bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms Involved in Governing Adherence ofVibrio choleraeto Granular Starch
Authors: Niderman-Meyer Orly , Zeidman Tal , Shimoni Eyal , Kashi Yechezkel
Published: December 2009

Vibrio choleraehas been shown to adhere to cornstarch granules. The present work explored the mechanisms involved in this adhesion and the possibility of its occurrencein vivo. The findings suggest that both specific and nonspecific interactions are involved in the adhesion. Nonspecific hydrophobic interactions may play a role, since bothV. choleraeand cornstarch granules exhibited hydrophobic properties when they were tested using a xylene-water system. In addition, the presence of bile acids reduced the adhesion. The adhesion also involves some specific carbohydrate-binding moieties on the cell surface, as reflected by reduced adhesion following pretreatment of the bacteria with proteinase K and sodiumm-periodate. Further investigations supported these observations and showed that media containing low-molecular-weight carbohydrates had a significant inhibitory effect. Binding cell lysate to starch granules and removing the adhered proteins using either glucose or bile acids led to identification (by liquid chromatography-tandem mass spectrometry analysis) of several candidateV. choleraeouter membrane-associated starch-binding proteins. Different sets of proteins were isolated by removal in a glucose solution or bile acids. When the upper gastrointestinal tract conditions were simulatedin vitro, both bile salts and the amylolytic activity of the pancreatic juices were found to have an inhibitory effect on the adherence ofV. choleraeto starch. However, during acute diarrhea, this inhibitory effect may be significantly reduced due to dilution, suggesting that adhesion does occurin vivo. Such adhesion may contribute to the beneficial effects observed following administration of granular starch-based oral rehydration solutions to cholera patients.
Yeast Surface Display of Trifunctional Minicellulosomes for Simultaneous Saccharification and Fermentation of Cellulose to Ethanol
Authors: Wen Fei , Sun Jie , Zhao Huimin
Published: December 2009

By combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step, consolidated bioprocessing (CBP) represents a promising technology for biofuel production. Here we report engineering of
Saccharomyces cerevisiae
strains displaying a series of uni-, bi-, and trifunctional minicellulosomes. These minicellulosomes consist of (i) a miniscaffoldin containing a cellulose-binding domain and three cohesin modules, which was tethered to the cell surface through the yeast a-agglutinin adhesion receptor, and (ii) up to three types of cellulases, an endoglucanase, a cellobiohydrolase, and a β-glucosidase, each bearing a C-terminal dockerin. Cell surface assembly of the minicellulosomes was dependent on expression of the miniscaffoldin, indicating that formation of the complex was dictated by the high-affinity interactions between cohesins and dockerins. Compared to the unifunctional and bifunctional minicellulosomes, the quaternary trifunctional complexes showed enhanced enzyme-enzyme synergy and enzyme proximity synergy. More importantly, surface display of the trifunctional minicellulosomes gave yeast cells the ability to simultaneously break down and ferment phosphoric acid-swollen cellulose to ethanol with a titer of ∼1.8 g/liter. To our knowledge, this is the first report of a recombinant yeast strain capable of producing cell-associated trifunctional minicellulosomes. The strain reported here represents a useful engineering platform for developing CBP-enabling microorganisms and elucidating principles of cellulosome construction and mode of action.
Metabolic Differentiation in Biofilms as Indicated by Carbon Dioxide Production Rates
Authors: Bester Elanna , Kroukamp Otini , Wolfaardt Gideon M. , Boonzaaier Leandro , Liss Steven N.
Published: December 2009

The measurement of carbon dioxide production rates as an indication of metabolic activity was applied to study biofilm development and response of
Pseudomonas
sp. biofilms to an environmental disturbance in the form of a moving air-liquid interface (i.e., shear). A differential response in biofilm cohesiveness was observed after bubble perturbation, and the biofilm layers were operationally defined as either shear-susceptible or non-shear-susceptible. Confocal laser scanning microscopy and image analysis showed a significant reduction in biofilm thickness and biomass after the removal of the shear-susceptible biofilm layer, as well as notable changes in the roughness coefficient and surface-to-biovolume ratio. These changes were accompanied by a 72% reduction of whole-biofilm CO
2
production; however, the non-shear-susceptible region of the biofilm responded rapidly after the removal of the overlying cells and extracellular polymeric substances (EPS) along with the associated changes in nutrient and O
2
flux, with CO
2
production rates returning to preperturbation levels within 24 h. The adaptable nature and the ability of bacteria to respond to environmental conditions were further demonstrated by the outer shear-susceptible region of the biofilm; the average CO
2
production rate of cells from this region increased within 0.25 h from 9.45 ± 5.40 fmol of CO
2
·cell
−1
·h
−1
to 22.6 ± 7.58 fmol of CO
2
·cell
−1
·h
−1
when cells were removed from the biofilm and maintained in suspension without an additional nutrient supply. These results also demonstrate the need for sufficient monitoring of biofilm recovery at the solid substratum if mechanical methods are used for biofouling control.
Seasonal Retention and Release of
Cryptosporidium parvum
Oocysts by Environmental Biofilms in the Laboratory
Authors: Wolyniak E. A. , Hargreaves B. R. , Jellison K. L.
Published: December 2009

Cryptosporidium
is a genus of waterborne protozoan parasites that causes significant gastrointestinal disease in humans. These parasites can accumulate in environmental biofilms and be subsequently released to contaminate water supplies. Natural microbial assemblages were collected each season from an eastern Pennsylvania stream and used to grow biofilms in laboratory microcosms in which influx, efflux, and biofilm retention were determined from daily oocyst counts. For each seasonal biofilm, oocysts attached to the biofilm quickly during oocyst dosing. Upon termination of oocyst dosing, the percentage of oocysts retained within the biofilm decreased to a new steady state within 5 days. Seasonal differences in biofilm retention of oocysts were observed. The spring biofilm retained the greatest percentage of oocysts, followed (in decreasing order) by the winter, summer, and fall biofilms. There was no statistically significant correlation between the percentage of oocysts attached to the biofilm and (i) any measured stream water quality parameter (including temperature, pH, conductivity, and dissolved organic carbon concentration) or (ii) experimental temperature. Seasonal differences in oocyst retention persisted when biofilms were tested with stream water from a different season. These data suggest that seasonal variation in the microbial community and resulting biofilm architecture may be more important to oocyst transport in this stream site than water quality. The biofilm attachment and detachment dynamics of
C. parvum
oocysts have implications for public health, and the drinking water industry should recognize that the potential exists for pathogen-free water to become contaminated during the distribution process as a result of biofilm dynamics.
Use of a Foam Spatula for Sampling Surfaces after Bioaerosol Deposition
Authors: Lewandowski Rafał , Kozłowska Krystyna , Szpakowska Małgorzata , Stępińska Małgorzata , Trafny Elżbieta A.
Published: December 2009

The present study had three goals: (i) to evaluate the relative quantities of aerosolized
Bacillus atrophaeus
spores deposited on the vertical, horizontal top, and horizontal bottom surfaces in a chamber; (ii) to assess the relative recoveries of the aerosolized spores from glass and stainless steel surfaces with a polyester swab and a macrofoam sponge wipe; and (iii) to estimate the relative recovery efficiencies of aerosolized
B. atrophaeus
spores and
Pantoea agglomerans
using a foam spatula at several different bacterial loads by aerosol distribution on glass surfaces. The majority of spores were collected from the bottom horizontal surface regardless of which swab type and extraction protocol were used. Swabbing with a macrofoam sponge wipe was more efficient in recovering spores from surfaces contaminated with high bioaerosol concentrations than swabbing with a polyester swab.
B. atrophaeus
spores and
P. agglomerans
culturable cells were detected on glass surfaces using foam spatulas when the theoretical surface bacterial loads were 2.88 × 10
4
CFU and 8.09 × 10
6
CFU per 100-cm
2
area, respectively. The median recovery efficiency from the surfaces using foam spatulas was equal to 9.9% for
B. atrophaeus
spores when the recovery was calculated relative to the theoretical surface spore load. Using a foam spatula permits reliable sampling of spores on the bioaerosol-exposed surfaces in a wide measuring range. The culturable
P. agglomerans
cells were recovered with a median efficiency of 0.001%, but staining the swab extracts with fluorescent dyes allowed us to observe that the viable cell numbers were higher by 1.83 log units than culturable organisms. However, additional work is needed to improve the analysis of the foam extracts in order to decrease the limit of detection of
Bacillus
spores and Gram-negative bacteria on contaminated surfaces.
Prolonged Survival ofCampylobacterSpecies in Bovine Manure Compost
Authors: Inglis G. Douglas , McAllister Tim A. , Larney Francis J. , Topp Edward
Published: December 2009

The persistence of naturally occurring campylobacteria in aerobic compost constructed of manure from beef cattle that were administered chlortetracycline and sulfamethazine (AS700) or from cattle not administered antibiotics (control) was examined. Although there were no differences in population sizes of heterotrophic bacteria, the temperature of AS700 compost was more variable and did not become as high as that of control compost. There were significant differences in water content, total carbon (C), total nitrogen (N), and electrical conductivity but not in the C/N ratio or pH between the two compost treatments. Campylobacteria were readily isolated from pen manure, for up to day 15 from control compost, and throughout the active phase of AS700 compost.CampylobacterDNA (includingCampylobacter coli,Campylobacter fetus,Campylobacter hyointestinalis, andCampylobacter jejuni) was detected over the ca. 10-month composting period, and no reductions in quantities ofC. jejuniDNA were observed over the duration of the active phase. The utilization of centrifugation in combination with ethidium monoazide (EMA) significantly reduced (>90%) the amplification ofC. jejuniDNA that did not originate from cells with intact cell membranes. No differences were observed in the frequency ofCampylobacterDNA detection between EMA- and non-EMA-treated samples, suggesting thatCampylobacterDNA amplified from compost was extracted from cells with intact cell membranes (i.e., from viable cells). The findings of this study indicate that campylobacteria excreted in cattle feces persist for long periods in compost and call into question the common belief that these bacteria do not persist in manure.
Optimization of RNA Extraction for PCR Quantification of Aromatic Compound Degradation Genes
Authors: Kong Weidong , Nakatsu Cindy H.
Published: December 2009

Seven different bacterial strains and primer sets and a mixed community were used to evaluate the use of reverse transcriptase quantitative PCR (Q-PCR) and Q-PCR of oxygenase genes to assess various approaches for monitoring the bioremediation of polluted sites. Differences in maximum activity were seen when different RNA extraction kits were compared.
Regulation of Arabinose and Xylose Metabolism inEscherichia coli
Authors: Desai Tasha A. , Rao Christopher V.
Published: December 2009

Bacteria such asEscherichia coliwill often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, whereE. coliwill first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy inE. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses,E. coliwill consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization inE. coliinvolves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering ofE. colistrains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.
Shear-Enhanced Oral Microbial Adhesion
Authors: Ding Albert M. , Palmer Robert J. , Cisar John O. , Kolenbrander Paul E.
Published: December 2009

Shear-enhanced adhesion, although not observed for fimbria-mediated adhesion of oral
Actinomyces
spp., was noted for Hsa-mediated adhesion of
Streptococcus gordonii
to sialic acid-containing receptors, an interaction implicated in the pathogenesis of infective endocarditis.
Association of Hemolytic Activity ofPseudomonas entomophila, a Versatile Soil Bacterium, with Cyclic Lipopeptide Production
Authors: Vallet-Gely Isabelle , Novikov Alexey , Augusto Luis , Liehl Peter , Bolbach Gérard , Péchy-Tarr Maria , Cosson Pierre , Keel Christoph , Caroff Martine , Lemaitre Bruno
Published: December 2009

Pseudomonas entomophilais an entomopathogenic bacterium that is able to infect and killDrosophila melanogasterupon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related toPseudomonas putida. The GacS/GacA two-component system plays a key role inP. entomophilapathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response.P. entomophilasecretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C10OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. TheP. entomophilaGacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence ofP. entomophilaforDrosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated withP. entomophila, including strong biocontrol abilities.
Improved Electrotransformation and Decreased Antibiotic Resistance of the Cystic Fibrosis Pathogen
Burkholderia cenocepacia
Strain J2315
Authors: Dubarry Nelly , Du Wenli , Lane David , Pasta Franck
Published: December 2009

The bacterium
Burkholderia cenocepacia
is pathogenic for sufferers from cystic fibrosis (CF) and certain immunocompromised conditions. The
B. cenocepacia
strain most frequently isolated from CF patients, and which serves as the reference for CF epidemiology, is J2315. The J2315 genome is split into three chromosomes and one plasmid. The strain was sequenced several years ago, and its annotation has been released recently. This information should allow genetic experimentation with J2315, but two major impediments appear: the poor potential of J2315 to act as a recipient in transformation and conjugation and the high level of resistance it mounts to nearly all antibiotics. Here, we describe modifications to the standard electroporation procedure that allow routine transformation of J2315 by DNA. In addition, we show that deletion of an efflux pump gene and addition of spermine to the medium enhance the sensitivity of J2315 to certain commonly used antibiotics and so allow a wider range of antibiotic resistance genes to be used for selection.
Adaptation of the Highly Productive T7 Expression System to
Streptomyces lividans
Authors: Lussier François-Xavier , Denis François , Shareck François
Published: December 2009

Streptomyces lividans
is a Gram-positive bacterium known for its remarkable secretion efficiency and low extracellular protease activity. In the present work, we adapted the highly productive T7 expression system to
S. lividans
. A codon-optimized T7 RNA polymerase gene was chromosomally integrated, and a bifunctional T7 expression vector was constructed.
Type II Diacylglycerol Acyltransferase fromClaviceps purpureawith Ricinoleic Acid, a Hydroxyl Fatty Acid of Industrial Importance, as Preferred Substrate
Authors: Mavraganis Ioannis , Meesapyodsuk Dauenpen , Vrinten Patricia , Smith Mark , Qiu Xiao
Published: December 2009

Claviceps purpurea, the fungal pathogen that causes the cereal disease ergot, produces glycerides that contain high levels of ricinoleic acid [(R)-12-hydroxyoctadec-cis-9-enoic acid] in its sclerotia. Recently, a fatty acid hydroxylase (C. purpureaFAH [CpFAH]) involved in the biosynthesis of ricinoleic acid was identified from this fungus (D. Meesapyodsuk and X. Qiu, Plant Physiol.147:1325-1333, 2008). Here, we describe the cloning and biochemical characterization of aC. purpureatype II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of ricinoleic acid into triglycerides. TheCpDGAT2gene was cloned by degenerate RT-PCR (reverse transcription-PCR). The expression of this gene restored thein vivosynthesis of triacylglycerol (TAG) in the quadruple mutant strainSaccharomyces cerevisiaeH1246, in which all four TAG biosynthesis genes (DGA1,LRO1,ARE1, andARE2) are disrupted.In vitroenzymatic assays using microsomal preparations from the transformed yeast strain indicated that CpDGAT2 prefers ricinoleic acid as an acyl donor over linoleic acid, oleic acid, or linolenic acid, and it prefers 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as an acyl acceptor. The coexpression of CpFAH with CpDGAT2 in yeast resulted in an increased accumulation of ricinoleic acid compared to the coexpression of CpFAH with the native yeast DGAT2 (S. cerevisiaeDGA1 [ScDGA1]) or the expression of CpFAH alone. Northern blot analysis indicated thatCpFAHis expressed solely in sclerotium cells, with no transcripts of this gene being detected in mycelium or conidial cells. CpDGAT2 was more widely expressed among the cell types examined, although expression was low in conidiospores. The high expression of CpDGAT2 and CpFAH in sclerotium cells, where high levels of ricinoleate glycerides accumulate, provided further evidence supporting the roles of CpDGAT2 and CpFAH as key enzymes for the synthesis and assembly of ricinoleic acid inC. purpurea.
Musca domestica
Salivary Gland Hypertrophy Virus, a Globally Distributed Insect Virus That Infects and Sterilizes Female Houseflies
Authors: Prompiboon Pannipa , Lietze Verena-Ulrike , Denton John S. S. , Geden Christopher J. , Steenberg Tove , Boucias Drion G.
Published: December 2009

The housefly,
Musca domestica
, is a cosmopolitan pest of livestock and poultry and is of economic, veterinary, and public health importance. Populations of
M. domestica
are naturally infected with
M. domestica
salivary gland hypertrophy virus (MdSGHV), a nonoccluded double-stranded DNA virus that inhibits egg production in infected females and is characterized by salivary gland hypertrophy (SGH) symptoms. MdSGHV has been detected in housefly samples from North America, Europe, Asia, the Caribbean, and the southwestern Pacific. In this study, houseflies were collected from various locations and dissected to observe SGH symptoms, and infected gland pairs were collected for MdSGHV isolation and amplification in laboratory-reared houseflies. Differences among the MdSGHV isolates were examined by using molecular and bioassay approaches. Approximately 600-bp nucleotide sequences from each of five open reading frames having homology to genes encoding DNA polymerase and partial homology to the genes encoding four
per os
infectivity factor proteins (p74, pif-1, pif-2, and pif-3) were selected for phylogenetic analyses. Nucleotide sequences from 16 different geographic isolates were highly homologous, and the polymorphism detected was correlated with geographic source. The virulence of the geographic MdSGHV isolates was evaluated by
per os
treatment of newly emerged and 24-h-old houseflies with homogenates of infected salivary glands. In all cases, 24-h-old flies displayed a resistance to oral infection that was significantly greater than that displayed by newly eclosed adults. Regardless of the MdSGHV isolate tested, all susceptible insects displayed similar degrees of SGH and complete suppression of oogenesis.
CalA, a Cyanobacterial AbrB Protein, Interacts with the Upstream Region of
hypC
and Acts as a Repressor of Its Transcription in the Cyanobacterium
Nostoc
sp. Strain PCC 7120
Authors: Agervald Åsa , Zhang Xiaohui , Stensjö Karin , Devine Ellenor , Lindblad Peter
Published: December 2009

The filamentous, heterocystous, nitrogen-fixing cyanobacterium
Nostoc
sp. strain PCC 7120 may contain, depending on growth conditions, up to two hydrogenases directly involved in hydrogen metabolism. HypC is one out of at least seven auxiliary gene products required for synthesis of a functional hydrogenase, specifically involved in the maturation of the large subunit. In this study we present a protein, CalA (Alr0946 in the genome), belonging to the transcription regulator family AbrB, which in protein-DNA assays was found to interact with the upstream region of
hypC
. Transcriptional investigations showed that
calA
is cotranscribed with the downstream gene
alr0947
, which encodes a putative protease from the abortive infection superfamily, Abi. CalA was shown to interact specifically not only with the upstream region of
hypC
but also with its own upstream region, acting as a repressor on
hypC
. The bidirectional hydrogenase activity was significantly downregulated when CalA was overexpressed, demonstrating a correlation with the transcription factor, either direct or indirect.
In silico
studies showed that homologues to both CalA and Alr0947 are highly conserved proteins within cyanobacteria with very similar physical organizations of the corresponding structural genes. Possible functions of the cotranscribed downstream protein Alr0947 are presented. In addition, we present a three-dimensional (3D) model of the DNA binding domain of CalA and putative DNA binding mechanisms are discussed.
A
m
ariner
-Based Transposon System for
In Vivo
Random Mutagenesis of
Clostridium difficile
Authors: Cartman Stephen T. , Minton Nigel P.
Published: December 2009

Understanding the molecular basis of
Clostridium difficile
infection is a prerequisite to the development of effective countermeasures. Although there are methods for constructing gene-specific mutants of
C. difficile
, currently there is no effective method for generating libraries of random mutants. In this study, we developed a novel
mariner
-based transposon system for
in vivo
random mutagenesis of
C. difficile
R20291, the BI/NAP1/027 epidemic strain at the center of the
C. difficile
outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. Transposition occurred at a frequency of 4.5 (±0.4) × 10
−4
per cell to give stable insertions at random genomic loci, which were defined only by the nucleotide sequence TA. Furthermore, mutants with just a single transposon insertion were generated in an overwhelming majority (98.3% in this study). Phenotypic screening of a
C. difficile
R20291 random mutant library yielded a sporulation/germination-defective clone with an insertion in the germination-specific protease gene
cspBA
and an auxotroph with an insertion in the pyrimidine biosynthesis gene
pyrB
. These results validate our
mariner
-based transposon system for use in forward genetic studies of
C. difficile
.
Soil Microbial Community Responses to Multiple Experimental Climate Change Drivers
Authors: Castro Hector F. , Classen Aimée T. , Austin Emily E. , Norby Richard J. , Schadt Christopher W.
Published: December 2009

Researchers agree that climate change factors such as rising atmospheric [CO
2
] and warming will likely interact to modify ecosystem properties and processes. However, the response of the microbial communities that regulate ecosystem processes is less predictable. We measured the direct and interactive effects of climatic change on soil fungal and bacterial communities (abundance and composition) in a multifactor climate change experiment that exposed a constructed old-field ecosystem to different atmospheric CO
2
concentration (ambient, +300 ppm), temperature (ambient, +3°C), and precipitation (wet and dry) might interact to alter soil bacterial and fungal abundance and community structure in an old-field ecosystem. We found that (i) fungal abundance increased in warmed treatments; (ii) bacterial abundance increased in warmed plots with elevated atmospheric [CO
2
] but decreased in warmed plots under ambient atmospheric [CO
2
]; (iii) the phylogenetic distribution of bacterial and fungal clones and their relative abundance varied among treatments, as indicated by changes in 16S rRNA and 28S rRNA genes; (iv) changes in precipitation altered the relative abundance of
Proteobacteria
and
Acidobacteria
, where
Acidobacteria
decreased with a concomitant increase in the
Proteobacteria
in wet relative to dry treatments; and (v) changes in precipitation altered fungal community composition, primarily through lineage specific changes within a recently discovered group known as soil clone group I. Taken together, our results indicate that climate change drivers and their interactions may cause changes in bacterial and fungal overall abundance; however, changes in precipitation tended to have a much greater effect on the community composition. These results illustrate the potential for complex community changes in terrestrial ecosystems under climate change scenarios that alter multiple factors simultaneously.
Distinct Subcellular Localization of a Group of Synaptobrevin-Like SNAREs in Paramecium tetraurelia and Effects of Silencing SNARE-Specific Chaperone NSF
Authors: Schilde Christina , Schönemann Barbara , Sehring Ivonne M. , Plattner Helmut
Published: December 2009

We have identified new synaptobrevin-like SNAREs and localized the corresponding gene products with green fluorescent protein (GFP)-fusion constructs and specific antibodies at the light and electron microscope (EM) levels. These SNAREs, named
Paramecium tetraurelia
synaptobrevins 8 to 12 (PtSyb8 to PtSyb12), showed mostly very restricted, specific localization, as they were found predominantly on structures involved in endo- or phagocytosis. In summary, we found PtSyb8 and PtSyb9 associated with the nascent food vacuole, PtSyb10 near the cell surface, at the cytostome, and in close association with ciliary basal bodies, and PtSyb11 on early endosomes and on one side of the cytostome, while PtSyb12 was found in the cytosol. PtSyb4 and PtSyb5 (identified previously) were localized on small vesicles, PtSyb5 probably being engaged in trichocyst (dense core secretory vesicle) processing. PtSyb4 and PtSyb5 are related to each other and are the furthest deviating of all SNAREs identified so far. Because they show no similarity with any other R-SNAREs outside ciliates, they may represent a ciliate-specific adaptation. PtSyb10 forms small domains near ciliary bases, and silencing slows down cell rotation during depolarization-induced ciliary reversal.
NSF
silencing supports a function of cell surface SNAREs by revealing vesicles along the cell membrane at sites normally devoid of vesicles. The distinct distributions of these SNAREs emphasize the considerable differentiation of membrane trafficking, particularly along the endo-/phagocytic pathway, in this protozoan.
tRNA Gene Sequences Are Required for Transcriptional Silencing in Entamoeba histolytica
Authors: Irmer Henriette , Hennings Ina , Bruchhaus Iris , Tannich Egbert
Published: December 2009

Transcriptional silencing by
trans
inactivation can contribute to the regulation of gene expression in eukaryotic cells. In the human intestinal protozoan parasite
Entamoeba histolytica
,
trans
inactivation of the amoebapore-A gene (
AP-A
) was recently achieved by episomal transfection of
E. histolytica
trophozoites with the plasmid psAP1. The mechanism of
AP-A trans
inactivation is largely unknown, though it was suggested that a partial short interspersed transposable element (SINE) is required. By systematic assessment of various
E. histolytica
isolates transfected with psAP1 derivates,
trans
inactivation of
AP-A
was restricted to the strain HM-1:IMSS (2411) but could not be achieved in other standard laboratory strains. Importantly, sequences of an
E. histolytica
tRNA array that were located on psAP1 in close proximity to the
AP-A
upstream region and comprising the glutamic acid (TTC) (E) and tyrosine (GTA) (Y) tRNA genes were indispensable for
AP-A
silencing. In contrast to the case described in previous reports, SINE was not required for
AP-A trans
inactivation.
AP-A
expression could be regained in silenced cells by episomal transfection under the control of a heterologous
E. histolytica
promoter, opening a way toward future silencing of individual genes of interest in
E. histolytica
. Our results indicate that tRNA gene-mediated silencing is not restricted to
Saccharomyces cerevisiae
.
Comparative Transcript Profiling of Candida albicans and Candida dubliniensis Identifies
SFL2
, a C. albicans Gene Required for Virulence in a Reconstituted Epithelial Infection Model
Authors: Spiering Martin J. , Moran Gary P. , Chauvel Murielle , MacCallum Donna M. , Higgins Judy , Hokamp Karsten , Yeomans Tim , d'Enfert Christophe , Coleman David C. , Sullivan Derek J.
Published: December 2009

Candida albicans
and
Candida dubliniensis
are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel
C. albicans
virulence genes.
C. albicans
gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE).
C. albicans
(SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage.
C. dubliniensis
(CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in
C. dubliniensis
were upregulated in
C. albicans
. One such gene,
SFL2
(
orf19.3969
), encoding a putative heat shock factor, was deleted in
C. albicans
. ΔΔ
sfl2
cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of
SFL2
into the ΔΔ
sfl2
strain. Moreover,
SFL2
overexpression in
C. albicans
triggered hyphal morphogenesis. Although
SFL2
deletion had no apparent effect on host survival in the murine model of systemic infection, ΔΔ
sfl2
strain-infected kidney tissues contained only yeast cells. These results suggest a role for
SFL2
in morphogenesis and an indirect role in
C. albicans
pathogenesis in epithelial tissues.
Hgc1 Mediates Dynamic Candida albicans-Endothelium Adhesion Events during Circulation
Authors: Wilson Duncan , Hube Bernhard
Published: December 2009

Common iatrogenic procedures can result in translocation of the human pathogenic fungus
Candida albicans
from mucosal surfaces to the bloodstream. Subsequent disseminated candidiasis and infection of deep-seated organs may occur if the fungus is not eliminated by blood cells. In these cases, fungal cells adhere to the endothelial cells of blood vessels, penetrate through endothelial layers, and invade deeper tissue. In this scenario, endothelial adhesion events must occur during circulation under conditions of physiological blood pressure. To investigate the fungal and host factors which contribute to this essential step of disseminated candidiasis, we have developed an
in vitro
circulatory
C. albicans
-endothelium interaction model. We demonstrate that both
C. albicans
yeast and hyphae can adhere under flow at a pressure similar to capillary blood pressure. Serum factors significantly enhanced the adhesion potential of viable but not killed
C. albicans
cells to endothelial cells. During circulation,
C. albicans
cells produced hyphae and the adhesion potential first increased, then decreased with time. We provide evidence that a specific temporal event in the yeast-to-hyphal transition, regulated by the G
1
cyclin Hgc1, is critical for
C. albicans
-endothelium adhesion during circulation.
Phospholipid-Binding Protein EhC2A Mediates Calcium-Dependent Translocation of Transcription Factor URE3-BP to the Plasma Membrane of
Entamoeba histolytica
Authors: Moreno Heriberto , Linford Alicia S. , Gilchrist Carol A. , Petri William A.
Published: December 2009

The
Entamoeba histolytica
upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds DNA in a Ca
2+
-inhibitable manner. The protein is located in both the nucleus and the cytoplasm but has also been found to be enriched in the plasma membrane of amebic trophozoites. We investigated the reason for the unusual localization of URE3-BP at the amebic plasma membrane. Here we identify and characterize a 22-kDa Ca
2+
-dependent binding partner of URE3-BP, EhC2A, a novel member of the C2-domain superfamily. Immunoprecipitations of URE3-BP and EhC2A showed that the proteins interact and that such interaction was enhanced in the presence of Ca
2+
. Recombinant and native EhC2A bound phospholipid liposomes in a Ca
2+
-dependent manner, with half-maximal binding occurring at 3.4 μM free Ca
2+
. A direct interaction between EhC2A and URE3-BP was demonstrated by the ability of recombinant EhC2A to recruit recombinant URE3-BP to phospholipid liposomes in a Ca
2+
-dependent manner. URE3-BP and EhC2A were observed to translocate to the amebic plasma membrane upon an increase in the intracellular Ca
2+
concentration of trophozoites, as revealed by subcellular fractionation and immunofluorescent staining. Short hairpin RNA-mediated knockdown of EhC2A protein expression significantly modulated the mRNA levels of URE3-BP-regulated transcripts. Based on these results, we propose a model for EhC2A-mediated regulation of the transcriptional activities of URE3-BP via Ca
2+
-dependent anchoring of the transcription factor to the amebic plasma membrane.
Nucleases Encoded by the Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of
Campylobacter jejuni
Authors: Gaasbeek Esther J. , Wagenaar Jaap A. , Guilhabert Magalie R. , van Putten Jos P. M. , Parker Craig T. , van der Wal Fimme J.
Published: December 2009

The species
Campylobacter jejuni
is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by
dns
, inhibits natural transformation in
C. jejuni
. In the present study, genetic factors coding for DNase activity in the absence of
dns
were identified. DNA arrays indicated that nonnaturally transformable
dns
-negative strains contain putative DNA/RNA nonspecific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on
C. jejuni
integrated elements 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative
Escherichia coli
and
C. jejuni
strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable
C. jejuni
strain resulted in a decreased efficiency of natural transformation. Modeling suggests that the
C. jejuni
DNases belong to the
Serratia
nuclease family. Overall, the data indicate that the acquisition of prophage-encoded DNA/RNA nonspecific endonucleases inhibits the natural transformability of
C. jejuni
through hydrolysis of DNA.
The MG1363 and IL1403 Laboratory Strains of
Lactococcus lactis
and Several Dairy Strains Are Diploid
Authors: Michelsen Ole , Hansen Flemming G. , Albrechtsen Bjarne , Jensen Peter Ruhdal
Published: December 2009

Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of
Lactococcus lactis
, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.
Genome-Wide Identification of Targets for the Archaeal Heat Shock Regulator Phr by Cell-Free Transcription of Genomic DNA
Authors: Keese Annette M. , Schut Gerrit J. , Ouhammouch Mohamed , Adams Michael W. W. , Thomm Michael
Published: December 2009

The hyperthermophilic archaeon
Pyrococcus furiosus
grows optimally near 100°C and undergoes a heat shock response at 105°C, mediated at least in part by the heat shock regulator Phr. Genes encoding a small heat shock protein (HSP20) and a member of the AAA
+
ATPase are the only known targets of the regulator, but a genetic mutant of Phr has yet to be characterized. We describe here an alternative approach for the identification of the regulon of Phr based on cell-free transcription of fragmented chromosomal DNA in the presence or absence of the regulator and hybridization of
in vitro
RNA to
P. furiosus
whole-genome microarrays. Our results confirmed the
phr
, the
hsp20
, and the
aaa
+
ATPase genes as targets of Phr and also identified six additional open reading frames, PF0624, PF1042, PF1291, PF1292, PF1488, and PF1616, as Phr-responsive genes, which include that encoding di-myo-inositol phosphate synthase. Transcription of the identified novel genes was inhibited by Phr in standard transcription assays, and the novel consensus sequence 5′-TTTAnnnACnnnnnGTnAnnAAAA-3′ (uppercase letters denote a high conservation of the bases) was inferred from our data as the Phr recognition motif. Mutational evidence for the significance of this sequence as Phr recognition was provided in DNA-binding experiments.
The
Mycobacterium tuberculosis
DosR Regulon Assists in Metabolic Homeostasis and Enables Rapid Recovery from Nonrespiring Dormancy
Authors: Leistikow Rachel L. , Morton Russell A. , Bartek Iona L. , Frimpong Isaac , Wagner Karleen , Voskuil Martin I.
Published: December 2009

Mycobacterium tuberculosis
survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The
M. tuberculosis
DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for
M. tuberculosis
survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once
M. tuberculosis
exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for
M. tuberculosis
to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability.
Rhizobium leguminosarum hupE
Encodes a Nickel Transporter Required for Hydrogenase Activity
Authors: Brito Belén , Prieto Rosa-Isabel , Cabrera Ezequiel , Mandrand-Berthelot Marie-Andrée , Imperial Juan , Ruiz-Argüeso Tomás , Palacios José-Manuel
Published: December 2009

Synthesis of the hydrogen uptake (Hup) system in
Rhizobium leguminosarum
bv.
viciae
requires the function of an 18-gene cluster (
hupSLCDEFGHIJK
-
hypABFCDEX
). Among them, the
hupE
gene encodes a protein showing six transmembrane domains for which a potential role as a nickel permease has been proposed. In this paper, we further characterize the nickel transport capacity of HupE and that of the translated product of
hupE2
, a hydrogenase-unlinked gene identified in the
R. leguminosarum
genome. HupE2 is a potential membrane protein that shows 48% amino acid sequence identity with HupE. Expression of both genes in the
Escherichia coli nikABCDE
mutant strain HYD723 restored hydrogenase activity and nickel transport. However, nickel transport assays revealed that HupE and HupE2 displayed different levels of nickel uptake. Site-directed mutagenesis of histidine residues in HupE revealed two motifs (HX
5
DH and FHGX[AV]HGXE) that are required for HupE functionality. An
R. leguminosarum
double mutant, SPF22A (
hupE hupE2
), exhibited reduced levels of hydrogenase activity in free-living cells, and this phenotype was complemented by nickel supplementation. Low levels of symbiotic hydrogenase activity were also observed in SPF22A bacteroid cells from lentil (
Lens culinaris
L.) root nodules but not in pea (
Pisum sativum
L.) bacteroids. Moreover, heterologous expression of the
R. leguminosarum hup
system in bacteroid cells of
Rhizobium tropici
and
Mesorhizobium loti
displayed reduced levels of hydrogen uptake in the absence of
hupE
. These data support the role of
R. leguminosarum
HupE as a nickel permease required for hydrogen uptake under both free-living and symbiotic conditions.
Purification of
Helicobacter pylori
NCTC 11637 Cytochrome
bc
1
and Respiration with
d
-Proline as a Substrate
Authors: Tanigawa Minoru , Shinohara Tomomitsu , Nishimura Katsushi , Nagata Kumiko , Ishizuka Morio , Nagata Yoko
Published: December 2009

Helicobacter pylori
is a microaerophilic bacterium associated with gastric inflammation and peptic ulcers. Knowledge of how pathogenic organisms produce energy is important from a therapeutic point of view. We found
d
-amino acid dehydrogenase-mediated electron transport from
d
-proline or
d
-alanine to oxygen via the respiratory chain in
H. pylori
. Coupling of the electron transport to ATP synthesis was confirmed by using uncoupler reagents. We reconstituted the electron transport chain to demonstrate the electron flow from the
d
-amino acids to oxygen using the recombinant cytochrome
bc
1
complex, cytochrome
c
-553, and the terminal oxidase cytochrome
cbb
3
complex. Upon addition of the recombinant
d
-amino acid dehydrogenase and
d
-proline or
d
-alanine to the reconstituted electron transport system, reduction of cytochrome
cbb
3
and oxygen consumption was revealed spectrophotometrically and polarographically, respectively. Among the constituents of
H. pylori
's electron transport chain, only the cytochrome
bc
1
complex had been remained unpurified. Therefore, we cloned and sequenced the
H. pylori
NCTC 11637 cytochrome
bc
1
gene clusters encoding Rieske Fe-S protein, cytochrome
b
, and cytochrome
c
1
, with calculated molecular masses of 18 kDa, 47 kDa, and 32 kDa, respectively, and purified the recombinant monomeric protein complex with a molecular mass of 110 kDa by gel filtration. The absorption spectrum of the recombinant cytochrome
bc
1
complex showed an α peak at 561 nm with a shoulder at 552 nm.
Characterization of CamH from
Methanosarcina thermophila
, Founding Member of a Subclass of the γ Class of Carbonic Anhydrases
Authors: Zimmerman Sabrina A. , Tomb Jean-Francois , Ferry James G.
Published: December 2009

The homotrimeric enzyme Mt-Cam from
Methanosarcina thermophila
is the archetype of the γ class of carbonic anhydrases. A search of databases queried with Mt-Cam revealed that a majority of the homologs comprise a putative subclass (CamH) in which there is major conservation of all of the residues essential for the archetype Mt-Cam except Glu62 and an acidic loop containing the essential proton shuttle residue Glu84. The CamH homolog from
M. thermophila
(Mt-CamH) was overproduced in
Escherichia coli
and characterized to validate its activity and initiate an investigation of the CamH subclass. The Mt-CamH homotrimer purified from
E. coli
cultured with supplemental zinc (Zn-Mt-CamH) contained 0.71 zinc and 0.15 iron per monomer and had
k
cat
and

k
cat

/

K
m

values that were substantially lower than those for the zinc form of Mt-Cam (Zn-Mt-Cam). Mt-CamH purified from
E. coli
cultured with supplemental iron (Fe-Mt-CamH) was also a trimer containing 0.15 iron per monomer and only a trace amount of zinc and had an effective
k
cat
(
k
cat
eff
) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the
k
cat
/

K
m

eff
was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the
k
cat
eff
of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H
2
O
2
, which supports the hypothesis that Fe
2+
has a role in the active site. The
k
cat
for Fe-Mt-CamH was dependent on the concentration of buffer in a way that indicates that it acts as a second substrate in a “ping-pong” mechanism accepting a proton. The
k
cat
/

K
m

was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO
2
and HCO
3
−
is separate from intermolecular proton transfer.
Characterization of a Glutamate Transporter Operon,
glnQHMP
, in
Streptococcus mutans
and Its Role in Acid Tolerance
Authors: Krastel Kirsten , Senadheera Dilani B. , Mair Richard , Downey Jennifer S. , Goodman Steven D. , Cvitkovitch Dennis G.
Published: December 2009

Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium
Streptococcus mutans
is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated
glnQHMP
(Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of
glnQHMP
resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the
glnQHMP
mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the
glnQHMP
operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its
glnQHMP
-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon
glnQHMP
in the acid tolerance response in
S. mutans
.