bluuebunny/crossref_metadata_2025 huggingface dataset:
Contrasting Effects of Heat Treatment and Incubation Temperature on Germination and Outgrowth of Individual Spores of Nonproteolytic
Clostridium botulinum
Bacteria
Authors: Stringer Sandra C. , Webb Martin D. , Peck Michael W.
Published: March 2009

In this study, we determined the effects of incubation temperature and prior heat treatment on the lag-phase kinetics of individual spores of nonproteolytic
Clostridium botulinum
Eklund 17B. The times to germination (
t
germ
), one mature cell (
t
C1
), and two mature cells (
t
C2
) were measured for individual unheated spores incubated at 8, 10, 15, or 22°C and used to calculate the
t
germ
, the outgrowth time (
t
C1
−
t
germ
), and the first doubling time (
t
C2
−
t
C1
). Measurements were also made at 22°C of spores that had previously been heated at 80°C for 20 s. For unheated spores, outgrowth made a greater contribution to the duration and variability of the lag phase than germination. Decreasing incubation temperature affected germination less than outgrowth; thus, the proportion of lag associated with germination was less at lower incubation temperatures. Heat treatment at 80°C for 20 s increased the median germination time of surviving spores 16-fold and greatly increased the variability of spore germination times. The shape of the lag-time (
t
C1
) and outgrowth (
t
C1
−
t
germ
) distributions were the same for unheated spores, but heat treatment altered the shape of the lag-time distribution, so it was no longer homogeneous with the outgrowth distribution. Although heat treatment mainly extended germination, there is also evidence of damage to systems required for outgrowth. However, this damage was quickly repaired and was not evident by the time the cells started to double. The results presented here combined with previous findings show that the stage of lag most affected, and the extent of any effect in terms of duration or variability, differs with both historical treatment and the growth conditions.
Purification, Characterization, and Potential Bacterial Wax Production Role of an NADPH-Dependent Fatty Aldehyde Reductase from
Marinobacter aquaeolei
VT8
Authors: Wahlen Bradley D. , Oswald Whitney S. , Seefeldt Lance C. , Barney Brett M.
Published: March 2009

Wax esters, ester-linked fatty acids and long-chain alcohols, are important energy storage compounds in select bacteria. The synthesis of wax esters from fatty acids is proposed to require the action of a four-enzyme pathway. An essential step in the pathway is the reduction of a fatty aldehyde to the corresponding fatty alcohol, although the enzyme responsible for catalyzing this reaction has yet to be identified in bacteria. We report here the purification and characterization of an enzyme from the wax ester-accumulating bacterium
Marinobacter aquaeolei
VT8, which is a proposed fatty aldehyde reductase in this pathway. The enzyme, a 57-kDa monomer, was expressed in
Escherichia coli
as a fusion protein with the maltose binding protein on the N terminus and was purified to near homogeneity by using amylose affinity chromatography. The purified enzyme was found to reduce a number of long-chain aldehydes to the corresponding alcohols coupled to the oxidation of NADPH. The highest specific activity was observed for the reduction of decanal (85 nmol decanal reduced/min/mg). Short-chain and aromatic aldehydes were not substrates. The enzyme showed no detectable catalysis of the reverse reaction, the oxidation of decanol by NADP
+
. The mechanism of the enzyme was probed with several site-specific chemical probes. The possible uses of this enzyme in the production of wax esters are discussed.
Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas
Authors: Hodgetts Jennifer , Boonham Neil , Mumford Rick , Dickinson Matthew
Published: March 2009

Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays.
Striking Diversity of
vmp1
, a Variable Gene Encoding a Putative Membrane Protein of the Stolbur Phytoplasma
Authors: Cimerman Agnès , Pacifico Davide , Salar Pascal , Marzachì Cristina , Foissac Xavier
Published: March 2009

Studies of phytoplasma-insect vector interactions and epidemiological surveys of plant yellows associated with the stolbur phytoplasma (StolP) require the identification of relevant candidate genes and typing markers. A recent StolP genome survey identified a partial coding sequence, SR01H10, having no homologue in the “
Candidatus
Phytoplasma asteris” genome but sharing low similarity with a variable surface protein of animal mycoplasmas. The complete coding sequence and its genetic environment have been fully characterized by chromosome walking. The
vmp1
gene encodes a protein of 557 amino acids predicted to possess a putative signal peptide and a potential C-terminal transmembrane domain. The mature 57.8-kDa VMP1 protein is likely to be anchored in the phytoplasma membrane with a large N-terminal hydrophilic part exposed to the phytoplasma cell surface. Southern blotting experiments detected multiple sequences homologous to
vmp1
in the genomes of nine StolP isolates.
vmp1
is variable in size, and eight different
vmp1
RsaI restriction fragment length polymorphism types could be distinguished among 12 StolP isolates. Comparison of
vmp1
sequences revealed that insertions in largest forms of the gene encode an additional copy of a repeated domain of 81 amino acids, while variations in 11-bp repeats led to gene disruption in two StolP isolates.
vmp1
appeared to be much more variable than three housekeeping genes involved in protein translation, maturation, and secretion and may therefore be involved in phytoplasma-host interactions.
Rapid Identification of Genes Encoding DNA Polymerases by Function-Based Screening of Metagenomic Libraries Derived from Glacial Ice
Authors: Simon Carola , Herath Judith , Rockstroh Stephanie , Daniel Rolf
Published: March 2009

Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an
Escherichia coli polA
mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.
Phylogenetic and Chemical Diversity of Three Chemotypes of Bloom-Forming
Lyngbya
Species (
Cyanobacteria
:
Oscillatoriales
) from Reefs of Southeastern Florida
Authors: Sharp Koty , Arthur Karen E. , Gu Liangcai , Ross Cliff , Harrison Genelle , Gunasekera Sarath P. , Meickle Theresa , Matthew Susan , Luesch Hendrik , Thacker Robert W. , Sherman David H. , Paul Valerie J.
Published: March 2009

The cyanobacterial genus
Lyngbya
includes free-living, benthic, filamentous cyanobacteria that form periodic nuisance blooms in lagoons, reefs, and estuaries.
Lyngbya
spp. are prolific producers of biologically active compounds that deter grazers and help blooms persist in the marine environment. Here, our investigations reveal the presence of three distinct
Lyngbya
species on nearshore reefs in Broward County, FL, sampled in 2006 and 2007. With a combination of morphological measurements, molecular biology techniques, and natural products chemistry, we associated these three
Lyngbya
species with three distinct
Lyngbya
chemotypes. One species, identified as
Lyngbya
cf.
confervoides
via morphological measurements and 16S rRNA gene sequencing, produces a diverse array of bioactive peptides and depsipeptides. Our results indicate that the other two
Lyngbya
species produce either microcolins A and B or curacin D and dragonamides C and D. Results from screening for the biosynthetic capacity for curacin production among the three
Lyngbya
chemotypes in this study correlated that capacity with the presence of curacin D. Our work on these bloom-forming
Lyngbya
species emphasizes the significant phylogenetic and chemical diversity of the marine cyanobacteria on southern Florida reefs and identifies some of the genetic components of those differences.
The Ultramicrobacterium “
Elusimicrobium minutum
” gen. nov., sp. nov., the First Cultivated Representative of the Termite Group 1 Phylum
Authors: Geissinger Oliver , Herlemann Daniel P. R. , Mörschel Erhard , Maier Uwe G. , Brune Andreas
Published: March 2009

Insect intestinal tracts harbor several novel, deep-rooting clades of as-yet-uncultivated bacteria whose biology is typically completely unknown. Here, we report the isolation of the first representative of the termite group 1 (TG1) phylum from sterile-filtered gut homogenates of a humivorous scarab beetle larva. Strain Pei191
T
is a mesophilic, obligately anaerobic ultramicrobacterium with a gram-negative cell envelope. Cells are typically rod shaped, but cultures are pleomorphic in all growth phases (0.3 to 2.5 μm long and 0.17 to 0.3 μm wide). The isolate grows heterotrophically on sugars and ferments
d
-galactose,
d
-glucose,
d
-fructose,
d
-glucosamine, and
N
-acetyl-
d
-glucosamine to acetate, ethanol, hydrogen, and alanine as major products but only if amino acids are present in the medium. PCR-based screening and comparative 16S rRNA gene sequence analysis revealed that strain Pei191
T
belongs to the “intestinal cluster,” a lineage of hitherto uncultivated bacteria present in arthropod and mammalian gut systems. It is only distantly related to the previously described so-called “endomicrobia” lineage, which comprises mainly uncultivated endosymbionts of termite gut flagellates. We propose the name “
Elusimicrobium minutum
” gen. nov., sp. nov. (type strain, Pei191
T
= ATCC BAA-1559
T
= JCM 14958
T
) for the first isolate of this deep-branching lineage and the name “
Elusimicrobia
” phyl. nov. for the former TG1 phylum.
Genomic Analysis of “
Elusimicrobium minutum
,” the First Cultivated Representative of the Phylum “
Elusimicrobia
” (Formerly Termite Group 1)
Authors: Herlemann D. P. R. , Geissinger O. , Ikeda-Ohtsubo W. , Kunin V. , Sun H. , Lapidus A. , Hugenholtz P. , Brune A.
Published: March 2009

Organisms of the candidate phylum termite group 1 (TG1) are regularly encountered in termite hindguts but are present also in many other habitats. Here, we report the complete genome sequence (1.64 Mbp) of “
Elusimicrobium minutum
” strain Pei191
T
, the first cultured representative of the TG1 phylum. We reconstructed the metabolism of this strictly anaerobic bacterium isolated from a beetle larva gut, and we discuss the findings in light of physiological data.
E. minutum
has all genes required for uptake and fermentation of sugars via the Embden-Meyerhof pathway, including several hydrogenases, and an unusual peptide degradation pathway comprising transamination reactions and leading to the formation of alanine, which is excreted in substantial amounts. The presence of genes encoding lipopolysaccharide biosynthesis and the presence of a pathway for peptidoglycan formation are consistent with ultrastructural evidence of a gram-negative cell envelope. Even though electron micrographs showed no cell appendages, the genome encodes many genes putatively involved in pilus assembly. We assigned some to a type II secretion system, but the function of 60
pilE
-like genes remains unknown. Numerous genes with hypothetical functions, e.g., polyketide synthesis, nonribosomal peptide synthesis, antibiotic transport, and oxygen stress protection, indicate the presence of hitherto undiscovered physiological traits. Comparative analysis of 22 concatenated single-copy marker genes corroborated the status of “
Elusimicrobia
” (formerly TG1) as a separate phylum in the bacterial domain, which was so far based only on 16S rRNA sequence analysis.
Establishment of Oxidative
d
-Xylose Metabolism in
Pseudomonas putida
S12
Authors: Meijnen Jean-Paul , de Winde Johannes H. , Ruijssenaars Harald J.
Published: March 2009

The oxidative
d
-xylose catabolic pathway of
Caulobacter crescentus
, encoded by the
xylXABCD
operon, was expressed in the gram-negative bacterium
Pseudomonas putida
S12. This engineered transformant strain was able to grow on
d
-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g
d
-xylose
−1
) and a maximum growth rate of 0.21 h
−1
. Remarkably, most of the genes of the
xylXABCD
operon appeared to be dispensable for growth on
d
-xylose. Only the
xylD
gene, encoding
d
-xylonate dehydratase, proved to be essential for establishing an oxidative
d
-xylose catabolic pathway in
P. putida
S12. The growth performance on
d
-xylose was, however, greatly improved by coexpression of
xylXA
, encoding 2-keto-3-deoxy-
d
-xylonate dehydratase and α-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of
P. putida
S12 was found to play a key role in efficient oxidative
d
-xylose utilization. Gcd activity not only contributes to
d
-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on
d
-xylose.
Effect of Iron Concentration on the Growth Rate of
Pseudomonas syringae
and the Expression of Virulence Factors in
hrp
-Inducing Minimal Medium
Authors: Kim Beum Jun , Park Joon Ho , Park Tai Hyun , Bronstein Philip A. , Schneider David J. , Cartinhour Samuel W. , Shuler Michael L.
Published: March 2009

Although chemically defined media have been developed and widely used to study the expression of virulence factors in the model plant pathogen
Pseudomonas syringae
, it has been difficult to link specific medium components to the induction response. Using a chemostat system, we found that iron is the limiting nutrient for growth in the standard
hrp
-inducing minimal medium and plays an important role in inducing several virulence-related genes in
Pseudomonas syringae
pv.
tomato
DC3000. With various concentrations of iron oxalate, growth was found to follow Monod-type kinetics for low to moderate iron concentrations. Observable toxicity due to iron began at 400 μM Fe
3+
. The kinetics of virulence factor gene induction can be expressed mathematically in terms of supplemented-iron concentration. We conclude that studies of induction of virulence-related genes in
P. syringae
should control iron levels carefully to reduce variations in the availability of this essential nutrient.
FvtA Is the Receptor for the Siderophore Vanchrobactin inVibrio anguillarum: Utility as a Route of Entry for Vanchrobactin Analogues
Authors: Balado Miguel , Osorio Carlos R. , Lemos Manuel L.
Published: March 2009

Some strains ofVibrio anguillarum, the causative agent of vibriosis in a variety of marine animals, produce a catechol-type siderophore named vanchrobactin. The biosynthetic pathway and regulation of vanchrobactin are quite well understood. However, aspects concerning its entry into the cell have remained uncharacterized. In the present study we characterized two genes,fvtAandorf13, encoding potential TonB-dependent ferric-vanchrobactin receptors in serotype O2V. anguillarumstrain RV22. We found that anfvtAmutant was defective for growth under iron limitation conditions and for utilization of vanchrobactin, suggesting thatfvtAencodes the vanchrobactin receptor ofV. anguillarum. Interestingly, anorf13mutant was not significantly affected, and results of reverse transcriptase PCR, as well as analysis of outer membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggested that this gene is not expressed. Furthermore,fatA, a plasmid gene coding for the anguibactin receptor in plasmid pJM1-harboring strains, is also present in the chromosome of RV22, although it is inactivated by insertion of transposases. In addition, we found that FvtA is the route of entry for vanchrobactin analogues, and there is evidence that it recognizes primarily the catechol-iron center. These analogues are potential candidate vectors for a Trojan horse strategy aimed at generating antimicrobial compounds exploiting the same route of entry for native siderophores. We found thatfvtAand vanchrobactin biosynthesis genes are ubiquitous in both vanchrobactin- and anguibactin-producingV. anguillarumstrains, which reinforces the utility of the vanchrobactin route of entry for the design of future strategies for the control of vibriosis.
Depletion of the Cullin Cdc53p Induces Morphogenetic Changes in
Candida albicans
Authors: Trunk Katharina , Gendron Patrick , Nantel André , Lemieux Sébastien , Roemer Terry , Raymond Martine
Published: March 2009

Candida albicans
is an important opportunistic human fungal pathogen that can cause both mucosal and systemic infections in immunocompromised patients. Critical for the virulence of
C. albicans
is its ability to undergo a morphological transition from yeast to hyphal growth mode. Proper induction of filamentation is dependent on the ubiquitination pathway, which targets proteins for proteasome-mediated protein degradation or activates them for signaling events. In the present study, we evaluated the role of ubiquitination in
C. albicans
by impairing the function of the major ubiquitin-ligase complex SCF. This was done by depleting its backbone, the cullin Cdc53p (orf19.1674), using a tetracycline downregulatable promoter system. Cdc53p-depleted cells displayed an invasive phenotype and constitutive filamentation under conditions favoring yeast growth mode, both on solid and in liquid media. In addition, these cells exhibited an early onset of cell death, as judged from propidium iodide staining, suggesting that
CDC53
is an essential gene in
C. albicans
. To identify Cdc53p-dependent pathways in
C. albicans
, a genome-wide expression analysis was carried out that revealed a total of 425 differentially expressed genes (fold change, ≥2;
P
≤ 0.05) with 192 up- and 233 downregulated genes in the
CDC53
-repressed mutant compared to the control strain. GO term analysis identified biological processes significantly affected by Cdc53p depletion, including amino acid starvation response, with 14 genes being targets of the transcriptional regulator Gcn4p, and reductive iron transport. These results indicate that Cdc53p enables
C. albicans
to adequately respond to environmental signals.
Surface-Expressed Enolase Contributes to the Pathogenesis of Clinical Isolate SSU ofAeromonas hydrophila
Authors: Sha Jian , Erova Tatiana E. , Alyea Rebecca A. , Wang Shaofei , Olano Juan P. , Pancholi Vijay , Chopra Ashok K.
Published: March 2009

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU ofAeromonas hydrophilabound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease α2-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT)A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability ofA. hydrophilaSSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied intrans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase inA. hydrophilaSSU; the mutated forms of enolase were hyperexpressed inEscherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues (252FYDAEKKEY260) in theA. hydrophilaSSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis ofA. hydrophilaSSU infections and of any gram-negative bacteria in general.
Genetic Variation and Evolution of the Pathogenicity Island of
Enterococcus faecalis
Authors: McBride Shonna M. , Coburn Phillip S. , Baghdayan Arto S. , Willems Rob J. L. , Grande Maria J. , Shankar Nathan , Gilmore Michael S.
Published: March 2009

Enterococcus faecalis
is a leading cause of nosocomial infections and is known for its ability to acquire and transfer virulence and antibiotic resistance determinants from other organisms. A 150-kb pathogenicity island (PAI) encoding several genes that contribute to pathogenesis was identified among antibiotic-resistant clinical isolates. In the current study, we examined the structure of the PAI in a collection of isolates from diverse sources in order to gain insight into its genesis and dynamics. Using multilocus sequence typing to assess relatedness at the level of strain background and microarray analysis to identify variations in the PAI, we determined the extent to which structural variations occur within the PAI and also the extent to which these variations occur independently of the chromosome. Our findings provide evidence for a modular gain of defined gene clusters by the PAI. These results support horizontal transfer as the mechanism for accretion of genes into the PAI and highlight a likely role for mobile elements in the evolution of the
E. faecalis
PAI.
Potassium Transport in
Corynebacterium glutamicum
Is Facilitated by the Putative Channel Protein CglK, Which Is Essential for pH Homeostasis and Growth at Acidic pH
Authors: Follmann Martin , Becker Markus , Ochrombel Ines , Ott Vera , Krämer Reinhard , Marin Kay
Published: March 2009

We studied the requirement for potassium and for potassium transport activity for the biotechnologically important bacterium
Corynebacterium glutamicum
, which is used for large-scale production of amino acids. Different from many other bacteria, at alkaline or neutral pH,
C. glutamicum
is able to grow without the addition of potassium, resulting in very low cytoplasmic potassium concentrations. In contrast, at acidic pH, the ability for growth was found to depend on the presence of K
+
. For the first time, we provide experimental evidence that a potential potassium channel (CglK) acts as the major potassium uptake system in a bacterium and proved CglK's function directly in its natural membrane environment. A full-length CglK protein and a separate soluble protein harboring the RCK domain can be translated from the
cglK
gene, and both are essential for full CglK functionality. As a reason for potassium-dependent growth limitation at acidic pH, we identified the impaired capacity for internal pH homeostasis, which depends on the availability and internal accumulation of potassium. Potassium uptake via CglK was found to be relevant for major physiological processes, like the activity of the respiratory chain, and to be crucial for maintenance of the internal pH, as well as for the adjustment of the membrane potential in
C. glutamicum
.
Regulation of
Corynebacterium glutamicum
Heat Shock Response by the Extracytoplasmic-Function Sigma Factor SigH and Transcriptional Regulators HspR and HrcA
Authors: Ehira Shigeki , Teramoto Haruhiko , Inui Masayuki , Yukawa Hideaki
Published: March 2009

Heat shock response in
Corynebacterium glutamicum
was characterized by whole-genome expression analysis using a DNA microarray. It was indicated that heat shock response of
C. glutamicum
included not only upregulation of heat shock protein (HSP) genes encoding molecular chaperones and ATP-dependent proteases, but it also increased and decreased expression of more than 300 genes related to disparate physiological functions. An extracytoplasmic-function sigma factor, SigH, was upregulated by heat shock. The SigH regulon was defined by gene expression profiling using
sigH
-disrupted and overexpressing strains in conjunction with mapping of transcription initiation sites. A total of 45 genes, including HSP genes and genes involved in oxidative stress response, were identified as the SigH regulon. Expression of some HSP genes was also upregulated by deletion of the transcriptional regulators HspR and HrcA. HspR represses expression of the
clpB
and
dnaK
operons, and HrcA represses expression of
groESL1
and
groEL2
. SigH was shown to play an important role in regulation of heat shock response in concert with HspR and HrcA, but its role is likely restricted to only a part of the regulation of
C. glutamicum
heat shock response. Upregulation of 18 genes encoding transcriptional regulators by heat shock suggests a complex regulatory network of heat shock response in
C. glutamicum
.
Plasmid pAMS1-Encoded, Bacteriocin-Related “Siblicide” in
Enterococcus faecalis
Authors: Sedgley Christine M. , Clewell Don B. , Flannagan Susan E.
Published: March 2009

The
Enterococcus faecalis
class IIa bacteriocin MC4-1 encoded by the sex pheromone-responding, multiple-antibiotic resistance plasmid pAMS1 exhibits “siblicidal” (sibling-killing) activity under certain conditions. Stabs of plasmid-containing cells on solid medium containing lawns of bacteria of the same (plasmid-containing) strain give rise to zones of inhibition. If the plasmid-containing host also produces gelatinase, bacteriocin cannot be detected.
Pseudomonas aeruginosaAlgR Controls Cyanide Production in an AlgZ-Dependent Manner
Authors: Cody William L. , Pritchett Christopher L. , Jones Adriana K. , Carterson Alexander J. , Jackson Debra , Frisk Anders , Wolfgang Matthew C. , Schurr Michael J.
Published: March 2009

Pseudomonas aeruginosais an opportunistic pathogen that causes chronic infections in individuals suffering from the genetic disorder cystic fibrosis. InP. aeruginosa, the transcriptional regulator AlgR controls a variety of virulence factors, including alginate production, twitching motility, biofilm formation, quorum sensing, and hydrogen cyanide (HCN) production. In this study, the regulation of HCN production was examined. Strains lacking AlgR or the putative AlgR sensor AlgZ produced significantly less HCN than did a nonmucoid isogenic parent. In contrast,algRandalgZmutants showed increased HCN production in an alginate-producing (mucoid) background. HCN production was optimal in a 5% O2environment. In addition, cyanide production was elevated in bacteria grown on an agar surface compared to bacteria grown in planktonic culture. A conserved AlgR phosphorylation site (aspartate at amino acid position 54), which is required for surface-dependent twitching motility but not alginate production, was found to be critical for cyanide production. Nuclease protection mapping of thehcnApromoter identified a new transcriptional start site required for HCN production. A subset of clinical isolates that lack this start site produced small amounts of cyanide. Taken together, these data show that theP. aeruginosa hcnApromoter contains three transcriptional start sites and that HCN production is regulated by AlgZ and AlgR and is maximal under microaerobic conditions when the organism is surface attached.
Influences of Capsule on Cell Shape and Chain Formation of Wild-Type andpcsBMutants of Serotype 2Streptococcus pneumoniae
Authors: Barendt Skye M. , Land Adrian D. , Sham Lok-To , Ng Wai-Leung , Tsui Ho-Ching T. , Arnold Randy J. , Winkler Malcolm E.
Published: March 2009

PcsB is a protein of unknown function that plays a critical role in cell division inStreptococcus pneumoniaeand other ovococcus species ofStreptococcus. We constructed isogenic sets of mutants expressing different amounts of PcsB in laboratory strain R6 and virulent serotype 2 strain D39 to evaluate its cellular roles. Insertion mutagenesis in parent andpcsB+merodiploid strains indicated thatpcsBis essential in serotype 2S. pneumoniae.Quantitative Western blotting of wild-type and epitope-tagged PcsB showed that all PcsB was processed into cell-associated and secreted forms of the same molecular mass and that cell-associated PcsB was moderately abundant and present at ≈4,900 monomers per cell. Controlled expression and complementation experiments indicated that there was a causative relationship between the severity of defects in cell division and decreasing PcsB amount. These experiments also showed that perturbations of expression of the upstreammreCDgenes did not contribute to the cell division defects ofpcsBmutants and thatmreCDcould be deleted. Unexpectedly, capsule influenced the cell shape and chain formation phenotypes of the wild-type D39 strain and mutants underexpressing PcsB or deleted for other genes involved in peptidoglycan biosynthesis, such asdacA. Underexpression of PcsB did not result in changes in the amounts or composition of lactoyl-peptides, which were markedly different in the R6 and D39 strains, and there was no correlation between decreased PcsB amount and sensitivity to penicillin. Finally, microarray analyses indicated that underexpression of PcsB may generate a signal that increases expression of the VicRK regulon, which includespcsB.
Isolation and Characterization of
Azotobacter vinelandii
Mutants Impaired in Alkylresorcinol Synthesis: Alkylresorcinols Are Not Essential for Cyst Desiccation Resistance
Authors: Segura Daniel , Vite Odon , Romero Yanet , Moreno Soledad , Castañeda Miguel , Espín Guadalupe
Published: March 2009

During encystment of
Azotobacter vinelandii
, a family of alkylresorcinols (ARs) and alkylpyrones (APs) are synthesized. In the mature cyst, these lipids replace the membrane phospholipids and are also components of the layers covering the cyst. In this study,
A. vinelandii
strains unable to synthesize ARs were isolated after mini-Tn
5
mutagenesis. Cloning and nucleotide sequencing of the affected loci revealed the presence of the transposons within the
arsA
gene of the previously reported
arsABCD
gene cluster, which encodes a type I fatty acid synthase. A mutant strain (SW-A) carrying an
arsA
mutation allowing transcription of
arsBCD
was constructed and shown to be unable to produce ARs, indicating that the ArsA protein is essential for the synthesis of these phenolic lipids. Transcription of
arsA
was induced 200-fold in cells undergoing encystment, but only 14-fold in aged cultures of
A. vinelandii
, in accordance with AR synthesis and cyst formation percentages under the two conditions. Although it was previously reported that the inactivation of
arsB
abolishes AR synthesis and results in a failure in encystment, the
arsA
mutants were able to form cysts resistant to desiccation. These data indicate that ARs play a structural role in the exine layer of the cysts, but they are not essential for either cyst formation or for desiccation resistance.
Characterization of the
Streptococcus pneumoniae
BgaC Protein as a Novel Surface β-Galactosidase with Specific Hydrolysis Activity for the Galβ1-3GlcNAc Moiety of Oligosaccharides
Authors: Jeong Jae Kap , Kwon Ohsuk , Lee Yun Mi , Oh Doo-Byoung , Lee Jung Mi , Kim Seonghun , Kim Eun-Hye , Le Tu Nhat , Rhee Dong-Kwon , Kang Hyun Ah
Published: March 2009

Streptococcus pneumoniae
is a causative agent of high morbidity and mortality. Although sugar moieties have been recognized as ligands for initial contact with the host, only a few exoglycosidases have been reported to occur in
S. pneumoniae
. In this study, a putative β-galactosidase, encoded by the
bgaC
gene of
S. pneumoniae
, was characterized for its enzymatic activity and virulence. The recombinant BgaC protein, expressed and purified from
Escherichia coli
, was found to have a highly regiospecific and sugar-specific hydrolysis activity for the Galβ1-3-GlcNAc moiety of oligosaccharides. Interestingly, the BgaC hydrolysis activity was localized at the cell surface of
S. pneumoniae
, indicating that BgaC is expressed as a surface protein although it does not have a typical signal sequence or membrane anchorage motif. The surface localization of BgaC was further supported by immunofluorescence microscopy analysis using an antibody raised against BgaC and by a reassociation assay with fluorescein isothiocyanate-labeled BgaC. Although the
bgaC
deletion mutation did not significantly attenuate the virulence of
S. pneumoniae
in vivo, the
bgaC
mutant strain showed relatively low numbers of viable cells compared to the wild type after 24 h of infection in vivo, whereas the mutant showed higher colonization levels at 6 and 24 h postinfection in vivo. Our data strongly indicate for the first time that
S. pneumoniae bgaC
encodes a surface β-galactosidase with high substrate specificity that is significantly associated with the infection activity of pneumococci.
Residues of Heat-Labile Enterotoxin Involved in Bacterial Cell Surface Binding
Authors: Mudrak Benjamin , Rodriguez Daniel L. , Kuehn Meta J.
Published: March 2009

Enterotoxigenic
Escherichia coli
(ETEC) is a leading cause of traveler's diarrhea worldwide. One major virulence factor released by this pathogen is the heat-labile enterotoxin LT, which upsets the balance of electrolytes in the intestine. After export, LT binds to lipopolysaccharide (LPS) on the bacterial surface. Although the residues responsible for LT's binding to its host receptor are known, the portion of the toxin which mediates LPS binding has not been defined previously. Here, we describe mutations in LT that impair the binding of the toxin to the external surface of
E. coli
without altering holotoxin assembly. One mutation in particular, T47A, nearly abrogates surface binding without adversely affecting expression or secretion in ETEC. Interestingly, T47A is able to bind mutant
E. coli
expressing highly truncated forms of LPS, indicating that LT binding to wild-type LPS may be due primarily to association with an outer core sugar. Consequently, we have identified a region of LT distinct from the pocket involved in eukaryotic receptor binding that is responsible for binding to the surface of
E. coli
.
Pseudomonas syringae
HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein
Authors: Morello Joanne E. , Collmer Alan
Published: March 2009

Pseudomonas syringae
delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS).
P. syringae
pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like
Yersinia
YscP. A Δ
hrpP
DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in
trans
. Though HrpP is a relatively divergent protein in the T3SS of different
P. syringae
pathovars,
hrpP
from
P. syringae
pv. syringae 61 and
P. syringae
pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 Δ
hrpP
. HrpP was translocated into
Nicotiana benthamiana
cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the Δ
hrpP
mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the Δ
hrpP
mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 Δ
hrpP
except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the
P. syringae
T3SS pilus likely varies in length due to differing plant cell walls.
ThecinandraiQuorum-Sensing Regulatory Systems inRhizobium leguminosarumAre Coordinated by ExpR and CinS, a Small Regulatory Protein Coexpressed with CinI
Authors: Edwards Anne , Frederix Marijke , Wisniewski-Dyé Florence , Jones Jacob , Zorreguieta Angeles , Downie J. Allan
Published: March 2009

To understand how theRhizobium leguminosarum raiI-raiRquorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-madeN-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required forraiRexpression, and RaiR is required to induceraiI. SinceraiR(andraiI) expression is also reduced incinIandcinRquorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induceraiR. However, added CinI-made AHLs did not induceraiRexpression in acinImutant. The reducedraiRexpression incinIandcinRmutants was due to lack of expression ofcinSimmediately downstream ofcinI. cinSencodes a 67-residue protein, translationally coupled to CinI, andcinSacts downstream ofexpRforraiRinduction. ClonedcinSinR. leguminosarumcaused an unusual collapse of colony structure, and this was delayed by mutation ofexpR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations incinI, cinR, cinS, andexpRall reduced expression ofplyB, encoding an EPS glycanase, and mutation ofplyBabolished the effect of clonedcinSon colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, includingRhizobium etli; the previously observed effect ofcinIandcinRmutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs.
vfr
, a Novel Locus Affecting Cysteine Protease Production in
Streptococcus pyogenes
Authors: Ma Yongsheng , Bryant Amy E. , Salmi Dan B. , McIndoo Eric , Stevens Dennis L.
Published: March 2009

A gene unique to
Streptococcus pyogenes
, called
vfr
, that negatively regulates
speB
, an important extracellular proteinase, has been identified. Disruption of
vfr
markedly increased SpeB production in a clinical strain of
S. pyogenes
and relieved its growth phase dependency. These findings may provide important insights into the pathogenesis of invasive
S. pyogenes
infections.
The
Bacillus subtilis ywjI
(
glpX
) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme
Authors: Jules Matthieu , Le Chat Ludovic , Aymerich Stéphane , Le Coq Dominique
Published: March 2009

We present here experimental evidence that the
Bacillus subtilis ywjI
gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the
fbp
-encoded class III enzyme, and constitutes with the upstream gene,
murAB
, an operon transcribed at the same level under glycolytic or gluconeogenic conditions.
Molecular Analysis of an Extrachromosomal Element Containing the C2 Toxin Gene Discovered inClostridium botulinumType C
Authors: Sakaguchi Yoshihiko , Hayashi Tetsuya , Yamamoto Yumiko , Nakayama Keisuke , Zhang Kai , Ma Shaobo , Arimitsu Hideyuki , Oguma Keiji
Published: March 2009

Clostridium botulinumcultures are classified into seven types, types A to G, based on the antigenicity of the neurotoxins produced. Of these seven types, only types C and D produce C2 toxin in addition to the neurotoxin. The C2 toxin consists of two components designated C2I and C2II. The genes encoding the C2 toxin components have been cloned, and it has been stated that they might be on the cell chromosome. The present study confirmed by using pulsed-field gel electrophoresis and subsequent Southern hybridization that these genes are on a large plasmid. The complete nucleotide sequence of this plasmid was determined by using a combination of inverse PCR and primer walking. The sequence was 106,981 bp long and contained 123 potential open reading frames, including thec2Iandc2IIgenes. The 57 products of these open reading frames had sequences similar to those of well-known proteins. It was speculated that 9 these 57 gene products were related to DNA replication, 2 were responsible for the two-component regulatory system, and 3 were σ factors. In addition, a total of 20 genes encoding proteins related to diverse processes in purine catabolism were found in two regions. In these regions, there were 9 and 11 genes rarely found in plasmids, indicating that this plasmid plays an important role in purine catabolism, as well as in C2 toxin production.
The French Musculoskeletal Disorders Surveillance Program: Pays de la Loire network
Authors: Ha C , Roquelaure Y , Leclerc A , Touranchet A , Goldberg M , Imbernon E
Published: March 2009

Objectives:

An epidemiological surveillance system for work-related musculoskeletal disorders (MSDs) was implemented in 2002 in France’s Pays de la Loire region to assess the incidence and prevalence of MSDs in the general and working populations, identify levels of exposure to occupational risk factors and investigate the proportion of cases attributable to work exposure.

Methods:

The program combines (1) surveillance of sentinel health events in the general population (carpal tunnel syndrome (CTS) was the sentinel event for upper limb MSDs), (2) assessment of the prevalence of the main upper limb MSDs and their risk factors in the workplace based on a network of occupational physicians and (3) registration of the notification of work-related diseases (WRDs).

Results:

1168 incident cases of CTS were included over a 3 year period. The estimated incidence of CTS was 1.00 per 1000 person-years in those aged 20–59 years (0.60 in men and 1.40 in women). The incidence rate was higher in employed than unemployed persons in the year of diagnosis (0.6 per 1000 vs 0.3 in men and 1.7 vs 0.8 in women). The occupational physician network noted high prevalence rates: 11% of men and 15% of women had at least one of the six main upper limb clinically-diagnosed MSDs. The WRD survey showed that MSDs represented 65% of notified WRDs.

Conclusion:

The Pays de la Loire program plays a significant role in informing the authorities and the public about the state of current MSDs. It is planned to extend it to a routine national surveillance program.
UNIFIED TECHNIQUE FOR ON-LINE TESTING OF DIGITAL CIRCUITS: DELAY AND STUCK-AT FAULT MODELS
Authors: BISWAS S. , MUKHOPADHYAY S. , PATRA A. , SARKAR D.
Published: March 2009

This work is concerned with the development of generic, nonintrusive, and flexible algorithms for the design of digital circuits with on-line testing (OLT) capability. Most of the works presented in the literature on OLT have used single stuck at (s - a) fault models. However, in the deep submicron technology, single s - a fault models may not capture more than a fraction of the real defects. To cater to the problem it is now advocated that additional fault models such as bridging faults, transition faults, delay faults, etc., are also used. The proposed technique is one of the first works that facilitates a unified scheme for on-line detection of delay faults and s - a faults with a high value of n for n-Detect tests. The technique can handle generic digital circuits with cell count as high as 15,000 and having the order of 2500 states. Results for the design of on-line detectors for various ISCAS89 benchmark circuits are provided. The results illustrate that with marginal increase in the area overhead, if compared to the ones with single s - a fault coverage only, the proposed scheme also provides coverage for the delay faults.
TUNABLE WAVEFORM GENERATORS USING SINGLE DUAL-CURRENT OUTPUT OTAs
Authors: LO YU-KANG , CHIEN HUNG-CHUN , CHIU HUANG-JEN
Published: March 2009

Two novel electronic-tunable waveform generators using a single dual-current output operational transconductance amplifier (DO-OTA) with a few passive components are proposed in this paper. The first one can produce symmetrical square and triangular waveforms simultaneously. Based on the first topology, an additional N-MOS is adopted as a switching element to construct the second proposed circuit, which can generate a pulse waveform by a negative triggering signal. The repetitive frequency and pulse width of the presented circuits are adjustable by external passive components. Moreover, the output levels of the proposed circuits can be controlled by the DO-OTA's bias currents. These waveform generators feature more compact configurations compared to the existing designs. Experimental results are close to the theoretical analyses.
A FRAMEWORK FOR OBJECT-ORIENTED EMBEDDED SYSTEM DEVELOPMENT BASED ON OO-ASIPS
Authors: MOHAMMADZADEH NASER , HESSABI SHAAHIN , GOUDARZI MAZIAR , MALAKI MAHDI
Published: March 2009

The growing complexity of today's embedded systems demands new methodologies and tools to manage the problems of analysis, design, implementation, and validation of complex-embedded systems. Focusing on this issue, this paper describes a design and implementation toolset using our ODYSSEY methodology, which advocates object-oriented (OO) modeling of embedded systems and its ASIP-based implementation. The proposed approach promotes a smooth transition from high-level object-oriented specification to the final embedded system, which is composed of hardware and software components. The transition from higher to lower abstraction levels is facilitated by the use of our GUI, which supports the intermediate steps of the design and implementation process. In order to illustrate the proposed approach and related toolset, we apply this top-down design and implementation framework to real-world embedded systems, namely JPEG codec and Motion JPEG codec. Experimental results show that the developed tool remarkably decreases the design and verification time with modest performance penalty.
CLOCK DISTRIBUTION NETWORKS WITH GRADUAL SIGNAL TRANSITION TIME RELAXATION FOR REDUCED POWER CONSUMPTION
Authors: TAWFIK SHERIF A. , KURSUN VOLKAN
Published: March 2009

Clock distribution network consumes a significant portion of the total chip power since the clock signal has the highest activity factor and drives the largest capacitive load in a synchronous integrated circuit. A new methodology is proposed in this paper for buffer insertion and sizing in an H-tree clock distribution network. The objective of the algorithm is to minimize the total power consumption while satisfying the maximum acceptable clock transition time constraints at the leaves of the clock distribution network for maintaining high performance. The new methodology employs nonuniform buffer insertion and progressive relaxation of the transition time requirements from the leaves to the root of the clock distribution network. The proposed algorithm provides up to 30% savings in the total power consumption without sacrificing clock skew as compared to a standard algorithm with uniform buffer insertion aimed at maintaining uniform transition time constraints at all the nodes of a clock tree in a 180 nm CMOS technology.