bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interactions between Food-Borne Pathogens and Protozoa Isolated from Lettuce and Spinach
Authors: Gourabathini Poornima , Brandl Maria T. , Redding Katherine S. , Gunderson John H. , Berk Sharon G. 
Published: March 2008

ABSTRACT

The survival of
Salmonella enterica
was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of
Tetrahymena
. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with
S. enterica, Escherichia coli
O157:H7, and
Listeria monocytogenes. Glaucoma
sp.,
Colpoda steinii
, and
Acanthamoeba palestinensis
were cultured from store-bought spinach and lettuce and used in our study. A strain of
Tetrahymena pyriformis
previously isolated from spinach and a soil-borne
Tetrahymena
sp. were also used. Washed protozoa were allowed to graze on green fluorescent protein- or red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by
Glaucoma
with all of the bacterial strains, although
L. monocytogenes
resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of
Tetrahymena
on
E. coli
O157:H7 and
S. enterica
but not during grazing on
L. monocytogenes
, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast,
C. steinii
and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of
E. coli
O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.
Taxonomic Structure and Stability of the Bacterial Community in Belgian Sourdough Ecosystems as Assessed by Culture and Population Fingerprinting
Authors: Scheirlinck Ilse , Van der Meulen Roel , Van Schoor Ann , Vancanneyt Marc , De Vuyst Luc , Vandamme Peter , Huys Geert 
Published: March 2008

ABSTRACT

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and
pheS
genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species
Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum
, and
Lactobacillus pontis
dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of
Acetobacter
sp. and a member of the
Erwinia
/
Enterobacter
/
Pantoea
group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.
Dramatic Activation of Antibiotic Production in
Streptomyces coelicolor
by Cumulative Drug Resistance Mutations
Authors: Wang Guojun , Hosaka Takeshi , Ochi Kozo 
Published: March 2008

ABSTRACT

We recently described a new method to activate antibiotic production in bacteria by introducing a mutation conferring resistance to a drug such as streptomycin, rifampin, paromomycin, or gentamicin. This method, however, enhanced antibiotic production by only up to an order of magnitude. Working with
Streptomyces coelicolor
A3(2), we established a method for the dramatic activation of antibiotic production by the sequential introduction of multiple drug resistance mutations. Septuple and octuple mutants, C7 and C8, thus obtained by screening for resistance to seven or eight drugs, produced huge amounts (1.63 g/liter) of the polyketide antibiotic actinorhodin, 180-fold higher than the level produced by the wild type. This dramatic overproduction was due to the acquisition of mutant ribosomes, with aberrant protein and ppGpp synthesis activity, as demonstrated by in vitro protein synthesis assays and by the abolition of antibiotic overproduction with
relA
disruption. This new approach, called “ribosome engineering,” requires less time, cost, and labor than other methods and may be widely utilized for bacterial strain improvement.
Development of a Novel Screening Method for the Isolation of “
Cronobacter
” spp. (
Enterobacter sakazakii
)
Authors: Iversen Carol , Druggan Patrick , Schumacher Sandra , Lehner Angelika , Feer Claudia , Gschwend Karl , Joosten Han , Stephan Roger 
Published: March 2008

ABSTRACT

A differential medium, “
Cronobacter
” screening broth, has been designed to complement agars based on hydrolysis of chromogenic α-glucopyranoside substrates. The broth was evaluated using 329
Enterobacteriaceae
strains (229 target isolates), spiked/naturally contaminated samples, and a parallel comparison with current methods for raw materials, line/end products, and factory environment samples.
Indirect Transmission of
Escherichia coli
O157:H7 Occurs Readily among Swine but Not among Sheep
Authors: Cornick Nancy A. , VuKhac Hung 
Published: March 2008

ABSTRACT

Transmission of
Escherichia coli
O157:H7 among reservoir animals is generally thought to occur either by direct contact between a naïve animal and an infected animal or by consumption of food or water containing the organism. Although ruminants are considered the major reservoir, there are two reports of human infections caused by
E. coli
O157:H7 linked to the consumption of pork products or to the contamination of fresh produce by swine manure. The objective of this study was to determine whether
E. coli
O157:H7 could be transmitted to naïve animals, both sheep and swine, that did not have any direct contact with an infected donor animal. We recovered
E. coli
O157:H7 from 10/10 pigs with nose-to-nose contact with the infected donor or animals adjacent to the donor and from 5/6 naïve pigs that were penned in the same room as the donor pig but 10 to 20 ft away. In contrast, when the experiment was repeated with sheep,
E. coli
O157:H7 was recovered from 4/6 animals that had nose-to-nose contact with the infected donor or adjacent animals and from 0/6 naïve animals penned 10 to 20 ft away from the donor. These results suggest that
E. coli
O157:H7 is readily transmitted among swine and that transmission can occur by the creation of contaminated aerosols.
Targeted Inactivation ofFrancisella tularensisGenes by Group II Introns
Authors: Rodriguez Stephen A. , Yu Jieh-Juen , Davis Greg , Arulanandam Bernard P. , Klose Karl E. 
Published: March 2008

ABSTRACTStudies of the molecular mechanisms of pathogenesis ofFrancisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption inF. tularensisutilizing mobile group II introns (targetrons) specifically optimized forF. tularensis. Utilizing a targetron targeted toblaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies:F. tularensissubsp.tularensis, F. tularensissubsp.holarctica, and “F. tularensissubsp.novicida.” A targetron was also utilized to inactivateF. tularensissubsp.holarctica iglC, a gene required for virulence. TheiglCgene is located within theFrancisellapathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, theiglCtargetron targeted both copies simultaneously, resulting in a strain mutated in bothiglCgenes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.
Identification of Hexose Transporter-Like Sensor
HXS1
and Functional Hexose Transporter
HXT1
in the Methylotrophic Yeast
Hansenula polymorpha
Authors: Stasyk Olena G. , Maidan Mykola M. , Stasyk Oleh V. , Van Dijck Patrick , Thevelein Johan M. , Sibirny Andriy A. 
Published: March 2008

ABSTRACT

We identified in the methylotrophic yeast
Hansenula polymorpha
(syn.
Pichia angusta
) a novel hexose transporter homologue gene,
HXS1
(
h
e
x
ose
s
ensor), involved in transcriptional regulation in response to hexoses, and a regular hexose carrier gene,
HXT1
(
h
e
x
ose
t
ransporter). The Hxs1 protein exhibits the highest degree of primary sequence similarity to the
Saccharomyces cerevisiae
transporter-like glucose sensors, Snf3 and Rgt2. When heterologously overexpressed in an
S. cerevisiae
hexose transporter-less mutant, Hxt1, but not Hxs1, restores growth on glucose or fructose, suggesting that Hxs1 is nonfunctional as a carrier. In its native host,
HXS1
is expressed at moderately low level and is required for glucose induction of the
H. polymorpha
functional low-affinity glucose transporter Hxt1. Similarly to other yeast sensors, one conserved amino acid substitution in the Hxs1 sequence (R203K) converts the protein into a constitutively signaling form and the C-terminal region of Hxs1 is essential for its function in hexose sensing. Hxs1 is not required for glucose repression or catabolite inactivation that involves autophagic degradation of peroxisomes. However,
HXS1
deficiency leads to significantly impaired transient transcriptional repression in response to fructose, probably due to the stronger defect in transport of this hexose in the
hxs1
Δ deletion strain. Our combined results suggest that in the Crabtree-negative yeast
H. polymorpha
, the single transporter-like sensor Hxs1 mediates signaling in the hexose induction pathway, whereas the rate of hexose uptake affects the strength of catabolite repression.
Unconventional Genomic Architecture in the Budding Yeast
Saccharomyces cerevisiae
Masks the Nested Antisense Gene
NAG1
Authors: Ma Jun , Dobry Craig J. , Krysan Damian J. , Kumar Anuj 
Published: March 2008

ABSTRACT

The genomic architecture of the budding yeast
Saccharomyces cerevisiae
is typical of other eukaryotes in that genes are spatially organized into discrete and nonoverlapping units. Inherent in this organizational model is the assumption that protein-coding sequences do not overlap completely. Here, we present evidence to the contrary, defining a previously overlooked yeast gene,
NAG1
(for
n
ested
a
ntisense
g
ene) nested entirely within the coding sequence of the YGR031W open reading frame in an antisense orientation on the opposite strand.
NAG1
encodes a 19-kDa protein, detected by Western blotting of hemagglutinin (HA)-tagged Nag1p with anti-HA antibodies and by β-galactosidase analysis of a
NAG1-lacZ
fusion.
NAG1
is evolutionarily conserved as a unit with YGR031W in bacteria and fungi. Unlike the YGR031WP protein product, however, which localizes to the mitochondria, Nag1p localizes to the cell periphery, exhibiting properties consistent with those of a plasma membrane protein. Phenotypic analysis of a site-directed mutant (
nag1
-
1
) disruptive for
NAG1
but silent with respect to YGR031W, defines a role for
NAG1
in yeast cell wall biogenesis; microarray profiling of
nag1
-
1
indicates decreased expression of genes contributing to cell wall organization, and the
nag1
-
1
mutant is hypersensitive to the cell wall-perturbing agent calcofluor white. Furthermore, production of Nag1p is dependent upon the presence of the cell wall integrity pathway mitogen-activated protein kinase Slt2p and its downstream transcription factor Rlm1p. Thus,
NAG1
is important for two reasons. First, it contributes to yeast cell wall biogenesis. Second, its genomic context is novel, raising the possibility that other nested protein-coding genes may exist in eukaryotic genomes.
Microneme Rhomboid Protease TgROM1 Is Required for Efficient Intracellular Growth of
Toxoplasma gondii
Authors: Brossier Fabien , Starnes G. Lucas , Beatty Wandy L. , Sibley L. David 
Published: March 2008

ABSTRACT

Rhomboids are serine proteases that cleave their substrates within the transmembrane domain.
Toxoplasma gondii
contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments.
Toxoplasma
rhomboid protein 1 (TgROM1) has previously been shown to be active in vitro, and the orthologue in
Plasmodium falciparum
processes the essential microneme protein AMA1 in a heterologous system. We investigated the role of TgROM1 to determine its role during in vitro growth of
T. gondii
. TgROM1 was localized in the secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contain adhesive proteins involved in invasion of host cells. However, unlike other micronemal proteins, TgROM1 was not released onto the parasite surface during cell invasion, suggesting it does not play a critical role in cell invasion. Suppression of
TgROM1
using the tetracycline-regulatable system revealed that ROM1-deficient parasites were outcompeted by wild-type
T. gondii
. ROM1-deficient parasites showed only modest decrease in invasion but replicated more slowly than wild-type cells. Collectively, these results indicate that ROM1 is required for efficient intracellular growth by
T. gondii
.
Anastomosis Is Required for Virulence of the Fungal NecrotrophAlternaria brassicicola
Authors: Craven Kelly D. , Vélëz Heriberto , Cho Yangrae , Lawrence Christopher B. , Mitchell Thomas K. 
Published: March 2008

ABSTRACTA fungal mycelium is typically composed of radially extending hyphal filaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination of nutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogenAlternaria brassicicolaand two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of theA. brassicicolaisolates tested, as well asA. mimiculaandA. japonica, were capable of self-fusion, with two isolates ofA. brassicicolabeing capable of non-self-fusion. Disruption of the anastomosis gene homolog (Aso1) inA. brassicicolaresulted in both the loss of self-anastomosis and pathogenicity on cabbage. This finding, combined with our discovery that a previously described nonpathogenicA. brassicicolamutant defective for a mitogen-activated protein kinase gene (amk1) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity inA. brassicicola.
The Peptidoglycan-Associated Lipoprotein OprL Helps Protect a
Pseudomonas aeruginosa
Mutant Devoid of the Transactivator OxyR from Hydrogen Peroxide-Mediated Killing during Planktonic and Biofilm Culture
Authors: Panmanee Warunya , Gomez Francisco , Witte David , Pancholi Vijay , Britigan Bradley E. , Hassett Daniel J. 
Published: March 2008

ABSTRACT

OxyR controls H
2
O
2
-dependent gene expression in
Pseudomonas aeruginosa
. Without OxyR, diluted (<10
7
/ml) organisms are easily killed by micromolar H
2
O
2
. The goal of this study was to define proteins that contribute to
oxyR
mutant survival in the presence of H
2
O
2
. We identified proteins in an
oxyR
mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double
oxyR oprL
mutant was constructed and was found to be more sensitive to H
2
O
2
than the
oxyR
mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H
2
O
2
. Given the sensitivity of
oxyR oprL
bacteria to planktonic H
2
O
2
, we next tested the hypothesis that the biofilm mode of growth might protect such organisms from H
2
O
2
-mediated killing. Surprisingly, biofilm-grown
oxyR oprL
mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the
oxyR
mutant, were sensitive to killing by as little as 0.5 mM H
2
O
2
. Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of
oxyR
and
oxyR oprL
mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H
2
O
2
in the exquisitely sensitive
oxyR
mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofilms.
aro
Mutations in
Salmonella enterica
Cause Defects in Cell Wall and Outer Membrane Integrity
Authors: Sebkova Alena , Karasova Daniela , Crhanova Magdalena , Budinska Eva , Rychlik Ivan 
Published: March 2008

ABSTRACT

In this study we characterized
aro
mutants of
Salmonella enterica
serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the
aroA
,
aroD
, and
aroC
mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate
aro
gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the
aroD
mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of
murA
. To confirm this, we artificially overexpressed
murA
from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of
aro
mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response.
Identification of AglE, a Second Glycosyltransferase Involved in N Glycosylation of the
Haloferax volcanii
S-Layer Glycoprotein
Authors: Abu-Qarn Mehtap , Giordano Assunta , Battaglia Francesca , Trauner Andrej , Hitchen Paul G. , Morris Howard R. , Dell Anne , Eichler Jerry 
Published: March 2008

ABSTRACT

Archaea
, like
Eukarya
and
Bacteria
, are able to N glycosylate select protein targets. However, in contrast to relatively advanced understanding of the eukaryal N glycosylation process and the information being amassed on the bacterial process, little is known of this posttranslational modification in
Archaea
. Toward remedying this situation, the present report continues ongoing efforts to identify components involved in the N glycosylation of the
Haloferax volcanii
S-layer glycoprotein. By combining gene deletion together with mass spectrometry, AglE, originally identified as a homologue of murine Dpm1, was shown to play a role in the addition of the 190-Da sugar subunit of the novel pentasaccharide decorating the S-layer glycoprotein. Topological analysis of an AglE-based chimeric reporter assigns AglE as an integral membrane protein, with its N terminus and putative active site facing the cytoplasm. These finding, therefore, contribute to the developing picture of the N glycosylation pathway in
Archaea
.
Identification of Novel Genes and Pathways Affecting
Salmonella
Type III Secretion System 1 Using a Contact-Dependent Hemolysis Assay
Authors: Field Terry R. , Layton Abigail N. , Bispham Jennie , Stevens Mark P. , Galyov Edouard E. 
Published: March 2008

ABSTRACT

We screened 5,700
Salmonella enterica
serovar Typhimurium mutants for defects in type III secretion system 1 (T3SS-1)-mediated contact-dependent hemolysis to identify novel genes and pathways affecting the activity of T3SS-1. Our data suggest that previously unrecognized factors such as type I fimbriae may modulate the expression, activity, or deployment of this key virulence factor.
SOS Induction by Stabilized Topoisomerase IA Cleavage Complex Occurs via the RecBCD Pathway
Authors: Sutherland Jeanette H. , Cheng Bokun , Liu I-Fen , Tse-Dinh Yuk-Ching 
Published: March 2008

ABSTRACT

Accumulation of mutant topoisomerase I cleavage complex can lead to SOS induction and cell death in
Escherichia coli
. The single-stranded break associated with mutant topoisomerase I cleavage complex is converted to double-stranded break, which then is processed by the RecBCD pathway, followed by association of RecA with the single-stranded DNA.
Functional Mapping of an Oligomeric Autotransporter Adhesin of
Aggregatibacter actinomycetemcomitans
Authors: Yu Chunxiao , Ruiz Teresa , Lenox Christopher , Mintz Keith P. 
Published: March 2008

ABSTRACT

Extracellular matrix protein adhesin A (EmaA) is a 202-kDa nonfimbrial adhesin, which mediates the adhesion of the oral pathogen
Aggregatibacter actinomycetemcomitans
to collagen. EmaA oligomers form surface antenna-like protrusions consisting of a long helical rod with an ellipsoidal ending. The functional analysis of in-frame
emaA
deletion mutants has located the collagen binding activity to the amino terminus of the protein corresponding to amino acids 70 to 386. The level of collagen binding of this deletion mutant was comparable to the
emaA
mutant strain. Transmission electron microscopy studies indicate that the first 330 amino acids of the mature protein form the ellipsoidal ending of the EmaA protrusions, where the activity resides. Amino acid substitution analysis within this sequence has identified a critical amino acid, which is essential for the formation of the ellipsoidal ending and for collagen binding activity.