidine; OpdB, proline; OpdT, tyrosine; OpdH,
cis
-aconitate; and OpdO, pyroglutamate. Thus, members of the OprD subfamily took up amino acids and related molecules, and those characterized members most similar to PhaK were responsible for the uptake of a diverse array of organic acids. These results imply that there is a functional basis for the phylogenetic clustering of these proteins and provide a framework for studying OprD homologues in other organisms.
Transcription of Virulence Factors in
Staphylococcus aureus
Small-Colony Variants Isolated from Cystic Fibrosis Patients Is Influenced by SigB
Authors: Moisan Hélène , Brouillette Eric , Jacob Christian Lebeau , Langlois-Bégin Philippe , Michaud Sophie , Malouin François
Published: December 2005

Staphylococcus aureus
small-colony variants (SCVs) are believed to account in part for the persistence of
S. aureus
during chronic infections. Little is understood about the gene expression profile that may explain the phenotype and distinguish SCVs from prototype
S. aureus
strains. In this study, DNA array transcriptional profiles of clinical SCVs isolated from the airways of cystic fibrosis patients were obtained and compared to those obtained from a laboratory-derived SCV strain (i.e., a respiratory-deficient
hemB
mutant) and prototype
S. aureus
strains. The genes commonly up-regulated in both
hemB
and clinical SCVs were found to be implicated in fermentation and glycolysis pathways. The well-known virulence regulator
agr
was not activated in SCVs, and such strains had low levels of alpha-toxin (
hla
) gene expression. Clinical SCVs also had a transcriptional signature of their own. Of striking interest is that many genes, most of them under the positive control of the alternate sigma factor SigB, were specifically up-regulated and differed in that way from that seen in prototype
S. aureus
and the
hemB
mutant. Since SigB influences up-regulation of adhesin type genes while indirectly down-regulating exoproteins and toxins, we evaluated the internalization and persistence of SCVs in mammalian cells. Results showed that clinical SCVs persisted much more efficiently in cells than the
hemB
and prototype strains and that a
sigB
mutant was a poor persister. Thus, it appears that the
agr
locus plays a minor role in the regulation of the virulon of SCVs, unlike SigB, which may have a key role in intracellular persistence.
Soluble V Domain of Nectin-1/HveC Enables Entry of Herpes Simplex Virus Type 1 (HSV-1) into HSV-Resistant Cells by Binding to Viral Glycoprotein D
Authors: Kwon Heechung , Bai Qing , Baek Hyun-Jung , Felmet Kelly , Burton Edward A. , Goins William F. , Cohen Justus B. , Glorioso Joseph C.
Published: December 2005

Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1
123
), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1
123
, approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1
123
did not associate with the cell surface. sNec1
123
-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.
Characterization of Early Steps in the Poliovirus Infection Process: Receptor-Decorated Liposomes Induce Conversion of the Virus to Membrane-Anchored Entry-Intermediate Particles
Authors: Tuthill Tobias J. , Bubeck Doryen , Rowlands David J. , Hogle James M.
Published: December 2005

The mechanism by which poliovirus infects the cell has been characterized by a combination of biochemical and structural studies, leading to a working model for cell entry. Upon receptor binding at physiological temperature, native virus (160S) undergoes a conformational change to a 135S particle from which VP4 and the N terminus of VP1 are externalized. These components interact with the membrane and are proposed to form a membrane pore. An additional conformational change in the particle is accompanied by release of the infectious viral RNA genome from the particle and its delivery, presumably through the membrane pore into the cytoplasm, leaving behind an empty 80S particle. In this report, we describe the generation of a receptor-decorated liposome system, comprising nickel-chelating nitrilotriacetic acid (NTA) liposomes and His-tagged poliovirus receptor, and its use in characterizing the early events in poliovirus infection. Receptor-decorated liposomes were able to capture virus and induce a temperature-dependent virus conversion to the 135S particle. Upon conversion, 135S particles became tethered to the liposome independently of receptor by a membrane interaction with the N terminus of VP1. Converted particles had lost VP4, which partitioned with the membrane. The development of a simple model membrane system provides a novel tool for studying poliovirus entry. The liposome system bridges the gap between previous studies using either soluble receptor or whole cells and offers a flexible template which can be extrapolated to electron microscopy experiments that analyze the structural biology of nonenveloped virus entry.
ICP0 Prevents RNase L-Independent rRNA Cleavage in Herpes Simplex Virus Type 1-Infected Cells
Authors: Sobol Paul T. , Mossman Karen L.
Published: December 2005

The classical interferon (IFN)-dependent antiviral response to viral infection involves the regulation of IFN-stimulated genes (ISGs), one being the gene encoding cellular endoribonuclease RNase L, which arrests protein synthesis and induces apoptosis by nonspecifically cleaving rRNA. Recently, the herpes simplex virus type 1 (HSV-1) protein ICP0 has been shown to block the induction of ISGs by subverting the IFN pathway upstream of the 2′-5′-oligoadenylate synthetase (OAS)/RNase L pathway. We report that ICP0 also prevents rRNA degradation at late stages of HSV-1 infection, independent of its E3 ubiquitin ligase activity, and that the resultant rRNA degradation is independent of the classical RNase L antiviral pathway. Moreover, the degradation is independent of the viral RNase vhs and is independent of IFN response factor 3. These studies indicate the existence of another, previously unidentified, RNase that is part of the host antiviral response to viral infection.
Identification of a Novel Pathway Essential for the Immediate-Early, Interferon-Independent Antiviral Response to Enveloped Virions
Authors: Noyce Ryan S. , Collins Susan E. , Mossman Karen L.
Published: December 2005

Viral infection elicits the activation of numerous cellular signal transduction pathways, leading to the induction of both innate and adaptive immunity. Previously we showed that entry of virion particles from a diverse array of enveloped virus families was capable of eliciting an interferon regulatory factor 3 (IRF-3)-mediated antiviral state in human fibroblasts in the absence of interferon production. Here we show that extracellular regulated kinase 1/2, p38 mitogen-activated protein kinase, and Jun N-terminal kinase/stress-activated protein kinase activities are not required for antiviral state induction. In contrast, treatment of cells with LY294002, an inhibitor of the phosphoinositide 3-kinase (PI3 kinase) family, prevents the induction of interferon-stimulated gene 56 (ISG56) and an antiviral response upon entry of virus particles. However, the prototypic class I p85/p110 PI3 kinase and its downstream effector Akt/PKB are dispensable for ISG and antiviral state induction. Furthermore, DNA-PK and PAK1, LY294002-sensitive members of the PI3 kinase family shown previously to be involved in IRF-3 activation, are also dispensable for ISG and antiviral state induction. The LY294002 inhibitor fails to prevent IRF-3 homodimerization or nuclear translocation upon virus particle entry. Together, these data suggest that virus entry triggers an innate antiviral response that requires the activity of a novel PI3 kinase family member.
Defective Assembly of Influenza A Virus due to a Mutation in the Polymerase Subunit PA
Authors: Regan John F. , Liang Yuying , Parslow Tristram G.
Published: December 2005

The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.
Interaction of Moloney Murine Leukemia Virus Capsid with Ubc9 and PIASy Mediates SUMO-1 Addition Required Early in Infection
Authors: Yueh Andrew , Leung Juliana , Bhattacharyya Subarna , Perrone Lucy A. , de los Santos Kenia , Pu Szy-yuan , Goff Stephen P.
Published: December 2005

Yeast two-hybrid screens led to the identification of Ubc9 and PIASy, the E2 and E3 small ubiquitin-like modifier (SUMO)-conjugating enzymes, as proteins interacting with the capsid (CA) protein of the Moloney murine leukemia virus. The binding site in CA for Ubc9 was mapped by deletion and alanine-scanning mutagenesis to a consensus motif for SUMOylation at residues 202 to 220, and the binding site for PIASy was mapped to residues 114 to 176, directly centered on the major homology region. Expression of CA and a tagged SUMO-1 protein resulted in covalent transfer of SUMO-1 to CA in vivo. Mutations of lysine residues to arginines near the Ubc9 binding site and mutations at the PIASy binding site reduced or eliminated CA SUMOylation. Introduction of these mutations into the complete viral genome blocked virus replication. The mutants exhibited no defects in the late stages of viral gene expression or virion assembly. Upon infection, the mutant viruses were able to carry out reverse transcription to synthesize normal levels of linear viral DNA but were unable to produce the circular viral DNAs or integrated provirus normally found in the nucleus. The results suggest that the SUMOylation of CA mediated by an interaction with Ubc9 and PIASy is required for early events of infection, after reverse transcription and before nuclear entry and viral DNA integration.
Herpes Simplex Virus DNA Synthesis Is Not a Decisive Regulatory Event in the Initiation of Lytic Viral Protein Expression in Neurons In Vivo during Primary Infection or Reactivation from Latency
Authors: Sawtell N. M. , Thompson R. L. , Haas R. L.
Published: December 2005

The herpes simplex virus genome can enter a repressed transcriptional state (latency) in sensory neurons of the host nervous system. Although reduced permissiveness of the neuronal environment is widely accepted as a causal factor, the molecular pathway(s) directing and maintaining the viral genome in the latent state remains undefined. Over the past decade, the field has been strongly influenced by the observations of Kosz-Vnenchak et al., which have been interpreted to indicate that, in sensory neurons in vivo, a critical level of viral DNA synthesis within the neuron is required for sufficient viral immediate-early (IE) and early (E) gene expression (M. Kosz-Vnenchak, J. Jacobson, D. M. Coen, and D. M. Knipe, J. Virol. 67:5383-5393, 1993). The levels of IE and E genes are, in turn, thought to regulate the decision to enter the lytic cycle or latency. We have reexamined this issue using new strategies for in situ detection and quantification of viral gene expression in whole tissues. Our results using thymidine kinase-null and rescued mutants as well as wild-type strains in conjunction with viral DNA synthesis blockers demonstrate that (i) despite inhibition of viral DNA replication, many neurons express lytic viral proteins, including IE proteins, during acute infection in the ganglion; (ii) at early times postinoculation, the number of neurons expressing viral proteins in the ganglion is not reduced by inhibition of viral DNA replication; and (iii) following a reactivation stimulus, the numbers of neurons and apparent levels of lytic viral proteins, including IE proteins, are not reduced by inhibition of viral DNA replication. We conclude that viral DNA replication in the neuron per se does not regulate IE gene expression or entry into the lytic cycle.
Liver Transduction with Recombinant Adeno-Associated Virus Is Primarily Restricted by Capsid Serotype Not Vector Genotype
Authors: Grimm Dirk , Pandey Kusum , Nakai Hiroyuki , Storm Theresa A. , Kay Mark A.
Published: December 2005

We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a
gfp
gene or the human factor IX (
hfIX
) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of
gfp
vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six
hfIX
variants into AAV-8 and infused mice via the portal vein with doses of 5 × 10
10
to 1.8 × 10
12
particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in ∼80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.