bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evolution of the Stx2-Encoding Prophage in Persistent Bovine Escherichia coli O157:H7 Strains
Authors: Park Dongjin , Stanton Eliot , Ciezki Kristin , Parrell Daniel , Bozile Matthew , Pike Daniel , Forst Steven A. , Jeong Kwang Cheol , Ivanek Renata , Döpfer Dörte , Kaspar Charles W. 
Published: December 2012

ABSTRACT

Escherichia coli
O157:H7 is a human pathogen that resides asymptomatically in its bovine host. The level of Shiga toxin (Stx) produced is variable in bovine-derived strains in contrast to human isolates that mostly produce high levels of Stx. To understand the genetic basis for varied Stx production, chronological collections of bovine isolates from Wisconsin dairy farms, R and X, were analyzed for multilocus prophage polymorphisms,
stx
2
subtypes, and the levels of
stx
2
transcript and toxin. The
E. coli
O157:H7 that persisted on both farms were phylogenetically distinct and yet produced little to no Stx2 due to gene deletions in Stx2c-encoding prophage (farm R) or insertional inactivation of
stx
2a
by IS
1203v
(farm X). Loss of key regulatory and lysis genes in Stx2c-encoding prophage abolished
stx
2c
transcription and induction of the prophage and
stx
2a
::IS
1203v
in Stx2a-encoding prophage generated a truncated
stx
2a
mRNA without affecting phage production. Stx2-producing strains were transiently present (farm R) and became Stx2 negative on farm X (i.e.,
stx
2a
::IS
1203v
). To our knowledge, this is the first study that details the evolution of
E. coli
O157:H7 and its Stx2-encoding prophage in a chronological collection of natural isolates. The data suggest the bovine and farm environments can be niches where Stx2-negative
E. coli
O157:H7 emerge and persist, which explains the Stx variability in bovine isolates and may be part of an evolutionary step toward becoming bovine specialists.
Carbon Isotope Fractionation of 11 Acetogenic Strains Grown on H
2
and CO
2
Authors: Blaser Martin B. , Dreisbach Lisa K. , Conrad Ralf 
Published: December 2012

ABSTRACT

Acetogenic bacteria are able to grow autotrophically on hydrogen and carbon dioxide by using the acetyl coenzyme A (acetyl-CoA) pathway. Acetate is the end product of this reaction. In contrast to the fermentative route of acetate production, which shows almost no fractionation of carbon isotopes, the acetyl-CoA pathway has been reported to exhibit a preference for light carbon. In
Acetobacterium woodii
the isotope fractionation factor (ε) for
13
C and
12
C has previously been reported to be ε = −58.6‰. To investigate whether such a strong fractionation is a general feature of acetogenic bacteria, we measured the stable carbon isotope fractionation factor of 10 acetogenic strains grown on H
2
and CO
2
. The average fractionation factor was ε
TIC
= −57.2‰ for utilization of total inorganic carbon and ε
acetate
= −54.6‰ for the production of acetate. The strongest fractionation was found for
Sporomusa sphaeroides
(ε
TIC
= −68.3‰), the lowest fractionation for
Morella thermoacetica
(ε
TIC
= −38.2‰). To investigate the reproducibility of our measurements, we determined the fractionation factor of 21 biological replicates of
Thermoanaerobacter kivui
. In general, our study confirmed the strong fractionation of stable carbon during chemolithotrophic acetate formation in acetogenic bacteria. However, the specific characteristics of the bacterial strain, as well as the cultural conditions, may have a moderate influence on the overall fractionation.
MiniUIB, a Novel Minitransposon-Based System for Stable Insertion of Foreign DNA into the Genomes of Gram-Negative and Gram-Positive Bacteria
Authors: Christie-Oleza Joseph Alexander , Brunet-Galmés Isabel , Lalucat Jorge , Nogales Balbina , Bosch Rafael 
Published: December 2012

ABSTRACT

Transposition of the insertion sequence (IS) IS
Ppu12
is actively induced after conjugative interaction. The transposase of this IS can act in
trans
on structures flanked by inverted repeats similar to those of the transposon. Based on that fact, an IS
Ppu12
-based minitransposon, miniUIB, has been constructed in order to biotechnologically exploit the self-regulation of IS
Ppu12
and its increased activity after conjugative interaction. Mobilization of the miniUIB structure into the genome of
Pseudomonas stutzeri
AN10 after conjugative interaction was demonstrated. A single gene, i.e., the kanamycin resistance determinant, or large genetic structures of >12 kb, i.e.,
alkBFGHJKL
and
alkST
operons of
Pseudomonas putida
TF4-1L (GPo1), have been easily integrated in
P. stutzeri
AN10 by an RP4-based delivery system. Therefore, the integration of the
alk
determinants by use of the miniUIB system has extended the biodegradation capabilities of this strain. Plasmid pJOC100, containing the transposase and regulator genes of IS
Ppu12
adjacent to the miniUIB structure, was constructed in order to extend the host range of this biotechnologically useful genetic tool to other model and real-world bacteria. The effectiveness of the system for random mutagenesis in a phylogenetic wide range of bacteria and for the insertion of novel functions has been demonstrated, even in successive steps.
Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase
Authors: Parker Josie E. , Warrilow Andrew G. S. , Cools Hans J. , Fraaije Bart A. , Lucas John A. , Rigdova Katarina , Griffiths William J. , Kelly Diane E. , Kelly Steven L. 
Published: December 2012

ABSTRACT

Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used
Candida albicans
CYP51 (CaCYP51) to investigate the
in vitro
activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of
C. albicans
cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from
C. albicans
grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.
Phytophthora infestans Cholinephosphotransferase with Substrate Specificity for Very-Long-Chain Polyunsaturated Fatty Acids
Authors: Chen Yan , Chi Hsiang-yun , Meesapyodsuk Dauenpen , Qiu Xiao 
Published: December 2012

ABSTRACT

The effective flux between phospholipids and neutral lipids is critical for a high level of biosynthesis and accumulation of very-long-chain polyunsaturated fatty acids (VLCPUFAs), such as arachidonic acid (ARA; 20:4n-6), eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic acid (DHA; 22:6n-3). Here we describe a cDNA (
PiCPT1
) from
Phytophthora infestans
, a VLCPUFA-producing oomycete, that may have a role in acyl trafficking between diacylglycerol (DAG) and phosphatidylcholine (PC) during the biosynthesis of VLCPUFAs. The cDNA encodes a polypeptide of 393 amino acids with a conserved CDP-alcohol phosphotransferase motif and approximately 27% amino acid identity to the
Saccharomyces cerevisiae
cholinephosphotransferase (ScCPT1).
In vitro
assays indicate that PiCPT1 has high cholinephosphotransferase (CPT) activity but no ethanolaminephosphotransferase (EPT) activity. Substrate specificity assays show that it prefers VLCPUFA-containing DAGs, such as ARA DAG and DHA DAG, as substrates. Real-time PCR analysis reveals that expression of
PiCPT1
was upregulated in
P. infestans
organisms fed with exogenous VLCPUFAs. These results lead us to conclude that PiCPT1 is a VLCPUFA-specific CPT which may play an important role in shuffling VLCPUFAs from DAG to PC in the biosynthesis of VLCPUFAs in
P. infestans
.
Molecular Analysis of theIn SituGrowth Rates of Subsurface Geobacter Species
Authors: Holmes Dawn E. , Giloteaux Ludovic , Barlett Melissa , Chavan Milind A. , Smith Jessica A. , Williams Kenneth H. , Wilkins Michael , Long Philip , Lovley Derek R. 
Published: December 2012

ABSTRACTMolecular tools that can provide an estimate of thein situgrowth rate ofGeobacterspecies could improve understanding of dissimilatory metal reduction in a diversity of environments. Whole-genome microarray analyses of a subsurface isolate ofGeobacter uraniireducens, grown under a variety of conditions, identified a number of genes that are differentially expressed at different specific growth rates. Expression of two genes encoding ribosomal proteins,rpsCandrplL, was further evaluated with quantitative reverse transcription-PCR (qRT-PCR) in cells with doubling times ranging from 6.56 h to 89.28 h. Transcript abundance ofrpsCcorrelated best (r2= 0.90) with specific growth rates. Therefore, expression patterns ofrpsCwere used to estimate specific growth rates ofGeobacterspecies during anin situuranium bioremediation field experiment in which acetate was added to the groundwater to promote dissimilatory metal reduction. Initially, increased availability of acetate in the groundwater resulted in higher expression ofGeobacter rpsC, and the increase in the number ofGeobactercells estimated with fluorescentin situhybridization compared well with specific growth rates estimated from levels ofin situ rpsCexpression. However, in later phases, cell number increases were substantially lower than predicted fromrpsCtranscript abundance. This change coincided with a bloom of protozoa and increased attachment ofGeobacterspecies to solid phases. These results suggest that monitoringrpsCexpression may better reflect the actual rate thatGeobacterspecies are metabolizing and growing duringin situuranium bioremediation than changes in cell abundance.
Engineering Synechococcus elongatus PCC 7942 for Continuous Growth under Diurnal Conditions
Authors: McEwen Jordan T. , Machado Iara M. P. , Connor Michael R. , Atsumi Shota 
Published: December 2012

ABSTRACT

Synechococcus elongatus
strain PCC 7942 strictly depends upon the generation of photosynthetically derived energy for growth and is incapable of biomass increase in the absence of light energy. Obligate phototrophs' core metabolism is very similar to that of heterotrophic counterparts exhibiting diverse trophic behavior. Most characterized cyanobacterial species are obligate photoautotrophs under examined conditions. Here we determine that sugar transporter systems are the necessary genetic factors in order for a model cyanobacterium,
Synechococcus elongatus
PCC 7942, to grow continuously under diurnal (light/dark) conditions using saccharides such as glucose, xylose, and sucrose. While the universal causes of obligate photoautotrophy may be diverse, installing sugar transporters provides new insight into the mode of obligate photoautotrophy for cyanobacteria. Moreover, cyanobacterial chemical production has gained increased attention. However, this obligate phototroph is incapable of product formation in the absence of light. Thus, converting an obligate photoautotroph to a heterotroph is desirable for more efficient, economical, and controllable production systems.
Mycobacterium gilvum Illustrates Size-Correlated Relationships between Mycobacteria and Acanthamoeba polyphaga
Authors: Lamrabet Otmane , Drancourt Michel 
Published: December 2012

ABSTRACT

Mycobacteria are isolated from soil and water environments, where free-living amoebae live. Free-living amoebae are bactericidal, yet some rapidly growing mycobacteria are amoeba-resistant organisms that survive in the amoebal trophozoites and cysts. Such a capacity has not been studied for the environmental rapidly growing organism
Mycobacterium gilvum
. We investigated the ability of
M. gilvum
to survive in the trophozoites of
Acanthamoeba polyphaga
strain Linc-AP1 by using optical and electron microscopy and culture-based microbial enumerations in the presence of negative controls. We observed that 29% of
A. polyphaga
cells were infected by
M. gilvum
mycobacteria by 6 h postinfection. Surviving
M. gilvum
mycobacteria did not multiply and did not kill the amoebal trophozoites during a 5-day coculture. Extensive electron microscopy observations indicated that
M. gilvum
measured 1.4 ± 0.5 μm and failed to find
M. gilvum
organisms in the amoebal cysts. Further experimental study of two other rapidly growing mycobacteria,
Mycobacterium rhodesiae
and
Mycobacterium thermoresistibile
, indicated that both measured <2 μm and exhibited the same amoeba-mycobacterium relationships as
M. gilvum
. In general, we observed that mycobacteria measuring <2 μm do not significantly grow within and do not kill amoebal trophozoites, in contrast to mycobacteria measuring >2 μm (
P
< 0.05). The mechanisms underlying such an observation remain to be determined.
Role of Nitrogen Limitation in Transformation of RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine) by Gordonia sp. Strain KTR9
Authors: Indest Karl J. , Hancock Dawn E. , Jung Carina M. , Eberly Jed O. , Mohn William W. , Eltis Lindsay D. , Crocker Fiona H. 
Published: December 2012

ABSTRACT

The transcriptome of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)-degrading strain
Gordonia
sp. strain KTR9 and its
glnR
mutant were studied as a function of nitrogen availability to further investigate the observed ammonium-mediated inhibition of RDX degradation. The results indicate that nitrogen availability is a major determinant of RDX degradation and
xplA
gene expression in KTR9.
Sequencing and Functional Annotation of Avian Pathogenic Escherichia coli Serogroup O78 Strains Reveal the Evolution of E. coli Lineages Pathogenic for Poultry via Distinct Mechanisms
Authors: Dziva Francis , Hauser Heidi , Connor Thomas R. , van Diemen Pauline M. , Prescott Graham , Langridge Gemma C. , Eckert Sabine , Chaudhuri Roy R. , Ewers Christa , Mellata Melha , Mukhopadhyay Suman , Curtiss Roy , Dougan Gordon , Wieler Lothar H. , Thomson Nicholas R. , Pickard Derek J. , Stevens Mark P. 
Published: December 2012

ABSTRACT

Avian pathogenic
Escherichia coli
(APEC) causes respiratory and systemic disease in poultry. Sequencing of a multilocus sequence type 95 (ST95) serogroup O1 strain previously indicated that APEC resembles
E. coli
causing extraintestinal human diseases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains χ7122 and IMT2125) and compared them to each other and to the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenic
E. coli
(ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to human ST23 ETEC than to APEC O1, indicating that separation of pathotypes on the basis of their extraintestinal or diarrheagenic nature is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2,185 signature-tagged transposon mutants of χ7122 following intra-air sac inoculation of turkeys. This procedure identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for the virulence of χ7122 and were conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity of
E. coli
strains adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for the evolution of
E. coli
strains adapted to cause avian or human disease via acquisition of distinct virulence genes.
Disruption of Francisella tularensis Schu S4
iglI
,
iglJ
, and
pdpC
Genes Results in Attenuation for Growth in Human Macrophages and
In Vivo
Virulence in Mice and Reveals a Unique Phenotype for
pdpC
Authors: Long Matthew E. , Lindemann Stephen R. , Rasmussen Jed A. , Jones Bradley D. , Allen Lee-Ann H. 
Published: December 2012

ABSTRACT

Francisella tularensis
is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of
Francisella tularensis
subsp.
tularensis
strain Schu S4 mutants that lack functional
iglI
,
iglJ
, or
pdpC
, three genes of the
Francisella
pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The
iglI
and
iglJ
mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the
pdpC
mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by
trans
complementation.
In vivo
virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10
8
CFU
iglJ-
or
pdpC
-deficient bacteria. Nevertheless, the
pdpC
mutant disseminated to the liver and spleen before being eliminated, whereas the
iglJ
mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for
F. tularensis
Schu S4 virulence and further suggest that
pdpC
may play a unique role in this process, as indicated by its distinct intermediate phenotype.
Helicobacter pylori Infection Impairs the Mucin Production Rate and Turnover in the Murine Gastric Mucosa
Authors: Navabi Nazanin , Johansson Malin E. V. , Raghavan Sukanya , Lindén Sara K. 
Published: December 2012

ABSTRACT

To protect the surface of the stomach, the epithelial cells secrete a mucus layer, which is mainly comprised of the MUC5AC mucin. Further protection is provided by a thick glycocalyx on the apical surface of the epithelial cell, with the cell surface mucin MUC1 as a major component. Here, we investigate the production rate and turnover of newly synthesized mucin in mice and analyze the effects of early colonization and chronic infection with
H. pylori
. Metabolic incorporation of an azido GalNAc analog (GalNAz) was used as a nonradioactive method to perform pulse experiments in the whole animal. First, the subcellular movement of newly synthesized mucin and mucin turnover was determined in uninfected mice. Based on the time line for mucin transport and dissemination, 2, 6, and 12 h after GalNAz injection was selected to collect the stomachs from mice infected with
H. pylori
strain SS1 during early colonization (7 days) and chronic infection (90 days). The results demonstrated that the speed from the start of glycosylation to the final destination is faster for the membrane-bound mucin to reach the glycocalyx (2 h) than for the secretory mucins to become secreted into the mucus layer (5 h). Furthermore, infection with
H. pylori
reduces the rate of mucin turnover and decreases the levels of Muc1. Since
H. pylori
colonizes this mucus niche, the decreased turnover rate indicates that
H. pylori
creates a more stable and favorable environment for itself by impairing the defense mechanism for clearing the mucosal surface of pathogens by mucus flow.
Deletion of the Braun Lipoprotein-Encoding Gene and Altering the Function of Lipopolysaccharide Attenuate the Plague Bacterium
Authors: Sha Jian , Kirtley Michelle L. , van Lier Christina J. , Wang Shaofei , Erova Tatiana E. , Kozlova Elena V. , Cao Anthony , Cong Yingzi , Fitts Eric C. , Rosenzweig Jason A. , Chopra Ashok K. 
Published: December 2012

ABSTRACTBraun (murein) lipoprotein (Lpp) and lipopolysaccharide (LPS) are major components of the outer membranes ofEnterobacteriaceaefamily members that are capable of triggering inflammatory immune responses by activating Toll-like receptors 2 and 4, respectively. Expanding on earlier studies that demonstrated a role played by Lpp inYersinia pestisvirulence in mouse models of bubonic and pneumonic plague, we characterized anmsbBin-frame deletion mutant incapable of producing an acyltransferase that is responsible for the addition of lauric acid to the lipid A moiety of LPS, as well as a ΔlppΔmsbBdouble mutant of the highly virulentY. pestisCO92 strain. Although the ΔmsbBsingle mutant was minimally attenuated, the Δlppsingle mutant and the ΔlppΔmsbBdouble mutant were significantly more attenuated than the isogenic wild-type (WT) bacterium in bubonic and pneumonic animal models (mouse and rat) of plague. These data correlated with greatly reduced survivability of the aforementioned mutants in murine macrophages. Furthermore, the ΔlppΔmsbBdouble mutant was grossly compromised in its ability to disseminate to distal organs in mice and in evoking cytokines/chemokines in infected animal tissues. Importantly, mice that survived challenge with the ΔlppΔmsbBdouble mutant, but not the Δlppor ΔmsbBsingle mutant, in a pneumonic plague model were significantly protected against a subsequent lethal WT CO92 rechallenge. These data were substantiated by the fact that the ΔlppΔmsbBdouble mutant maintained an immunogenicity comparable to that of the WT strain and induced long-lasting T-cell responses against heat-killed WT CO92 antigens. Taken together, the data indicate that deletion of themsbBgene augmented the attenuation of the Δlppmutant by crippling the spread of the double mutant to the peripheral organs of animals and by inducing cytokine/chemokine responses. Thus, the ΔlppΔmsbBdouble mutant could provide a new live-attenuated background vaccine candidate strain, and this should be explored in the future.