bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis of the Osmoprotectant Ectoine, but Not Glycine Betaine, Is Critical for Survival of Osmotically Stressed Vibrio parahaemolyticus Cells
Authors: Ongagna-Yhombi Serge Y. , Boyd E. Fidelma
Published: June 2013

Vibrio parahaemolyticus
is a halophile present in marine and estuarine environments, ecosystems characterized by fluctuations in salinity and temperature. One strategy to thrive in such environments is the synthesis and/or uptake of compatible solutes. The
V. parahaemolyticus
genome contains biosynthesis systems for both ectoine and glycine betaine, which are known to act as compatible solutes in other species. We showed that
V. parahaemolyticus
had a 6% NaCl tolerance when grown in M9 minimal medium with 0.4% glucose (M9G) with a >5-h lag phase. By using
1
H nuclear magnetic resonance spectroscopy (
1
H-NMR) analysis, we determined that cells synthesized ectoine and glutamate in a NaCl-dependent manner. The most effective compatible solutes as measured by a reduction in lag-phase growth in M9G with 6% NaCl (M9G 6% NaCl) were in the order glycine betaine > choline > proline = glutamate > ectoine. However,
V. parahaemolyticus
could use glutamate or proline as the sole carbon source, but not ectoine or glycine betaine, which suggests that these are bona fide compatible solutes. Expression analysis showed that the
ectA
and
betA
genes were more highly expressed in log-phase cells, and expression of both genes was induced by NaCl up-shock. Under all conditions examined, the
ectA
gene was more highly expressed than the
betA
gene. Analysis of in-frame deletions in
betA
and
ectB
and in a double mutant showed that the
ectB
mutant was defective for growth, and this defect was rescued by the addition of glycine betaine, proline, ectoine, and glutamate, indicating that these compounds are compatible solutes for this species. The presence of both synthesis systems was the predominant distribution pattern among members of the
Vibrionaceae
family, suggesting this is the ancestral state.
Evidence of Metabolic Switching and Implications for Food Safety from the Phenome(s) of Salmonella enterica Serovar Typhimurium DT104 Cultured at Selected Points across the Pork Production Food Chain
Authors: Martins Marta , McCusker Matthew P. , McCabe Evonne M. , O'Leary Denis , Duffy Geraldine , Fanning Séamus
Published: June 2013

Salmonella enterica
serovar Typhimurium DT104 is a recognized food-borne pathogen that displays a multidrug-resistant phenotype and that is associated with systemic infections. At one extreme of the food chain, this bacterium can infect humans, limiting the treatment options available and thereby contributing to increased morbidity and mortality. Although the antibiotic resistance profile is well defined, little is known about other phenotypes that may be expressed by this pathogen at key points across the pork production food chain. In this study, 172
Salmonella enterica
serovar Typhimurium DT104/DT104b isolated from an extensive “farm-to-fork” surveillance study, focusing on the pork food chain, were characterized in detail. Isolates were cultured from environmental, processing, retail, and clinical sources, and the study focused on phenotypes that may have contributed to persistence/survival in these different niches. Molecular subtypes, along with antibiotic resistance profiles, tolerance to biocides, motility, and biofilm formation, were determined. As a basis for human infection, acid survival and the ability to utilize a range of energy sources and to adhere to and/or invade Caco-2 cells were also studied. Comparative alterations to biocide tolerance were observed in isolates from retail.
l
-Tartaric acid and
d
-mannose-1-phosphate induced the formation of biofilms in a preselected subset of strains, independent of their origin. All clinical isolates were motile and demonstrated an enhanced ability to survive in acidic conditions. Our data report on a diverse phenotype, expressed by
S
. Typhimurium isolates cultured from the pork production food chain. Extending our understanding of the means by which this pathogen adapts to environmental niches along the “farm-to-fork” continuum will facilitate the protection of vulnerable consumers through targeted improvements in food safety measures.
Adherence to and Invasion of Human Intestinal Cells by Arcobacter Species and Their Virulence Genotypes
Authors: Levican Arturo , Alkeskas Aldukali , Günter Claudia , Forsythe Stephen J. , Figueras María José
Published: June 2013

The genus
Arcobacter
is composed of 17 species which have been isolated from various sources. Of particular interest are
A. butzleri
,
A. cryaerophilus
, and
A. skirrowii
, as these have been associated with human cases of diarrhea, the probable transmission routes being through the ingestion of contaminated drinking water and food. To date, only limited studies of virulence traits in this genus have been undertaken. The present study used 60
Arcobacter
strains isolated from different sources, representing 16 of the 17 species of the genus, to investigate their ability to adhere to and invade the human intestinal cell line Caco-2. In addition, the presence of five putative virulence genes (
ciaB
,
cadF
,
cj1349
,
hecA
, and
irgA
) was screened for in these strains by PCR. All
Arcobacter
species except
A. bivalviorum
and
Arcobacter
sp. strain W63 adhered to Caco-2 cells, and most species (10/16) were invasive. The most invasive species were
A. skirrowii
,
A. cryaerophilus
,
A. butzleri
, and
A. defluvii
. All invasive strains were positive for
ciaB
(encoding a putative invasion protein). Other putative virulence genes were present in other species, i.e.,
A. butzleri
(
cadF
,
cj1349
,
irgA
, and
hecA
),
A. trophiarum
(
cj1349
),
A. ellisii
(
cj1349
), and
A. defluvii
(
irgA
). No virulence genes were detected in strains which showed little or no invasion of Caco-2 cells. These results indicate that many
Arcobacter
species are potential pathogens of humans and animals.
Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere
Authors: Stiefel Philipp , Zambelli Tomaso , Vorholt Julia A.
Published: June 2013

In their natural environment, bacteria often behave differently than they do under laboratory conditions. To gain insight into the physiology of bacteria
in situ
, dedicated approaches are required to monitor their adaptations and specific behaviors under environmental conditions. Optical microscopy is crucial for the observation of fundamental characteristics of bacteria, such as cell shape, size, and marker gene expression. Here, fluidic force microscopy (FluidFM) was exploited to isolate optically selected bacteria for subsequent identification and characterization. In this study, bacteriochlorophyll-producing bacteria, which can be visualized due to their characteristic fluorescence in the infrared range, were isolated from leaf washes. Bacterial communities from the phyllosphere were investigated because they harbor genes indicative of aerobic anoxygenic photosynthesis. Our data show that different species of
Methylobacterium
express their photosystem
in planta
, and they show a distinct pattern of bacteriochlorophyll production under laboratory conditions that is dependent on supplied carbon sources.
Sequence Variability of P2-Like Prophage Genomes Carrying the Cytolethal Distending Toxin V Operon in Escherichia coli O157
Authors: Sváb Domonkos , Horváth Balázs , Maróti Gergely , Dobrindt Ulrich , Tóth István
Published: June 2013

Cytolethal distending toxins (CDT) are potent cytotoxins of several Gram-negative pathogenic bacteria, including
Escherichia coli
, in which five types (CDT-I to CDT-V) have been identified so far. CDT-V is frequently associated with Shiga-toxigenic
E. coli
(STEC), enterohemorrhagic
E. coli
(EHEC) O157 strains, and strains not fitting any established pathotypes. In this study, we were the first to sequence and annotate a 31.2-kb-long, noninducible P2-like prophage carrying the
cdt-V
operon from an
stx
- and
eae
-negative
E. coli
O157:H43 strain of bovine origin. The
cdt-V
operon is integrated in the place of the
tin
and
old
phage immunity genes (termed the TO region) of the prophage, and the prophage itself is integrated into the bacterial chromosome between the housekeeping genes
cpxP
and
fieF
. The presence of P2-like genes (
n
= 20) was investigated in a further five CDT-V-positive bovine
E. coli
O157 strains of various serotypes, three EHEC O157:NM strains, four strains expressing other variants of CDT, and eight CDT-negative strains. All but one CDT-V-positive atypical O157 strain uniformly carried all the investigated genomic regions of P2-like phages, while the EHEC O157 strains missed three regions and the CDT-V-negative strains carried only a few P2-like sequences. Our results suggest that P2-like phages play a role in the dissemination of
cdt-V
between
E. coli
O157 strains and that after integration into the bacterial chromosome, they adapted to the respective hosts and became temperate.
Acetate Production from Oil under Sulfate-Reducing Conditions in Bioreactors Injected with Sulfate and Nitrate
Authors: Callbeck Cameron M. , Agrawal Akhil , Voordouw Gerrit
Published: June 2013

Oil production by water injection can cause souring in which sulfate in the injection water is reduced to sulfide by resident sulfate-reducing bacteria (SRB). Sulfate (2 mM) in medium injected at a rate of 1 pore volume per day into upflow bioreactors containing residual heavy oil from the Medicine Hat Glauconitic C field was nearly completely reduced to sulfide, and this was associated with the generation of 3 to 4 mM acetate. Inclusion of 4 mM nitrate inhibited souring for 60 days, after which complete sulfate reduction and associated acetate production were once again observed. Sulfate reduction was permanently inhibited when 100 mM nitrate was injected by the nitrite formed under these conditions. Pulsed injection of 4 or 100 mM nitrate inhibited sulfate reduction temporarily. Sulfate reduction resumed once nitrate injection was stopped and was associated with the production of acetate in all cases. The stoichiometry of acetate formation (3 to 4 mM formed per 2 mM sulfate reduced) is consistent with a mechanism in which oil alkanes and water are metabolized to acetate and hydrogen by fermentative and syntrophic bacteria (K. Zengler et al., Nature 401:266–269, 1999), with the hydrogen being used by SRB to reduce sulfate to sulfide. In support of this model, microbial community analyses by pyrosequencing indicated SRB of the genus
Desulfovibrio
, which use hydrogen but not acetate as an electron donor for sulfate reduction, to be a major community component. The model explains the high concentrations of acetate that are sometimes found in waters produced from water-injected oil fields.
Proteome of the Nematode-Trapping Cells of the Fungus Monacrosporium haptotylum
Authors: Andersson Karl-Magnus , Meerupati Tejashwari , Levander Fredrik , Friman Eva , Ahrén Dag , Tunlid Anders
Published: June 2013

Many nematophagous fungi use morphological structures called traps to capture nematodes by adhesion or mechanically. To better understand the cellular functions of adhesive traps, the trap cell proteome of the fungus
Monacrosporium haptotylum
was characterized. The trap of
M. haptotylum
consists of a unicellular structure called a knob that develops at the apex of a hypha. Proteins extracted from knobs and mycelia were analyzed using SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC–MS-MS). The peptide sequences were matched against predicted gene models from the recently sequenced
M. haptotylum
genome. In total, 336 proteins were identified, with 54 expressed at significantly higher levels in the knobs than in the mycelia. The upregulated knob proteins included peptidases, small secreted proteins with unknown functions, and putative cell surface adhesins containing carbohydrate-binding domains, including the WSC domain. Phylogenetic analysis showed that all upregulated WSC domain proteins belonged to a large, expanded cluster of paralogs in
M. haptotylum
. Several peptidases and homologs of experimentally verified proteins in other pathogenic fungi were also upregulated in the knob proteome. Complementary profiling of gene expression at the transcriptome level showed poor correlation between the upregulation of knob proteins and their corresponding transcripts. We propose that the traps of
M. haptotylum
contain many of the proteins needed in the early stages of infection and that the trap cells can tightly control the translation and degradation of these proteins to minimize the cost of protein synthesis.
Real-Time PCR for Quantitative Analysis of Human Commensal Escherichia coli Populations Reveals a High Frequency of Subdominant Phylogroups
Authors: Smati Mounira , Clermont Olivier , Le Gal Frédéric , Schichmanoff Olivier , Jauréguy Françoise , Eddi Alain , Denamur Erick , Picard Bertrand ,   for the Coliville Group
Published: June 2013

Escherichia coli
is divided into four main phylogenetic groups, which each exhibit ecological specialization. To understand the population structure of
E. coli
in its primary habitat, we directly assessed the relative proportions of these phylogroups from the stools of 100 healthy human subjects using a new real-time PCR method, which allows a large number of samples to be studied. The detection threshold for our technique was 0.1% of the
E. coli
population, i.e., 10
5
CFU/g of feces; in other methods based on individual colony analysis, the threshold is 10%. One, two, three, or four phylogenetic groups were simultaneously found in 21%, 48%, 21%, and 8% of the subjects, respectively. Phylogroups present at a threshold of less than 10% of the population were found in 40% of the subjects, revealing high within-individual diversity. Phylogroups A and B2 were detected in 74% and 70% of the subjects, respectively; phylogroups B1 and D were detected in 36% and 32%, respectively. When phylogroup B2 was dominant, it tended not to cooccur with other phylogroups. In contrast, other phylogroups were present when phylogroup A was dominant. These data indicate a complex pattern of interactions between the members of a single species within the human gut and identify a reservoir of clones that are present at a low frequency. The presence of these minor clones could explain the fluctuation in the composition of the
E. coli
microbiota within single individuals that may be seen over time. They could also constitute reservoirs of virulent and/or resistant strains.
Inhibition of Aspergillus niger Phosphate Solubilization by Fluoride Released from Rock Phosphate
Authors: Mendes Gilberto de Oliveira , Vassilev Nikolay Bojkov , Bonduki Victor Hugo Araújo , da Silva Ivo Ribeiro , Ribeiro José Ivo , Costa Maurício Dutra
Published: June 2013

The simultaneous release of various chemical elements with inhibitory potential for phosphate solubilization from rock phosphate (RP) was studied in this work. Al, B, Ba, Ca, F, Fe, Mn, Mo, Na, Ni, Pb, Rb, Si, Sr, V, Zn, and Zr were released concomitantly with P during the solubilization of Araxá RP (Brazil), but only F showed inhibitory effects on the process at the concentrations detected in the growth medium. Besides P solubilization, fluoride decreased fungal growth, citric acid production, and medium acidification by
Aspergillus niger
. At the maximum concentration found during Araxá RP solubilization (22.9 mg F
−
per liter), fluoride decreased P solubilization by 55%. These findings show that fluoride negatively affects RP solubilization by
A. niger
through its inhibitory action on the fungal metabolism. Given that fluoride is a common component of RPs, the data presented here suggest that most of the microbial RP solubilization systems studied so far were probably operated under suboptimal conditions.
Multilocus Genotype Analysis of Escherichia coli O157 Isolates from Australia and the United States Provides Evidence of Geographic Divergence
Authors: Mellor Glen E. , Besser Thomas E. , Davis Margaret A. , Beavis Brittany , Jung WooKyung , Smith Helen V. , Jennison Amy V. , Doyle Christine J. , Chandry P. Scott , Gobius Kari S. , Fegan Narelle
Published: June 2013

Escherichia coli
O157 is a food-borne pathogen whose major reservoir has been identified as cattle. Recent genetic information has indicated that populations of
E. coli
O157 from cattle and humans can differ genetically and that this variation may have an impact on their ability to cause severe human disease. In addition, there is emerging evidence that
E. coli
O157 strains from different geographical regions may also be genetically divergent. To investigate the extent of this variation, we used Shiga toxin bacteriophage insertion sites (SBI), lineage-specific polymorphisms (LSPA-6), multilocus variable-number tandem-repeat analysis (MLVA), and a
tir
255T>A polymorphism to examine 606 isolates representing both Australian and U.S. cattle and human populations. Both uni- and multivariate analyses of these data show a strong association between the country of origin and multilocus genotypes (
P
< 0.0001). In addition, our results identify factors that may play a role in virulence that also differed in isolates from each country, including the carriage of
stx
1
in the
argW
locus uniquely observed in Australian isolates and the much higher frequency of
stx
2
-positive (also referred to as
stx
2a
) strains in the U.S. isolates (4% of Australian isolates versus 72% of U.S. isolates). LSPA-6 lineages differed between the two continents, with the majority of Australian isolates belonging to lineage I/II (LI/II) (LI, 2%; LI/II, 85%; LII, 13%) and the majority of U.S. isolates belonging to LI (LI, 60%; LI/II, 16%; LII, 25%). The results of this study provide strong evidence of phylogeographic structuring of
E. coli
O157 populations, suggesting divergent evolution of enterohemorrhagic
E. coli
O157 in Australia and the United States.
Histidine Phosphotransfer Proteins in Fungal Two-Component Signal Transduction Pathways
Authors: Fassler Jan S. , West Ann H.
Published: June 2013

The histidine phosphotransfer (HPt) protein Ypd1 is an important participant in the
Saccharomyces cerevisiae
multistep two-component signal transduction pathway and, unlike the expanded histidine kinase gene family, is encoded by a single gene in nearly all model and pathogenic fungi. Ypd1 is essential for viability in both
S. cerevisiae
and in
Cryptococcus neoformans
. These and other aspects of Ypd1 biology, combined with the availability of structural and mutational data in
S. cerevisiae
, suggest that the essential interactions between Ypd1 and response regulator domains would be a good target for antifungal drug development. The goal of this minireview is to summarize the wealth of data on
S. cerevisiae
Ypd1 and to consider the potential benefits of conducting related studies in pathogenic fungi.
Candida albicans Forms a Specialized “Sexual” as Well as “Pathogenic” Biofilm
Authors: Park Yang-Nim , Daniels Karla J. , Pujol Claude , Srikantha Thyagarajan , Soll David R.
Published: June 2013

Candida albicans
forms two types of biofilm in RPMI 1640 medium, depending upon the configuration of the mating type locus. In the prevalent
a
/α configuration, cells form a biofilm that is impermeable, impenetrable by leukocytes, and fluconazole resistant. It is regulated by the Ras1/cyclic AMP (cAMP) pathway. In the
a/a
or α/α configuration, white cells form a biofilm that is architecturally similar to an
a
/α biofilm but, in contrast, is permeable, penetrable, and fluconazole susceptible. It is regulated by the mitogen-activated protein (MAP) kinase pathway. The
MTL
-homozygous biofilm has been shown to facilitate chemotropism, a step in the mating process. This has led to the hypothesis that specialized
MTL
-homozygous biofilms facilitate mating. If true, then
MTL
-homozygous biofilms should have an advantage over
MTL
-heterozygous biofilms in supporting mating. We have tested this prediction using a complementation strategy and show that minority opaque
a/a
and α/α cells seeded in
MTL
-homozygous biofilms mate at frequencies 1 to 2 orders of magnitude higher than in
MTL
-heterozygous biofilms. No difference in mating frequencies was observed between seeded patches of
MTL
-heterozygous and
MTL
-homozygous cells grown on agar at 28°C in air or 20% CO
2
and at 37°C. Mating frequencies are negligible in seeded patches of both
a
/α and
a/a
cells, in contrast to seeded biofilms. Together, these results support the hypothesis that
MTL
-homozygous (
a/a
or α/α) white cells form a specialized “sexual biofilm.”
Genetic and Physiological Responses of Bifidobacterium animalis subsp. lactis to Hydrogen Peroxide Stress
Authors: Oberg Taylor S. , Ward Robert E. , Steele James L. , Broadbent Jeff R.
Published: June 2013

Consumer interest in probiotic bifidobacteria is increasing, but industry efforts to secure high cell viability in foods is undermined by these anaerobes' sensitivity to oxidative stress. To address this limitation, we investigated genetic and physiological responses of two fully sequenced
Bifidobacterium animalis
subsp.
lactis
strains, BL-04 and DSM 10140, to hydrogen peroxide (H
2
O
2
) stress. Although the genome sequences for these strains are highly clonal, prior work showed that they differ in both intrinsic and inducible H
2
O
2
resistance. Transcriptome analysis of early-stationary-phase cells exposed to a sublethal H
2
O
2
concentration detected significant (
P
< 0.05) changes in expression of 138 genes in strain BL-04 after 5 min and 27 genes after 20 min. Surprisingly, no significant changes in gene expression were detected in DSM 10140 at either time. Genomic data suggested that differences in H
2
O
2
stress resistance might be due to a mutation in a BL-04 gene encoding long-chain fatty acid coenzyme A (CoA) ligase. To explore this possibility, membrane fatty acids were isolated and analyzed by gas chromatography-mass spectrometry (GC-MS). Results confirmed that the strains had significantly different lipid profiles: the BL-04 membrane contained higher percentages of C
14:0
and C
16:0
and lower percentages of C
18:1n9
. Alteration of the DSM 10140 membrane lipid composition using modified growth medium to more closely mimic that of BL-04 yielded cells that showed increased intrinsic resistance to lethal H
2
O
2
challenge but did not display an inducible H
2
O
2
stress response. The results show that deliberate stress induction or membrane lipid modification can be employed to significantly improve H
2
O
2
resistance in
B. animalis
subsp.
lactis
strains.
Isolation and Characterization of Mutant Sinorhizobium meliloti NodD1 Proteins with Altered Responses to Luteolin
Authors: Peck Melicent C. , Fisher Robert F. , Bliss Robert , Long Sharon R.
Published: June 2013

NodD1, a member of the NodD family of LysR-type transcriptional regulators (LTTRs), mediates nodulation (
nod
) gene expression in the soil bacterium
Sinorhizobium meliloti
in response to the plant-secreted flavonoid luteolin. We used genetic screens and targeted approaches to identify NodD1 residues that show altered responses to luteolin during the activation of
nod
gene transcription. Here we report four types of NodD1 mutants. Type I (NodD1 L69F, S104L, D134N, and M193I mutants) displays reduced or no activation of
nod
gene expression. Type II (NodD1 K205N) is constitutively active but repressed by luteolin. Type III (NodD1 L280F) demonstrates enhanced activity with luteolin compared to that of wild-type NodD1. Type IV (NodD1 D284N) shows moderate constitutive activity yet can still be induced by luteolin. In the absence of luteolin, many mutants display a low binding affinity for
nod
gene promoter DNA
in vitro
. Several mutants also show, as does wild-type NodD1, increased affinity for
nod
gene promoters with added luteolin. All of the NodD1 mutant proteins can homodimerize and heterodimerize with wild-type NodD1. Based on these data and the crystal structures of several LTTRs, we present a structural model of wild-type NodD1, identifying residues important for inducer binding, protein multimerization, and interaction with RNA polymerase at
nod
gene promoters.
Transcription of the Escherichia coli Fatty Acid Synthesis OperonfabHDGIs Directly Activated by FadR and Inhibited by ppGpp
Authors: My Laetitia , Rekoske Brian , Lemke Justin J. , Viala Julie P. , Gourse Richard L. , Bouveret Emmanuelle
Published: June 2013

InEscherichia coli, FadR and FabR are transcriptional regulators that control the expression of fatty acid degradation and unsaturated fatty acid synthesis genes, depending on the availability of fatty acids. In this report, we focus on the dual transcriptional regulator FadR. In the absence of fatty acids, FadR represses the transcription offadgenes required for fatty acid degradation. However, FadR is also an activator, stimulating transcription of the products of thefabAandfabBgenes responsible for unsaturated fatty acid synthesis. In this study, we show that FadR directly activates another fatty acid synthesis promoter, PfabH, which transcribes thefabHDGoperon, indicating that FadR is a global regulator of both fatty acid degradation and fatty acid synthesis. We also demonstrate that ppGpp and its cofactor DksA, known primarily for their role in regulation of the synthesis of the translational machinery, directly inhibit transcription from thefabHpromoter. ppGpp also inhibits thefadRpromoter, thereby reducing transcription activation offabHby FadR indirectly. Our study shows that both ppGpp and FadR have direct roles in the control of fatty acid promoters, linking expression in response to both translation activity and fatty acid availability.
Dual Mechanisms of Metabolite Acquisition by the Obligate Intracytosolic Pathogen Rickettsia prowazekii Reveal Novel Aspects of Triose Phosphate Transport
Authors: Frohlich Kyla M. , Audia Jonathon P.
Published: June 2013

Rickettsia prowazekii
is an obligate intracytosolic pathogen and the causative agent of epidemic typhus fever in humans. As an evolutionary model of intracellular pathogenesis, rickettsiae are notorious for their use of transport systems that parasitize eukaryotic host cell biochemical pathways. Rickettsial transport systems for substrates found only in eukaryotic cell cytoplasm are uncommon among free-living microorganisms and often possess distinctive mechanisms. We previously reported that
R. prowazekii
acquires triose phosphates for phospholipid biosynthesis via the coordinated activities of a novel dihydroxyacetone phosphate transport system and an
sn
-glycerol-3-phosphate dehydrogenase (K. M. Frohlich et al., J. Bacteriol. 192:4281–4288, 2010). In the present study, we have determined that
R. prowazekii
utilizes a second, independent triose phosphate acquisition pathway whereby
sn
-glycerol-3-phosphate is directly transported and incorporated into phospholipids. Herein we describe the
sn
-glycerol-3-phosphate and dihydroxyacetone phosphate transport systems in isolated
R. prowazekii
with respect to kinetics, energy coupling, transport mechanisms, and substrate specificity. These data suggest the existence of multiple rickettsial triose phosphate transport systems. Furthermore, the
R. prowazekii
dihydroxyacetone phosphate transport systems displayed unexpected mechanistic properties compared to well-characterized triose phosphate transport systems from plant plastids. Questions regarding possible roles for dual-substrate acquisition pathways as metabolic virulence factors in the context of a pathogen undergoing reductive evolution are discussed.
Role for Skp in LptD Assembly in Escherichia coli
Authors: Schwalm Jaclyn , Mahoney Tara F. , Soltes Garner R. , Silhavy Thomas J.
Published: June 2013

The periplasmic chaperone Skp has long been implicated in the assembly of outer membrane proteins (OMPs) in
Escherichia coli
. It has been shown to interact with unfolded OMPs, and the simultaneous loss of Skp and the main periplasmic chaperone in
E. coli
, SurA, results in synthetic lethality. However, a Δ
skp
mutant displays only minor OMP assembly defects, and no OMPs have been shown to require Skp for their assembly. Here, we report a role for Skp in the assembly of the essential OMP LptD. This role may be compensated for by other OMP assembly proteins; in the absence of both Skp and FkpA or Skp and BamB, LptD assembly is impaired. Overexpression of SurA does not restore LptD levels in a Δ
skp
Δ
fkpA
double mutant, nor does the overexpression of Skp or FkpA restore LptD levels in the Δ
surA
mutant, suggesting that Skp acts in concert with SurA to efficiently assemble LptD in
E. coli
. Other OMPs, including LamB, are less affected in the Δ
skp
Δ
fkpA
and Δ
skp bamB
::
kan
double mutants, suggesting that Skp is specifically necessary for the assembly of certain OMPs. Analysis of an OMP with a domain structure similar to that of LptD, FhuA, suggests that common structural features may determine which OMPs require Skp for their assembly.
Crippling the Essential GTPase Der Causes Dependence on Ribosomal Protein L9
Authors: Naganathan Anusha , Moore Sean D.
Published: June 2013

Ribosomal protein L9 is a component of all eubacterial ribosomes, yet deletion strains display only subtle growth defects. Although L9 has been implicated in helping ribosomes maintain translation reading frame and in regulating translation bypass, no portion of the ribosome-bound protein seems capable of contacting either the peptidyltransferase center or the decoding center, so it is a mystery how L9 can influence these important processes. To reveal the physiological roles of L9 that have maintained it in evolution, we identified mutants of
Escherichia coli
that depend on L9 for fitness. In this report, we describe a class of L9-dependent mutants in the ribosome biogenesis GTPase Der (EngA/YphC). Purified mutant proteins were severely compromised in their GTPase activities, despite the fact that the mutations are not present in GTP hydrolysis sites. Moreover, although L9 and YihI complemented the slow-growth
der
phenotypes, neither factor could rescue the GTPase activities
in vitro
. Complementation studies revealed that the N-terminal domain of L9 is necessary and sufficient to improve the fitness of these Der mutants, suggesting that this domain may help stabilize compromised ribosomes that accumulate when Der is defective. Finally, we employed a targeted degradation system to rapidly deplete L9 from a highly compromised
der
mutant strain and show that the L9-dependent phenotype coincides with a cell division defect.
The
agr
Locus Regulates Virulence and Colonization Genes in Clostridium difficile 027
Authors: Martin Melissa J. , Clare Simon , Goulding David , Faulds-Pain Alexandra , Barquist Lars , Browne Hilary P. , Pettit Laura , Dougan Gordon , Lawley Trevor D. , Wren Brendan W.
Published: June 2013

The transcriptional regulator AgrA, a member of the LytTR family of proteins, plays a key role in controlling gene expression in some Gram-positive pathogens, including
Staphylococcus aureus
and
Enterococcus faecalis
. AgrA is encoded by the
agrACDB
global regulatory locus, and orthologues are found within the genome of most
Clostridium difficile
isolates, including the epidemic lineage 027/BI/NAP1. Comparative RNA sequencing of the wild type and otherwise isogenic
agrA
null mutant derivatives of
C. difficile
R20291 revealed a network of approximately 75 differentially regulated transcripts at late exponential growth phase, including many genes associated with flagellar assembly and function, such as the major structural subunit, FliC. Other differentially regulated genes include several involved in bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) synthesis and toxin A expression.
C. difficile
027 R20291
agrA
mutant derivatives were poorly flagellated and exhibited reduced levels of colonization and relapses in the murine infection model. Thus, the
agr
locus likely plays a contributory role in the fitness and virulence potential of
C. difficile
strains in the 027/BI/NAP1 lineage.
Pilus Gene Pool Variation and the Virulence of Corynebacterium diphtheriae Clinical Isolates during Infection of a Nematode
Authors: Broadway Melissa M. , Rogers Elizabeth A. , Chang Chungyu , Huang I-Hsiu , Dwivedi Prabhat , Yildirim Suleyman , Schmitt Michael P. , Das Asis , Ton-That Hung
Published: June 2013

Toxigenic
Corynebacterium diphtheriae
strains cause diphtheria in humans. The toxigenic
C. diphtheriae
isolate NCTC13129 produces three distinct heterotrimeric pili that contain SpaA, SpaD, and SpaH, making up the shaft structure. The SpaA pili are known to mediate bacterial adherence to pharyngeal epithelial cells. However, to date little is known about the expression of different pili in various clinical isolates and their importance in bacterial pathogenesis. Here, we characterized a large collection of
C. diphtheriae
clinical isolates for their pilin gene pool by PCR and for the expression of the respective pilins by immunoblotting with antibodies against Spa pilins. Consistent with the role of a virulence factor, the SpaA-type pili were found to be prevalent among the isolates, and most significantly, corynebacterial adherence to pharyngeal epithelial cells was strictly correlated with isolates that were positive for the SpaA pili. By comparison, the isolates were heterogeneous for the presence of SpaD- and SpaH-type pili. Importantly, using
Caenorhabditis elegans
as a model host for infection, we show here that strain NCTC13129 rapidly killed the nematodes, the phenotype similar to isolates that were positive for toxin and all pilus types. In contrast, isogenic mutants of NCTC13129 lacking SpaA-type pili or devoid of toxin and SpaA pili exhibited delayed killing of nematodes with similar kinetics. Consistently, nontoxigenic or toxigenic isolates that lack one, two, or all three pilus types were also attenuated in virulence. This work signifies the important role of pili in corynebacterial pathogenesis and provides a simple host model to identify additional virulence factors.
A Multicopper Oxidase Is Required for Copper Resistance in Mycobacterium tuberculosis
Authors: Rowland Jennifer L. , Niederweis Michael
Published: June 2013

Mycobacterium tuberculosis
, the causative agent of tuberculosis, is one of the most important bacterial pathogens. Recent work has revealed that the natural bactericidal properties of copper are utilized by the host immune system to combat infections with bacteria, including
M. tuberculosis
. However,
M. tuberculosis
employs multiple mechanisms to reduce the internal copper amount by efflux and sequestration, which are required for virulence of
M. tuberculosis
. Here, we describe an alternative mechanism of copper resistance by
M. tuberculosis
. Deletion of the
rv0846c
gene increased the susceptibility of
M. tuberculosis
to copper at least 10-fold, establishing Rv0846c as a major component of copper resistance in
M. tuberculosis
.
In vitro
assays showed that Rv0846c oxidized organic substrates and Fe(II). Importantly, mutation of the predicted copper-coordinating cysteine 486 resulted in inactive Rv0846c protein which did not protect
M. tuberculosis
against copper stress. Hence, Rv0846c is a multicopper oxidase of
M. tuberculosis
and was renamed
m
ycobacterial
m
ulti
c
opper
o
xidase (MmcO). MmcO is membrane associated, probably by lipidation after export across the inner membrane by the twin-arginine translocation system. However, mutation of the lipidation site did not affect the oxidase activity or the copper protective function of MmcO. Our study revealed MmcO as an important copper resistance mechanism of
M. tuberculosis
, which possibly acts by oxidation of toxic Cu(I) in the periplasm.
Doctors’ self-reported physical activity, their counselling practices and their correlates in urban Trivandrum, South India: should a full-service doctor be a physically active doctor?
Authors: Patra Lipika , Mini G K , Mathews Elezebeth , Thankappan K R
Published: June 2013

Background

Doctors’ self-reported physical activity (PA) is associated with their propensity for prescribing PA.

Methods

We surveyed 146 doctors (median age 42 years; men 58.9%), selected by multistage random sampling. Information on demographic details, self-reported PA and counselling offered to their patients was collected using a pretested, structured, self-administered questionnaire. Multivariate logistic regression analysis was carried out to find the predictors of PA and PA counselling offered to the patients.

Results

Moderate PA was reported by 37.7% (95% CI 29.8 to 45.5) of the doctors and the remaining 62.3%reported being inactive. Doctors who were motivated to perform PA (OR 4.01, 95% CI 1.82 to 8.86), who used exercise equipment at home (OR 3.97, CI 1.68 to 9.36) and who used a neighbourhood facility for PA (OR 2.36, CI 1.11 to 5.02) were more likely to perform moderate PA compared with their counterparts. 25% of the doctors always asked and advised their patients on PA. Doctors who believed that their own healthy lifestyle influenced advice practices (OR 9.13, CI 2.49 to 33.41), who consulted less than 30 patients/day (OR 5.35, CI 1.41 to 20.25) and who reported previous participation in sports activities (OR 4.22, CI 1.77 to 10.04) were more likely to always ask and advise their patients on PA compared with their counterparts.

Conclusions

A majority of the doctors in our study were inactive and did not ask or advise their patients on PA. Measures are warranted to enhance doctors’ own PA and their counselling practices.
What physical activity surveillance needs: validity of a single-item questionnaire
Authors: Wanner Miriam , Probst-Hensch Nicole , Kriemler Susi , Meier Flurina , Bauman Adrian , Martin Brian W
Published: June 2013

Background

Self-report instruments to assess physical activity are still the most feasible option in many population-wide surveys, and often need to be very short owing to resource constraints. The aim of this study was to test the criterion validity of a single-item physical activity measure using accelerometers and to compare its measurement properties by gender, age group (including older adults) and language region.

Methods

A validation study was carried out within the second follow-up of a large Swiss cohort study (Swiss Cohort Study on Air Pollution and Lung and Heart Disease in Adults, SAPALDIA, n=208) and included an additional convenient sample (n=110). Participants wore an accelerometer over eight consecutive days and then completed the single-item measure. Spearman's rank-order correlations were used to assess the criterion validity.

Results

Physical activity levels were higher in men, younger individuals and those from the German-speaking part of Switzerland. Correlation coefficients for the number of days with at least 30 min of moderate-to-vigorous physical activity according to the single item and different accelerometer activity outcomes ranged from 0.40 to 0.54. Correlations were higher for women, younger individuals and participants from the French-speaking and the Italian-speaking parts.

Conclusions

The single-item physical activity measure performed at least as well as other physical activity questionnaires. The differences in criterion validity between sub groups indicate that factors such as gender and age should be taken into account when developing physical activity questionnaires and in future validation studies.
Recurrence of Achilles tendon injuries in elite male football players is more common after early return to play: an 11-year follow-up of the UEFA Champions League injury study
Authors: Gajhede-Knudsen Mariann , Ekstrand Jan , Magnusson Henrik , Maffulli Nicola
Published: June 2013

Background

There is limited information about Achilles tendon disorders in professional football.

Aims

To investigate the incidence, injury circumstances, lay-off times and reinjury rates of Achilles tendon disorders in male professional football.

Methods

A total of 27 clubs from 10 countries and 1743 players have been followed prospectively during 11 seasons between 2001 and 2012. The team medical staff recorded individual player exposure and time-loss injuries.

Results

A total of 203 (2.5% of all injuries) Achilles tendon disorders were registered. A majority (96%) of the disorders were tendinopathies, and nine were partial or total ruptures. A higher injury rate was found during the preseason compared with the competitive season, 0.25 vs 0.18/1000 h (rate ratio (RR) 1.4, 95% CI 1.1 to 2.0; p=0.027). The mean lay-off time for Achilles tendinopathies was 23±37 (median=10, Q1=4 and Q3=24) days, while a rupture of the Achilles tendon, on average, caused 161±65 (median=169, Q1=110 and Q3=189) days of absence. Players with Achilles tendon disorders were significantly older than the rest of the cohort, with a mean age of 27.2±4 years vs 25.6±4.6 years (p<0.001). 27% of all Achilles tendinopathies were reinjuries. A higher reinjury risk was found after short recovery periods (31%) compared with longer recovery periods (13%) (RR 2.4, 95% CI 2.1 to 2.8; p<0.001).

Conclusions

Achilles tendon disorders account for 3.8% of the total lay-off time and are more common in older players. Recurrence is common after Achilles tendinopathies and the reinjury risk is higher after short recovery periods.
Unravelling the grey zone: cardiac MRI volume to wall mass ratio to differentiate hypertrophic cardiomyopathy and the athlete's heart
Authors: Luijkx Tim , Cramer Maarten J , Buckens Constantinus F , Zaidi Abbas , Rienks Rienk , Mosterd Arend , Prakken Niek HJ , Dijkman Barbara , Mali Willem PThM , Velthuis Birgitta K
Published: June 2013

Background

Differentiating physiological left ventricular hypertrophy (LVH) in athletes from pathological hypertrophic cardiomyopathy (HCM) can be challenging. This study assesses the ability of cardiac MRI (CMR) to distinguish between physiological LVH (so-called athlete's heart) and HCM.

Methods

45 patients with HCM (71% men and 20% athletic) and 734 healthy control participants (60% men and 75% athletic) underwent CMR. Quantitative ventricular parameters were used for multivariate logistic regression with age, gender, sport status and left ventricular (LV) end-diastolic volume (EDV) to ED ventricular wall mass (EDM) ratio as covariates. A second model added the LV EDV : right ventricular (RV) EDV ratio. The performance of the model was subsequently tested.

Results

LV EDM was greater in patients with HCM (74 g/m2) compared with healthy athletes/non-athletes (53/41 g/m2), while LV EDV was largest in athletes (114 ml/m2) as compared with non-athletes (94 ml/m2) and patients with HCM (88 ml/m2). The LV EDV : EDM ratio was significantly lower in patients with HCM compared with healthy controls and athletes (1.30/2.39/2.25, p<0.05). The LV EDV : RV EDV ratio was significantly greater in patients with HCM (1.10) than in healthy participants (non-athletes/athletes 0.94/0.93). The regression model resulted in high sensitivity and specificity levels in all and borderline-LVH participants (as defined by septal wall thickness). Corresponding areas under the receiver operator characteristic (ROC) curves were 0.995 (all participants) and 0.992 (borderline-LVH participants only). Adding the LV EDV : RV EDV ratio yielded no additional improvement.

Conclusions

A model incorporating the LV EDV : EDM ratio can help distinguish HCM from physiological hypertrophy in athletes. This also applies to cases with borderline LVH, which present the greatest diagnostic challenge in clinical practice.
Gastrointestinal polyposis syndromes for the general gastroenterologist
Authors: Hurley Joanna J , Ewing Iain , Sampson Julian R , Dolwani Sunil
Published: June 2013

The occurrence of colonic polyps is a common phenomenon; however, where there are numerous adenomas or other polyps, and/or the patient is at a relatively young age, an inheritable form of gastrointestinal polyposis should be considered. Patients can present via different referral routes, for example, at colonoscopy where multiple polyps are detected, following a personal diagnosis of colorectal cancer, or by family screening. This article outlines the important considerations in the diagnosis of a polyposis syndrome and key diagnostic features to consider. It will also describe the underlying genetic factors associated with the common polyposis syndromes, including classical familial adenomatous polyposis (FAP), attenuated FAP, MUTYH-associated adenomatous polyposis, Peutz–Jeghers syndrome, juvenile polyposis syndrome, Cowden syndrome and serrated polyposis, and the subsequent management of each condition.
Safety and efficacy of ticagrelor for neuroendovascular procedures. A single center initial experience
Authors: Hanel Ricardo A , Taussky Philipp , Dixon Tanya , Miller David A , Sapin Merlie , Nordeen Jerah D , Tawk Rabih G , Navarro Ramon , Johns Gretchen , Freeman William D
Published: June 2013

Introduction

Although platelet response testing is controversial, up to one-third of neuroendovascular patients are ‘resistant’ to clopidogrel and are at risk for in stent thrombotic complications and may require alternative antiplatelet therapy. Ticagrelor is a new reversible ADP P2Y12 platelet receptor inhibitor with no known resistance. We describe the clinical experience with ticagrelor for neuroendovascular procedures as an alternative in clopidogrel P2Y12 platelet resistant patients.

Methods

We reviewed our cerebrovascular database for all patients who were non-responders to clopidogrel, defined as P2Y12% inhibition <30%, despite repeat clopidogrel loading dose of at least 600 mg, and who were then administered ticagrelor.

Results

18 patients were non-responders to clopidogrel; 10 (56%) were men, eight (44%) were women, with a median age of 61 years (range 38–84). All patients received loading doses of at least 600 mg of clopidogrel and showed P2Y12 levels below 20% prior to ticagrelor administration. Patients were loaded with 180 mg of ticagrelor, and all but one patient showed an initial P2Y12 response above 60%. 11 patients underwent stenting, two underwent coiling, and five underwent treatment by pipeline embolization device. No patient experienced any adverse effects in the postoperative period related to the use of ticagrelor.

Conclusions

Ticagrelor offers an effective alternative to clopidogrel non-responders. All of our patients showed immediate platelet inhibition after a loading dose of 180 mg of ticagrelor, with no adverse effects. The cost of medication, patient compliance (twice a day doses), and reversible inhibition should be taken into consideration when using ticagrelor.
The organisation, operational dynamics and structure of female sex work in Pakistan
Authors: Emmanuel Faran , Thompson Laura H , Athar Uzma , Salim Momina , Sonia Altaf , Akhtar Naeem , Blanchard James F
Published: June 2013

Background

Pakistan is known to have large populations of female sex workers (FSWs) with considerable geographic heterogeneity in their characteristics. In this paper, we describe the social organisation and structural patterns of female sex work in different geographic regions of Pakistan.

Methods

We report geographic and network mapping data collected among FSWs in 15 cities across Pakistan in 2011 as part of the Canada-Pakistan HIV/AIDS Surveillance Project.

Results

A total number of 89 178 FSWs were estimated in the target cities for an average of 7.2 FSWs per 1000 adult males. 55% of the estimated number of FSWs concentrated in Karachi and Lahore. Based on the operations of female sex work, two major typologies of FSWs were identified: establishment-based and non-establishment-based. FSWs were further subtyped into those operating through brothels, homes, kothikhanas, streets and by cell phone. Cities varied considerably in terms of predominance of different FSW typologies.

Conclusions

There is considerable heterogeneity among FSWs in Pakistan, geographically and in terms of operational typology. Understanding the social organisation of sex work and the influence of social-cultural and legal factors in Pakistan is essential for the design of HIV prevention programmes and other services for FSWs.