bluuebunny/crossref_metadata_2025 huggingface dataset:
Roles of DacB and Spm Proteins in
Clostridium perfringens
Spore Resistance to Moist Heat, Chemicals, and UV Radiation
Authors: Paredes-Sabja Daniel , Sarker Nahid , Setlow Barbara , Setlow Peter , Sarker Mahfuzur R.
Published: April 2008

Clostridium perfringens
food poisoning is caused mainly by enterotoxigenic type A isolates that typically possess high spore heat resistance. Previous studies have shown that α/β-type small, acid-soluble proteins (SASP) play a major role in the resistance of
Bacillus subtilis
and
C. perfringens
spores to moist heat, UV radiation, and some chemicals. Additional major factors in
B. subtilis
spore resistance are the spore's core water content and cortex peptidoglycan (PG) structure, with the latter properties modulated by the
spm
and
dacB
gene products and the sporulation temperature. In the current work, we have shown that the
spm
and
dacB
genes are expressed only during
C. perfringens
sporulation and have examined the effects of
spm
and
dacB
mutations and sporulation temperature on spore core water content and spore resistance to moist heat, UV radiation, and a number of chemicals. The results of these analyses indicate that for
C. perfringens
SM101 (i) core water content and, probably, cortex PG structure have little if any role in spore resistance to UV and formaldehyde, presumably because these spores’ DNA is saturated with α/β-type SASP; (ii) spore resistance to moist heat and nitrous acid is determined to a large extent by core water content and, probably, cortex structure; (iii) core water content and cortex PG cross-linking play little or no role in spore resistance to hydrogen peroxide; (iv) spore core water content decreases with higher sporulation temperatures, resulting in spores that are more resistant to moist heat; and (v) factors in addition to SpmAB, DacB, and sporulation temperature play roles in determining spore core water content and thus, spore resistance to moist heat.
Two Angular Dioxygenases Contribute to the Metabolic Versatility of Dibenzofuran-Degrading
Rhodococcus
sp. Strain HA01
Authors: Aly Hamdy A. H. , Huu Nguyen B. , Wray Victor , Junca Howard , Pieper Dietmar H.
Published: April 2008

Rhodococcus
sp. strain HA01, isolated through its ability to utilize dibenzofuran (DBF) as the sole carbon and energy source, was also capable, albeit with low activity, of transforming dibenzo-
p
-dioxin (DD). This strain could also transform 3-chlorodibenzofuran (3CDBF), mainly by angular oxygenation at the ether bond-carrying carbon (the angular position) and an adjacent carbon atom, to 4-chlorosalicylate as the end product. Similarly, 2-chlorodibenzofuran (2CDBF) was transformed to 5-chlorosalicylate. However, lateral oxygenation at the 3,4-positions was also observed and yielded the novel product 2-chloro-3,4-dihydro-3,4-dihydroxydibenzofuran. Two gene clusters encoding enzymes for angular oxygenation (
dfdA1A2A3A4
and
dbfA1A2
) were isolated, and expression of both was observed during growth on DBF. Heterologous expression revealed that both oxygenase systems catalyze angular oxygenation of DBF and DD but exhibited complementary substrate specificity with respect to CDBF transformation. While DfdA1A2A3A4 oxygenase, with high similarity to DfdA1A2A3A4 oxygenase from
Terrabacter
sp. strain YK3, transforms 3CDBF by angular dioxygenation at a rate of 29% ± 4% that of DBF, 2CDBF was not transformed. In contrast, DbfA1A2 oxygenase, with high similarity to the DbfA1A2 oxygenase from
Terrabacter
sp. strain DBF63, exhibited complementary activity with angular oxygenase activity against 2CDBF but negligible activity against 3CDBF. Thus,
Rhodococcus
sp. strain HA01 constitutes the first described example of a bacterial strain where coexpression of two angular dioxygenases was observed. Such complementary activity allows for the efficient transformation of chlorinated DBFs.
Increased Fitness of
Pseudomonas fluorescens
Pf0-1 Leucine Auxotrophs in Soil
Authors: Kim Wook , Levy Stuart B.
Published: April 2008

The annotation process of a newly sequenced bacterial genome is largely based on algorithms derived from databases of previously defined RNA and protein-encoding gene structures. This process generally excludes the possibility that the two strands of a given stretch of DNA can each harbor a gene in an overlapping manner. While the presence of such structures in eukaryotic genomes is considered to be relatively common, their counterparts in prokaryotic genomes are just beginning to be recognized. Application of an in vivo expression technology has previously identified 22 discrete genetic loci in
Pseudomonas fluorescens
Pf0-1 that were specifically activated in the soil environment, of which 10 were present in an antisense orientation relative to previously annotated genes. This observation led to the hypothesis that the physiological role of overlapping genetic structures may be relevant to growth conditions outside artificial laboratory media. Here, we examined the role of one of the overlapping gene pairs,
iiv19
and
leuA2
, in soil. Although
iiv19
was previously demonstrated to be preferentially activated in the soil environment, its absence did not alter the ability of
P. fluorescens
to colonize or survive in soil. Surprisingly, the absence of the
leuA2
gene conferred a fitness advantage in the soil environment when leucine was supplied exogenously. This effect was determined to be independent of the
iiv19
gene, and further analyses revealed that amino acid antagonism was the underlying mechanism behind the observed fitness advantage of the bacterium in soil. Our findings provide a potential mechanism for the frequent occurrence of auxotrophic mutants of
Pseudomonas
spp. in the lungs of cystic fibrosis patients.
Temporal Variations in the Dynamics of Potentially Microcystin-Producing Strains in a Bloom-Forming
Planktothrix agardhii
(Cyanobacterium) Population
Authors: Briand Enora , Gugger Muriel , François Jean-Christophe , Bernard Cécile , Humbert Jean-François , Quiblier Catherine
Published: April 2008

The concentration of microcystins (MCs) produced during blooms depends on variations in both the proportion of strains containing the genes involved in MC production and the MC cell quota (the ratio between the MC concentration and the density of cells with the
mcyA
genotype) for toxic strains. In order to assess the dynamics of MC-producing and non-MC-producing strains and to identify the impact of environmental factors on the relative proportions of these two subpopulations, we performed a 2-year survey of a perennial bloom of
Planktothrix agardhii
(cyanobacteria). Applying quantitative real-time PCR to the
mcyA
and phycocyanin genes, we found that the proportion of cells with the
mcyA
genotype varied considerably over time (ranging from 30 to 80% of the population). The changes in the proportion of cells with the
mcyA
genotype appeared to be inversely correlated to changes in the density of
P. agardhii
cells and also, to a lesser extent, to the availability of certain nutrients and the abundance of cladocerans. Among toxic cells, the MC cell quota varied throughout the survey. However, a negative correlation between the MC cell quota and the
mcyA
cell number during two short periods characterized by marked changes in the cyanobacterial biomass was found. Finally, only 54% of the variation in the MC concentrations measured in the lake can be explained by the dynamics of the density of cells with the MC producer genotype, suggesting that this measurement is not a satisfactory method for use in monitoring programs intended to predict the toxic risk associated with cyanobacterial proliferation.
Alternative Hand Contamination Technique To Compare the Activities of Antimicrobial and Nonantimicrobial Soaps under Different Test Conditions
Authors: Fuls Janice L. , Rodgers Nancy D. , Fischler George E. , Howard Jeanne M. , Patel Monica , Weidner Patrick L. , Duran Melani H.
Published: April 2008

Antimicrobial hand soaps provide a greater bacterial reduction than nonantimicrobial soaps. However, the link between greater bacterial reduction and a reduction of disease has not been definitively demonstrated. Confounding factors, such as compliance, soap volume, and wash time, may all influence the outcomes of studies. The aim of this work was to examine the effects of wash time and soap volume on the relative activities and the subsequent transfer of bacteria to inanimate objects for antimicrobial and nonantimicrobial soaps. Increasing the wash time from 15 to 30 seconds increased reduction of
Shigella flexneri
from 2.90 to 3.33 log
10
counts (
P
= 0.086) for the antimicrobial soap, while nonantimicrobial soap achieved reductions of 1.72 and 1.67 log
10
counts (
P
> 0.6). Increasing soap volume increased bacterial reductions for both the antimicrobial and the nonantimicrobial soaps. When the soap volume was normalized based on weight (∼3 g), nonantimicrobial soap reduced
Serratia marcescens
by 1.08 log
10
counts, compared to the 3.83-log
10
reduction caused by the antimicrobial soap (
P
< 0.001). The transfer of
Escherichia coli
to plastic balls following a 15-second hand wash with antimicrobial soap resulted in a bacterial recovery of 2.49 log
10
counts, compared to the 4.22-log
10
(
P
< 0.001) bacterial recovery on balls handled by hands washed with nonantimicrobial soap. This indicates that nonantimicrobial soap was less active and that the effectiveness of antimicrobial soaps can be improved with longer wash time and greater soap volume. The transfer of bacteria to objects was significantly reduced due to greater reduction in bacteria following the use of antimicrobial soap.
Assembly of Viral Metagenomes from Yellowstone Hot Springs
Authors: Schoenfeld Thomas , Patterson Melodee , Richardson Paul M. , Wommack K. Eric , Young Mark , Mead David
Published: April 2008

Thermophilic viruses were reported decades ago; however, knowledge of their diversity, biology, and ecological impact is limited. Previous research on thermophilic viruses focused on cultivated strains. This study examined metagenomic profiles of viruses directly isolated from two mildly alkaline hot springs, Bear Paw (74°C) and Octopus (93°C). Using a new method for constructing libraries from picograms of DNA, nearly 30 Mb of viral DNA sequence was determined. In contrast to previous studies, sequences were assembled at 50% and 95% identity, creating composite contigs up to 35 kb and facilitating analysis of the inherent heterogeneity in the populations. Lowering the assembly identity reduced the estimated number of viral types from 1,440 and 1,310 to 548 and 283, respectively. Surprisingly, the diversity of viral species in these springs approaches that in moderate-temperature environments. While most known thermophilic viruses have a chronic, nonlytic infection lifestyle, analysis of coding sequences suggests lytic viruses are more common in geothermal environments than previously thought. The 50% assembly included one contig with high similarity and perfect synteny to nine genes from
Pyrobaculum spherical virus
(PSV). In fact, nearly all the genes of the 28-kb genome of PSV have apparent homologs in the metagenomes. Similarities to thermoacidophilic viruses isolated on other continents were limited to specific open reading frames but were equally strong. Nearly 25% of the reads showed significant similarity between the hot springs, suggesting a common subterranean source. To our knowledge, this is the first application of metagenomics to viruses of geothermal origin.
Morphological and Genetic Evidence that the Cyanobacterium
Lyngbya wollei
(Farlow ex Gomont) Speziale and Dyck Encompasses at Least Two Species
Authors: Joyner Jennifer J. , Litaker R. Wayne , Paerl Hans W.
Published: April 2008

Dense blooms of the cyanobacterium
Lyngbya wollei
are increasingly responsible for declining water quality and habitat degradation in numerous springs, rivers, and reservoirs. This research represents the first molecular phylogenetic analysis of
L. wollei
in comparison with the traditional morphological characterization of this species. Specimens were collected from several springs in Florida and a reservoir in North Carolina. Segments of the small-subunit (SSU) rRNA and
nifH
genes were PCR amplified, cloned, and sequenced. The phylogenetic analysis of the SSU rRNA gene revealed sequences that fell into three distinct subclusters, each with >97% sequence similarity. These were designated operational taxonomic unit 1 (OTU1), OTU2, and OTU3. Similarly, the
nifH
sequences fell into three distinct subclusters named S1, S2, and S3. When either bulk samples or individual filaments were analyzed, we recovered OTU1 with S1, OTU2 with S2, and OTU3 with S3. The coherence between the three SSU rRNA gene and
nifH
subclusters was consistent with genetically distinct strains or species. Cells associated with subclusters OTU3 and S3 were significantly wider and longer than those associated with other subclusters. The combined molecular and morphological data indicate that the species commonly identified as
L. wollei
in the literature represents two or possibly more species. Springs containing OTU3 and S3 demonstrated lower ion concentrations than other collection sites. Geographical locations of
Lyngbya
subclusters did not correlate with residual dissolved inorganic nitrogen or phosphorus concentrations. This study emphasizes the need to complement traditional identification with molecular characterization to more definitively detect and characterize harmful cyanobacterial species or strains.
Comparison of Broth Microdilution, Etest, and Agar Disk Diffusion Methods for Antimicrobial Susceptibility Testing of
Lactobacillus acidophilus
Group Members
Authors: Mayrhofer Sigrid , Domig Konrad J. , Mair Christiane , Zitz Ulrike , Huys Geert , Kneifel Wolfgang
Published: April 2008

In recent years, the absence of acquired antimicrobial resistance has become an important criterion to evaluate the biosafety of lactobacilli used as industrial starter or probiotic cultures. At present, however, standards for susceptibility testing of
Lactobacillus
strains or approved guidelines for interpreting the test results are not available. Hence, this study was carried out to contribute to the establishment of a standardized procedure for antimicrobial susceptibility testing of lactobacilli. The results obtained by testing 104 strains of the
Lactobacillus acidophilus
group were compared based on broth microdilution, disk diffusion, and Etest. Except for some specific agent-related effects, agreement between MICs resulting from the broth microdilution method and the Etest was good. In addition, inhibition zone diameters determined with disk diffusion correlated well with MICs from Etest and broth microdilution.
Nodulation Gene Regulation and Quorum Sensing Control Density-Dependent Suppression and Restriction of Nodulation in theBradyrhizobium japonicum-Soybean Symbiosis
Authors: Jitacksorn Siriluck , Sadowsky Michael J.
Published: April 2008

The nodulation ofGlycine maxcv. Lambert and the nodulation-restricting plant introduction (PI) genotype PI 417566 by wild-typeBradyrhizobium japonicumUSDA110 is regulated in a population-density-dependent manner. Nodulation on both plant genotypes was suppressed (inhibited) when plants received a high-density inoculum (109cells/ml) of strain USDA110 grown in complex medium, and more nodules were produced on plants receiving a low-cell-density inoculum (105cells/ml). Since cell-free supernatants from strain USDA110 grown to high cell density in complex medium decreased the expression of annodY-lacZfusion, this phenomenon was attributed to bradyoxetin-induced repression ofnodgene expression. Inoculation of either the permissive soybean genotype (cv. Lambert) or PI 417566 with 109cells/ml of thenodD2,nolA,nodW, andnwsBmutants of USDA110 enhanced nodulation (up to 24%) relative to that seen with inoculations done with 105cells/ml of the mutants or the wild-type strain, indicating that these genes are involved in population-density-dependent nodulation of soybeans. In contrast, the number of nodules produced by annodD1mutant on either soybean genotype was less than those seen with the wild-type strain inoculated at a low inoculum density. ThenodD2mutant outcompetedB. japonicumstrain USDA123 for nodulation ofG. maxcv. Lambert at a high or low inoculum density, and the results of root-tip-marking and time-to-nodulate studies indicated that thenolAandnodD2mutants nodulated this soybean genotype faster than wild-type USDA110. Taken together, the results from these studies indicate that thenodD2mutant ofB. japonicummay be useful to enhance soybean nodulation at high inoculum densities and that NodD2 is a key repressor influencing host-controlled restriction of nodulation, density-dependent suppression of nodulation, perception of bradyoxetin, and competitiveness in the soybean-B. japonicumsymbiosis.
Loss of Regulators of Vacuolar ATPase Function and Ceramide Synthesis Results in Multidrug Sensitivity in
Schizosaccharomyces pombe
Authors: Dawson Keren , Toone W. Mark , Jones Nic , Wilkinson Caroline R. M.
Published: April 2008

We undertook a screen to isolate determinants of drug resistance in fission yeast and identified two genes that, when mutated, result in sensitivity to a range of structurally unrelated compounds, some of them commonly used in the clinic. One gene,
rav1
, encodes the homologue of a budding yeast protein which regulates the assembly of the vacuolar ATPase. The second gene,
lac1
, encodes a homologue of genes that are required for ceramide synthesis. Both mutants are sensitive to the chemotherapeutic agent doxorubicin, and using the naturally fluorescent properties of this compound, we found that both
rav1
and
lac1
mutations result in an increased accumulation of the drug in cells. The multidrug-sensitive phenotype of
rav1
mutants can be rescued by up-regulation of the
lag1
gene which encodes a homologue of
lac1
, whereas overexpression of either
lac1
or
lag1
confers multidrug resistance on wild-type cells. These data suggest that changing the amount of ceramide synthase activity in cells can influence innate drug resistance. The function of Rav1 appears to be conserved, as we show that SpRav1 is part of a RAVE-like complex in fission yeast and that loss of
rav1
results in defects in vacuolar (H
+
)-ATPase activity. Thus, we conclude that loss of normal V-ATPase function results in an increased sensitivity of
Schizosaccharomyces pombe
cells to drugs. The
rav1
and
lac1
genes are conserved in both higher eukaryotes and various pathogenic fungi. Thus, our data could provide the basis for strategies to sensitize tumor cells or drug-resistant pathogenic fungi to drugs.
Genomewide Recruitment Analysis of Rpb4, a Subunit of Polymerase II in
Saccharomyces cerevisiae
, Reveals Its Involvement in Transcription Elongation
Authors: Verma-Gaur Jiyoti , Rao Sudha Narayana , Taya Toshiki , Sadhale Parag
Published: April 2008

The Rpb4/Rpb7 subcomplex of yeast RNA polymerase II (Pol II) has counterparts in all multisubunit RNA polymerases from archaebacteria to higher eukaryotes. The Rpb4/7 subcomplex in
Saccharomyces cerevisiae
is unique in that it easily dissociates from the core, unlike the case in other organisms. The relative levels of Rpb4 and Rpb7 in yeasts affect the differential gene expression and stress response. Rpb4 is nonessential in
S. cerevisiae
and affects expression of a small number of genes under normal growth conditions. Here, using a chromatin immunoprecipitation (“ChIP on-chip”) technique, we compared genomewide binding of Rpb4 to that of a core Pol II subunit, Rpb3. Our results showed that in spite of being nonessential for survival, Rpb4 was recruited on coding regions of most transcriptionally active genes, similar to the case with the core Pol II subunit, Rpb3, albeit to a lesser extent. The extent of Rpb4 recruitment increased with increasing gene length. We also observed Pol II lacking Rpb4 to be defective in transcribing long, GC-rich transcription units, suggesting a role for Rpb4 in transcription elongation. This role in transcription elongation was supported by the observed 6-azauracil (6AU) sensitivity of the
rpb4
Δ mutant. Unlike most phenotypes of
rpb4
Δ, the 6AU sensitivity of the
rpb4
Δ strain was not rescued by overexpression of
RPB7
. This report provides the first instance of a distinct role for Rpb4 in transcription, which is independent of its interacting partner, Rpb7.
Functional Characterization of a Redundant
Plasmodium
TRAP Family Invasin, TRAP-Like Protein, by Aldolase Binding and a Genetic Complementation Test
Authors: Heiss Kirsten , Nie Hui , Kumar Sumit , Daly Thomas M. , Bergman Lawrence W. , Matuschewski Kai
Published: April 2008

Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum
Apicomplexa
are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although
TLP
is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of
TLP
, encoding a redundant TRAP family invasin, in the malaria parasite.
Modulation of Antioxidant Defense in
Aspergillus parasiticus
Is Involved in Aflatoxin Biosynthesis: a Role for the Ap
yapA
Gene
Authors: Reverberi Massimo , Zjalic Slaven , Ricelli Alessandra , Punelli Federico , Camera Emanuela , Fabbri Claudia , Picardo Mauro , Fanelli Corrado , Fabbri Anna A.
Published: April 2008

Oxidative stress is recognized as a trigger of different metabolic events in all organisms. Various factors correlated with oxidation, such as the β-oxidation of fatty acids and their enzymatic or nonenzymatic by-products (e.g., precocious sexual inducer factors and lipoperoxides) have been shown to be involved in aflatoxin formation. In the present study, we found that increased levels of reactive oxygen species (ROS) were correlated with increased levels of aflatoxin biosynthesis in
Aspergillus parasiticus
. To better understand the role of ROS formation in toxin production, we generated a mutant (ΔAp
yapA
) having the Ap
yapA
gene deleted, given that Ap
yapA
orthologs have been shown to be part of the antioxidant response in other fungi. Compared to the wild type, the mutant showed an increased susceptibility to extracellular oxidants, as well as precocious ROS formation and aflatoxin biosynthesis. Genetic complementation of the ΔAp
yapA
mutant restored the timing and quantity of toxin biosynthesis to the levels found in the wild type. The presence of putative AP1 (ApYapA orthologue) binding sites in the promoter region of the regulatory gene
aflR
further supports the finding that ApYapA plays a role in the regulation of aflatoxin biosynthesis. Overall, our results show that the lack of Ap
yapA
leads to an increase in oxidative stress, premature conidiogenesis, and aflatoxin biosynthesis.
RscS Functions Upstream of SypG To Control the
syp
Locus and Biofilm Formation in
Vibrio fischeri
Authors: Hussa Elizabeth A. , Darnell Cynthia L. , Visick Karen L.
Published: April 2008

Two-component signal transduction systems, composed of sensor kinase (SK) and response regulator (RR) proteins, allow bacterial cells to adapt to changes such as environmental flux or the presence of a host. RscS is an SK required for
Vibrio fischeri
to initiate a symbiotic partnership with the Hawaiian squid
Euprymna scolopes
, likely due to its role in controlling the symbiosis polysaccharide (
syp
) genes and thus biofilm formation. To determine which RR(s) functions downstream of RscS, we performed epistasis experiments with a library of 35 RR mutants. We found that several RRs contributed to RscS-mediated biofilm formation in
V. fischeri
. However, only the
syp
-encoded symbiosis regulator SypG was required for both biofilm phenotypes and
syp
transcription induced by RscS. These data support the hypothesis that RscS functions upstream of SypG to induce biofilm formation. In addition, this work also revealed a role for the
syp
-encoded RR SypE in biofilm formation. To our knowledge, no other study has used a large-scale epistasis approach to elucidate two-component signaling pathways. Therefore, this work both contributes to our understanding of regulatory pathways important for symbiotic colonization by
V. fischeri
and establishes a paradigm for evaluating two-component pathways in the genomics era.
Streptococcal Antagonism in Oral Biofilms:Streptococcus sanguinisandStreptococcus gordoniiInterference withStreptococcus mutans
Authors: Kreth Jens , Zhang Yongshu , Herzberg Mark C.
Published: April 2008

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species,Streptococcus gordonii, Streptococcus sanguinis, and cariogenicStreptococcus mutans. S. sanguinisandS. gordoniiused oxygen availability and the differential production of hydrogen peroxide (H2O2) to compete effectively againstS. mutans. Interspecies antagonism was influenced by glucose with reduced production of H2O2. Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV ofS. mutans, as well as the H2O2-dependent release of heterologous DNA from mixed cultures ofS. sanguinisandS. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms ofS. mutansin the oral biofilm.
Roles of Pneumococcal DivIB in Cell Division
Authors: Le Gouëllec Audrey , Roux Laure , Fadda Daniela , Massidda Orietta , Vernet Thierry , Zapun André
Published: April 2008

DivIB, also known as FtsQ in gram-negative organisms, is a division protein that is conserved in most eubacteria. DivIB is localized at the division site and forms a complex with two other division proteins, FtsL and DivIC/FtsB. The precise function of these three bitopic membrane proteins, which are central to the division process, remains unknown. We report here the characterization of adivIBdeletion mutant ofStreptococcus pneumoniae, which is a coccus that divides with parallel planes. Unlike its homologue FtsQ inEscherichia coli, pneumococcal DivIB is not required for growth in rich medium, but the ΔdivIBmutant forms chains of diplococci and a small fraction of enlarged cells with defective septa. However, the deletion mutant does not grow in a chemically defined medium. In the absence of DivIB and protein synthesis, the partner FtsL is rapidly degraded, whereas other division proteins are not affected, pointing to a role of DivIB in stabilizing FtsL. This is further supported by the finding that an additional copy offtsLrestores growth of the ΔdivIBmutant in defined medium. Functional mapping of the three distinct α, β, and γ domains of the extracellular region of DivIB revealed that a complete β domain is required to fully rescue the deletion mutant. DivIB with a truncated β domain reverts only the chaining phenotype, indicating that DivIB has distinct roles early and late in the division process. Most importantly, the deletion ofdivIBincreases the susceptibility to β-lactams, more evidently in a resistant strain, suggesting a function in cell wall synthesis.
Regulatory Network Controlling Extracellular Proteins inErwinia carotovorasubsp.carotovora: FlhDC, the Master Regulator of Flagellar Genes, ActivatesrsmBRegulatory RNA Production by AffectinggacAandhexA(lrhA) Expression
Authors: Cui Yaya , Chatterjee Asita , Yang Hailian , Chatterjee Arun K.
Published: April 2008

Erwinia carotovorasubsp.carotovoraproduces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal,N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA,rsmBRNA).rsmBRNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT inErwinia chrysanthemi; LrhA [LysR homolog A] inEscherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA withrsmBRNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to theflhDCoperon ofE. colialso controls extracellular enzyme production. A transposon insertion FlhDC−mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, andE. carotovorasubsp.carotovoraHarpin (HarpinEcc) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC+plasmid. Sequence analysis and transcript assays disclosed that theflhDoperon ofE. carotovorasubsp.carotovora, like those of other enterobacteria, consists offlhDandflhC. Complementation analysis revealed that the regulatory effect requires functions of bothflhDandflhCproducts. The data presented here show that FlhDC positively regulatesgacA,rsmC, andfliAand negatively regulateshexA(lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects onhexAandgacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC−mutant are responsible for the inhibition ofrsmBRNA production, a condition conducive to the accumulation of free RsmA. Indeed, studies with an RsmA−FlhDC−double mutant and multiple copies ofrsmB+DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation offliAhas no bearing on exoprotein production inE. carotovorasubsp.carotovora. Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA, andrsmBRNA in the context of the exoprotein production and virulence ofE. carotovorasubsp.carotovora.
A Genomic Islet Mediates Flagellar Phase Variation in
Escherichia coli
Strains Carrying the Flagellin-Specifying Locus
flk
Authors: Feng Lu , Liu Bin , Liu Yanqun , Ratiner Yuli A. , Hu Bo , Li Dan , Zong Xiaolin , Xiong Wei , Wang Lei
Published: April 2008

The occurrence of unilateral flagellar phase variation was previously demonstrated in
Escherichia coli
strains carrying the non-
fliC
flagellin-specifying locus
flk
. In this study, we investigated the mechanism involved in this process. By using sequencing and sequence analysis, the
flk
region between the chromosomal genes
yhaC
and
rnpB
was characterized in all described
flk
-positive
E. coli
strains, including the H35 strain identified in this study (the other strains used are H3, H36, H47, and H53 strains), and this region was found to contain a putative integrase gene and flanking direct repeats in addition to the
flk
flagellin-specifying gene
flkA
and a
fliC
repressor gene,
flkB
, indicating that there is a typical genomic islet (GI), which was designated the
flk
GI. The horizontal transfer potential of the
flk
GI was indicated by detection of the excised extrachromosomal circular form of the
flk
GI. By generating
fliC
-expressing variants of H3 and H47 strains, unilateral flagellar phase variation in
flk
-positive strains was shown to be mediated by excision of the
flk
GI. The function of the proposed integrase gene was confirmed by deletion and a complementation test. The potential integration sites of the
flk
GI were identified. A general model for flagellar phase variation in
flk
-positive
E. coli
strains can be expressed as
fliC
off
+
flkA
on
→
fliC
on
+
flkA
none
. This is the first time that a molecular mechanism for flagellar phase variation has been reported for
E. coli
.
Signal Pathway in Salt-Activated Expression of theSalmonellaPathogenicity Island 1 Type III Secretion System inSalmonella entericaSerovar Typhimurium
Authors: Mizusaki Hideaki , Takaya Akiko , Yamamoto Tomoko , Aizawa Shin-Ichi
Published: April 2008

Salmonella entericaserovar Typhimurium secretes virulence factors for invasion called Sip proteins or Sips into its hosts through a type III secretion system (T3SS). In the absence of a host,S. entericainduces Sip secretion in response to sucrose or simple salts, such as NaCl. We analyzed induction of host-independent Sip secretion by monitoring protein secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), assembly of needle complexes by electron microscopy, and transcription of virulence regulatory genes by quantitative reverse transcriptase PCR (real-time PCR). SDS-PAGE showed that addition of sucrose or simple salts, such as NaCl, to the growth medium induced Sip secretion without altering flagellar protein secretion, which requires a distinct T3SS. Electron microscopy confirmed that the amount of secreted Sips increased as the number of assembled needle complexes increased. Real-time PCR revealed that added sucrose or NaCl enhanced transcription ofhilA,hilC, andhilD, which encode known regulators ofSalmonellavirulence. However, epistasis analysis implicated HilD and HilA, but not HilC, in the direct pathway from the salt stimulus to the Sip secretion response. Further analyses showed that the BarA/SirA two-component signal transduction pathway, but not the two-component sensor kinase EnvZ, directly activatedhilDandhilAtranscription and thus Sip secretion in response to either sucrose or NaCl. Finally, real-time PCR showed that salt does not influence transcription of the BarA/SirA-dependentcsrBandcsrCgenes. A model is proposed for the major pathway in which sucrose or salt signals to enhance virulence gene expression.
Evidence for an Elongated Dimeric Structure of Heparin-Binding Hemagglutinin from
Mycobacterium tuberculosis
Authors: Esposito Carla , Pethoukov Maxim V. , Svergun Dmitri I. , Ruggiero Alessia , Pedone Carlo , Pedone Emilia , Berisio Rita
Published: April 2008

Heparin-binding hemagglutinin (HBHA) is a virulence factor of tuberculosis which is responsible for extrapulmonary dissemination of this disease. A thorough biochemical characterization of HBHA has provided experimental evidence of a coiled-coil nature of HBHA. These data, together with the low-resolution structures of a full-length form and a truncated form of HBHA obtained by small-angle X-ray scattering, have unambiguously indicated that HBHA has a dimeric structure with an elongated shape.
Clinical prioritisations of healthcare for the aged—professional roles
Authors: Nortvedt P , Pedersen R , Grøthe K H , Nordhaug M , Kirkevold M , Slettebø Å , Brinchmann B S , Andersen B
Published: April 2008

Background:

Although fair distribution of healthcare services for older patients is an important challenge, qualitative research exploring clinicians’ considerations in clinical prioritisation within this field is scarce.

Objectives:

To explore how clinicians understand their professional role in clinical prioritisations in healthcare services for old patients.

Design:

A semi-structured interview-guide was employed to interview 45 clinicians working with older patients. The interviews were analysed qualitatively using hermeneutical content analysis.

Participants:

20 physicians and 25 nurses working in public hospitals and nursing homes in different parts of Norway.

Results and interpretations:

The clinicians struggle with not being able to attend to the comprehensive needs of older patients, and being unfaithful to professional ideals and expectations. There is a tendency towards lowering the standards and narrowing the role of the clinician. This is done in order to secure the vital needs of the patient, but is at the expense of good practice and holistic role modelling. Increased specialisation, advances and increase in medical interventions, economical incentives, organisational structures, and biomedical paradigms, may all contribute to a narrowing of the clinicians’ role.

Conclusion:

Distributing healthcare services in a fair way is generally not described as integral to the clinicians’ role in clinical prioritisations. If considerations of justice are not included in clinicians’ role, it is likely that others will shape major parts of their roles and responsibilities in clinical prioritisations. Fair distribution of healthcare services for older patients is possible only if clinicians accept responsibility in these questions.
Stress path and steady state
Authors: Vaid Y. P. , Chung E. K. F. , Kuerbis R. H.
Published: April 2008

The effect of stress path on the steady state line of a liquefiable sand is investigated. Results from undrained triaxial compression and extension tests on water-deposited sands show that the steady state line of a given sand, though unique in the effective stress space, is not so in the void ratio – effective stress space. The sand is contractive over a much larger range of void ratios in extension than in compression. While a single steady state line emerges for compression loading, extension loading yields several lines, each characteristic to a given deposition void ratio. All these extension lines lie to the left of the compression line in void ratio – effective stress space. Thus at a given void ratio, steady state strength is smaller in extension than in compression, the difference increasing as the sand becomes looser. The implications of the results are discussed in relation to practical design. Key words: sand, liquefaction, steady state, stress path.
Behaviour of flexible piles under inclined loads
Authors: Sastry V. V. R. N. , Meyerhof G. G.
Published: April 2008

The lateral soil pressures, bending moments, pile displacements at ground surface, and bearing capacity of instrumented vertical single flexible model piles in homogeneous loose sand and soft clay under central inclined loads have been investigated. The results of these load tests are compared with theoretical estimates based on the concept of an effective embedment depth of equivalent rigid piles. Reasonable agreement has been found between the observed and predicted behaviour of flexible piles. The analyses are also compared with the results of some field case records. Key words: bending moments, clay, displacements, inclined loads, instrumentation, lateral soil pressure, model test, pile, sand.
Sand–bentonite liners: predicting permeability from laboratory tests
Authors: Chapuis Robert P.
Published: April 2008

Soil–bentonite mixes are frequently used as impervious blankets in waste disposal projects. Numerous results of laboratory permeability tests are presented for sands containing up to 33% bentonite. These results seem difficult to analyze because different testing methods have been used in which it is not easy to control certain parameters, such as hydration period, degree of saturation, and swelling under low confining pressure. Hydraulic conductivity, however, can be obtained by using empirical equations that take into account the bentonite content, porosity, and degree of saturation of the sand alone when tested at its optimum modified Proctor value in a permeameter. This preliminary evaluation helps to select the soil to be tested. Then, the laboratory test results can be used to predict the in situ hydraulic performance after due consideration of the variabilities in natural sand and bentonite content. Key words: liner, soil, bentonite, permeability, laboratory, field.
Evaluation of three polymerase chain reaction techniques for detection ofBrucellaDNA in peripheral human blood
Authors: Baddour Manal M. , Alkhalifa Dalal H.
Published: April 2008

Brucellosis is a widespread zoonosis. Currently the diagnosis of this zoonosis is based on microbiological and serological laboratory tests. Polymerase chain reaction (PCR) has been used to detect DNA from Brucella . Different target genes, primer pairs, PCR techniques, and extraction procedures have previously been published for Brucella detection. But only a few of these primers have been used in human samples, and only one study has been carried out to compare sensitivity between them. In the present study, 3 sets of primers and 3 different PCR protocols amplifying 3 different regions of the Brucella genome were compared for detection of Brucella DNA in a peripheral-blood PCR assay to conclude which is most suitable for the clinical diagnostic laboratory. These 3 pairs of primers amplify 3 different fragments included in (i) a gene encoding a 31 kDa Brucella abortus antigen (B4/B5), (ii) a sequence 16S rRNA of B. abortus (F4/R2), and (iii) a gene encoding an outer membrane protein (omp-2) (JPF/JPR). Some modifications on the reported techniques were applied during the present work to improve the outcome. The results showed that the B4/B5 primer pair had the highest sensitivity for detection of positive samples (98%), the JPF/JPR primer pair detected 88.4% of positive samples, whereas F4/R2 primer pair was the least sensitive, being able to detect only 53.1% of positive samples. The specificity of the 3 techniques was 100%. The B4/B5 primer pair was also able to detect the smallest number of bacteria (700 cfu/mL), whereas JPF/JPR was able to detect 7 × 105 cfu/mL and F4/R2 was able to detect 7 × 107cfu/mL. It is thus concluded that using the B4/B5 primer PCR with the suggested modifications is a robust assay, which meets the sensitivity requirements to be used for testing of human blood samples for brucellosis in the diagnostic laboratory.
Aspergillus flavus aflatoxin occurrence and expression of aflatoxin biosynthesis genes in soil
Authors: Accinelli Cesare , Abbas H.K. , Zablotowicz R.M. , Wilkinson J.R.
Published: April 2008

The carcinogen aflatoxin B1 (AFB1) produced by Aspergillus flavus is a major food safety concern in crops. However, information on AFB1 occurrence in soil and crop residue is scarce. A series of experiments investigated the occurrence of AFB1 in soil and corn residues and ascertained the ecology of A. flavus in a Dundee silt loam soil. Samples of untilled soil (0–2 cm) and residues were collected in March 2007 from plots previously planted with a corn isoline containing the Bacillus thuringiensis (Bt) endotoxin gene or the parental non-Bt isoline. AFB1 levels were significantly different in various corn residues. The highest AFB1 levels were observed in cobs containing grain, with 145 and 275 ng·g–1in Bt and non-Bt, respectively (P ≥ F = 0.001). Aflatoxin levels averaged 3.3 and 9.6 ng·g–1in leaves and (or) stalks and cobs without grain, respectively. All soils had AFB1 ranging from 0.6 to 5.5 ng·g–1with similar levels in plots from Bt and non-Bt corn. Based on cultural methods, soil contained from log103.1 to 4.5 A. flavus cfu·g–1with about 60% of isolates producing aflatoxin. Laboratory experiments demonstrated that AFB1 is rapidly degraded in soil at 28 °C (half-life ≤ 5 days). The potential of the soil A. flavus to produce aflatoxins was confirmed by molecular methods. Transcription of 5 aflatoxin biosynthesis genes, including aflD, aflG, aflP, aflR, and aflS, were detected by reverse transcription – polymerase chain reaction analysis in soil. Although AFB1 appears to be transient in soils, it is clear that AFB1 is produced in surface soil in the presence of corn residues, as indicated by A. flavus cfu levels, AFB1 detection, and expression of aflatoxin biosynthetic genes.
Safety evaluation of two bacterial strains used in asian probiotic products
Authors: Tompkins Thomas A. , Hagen Karen E. , Wallace Timothy D. , Fillion-Forté Valerie
Published: April 2008

Probiotics, known for their prophylactic and therapeutic properties, are routinely used by the medical community in various regions of the world. In some Asian countries, these products are controlled as pharmaceutical substances and must adhere to strict regulatory guidelines. However, outside of Europe where the European Food Safety Authority has recently adopted a Qualified Presumption of Safety approach for probiotics used in food and feed, current safety requirements do not necessitate screening for the presence of virulence and other risk factors, which may result in the inadvertent use of probiotic strains harboring harmful genes. A safety evaluation was conducted on Enterococcus faecium R0026 and Bacillus subtilis R0179 used in several commercial probiotic products marketed in Asia. Molecular techniques were used to verify the identity of each strain and antibiotic resistance profiles were determined towards clinically relevant antibiotics. Strains were subsequently screened for the presence of enterotoxins and virulence factors and were subjected to 28 days of repeated high-dose oral toxicity testing in rats. No risk factors or aberrant activities were identified using such a detailed approach. Thus, both microbes were deemed to pose low risk to the consumer and, therefore, safe for use as probiotics.
The Future of Site Investigation
Authors: Craig C.
Published: April 2008

A review of the recent past leads one to conclude that the UK has an over capacity in terms of consultancy, specialist contracting and professional staff. This will have to be brought into balance.

Factors likely to affect the future are discussed—technological breakthrough, legislation, certification, litigation, and market attitude.

The fragmented organisation of the five basic activities of SI are considered and it is suggested that there is much to recommend the appointment of an independent geotechnical consultant, who would accept total responsibility for the SI.
Effects of Stress Path and Stress History on the Stiffness of Reconstituted London Clay
Authors: Atkinson J. H. , Evans J. S. , Richardson D.
Published: April 2008

Soil behaviour is stress history dependent and stress path dependent and soil parameters, particularly those for stress-strain behaviour, measured in conventional triaxial tests may not represent the behaviour of soil in many civil engineering works.

To obtain more realistic parameters it may be necessary to conduct laboratory tests which more closely represent
in situ
conditions before and during construction.

The paper describes equipment developed at The City University to carry out stress path tests simply and economically. A series of CU triaxial tests and stress path tests on reconstituted soil illustrate the dependence of measured soil parameters on stress history and stress path.
An Assessment of the British Code of Practice for Site Investigations for Hong Kong Conditions
Authors: Borrie G. W. , Cipullo A. , Massey J. B.
Published: April 2008

The Geotechnical Control Office has undertaken the preparation of two separate, complementary guides on “Rock and Soil Description ” and “Site Investigation ”, as companion documents to BS 5930:1981 for use in Hong Kong. This paper examines the need for a separate guidance document on site investigation for Hong Kong conditions. The predominance of heterogeneous weathered soil profiles and colluvium in Hong Kong presents particular problems, both in their description and investigation, which are not covered adequately by BS 5930:1981.

Emphasis in this paper is placed on highlighting the shortcomings or inappropriateness of some of the BS 5930:1981 site investigation techniques when applied to Hong Kong conditions. For example, the British Code of Practice discusses techniques which are not used in Hong Kong, such as light cable percussive drilling; techniques and good practice associated with rotary core drilling are amplified instead. Similar treatment is given to the commonly installed open hydraulic (Casagrande) piezometers.

Particular problems and interpretation of frequently performed field tests such as dynamic probing, the standard penetration test,
in-situ
vane shear test and impression packer surveys are also discussed.