bluuebunny/crossref_metadata_2025 huggingface dataset:
Lifestyle, participation, and health‐related quality of life in adolescents and young adults with myelomeningocele
Authors: BUFFART LAURIEN M , BERG‐EMONS RITA J G VAN DEN , MEETEREN JETTY VAN , STAM HENK J , ROEBROECK MARIJ E
Published: March 2009

This study aimed to describe participation and health‐related quality of life (HRQoL) in adolescents and young adults with myelomeningocele and to explore their relationships with lifestyle‐related factors. Fifty‐one individuals with a mean age of 21 years 1 month (SD 4y 6mo) years participated (26 males, 25 females; 82% hydrocephalus, 55% wheelchair‐dependent). Participation was assessed using the Life Habits Questionnaire, and HRQoL was assessed using the Medical Outcomes Study 36‐item Short‐form Health Survey. Physical activity was measured using an accelerometry‐based activity monitor, fitness (peak oxygen uptake) was measured during a maximal exercise test, and the sum of four skin‐folds was assessed to indicate body fat. Relationships were studied using logistic regression analyses. Of the participants, 63% had difficulties in daily activities and 59% in social roles. Participants perceived lower physical HRQoL than a Dutch reference population. Participants with higher levels of physical activity and fitness had fewer difficulties in participating in daily activities (odds ratio [OR]=8.8, p=0.02 and OR=29.7, p=0.02 respectively) and a higher physical HRQoL (OR=4.8, p=0.02 and OR=30.2, p=0.006 respectively), but not mental HRQoL. Body fat was not related to participation or HRQoL. In conclusion, a large proportion of individuals with myelomeningocele had difficulties in participation and perceived low physical HRQoL. Higher levels of physical activity and fitness were related to fewer difficulties in participation and higher physical HRQoL.
Early motor repertoire is related to level of self‐mobility in children with cerebral palsy at school age
Authors: BRUGGINK JANNEKE LM , CIONI GIOVANNI , EINSPIELER CHRISTA , MAATHUIS CAREL GB , PASCALE Rosa , BOS AREND F
Published: March 2009

Aim  To determine the predictive value of the early motor repertoire for the level of self‐mobility in children with cerebral palsy (CP) at school age.

Method  Video recordings were made at 11 to 17 weeks post‐term of 37 preterm infants (20 males, 17 females) who later developed CP. The early motor repertoire was assessed by obtaining a motor optimality score. At 6 to 12 years, children were classified according to the Gross Motor Function Classification System (GMFCS).

Results  Of 37 children (mean gestational age 29.1wks, SD 1.9; mean birthweight 1273g, SD 324), nine had unilateral and 28 had bilateral spastic CP. Twelve children were in GMFCS level I, three level II, 10 level III, four level IV, and eight level V. The absence of the age‐adequate motor repertoire, a cramped motor repertoire, an abnormal kicking pattern, and a non‐flat supine posture were associated with lower levels of self‐mobility (χ2 for trend test, p<0.05). Predictive for a low level of self‐mobility was a cramped motor repertoire/non‐flat supine posture (positive predictive values [PPV] 100%, negative predictive values [NPV] 54%). Predictive for a high level of self‐mobility was a non‐cramped repertoire/flat supine posture (PPV 80%, NPV 74%).

Interpretation  Several aspects of the motor repertoire at 11 to 17 weeks post‐term predicted the degree of functional limitations in children with CP at school age.
Motor testing at 1 year improves the prediction of motor and mental outcome at 2 years after perinatal hypoxic–ischaemic encephalopathy
Authors: VAN SCHIE PETRA EM , BECHER JULES G , DALLMEIJER ANNET J , BARKHOF FREDERIK , VAN WEISSENBRUCH MIRJAM M , VERMEULEN R JEROEN
Published: March 2009

Aim  To investigate the predictive value of motor testing at 1 year for motor and mental outcome at 2 years after perinatal hypoxic‐ischaemic encephalopathy (HIE) in term neonates.

Method  Motor and mental outcome at 2 years was assessed with the Bayley Scales of Infant Development, 2nd edition (BSID‐II) in 32 surviving children (20 males, 12 females; mean gestational age 40.2 wk, SD 1.4; mean birthweight 3217g, SD 435) participating in a prospective cohort study of HIE. The predictive value of three motor tests (Alberta Infant Motor Scale [AIMS], BSID‐II, and the Neurological Optimality Score [NOS]) at 1 year was analysed, in addition to predictions based on neonatal Sarnat staging and magnetic resonance imaging (MRI). Poor motor test results were defined as an AIMS z‐score of <−2, a psychomotor developmental index of the BSID‐II of <70, or a NOS of <26. Poor motor and poor mental outcome at 2 years was defined as a psychomotor developmental index or mental developmental index of the BSID‐II of <70.

Results  Twelve children, all with Sarnat grade II, had a poor motor outcome and 12 children, of whom one had Sarnat grade I, had a poor mental outcome at 2 years. Nine children had cerebral palsy, of whom five had quadriplegia, three had dyskinesia, and one had hemiplegia. Poor motor tests at 1 year increased the probability of a poor motor outcome from 71% (range 92 to 100%), and a poor mental outcome from 59% (range 77 to 100%) in children with Sarnat grade II and abnormal MRI, assessed with the AIMS and BSID‐II or NOS respectively.

Interpretation  Additional motor testing at 1 year improves the prediction of motor and mental outcome at 2 years in children with Sarnat grade II and abnormal MRI.
Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors
Authors: Jindrichova Marie , Vavra Vojtech , Obsil Tomas , Stojilkovic Stanko S. , Zemkova Hana
Published: March 2009

J. Neurochem. (2009)109, 923–934.

The functional relevance of aromatic residues in the upper part of the transmembrane domain‐1 of purinergic P2X receptors (P2XRs) was examined. Replacement of the conserved Tyr residue with Ala had a receptor‐specific effect: the P2X1R was non‐functional, the P2X2R, P2X4R, and P2X3R exhibited enhanced sensitivity to ATP and αβ‐meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7R sensitivity for agonists was not affected, though decay of current was delayed. The replacement of the P2X4R‐Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2R, P2X3R, and P2X4R function. Double mutation of ipsilateral Tyr42 and Trp46 P2X4R residues restored receptor function, whereas the corresponding P2X2R double mutant was not functional. In contrast, mutation of the contralateral Phe48 residue in the P2X4R‐Y42A mutant had no effect. These results indicate that aromatic residues in the upper part of TM1 play important roles in the three‐dimensional structure of the P2XRs and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating.
The importance of samples and isolates for species–area relationships
Authors: Tjørve Even , Turner Will R.
Published: March 2009

Species–area relationships (SARs) are a key tool for understanding patterns of species diversity. A framework for the interpretation of SARs and their prediction under different landscape configurations remains elusive, however. This article addresses one of these configurations: how species’ minimum‐area requirements affect the shape of island or other isolate Species–area curves. We distinguish between two classes of SARs: sample‐area curves, compiled entirely within larger contiguous areas, and isolate curves, compiled between isolated areas. We develop this conceptual and graphic model in order to illuminate landscape‐scale diversity patterns, to discuss how various landscape and species characteristics affect outcomes, and to investigate the dynamics of local extinction under conditions of habitat fragmentation. Minimum‐area effects on actual islands and other isolates predictably cause Species–area curves either to be sigmoid in arithmetic space or to be lowered for smaller areas. In order to illustrate the inherent shape of isolate curves, this study fits convex and sigmoid regression models to empirical isolate (island) data sets that cover the small scales expected to include inflection points.
Botanical richness and endemicity patterns of Borneo derived from species distribution models
Authors: Raes Niels , Roos Marco C. , Slik J. W. Ferry , Van Loon E. Emiel , Steege Hans ter
Published: March 2009

This study provides a Borneo‐wide, quantitative assessment of botanical richness and endemicity at a high spatial resolution, and based on actual collection data. To overcome the bias in collection effort, and to be able to predict the presence and absence of species, even for areas where no collections have been made, we constructed species distribution models (SDMs) for all species taxonomically revised in Flora Malesiana. Species richness and endemicity maps were based on 1439 significant SDMs. Mapping of the residuals of the richness‐endemicity relationship identified areas with higher levels of endemicity than can be expected on the basis of species richness, the endemicity hotspots. We were able to identify one previously unknown region of high diversity, the high mountain peaks of East Kalimantan; and two additional endemicity hotspots, the Müller Mountains and the Sangkulirang peninsula. The areas of high diversity and endemicity were characterized by a relatively small range in annual temperature, but with seasonality in temperatures within that range. Furthermore, these areas were least affected by El Niño Southern Oscillation drought events. The endemicity hotspots were found in areas, which were ecologically distinct in altitude, edaphic conditions, annual precipitation, or a combination of these factors. These results can be used to guide conservation efforts of the highly threatened forests of Borneo.
Effects of repeated injections of fibroblast‐stimulating lipopeptide‐1 on fever, formation of cytokines, and on the responsiveness to endotoxin in guinea‐pigs
Authors: Greis A. , Murgott J. , Gerstberger R. , Hübschle T. , Roth J.
Published: March 2009

Aims:  We investigated, whether the Toll‐like receptors (TLRs)‐2/6‐agonist fibroblast‐stimulating lipopeptide‐1 (FSL‐1), like the TLR‐4 agonist lipopolysaccharide (LPS), induces a state of tolerance. We further tested the influence of repeated pre‐treatment with FSL‐1 on the animals’ responsiveness to LPS.

Methods:  Abdominal temperature was recorded in unrestrained guinea‐pigs with intra‐abdominally implanted radiotransmitters. Circulating concentrations of tumour necrosis factor (TNF) and interleukin‐6 (IL‐6) were measured with specific bioassays. We tested the effects of intra‐arterial (i.a.) or intraperitoneal (i.p.) injections of 100 μg kg−1 FSL‐1, repeated five times at intervals of 3 days. The animals’ responses to i.a. or i.p. injections of 10 μg kg−1 LPS were determined another 3 days later and compared to those of naïve guinea‐pigs.

Results:  The FSL‐1‐induced TNF peak was significantly attenuated starting with the third i.a. administration, while fever was unimpaired and the IL‐6‐peak just tended to decrease. Fever and IL‐6 in response to i.a. injections of LPS were identical in both groups, while circulating TNF was higher in naïve compared to FSL‐1 pre‐treated animals. The effects of repeated i.p. injections of FSL‐1 were more pronounced resulting in attenuation of fever as well as circulating TNF and IL‐6, the strongest reduction observed after the third stimulation with FSL‐1. Repeated i.p. pre‐treatment with FSL‐1 induced hyporesponsiveness to i.p. administration of LPS compared to naïve animals with regard to fever and especially with regard to LPS‐induced formation of cytokines.

Conclusions:  There is a development of tolerance to FSL‐1 and cross‐tolerance between FSL‐1 and LPS depending on the route of administration of the respective TLR‐2/6 and TLR‐4 agonists.
Myrip uses distinct domains in the cellular activation of myosin VA and myosin VIIA in melanosome transport
Authors: Ramalho José S. , Lopes Vanda S. , Tarafder Abul K. , Seabra Miguel C. , Hume Alistair N.
Published: March 2009

Myrip is a Rab27a and MyosinVIIa (MyoVIIa) linking protein that may regulate melanosome transport in the retinal pigment epithelium (RPE). Myrip also binds MyosinVa (MyoVa) in vitro however it is unclear whether this interaction is of sufficient affinity to be physiologically relevant. Here, we addressed the questions of whether Myrip interacts with MyoVa in cells and the molecular basis of cellular activation of MyoVa and MyoVIIa by Myrip. To answer these questions we used melanosome transport in skin melanocytes and RPE cells as read‐outs of MyoVa and MyoVIIa activity. We found that Myrip recruits and activates MyoVa on skin melanosomes with similar efficiency to the established MyoVa activator Melanophilin (Mlph). Mutagenesis showed that a Myrip–Mlph conserved amphipathic helix (MMAH) is essential for MyoVa interaction while other Myrip regions, including the MyoVa exon F binding domain equivalent, play non‐essential roles in this interaction. This suggests that, in contrast to Mlph, Myrip interacts with MyoVa lacking melanocyte‐specific exon F. Parallel studies of RPE melanosome transport reveal that Myrip‐specific inserts, but not the MMAH, are essential for MyoVIIa activation. We conclude that Myrip is a versatile Rab27a‐associated myosin‐activating protein that mediates cellular activation of MyoVa and MyoVIIa via non‐overlapping domains.
REDD in the red: palm oil could undermine carbon payment schemes
Authors: Butler Rhett A. , Koh Lian Pin , Ghazoul Jaboury
Published: March 2009

Mechanisms to reduce carbon emissions from deforestation and forest degradation (REDD) have been gaining momentum as a way to combat global warming, fund forest conservation, and deliver economic benefits to rural populations. However, the economic viability of REDD schemes will depend on the profitability of alternative land uses. Oil palm agriculture has become a major driver of tropical deforestation over the last few decades. Here, we model and compare the profitability of converting forest to oil palm versus conserving it for an REDD project. We show that converting a hectare of forest for palm oil production will be more profitable (yielding net present values of $3,835–$9,630) to land owners than preserving it for carbon credits ($614–$994), which are currently restricted to voluntary carbon markets. Giving REDD credits price parity with carbon credits traded in compliance markets would boost the profitability of avoided deforestation (up to $6,605). Unless post‐2012 global climate policies legitimize the trading of carbon credits from avoided deforestation, REDD will not be able to compete with oil palm agriculture or other similarly profitable human activities as an economically attractive land‐use option, in which case REDD will not be able to fulfill its primary function of avoiding deforestation.
How Perfect Can Protein Interactomes Be?
Authors: Levy Emmanuel D. , Landry Christian R. , Michnick Stephen W.
Published: March 2009

Evolutionary noise in protein-protein interactions implies that not all interactions must be functional.
The Chromosomal Toxin Gene
yafQ
Is a Determinant of Multidrug Tolerance for
Escherichia coli
Growing in a Biofilm
Authors: Harrison Joe J. , Wade William D. , Akierman Sarah , Vacchi-Suzzi Caterina , Stremick Carol A. , Turner Raymond J. , Ceri Howard
Published: March 2009

Escherichia coli
is refractory to elevated doses of antibiotics when it is growing in a biofilm, and this is potentially due to high numbers of multidrug-tolerant persister cells in the surface-adherent population. Previously, the chromosomal toxin-antitoxin loci
hipBA
and
relBE
have been linked to the frequency at which persister cells occur in
E. coli
populations. In the present study, we focused on the
dinJ
-
yafQ
-encoded toxin-antitoxin system and hypothesized that deletion of the toxin gene
yafQ
might influence cell survival in antibiotic-exposed biofilms. By using confocal laser scanning microscopy and viable cell counting, it was determined that a Δ
yafQ
mutant produced biofilms with a structure and a cell density equivalent to those of the parental strain. In-depth susceptibility testing identified that relative to wild-type
E. coli
, the Δ
yafQ
strain had up to a ∼2,400-fold decrease in cell survival after the biofilms were exposed to bactericidal concentrations of cefazolin or tobramycin. Corresponding to these data, controlled overexpression of
yafQ
from a high-copy-number plasmid resulted in up to a ∼10,000-fold increase in the number of biofilm cells surviving exposure to these bactericidal drugs. In contrast, neither the inactivation nor the overexpression of
yafQ
affected the tolerance of biofilms to doxycycline or rifampin (rifampicin). Furthermore, deletion of
yafQ
did not affect the tolerance of stationary-phase planktonic cells to any of the antibacterials tested. These results suggest that
yafQ
mediates the tolerance of
E. coli
biofilms to multiple but specific antibiotics; moreover, our data imply that this cellular pathway for persistence is likely different from that of multidrug-tolerant cells in stationary-phase planktonic cell cultures.
Construction of a Gene Knockout System for Application in
Paenibacillus alvei
CCM 2051
T
, Exemplified by the S-Layer Glycan Biosynthesis Initiation Enzyme WsfP
Authors: Zarschler Kristof , Janesch Bettina , Zayni Sonja , Schäffer Christina , Messner Paul
Published: March 2009

The gram-positive bacterium
Paenibacillus alvei
CCM 2051
T
is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [→3)-β-
d
-Gal
p
-(1[α-
d
-Glc
p
-(1→6)]→4)-β-
d
-Man
p
NAc-(1→] repeating units that is linked by an adaptor of -[GroA-2→OPO
2
→4-β-
d
-Man
p
NAc-(1→4)]→3)-α-
l
-Rha
p
-(1→3)-α-
l
-Rha
p
-(1→3)-α-
l
-Rha
p
-(1→3)-β-
d
-Gal
p
-(1→ to specific tyrosine residues of the S-layer protein. For elucidation of the mechanism governing S-layer glycan biosynthesis, a gene knockout system using bacterial mobile group II intron-mediated gene disruption was developed. The system is further based on the
sgsE
S-layer gene promoter of
Geobacillus stearothermophilus
NRS 2004/3a and on the
Geobacillus-Bacillus-Escherichia coli
shuttle vector pNW33N. As a target gene,
wsfP
, encoding a putative UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase, representing the predicted initiation enzyme of S-layer glycan biosynthesis, was disrupted. S-layer protein glycosylation was completely abolished in the insertional
P. alvei
CCM 2051
T
wsfP
mutant, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis evidence and carbohydrate analysis. Glycosylation was fully restored by plasmid-based expression of
wsfP
in the glycan-deficient
P. alvei
mutant, confirming that WsfP initiates S-layer protein glycosylation. This is the first report on the successful genetic manipulation of bacterial S-layer protein glycosylation in vivo, including transformation of and heterologous gene expression and gene disruption in the model organism
P. alvei
CCM 2051
T
.
Development of
cpn60
-Based Real-Time Quantitative PCR Assays for the Detection of 14
Campylobacter
Species and Application to Screening of Canine Fecal Samples
Authors: Chaban Bonnie , Musil Kristyna M. , Himsworth Chelsea G. , Hill Janet E.
Published: March 2009

Campylobacter
species are important organisms in both human and animal health. The identification of
Campylobacter
currently requires the growth of organisms from complex samples and biochemical identification. In many cases, the condition of the sample being tested and/or the fastidious nature of many
Campylobacter
species has limited the detection of campylobacters in a laboratory setting. To address this, we have designed a set of real-time quantitative PCR (qPCR) assays to detect and quantify 14
Campylobacter
species,
C. coli, C. concisus, C. curvus, C. fetus, C. gracilis, C. helveticus, C. hyointestinalis, C. jejuni, C. lari, C. mucosalis, C. rectus, C. showae, C. sputorum
, and
C. upsaliensis
, directly from DNA extracted from feces. By use of a region of the
cpn60
(also known as
hsp60
or
groEL
) gene, which encodes the universally conserved 60-kDa chaperonin, species-specific assays were designed and validated. These assays were then employed to determine the prevalence of
Campylobacter
species in fecal samples from dogs. Fecal samples were found to contain detectable and quantifiable levels of
C. fetus, C. gracilis, C. helveticus, C. jejuni, C. showae
, and
C. upsaliensis
, with the majority of samples containing multiple
Campylobacter
species. This study represents the first report of
C. fetus, C. gracilis, C. mucosalis
, and
C. showae
detection in dogs and implicates dogs as a reservoir for these species. The qPCR assays described offer investigators a new tool to study many
Campylobacter
species in a culture-independent manner.
Bile-Inducible Efflux Transporter from
Bifidobacterium longum
NCC2705, Conferring Bile Resistance
Authors: Gueimonde Miguel , Garrigues Christel , van Sinderen Douwe , de los Reyes-Gavilán Clara G. , Margolles Abelardo
Published: March 2009

Bifidobacteria are normal inhabitants of the human gut. Some strains of this genus are considered health promoting or probiotic, being included in numerous food products. In order to exert their health benefits, these bacteria must overcome biological barriers, including bile salts, to colonize and survive in specific parts of the intestinal tract. The role of multidrug resistance (MDR) transporters in bile resistance of probiotic bacteria and the effect of bile on probiotic gene expression are not fully understood. In the present study, the effect of subinhibitory concentrations of bile on the expression levels of predicted MDR genes from three different bifidobacterial strains, belonging to
Bifidobacterium longum
subsp.
longum, Bifidobacterium breve
, and
Bifidobacterium animalis
subsp.
lactis
, was tested. In this way, two putative MDR genes whose expression was induced by bile, BL0920 from
B. longum
and its homolog, Bbr0838, from
B. breve
, were identified. The expression of the BL0920 gene in
Escherichia coli
was shown to confer resistance to bile, likely to be mediated by active efflux from the cells. To the best of our knowledge, this represents the first identified bifidobacterial bile efflux pump whose expression is induced by bile.
Intestinal Endocellular Symbiotic Bacterium of the Macaque Louse
Pedicinus obtusus
: Distinct Endosymbiont Origins in Anthropoid Primate Lice and the Old World Monkey Louse
Authors: Fukatsu Takema , Hosokawa Takahiro , Koga Ryuichi , Nikoh Naruo , Kato Takuya , Hayama Shin-ichi , Takefushi Haruo , Tanaka Ichirou
Published: March 2009

A symbiotic bacterium of the macaque louse,
Pedicinus obtusus
, was characterized. The symbiont constituted a gammaproteobacterial lineage distinct from the symbionts of anthropoid primate lice, localized in the midgut epithelium and the ovaries and exhibiting AT-biased genes and accelerated molecular evolution. The designation “
Candidatus
Puchtella pedicinophila” was proposed for it.
Cloning and In Situ Expression Studies of the
Hydrogenobaculum
Arsenite Oxidase Genes
Authors: Clingenpeel Scott R. , D'Imperio Seth , Oduro Harry , Druschel Greg K. , McDermott Timothy R.
Published: March 2009

Novel arsenite [As(III)] oxidase structural genes (
aoxAB
) were cloned from
Hydrogenobaculum
bacteria isolated from an acidic geothermal spring. Reverse transcriptase PCR demonstrated expression throughout the outflow channel, and the
aoxB
cDNA clones exhibited distribution patterns relative to the physicochemical gradients in the spring. Microelectrode analyses provided evidence of quantitative As(III) transformation within the microbial mat.
Microbial Diversity of Septic Tank Effluent and a Soil Biomat
Authors: Tomaras Jill , Sahl Jason W. , Siegrist Robert L. , Spear John R.
Published: March 2009

Microbial diversity of septic tank effluent (STE) and the biomat that is formed as a result of STE infiltration on soil were characterized by 16S rRNA gene sequence analysis. Results indicate that microbial communities are different within control soil, STE, and the biomat and that microbes found in STE are not found in the biomat. The development of a stable soil biomat appears to provide the best on-site water treatment or protection for subsequent groundwater interactions of STE.
Empirical Leucine-to-Carbon Conversion Factors for Estimating Heterotrophic Bacterial Production: Seasonality and Predictability in a Temperate Coastal Ecosystem
Authors: Calvo-Díaz Alejandra , Morán Xosé Anxelu G.
Published: March 2009

Leucine-to-carbon conversion factors (CFs) are needed for converting substrate incorporation into biomass production of heterotrophic bacteria. During 2006 we performed 20 dilution experiments for determining the spatiotemporal variability of empirical CFs in temperate Atlantic coastal waters. Values (0.49 to 1.92 kg C mol Leu
−1
) showed maxima in autumn to early winter and minima in summer. Spatially averaged CFs were significantly negatively correlated with in situ leucine incorporation rates (
r
= −0.91) and positively correlated with phosphate concentrations (
r
= 0.76). These relationships, together with a strong positive covariation between cell-specific leucine incorporation rates and carbon contents (
r
= 0.85), were interpreted as a strategy to maximize survival through protein synthesis and low growth rates under nutrient limitation (low CFs) until favorable conditions stimulate cell division relative to protein synthesis (high CFs). A multiple regression with in situ leucine incorporation rates and cellular carbon contents explained 96% of CF variance in our ecosystem, suggesting their potential prediction from more easily measurable routine variables. The use of the theoretical CF of 1.55 kg C mol Leu
−1
would have resulted in a serious overestimation (73%) of annual bacterial production rates. Our results emphasize the need for considering the temporal scale in CFs for bacterial production studies.
Inactivation of the SauI Type I Restriction-Modification System Is Not Sufficient To Generate
Staphylococcus aureus
Strains Capable of Efficiently Accepting Foreign DNA
Authors: Veiga Helena , Pinho Mariana G.
Published: March 2009

Genetic manipulation of
Staphylococcus aureus
is limited by the availability of only a single strain, RN4220, that is capable of easily accepting foreign DNA. Inactivation of the
hsdR
gene of the SauI type I restriction-modification system was shown previously to be responsible for the high transformation efficiency of RN4220 (D. E. Waldron and J. A. Lindsay, J Bacteriol.
188:
5578-5585, 2006). However, deletion of this gene in three different
S. aureus
strains was not sufficient to make them readily transformable, which would be remarkably useful for genetic studies of this pathogenic organism. These results indicate that another unknown factor(s) is required for the transformable phenotype in
S. aureus
.
Stereospecific Biotransformation of Dihydrodaidzein into (3
S
)-Equol by the Human Intestinal Bacterium
Eggerthella
Strain Julong 732
Authors: Kim Mihyang , Kim Su-Il , Han Jaehong , Wang Xiu-Ling , Song Dae-Geun , Kim Soo-Un
Published: March 2009

Stereochemical course of isoflavanone dihydrodaidzein (DHD) reduction into the isoflavan (3
S
)-equol via tetrahydrodaidzein (THD) by the human intestinal anaerobic bacterium
Eggerthella
strain Julong 732 was studied. THD was synthesized by catalytic hydrogenation, and each stereoisomer was separated by chiral high-performance liquid chromatography. Circular dichroism spectroscopy was used to elucidate the absolute configurations of four synthetic THD stereoisomers. Rapid racemization of DHD catalyzed by Julong 732 prevented the substrate stereospecificity in the conversion of DHD into THD from being confirmed. The absolute configuration of THD, prepared by reduction of DHD in the cell-free incubation, was assigned as (3
R
,4
S
) via comparison of the retention time to that of the authentic THD by chiral chromatography. Dehydroequol (DE) was unable to produce the (3
S
)-equol both in the cell-free reaction and in the bacterial transformation, negating the possible intermediacy of DE. Finally, the intermediate (3
R
,4
S
)-THD was reduced into (3
S
)-equol by the whole cell, indicating the inversion of stereochemistry at C-3 during the reduction. A possible mechanism accounting for the racemization of DHD and the inversion of configuration of THD during reduction into (3
S
)-equol is proposed.
Diversity of Bacteria in the Marine Sponge
Aplysina fulva
in Brazilian Coastal Waters
Authors: Hardoim C. C. P. , Costa R. , Araújo F. V. , Hajdu E. , Peixoto R. , Lins U. , Rosado A. S. , van Elsas J. D.
Published: March 2009

Microorganisms can account for up to 60% of the fresh weight of marine sponges. Marine sponges have been hypothesized to serve as accumulation spots of particular microbial communities, but it is unknown to what extent these communities are directed by the organism or the site or occur randomly. To address this question, we assessed the composition of specific bacterial communities associated with
Aplysina fulva
, one of the prevalent sponge species inhabiting Brazilian waters. Specimens of
A. fulva
and surrounding seawater were collected in triplicate in shallow water at two sites, Caboclo Island and Tartaruga beach, Búzios, Brazil. Total community DNA was extracted from the samples using “direct” and “indirect” approaches. 16S rRNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the total bacterial community and of specific bacterial groups
—Pseudomonas
and
Actinobacteria—
revealed that the structure of these assemblages in
A. fulva
differed drastically from that observed in seawater. The DNA extraction methodology and sampling site were determinative for the composition of actinobacterial communities in
A. fulva
. However, no such effects could be gleaned from total bacterial and
Pseudomonas
PCR-DGGE profiles. Bacterial 16S rRNA gene clone libraries constructed from directly and indirectly extracted DNA did not differ significantly with respect to diversity and composition. Altogether, the libraries encompassed 15 bacterial phyla and the candidate division TM7. Clone sequences affiliated with the
Cyanobacteria
,
Chloroflexi
,
Gamma
- and
Alphaproteobacteria
,
Actinobacteria
,
Bacteroidetes
, and
Acidobacteria
were, in this order, most abundant. The bacterial communities associated with the
A. fulva
specimens were distinct and differed from those described in studies of sponge-associated microbiota performed with other sponge species.
Identification of Natural Target Proteins Indicates Functions of a Serralysin-Type Metalloprotease, PrtA, in Anti-Immune Mechanisms
Authors: Felföldi Gabriella , Marokházi Judit , Képiró Miklós , Venekei István
Published: March 2009

Serralysins are generally thought to function as pathogenicity factors of bacteria, but so far no hard evidence of this (e.g., specific substrate proteins that are sensitive to the cleavage by these proteases) has been found. We have looked for substrate proteins to a serralysin-type proteinase, PrtA, in a natural host-pathogen molecular interaction system involvingManduca sextaandPhotorhabdus luminescens. The exposure in vitro of hemolymph to PrtA digestion resulted in selective cleavage of 16 proteins, provisionally termed PAT (PrtA target) proteins. We could obtain sequence information for nine of these PrtA sensitive proteins, and by searching databases, we could identify six of them. Each has immune-related function involving every aspect of the immune defense: β-1,3 glucan recognition protein 2 (immune recognition), hemocyte aggregation inhibitor protein (HAIP), serine proteinase homolog 3, six serpin-1 variants, including serpin-1I (immune signaling and regulation), and scolexins A and B (coagulation cascade effector function). The functions of the identified PrtA substrate proteins shed new light on a possible participation of a serralysin in the virulence mechanism of a pathogen. Provided these proteins are targets of PrtA in vivo, this might represent, among others, a complex suppressive role on the innate immune response via interference with both the recognition and the elimination of the pathogen during the first, infective stage of the host-pathogen interaction. Our results also raise the possibility that the natural substrate proteins of serralysins of vertebrate pathogens might be found among the components of the innate immune system.
Genetic Diversity and Fitness of
Fusarium graminearum
Populations from Rice in Korea
Authors: Lee Jungkwan , Chang In-Young , Kim Hun , Yun Sung-Hwan , Leslie John F. , Lee Yin-Won
Published: March 2009

Fusarium graminearum
is an important fungal pathogen of cereal crops and produces mycotoxins, such as the trichothecenes nivalenol and deoxynivalenol. This species may be subdivided into a series of genetic lineages or phylogenetic species. We identified strains of
F. graminearum
from the Republic of Korea to lineage, tested their ability to produce nivalenol and deoxynivalenol, and determined the genetic composition and structure of the populations from which they were recovered. Based on amplified fragment length polymorphism (AFLP), PCR genotyping, and chemical analyses of trichothecenes, all 249 isolates from southern provinces belonged to lineage 6, with 241 having the nivalenol genotype and 8 having the deoxynivalenol genotype. In the eastern Korea province, we recovered 84 lineage 6 isolates with the nivalenol genotype and 23 lineage 7 isolates with the deoxynivalenol genotype. Among 333 lineage 6 isolates, 36% of the AFLP bands were polymorphic, and there were 270 multilocus haplotypes. Genetic identity among populations was high (>0.972), and genotype diversity was low (30 to 58%). To test the adaptation of lineage 6 to rice, conidial mixtures of strains from lineages 3, 6, and 7 were inoculated onto rice plants and then recovered from the rice grains produced. Strains representing lineages 6 and 7 were recovered from inoculated spikelets at similar frequencies that were much higher than those for the strain representing lineage 3. Abundant perithecia were produced on rice straw, and 247 single-ascospore isolates were recovered from 247 perithecia. Perithecia representing lineage 6 (87%) were the most common, followed by those representing lineage 7 (13%), with perithecia representing lineage 3 not detected. These results suggest that
F. graminearum
lineage 6 may have a host preference for rice and that it may be more fit in a rice agroecosystem than are the other lineages present in Korea.
Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System
Authors: Ferrari B. C. , Gillings M. R.
Published: March 2009

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium
Leifsonia xyli
, a plant pathogen that has never been isolated outside the host.
Development and Application of a
upp
-Based Counterselective Gene Replacement System for the Study of the S-Layer Protein SlpX of
Lactobacillus acidophilus
NCFM
Authors: Goh Yong Jun , Azcárate-Peril M. Andrea , O'Flaherty Sarah , Durmaz Evelyn , Valence Florence , Jardin Julien , Lortal Sylvie , Klaenhammer Todd R.
Published: March 2009

In silico genome analysis of
Lactobacillus acidophilus
NCFM coupled with gene expression studies have identified putative genes and regulatory networks that are potentially important to this organism's survival, persistence, and activities in the gastrointestinal tract. Correlation of key genotypes to phenotypes requires an efficient gene replacement system. In this study, use of the
upp
-encoded uracil phosphoribosyltransferase (UPRTase) of
L. acidophilus
NCFM was explored as a counterselection marker to positively select for recombinants that have resolved from chromosomal integration of pORI-based plasmids. An isogenic mutant carrying a
upp
gene deletion was constructed and was resistant to 5-fluorouracil (5-FU), a toxic uracil analog that is also a substrate for UPRTase. A 3.0-kb pORI-based counterselectable integration vector bearing a
upp
expression cassette, pTRK935, was constructed and introduced into the Δ
upp
host harboring the pTRK669 helper plasmid. Extrachromosomal replication of pTRK935 complemented the mutated chromosomal
upp
allele and restored sensitivity to 5-FU. This host background provides a platform for a two-step plasmid integration and excision strategy that can select for plasmid-free recombinants with either the wild-type or mutated allele of the targeted gene in the presence of 5-FU. The efficacy of the system was demonstrated by in-frame deletion of the
slpX
gene (LBA0512) encoding a novel 51-kDa secreted protein associated with the S-layer complex of
L. acidophilus
. The resulting Δ
slpX
mutant exhibited lower growth rates, increased sensitivity to sodium dodecyl sulfate, and greater resistance to bile. Overall, this improved gene replacement system represents a valuable tool for investigating the mechanisms underlying the probiotic functionality of
L. acidophilus
.
Changes in Benthic Denitrification, Nitrate Ammonification, and Anammox Process Rates and Nitrate and Nitrite Reductase Gene Abundances along an Estuarine Nutrient Gradient (the Colne Estuary, United Kingdom)
Authors: Dong Liang F. , Smith Cindy J. , Papaspyrou Sokratis , Stott Andrew , Osborn A. Mark , Nedwell David B.
Published: March 2009

Estuarine sediments are the location for significant bacterial removal of anthropogenically derived inorganic nitrogen, in particular nitrate, from the aquatic environment. In this study, rates of benthic denitrification (DN), dissimilatory nitrate reduction to ammonium (DNRA), and anammox (AN) at three sites along a nitrate concentration gradient in the Colne estuary, United Kingdom, were determined, and the numbers of functional genes (
narG
,
napA
,
nirS
, and
nrfA
) and corresponding transcripts encoding enzymes mediating nitrate reduction were determined by reverse transcription-quantitative PCR. In situ rates of DN and DNRA decreased toward the estuary mouth, with the findings from slurry experiments suggesting that the potential for DNRA increased while the DN potential decreased as nitrate concentrations declined. AN was detected only at the estuary head, accounting for ∼30% of N
2
formation, with 16S rRNA genes from anammox-related bacteria also detected only at this site. Numbers of
narG
genes declined along the estuary, while
napA
gene numbers were stable, suggesting that NAP-mediated nitrate reduction remained important at low nitrate concentrations.
nirS
gene numbers (as indicators of DN) also decreased along the estuary, whereas
nrfA
(an indicator for DNRA) was detected only at the two uppermost sites. Similarly, nitrate and nitrite reductase gene transcripts were detected only at the top two sites. A regression analysis of log(
n
+ 1) process rate data and log(
n
+ 1) mean gene abundances showed significant relationships between DN and
nirS
and between DNRA and
nrfA
. Although these log-log relationships indicate an underlying relationship between the genetic potential for nitrate reduction and the corresponding process activity, fine-scale environmentally induced changes in rates of nitrate reduction are likely to be controlled at cellular and protein levels.
Application of a Newly Identified and Characterized 18-
O
-Acyltransferase in Chemoenzymatic Synthesis of Selected Natural and Nonnatural Bioactive Derivatives of Phoslactomycins
Authors: Ghatge Mohini S. , Palaniappan Nadaraj , Alhamadsheh Ma'moun M. , DiBari Jessica , Reynolds Kevin A.
Published: March 2009

Phoslactomycins (PLMs) and related leustroducsins (LSNs) have been isolated from a variety of bacteria based on antifungal, anticancer, and other biological assays.
Streptomyces
sp. strain HK 803 produces five PLM analogs (PLM A and PLMs C to F) in which the C-18 hydroxyl substituent is esterified with a range of branched, short-alkyl-chain carboxylic acids. The proposed pathway intermediate, PLM G, in which the hydroxyl residue is not esterified has not been observed at any significant level in fermentation, and the only route to this potentially useful intermediate has been an enzymatic deacylation of other PLMs and LSNs. We report that deletion of
plmS
3
from the PLM biosynthetic cluster gives rise to a mutant which accumulates the PLM G intermediate. The 921-bp
plmS
3
open reading frame was cloned and expressed as an N-terminally polyhistidine-tagged protein in
Escherichia coli
and shown to be an 18-
O
acyltransferase, catalyzing conversion of PLM G to PLM A, PLM C, and PLM E using isobutyryl coenzyme A (CoA), 3-methylbutyryl-CoA, and cyclohexylcarbonyl-CoA, respectively. The efficiency of this process (
k
cat
of 28 ± 3 min
−1
and

K
m

of 88 ± 16 μM) represents a one-step chemoenzymatic alternative to a multistep synthetic process for selective chemical esterification of the C-18 hydroxy residue of PLM G. PlmS
3
was shown to catalyze esterification of PLM G with CoA and
N
-acetylcysteamine thioesters of various saturated, unsaturated, and aromatic carboxylic acids and thus also to provide an efficient chemoenzymatic route to new PLM analogs.
Metabolic Engineering of
Escherichia coli
for Enhanced Production of (
R
)- and (
S
)-3-Hydroxybutyrate
Authors: Tseng Hsien-Chung , Martin Collin H. , Nielsen David R. , Prather Kristala L. Jones
Published: March 2009

Synthetic metabolic pathways have been constructed for the production of enantiopure (
R
)- and (
S
)-3-hydroxybutyrate (3HB) from glucose in recombinant
Escherichia coli
strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (
R
)-enantiomer, and Hbd, producing the (
S
)-enantiomer, were utilized to control the 3HB chirality across two
E. coli
backgrounds, BL21Star(DE3) and MG1655(DE3), representing
E. coli
B- and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (
R
)-3HB and (
S
)-3HB as high as 2.92 g liter
−1
and 2.08 g liter
−1
, respectively, in shake flask cultures within 2 days. The NADPH/NADP
+
ratio was found to be two- to three-fold higher than the NADH/NAD
+
ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (
R
)-3HB than (
S
)-3HB. To the best of our knowledge, this study reports the highest (
S
)-3HB titer achieved in shake flask
E. coli
cultures to date.
Exopolyphosphatases PPX1 and PPX2 from
Corynebacterium glutamicum
Authors: Lindner Steffen N. , Knebel Sandra , Wesseling Hendrik , Schoberth Siegfried M. , Wendisch Volker F.
Published: March 2009

Corynebacterium glutamicum
accumulates up to 300 mM of inorganic polyphosphate (PolyP) in the cytosol or in granules. The gene products of cg0488 (
ppx1
) and cg1115 (
ppx2
) were shown to be active as exopolyphosphatases (PPX), as overexpression of either gene resulted in higher exopolyphosphatase activities in crude extracts and deletion of either gene with lower activities than those of the wild-type strain. PPX1 and PPX2 from
C. glutamicum
share only 25% identical amino acids and belong to different protein groups, which are distinct from enterobacterial, archaeal, and yeast exopolyphosphatases. In comparison to that in the wild type, more intracellular PolyP accumulated in the Δ
ppx1
and Δ
ppx2
deletion mutations but less when either
ppx1
or
ppx2
was overexpressed. When
C. glutamicum
was shifted from phosphate-rich to phosphate-limiting conditions, a growth advantage of the deletion mutants and a growth disadvantage of the overexpression strains compared to the wild type were observed. Growth experiments, exopolyphosphatase activities, and intracellular PolyP concentrations revealed PPX2 as being a major exopolyphosphatase from
C. glutamicum
. PPX2
His
was purified to homogeneity and shown to be active as a monomer. The enzyme required Mg
2+
or Mn
2+
cations but was inhibited by millimolar concentrations of Mg
2+
, Mn
2+
, and Ca
2+
. PPX2 from
C. glutamicum
was active with short-chain polyphosphates, even accepting pyrophosphate, and was inhibited by nucleoside triphosphates.
Distribution, Diversity, and Potential Mobility of Extrachromosomal Elements Related to the
Bacillus anthracis
pXO1 and pXO2 Virulence Plasmids
Authors: Hu Xiaomin , Van der Auwera Géraldine , Timmery Sophie , Zhu Lei , Mahillon Jacques
Published: March 2009

The presence of a pXO1- and/or pXO2-like plasmid(s) in clinical isolates of
Bacillus cereus
sensu stricto and in strains of the biopesticide
Bacillus thuringiensis
has been reported recently, and the pXO2-like plasmid pBT9727 and another pXO2-like plasmid, pAW63, were found to be conjugative. In this study, a total of 1,000
B. cereus
group isolates were analyzed for the presence of pXO1- and pXO2-like replicons and for the presence of pXO2-related conjugative modules. pXO1- and pXO2-like replicons were present in ca. 6.6% and 7.7% of random environmental samples, respectively, and ca. 1.54% of the strains were positive for pXO2-like transfer module genes. Only the strains harboring a pXO2-like replicon also contained the corresponding transfer genes. For the strains which contained a pXO1- and/or pXO2-like replicon(s), a large plasmid(s) whose size was similar to that of pXO1-like and/or pXO2-like plasmids was also observed, but none of these isolates were found to carry the
Bacillus anthracis
toxin or capsule virulence genes. Furthermore, 17 of 22 pXO2-like plasmids containing the transfer modules were able to self-transfer and to mobilize small plasmids. No pXO1- or pXO2-like plasmid lacking the cognate transfer modules has been found to have transfer potential. In the strains possessing the putative pXO2-like conjugative apparatus, variations in the presence of the group II introns B.th.I.1 and B.th.I.2 were observed, suggesting that there is important flexibility in the conjugation modules and their regulation. There was no consistent correlation between a pXO2-like
repA
dendrogram and the presence of the
tra
region or between a
virB4
dendrogram and transfer ability. Discrepancies between pXO2-like
repA
and
virB4
dendrograms were also observed, indicating that the evolution of pXO2 is an active process.
Light-Dependent Transcriptional Regulation of Genes of Biogeochemical Interest in the Diploid and Haploid Life Cycle Stages of
Emiliania huxleyi
Authors: Richier Sophie , Kerros Marie-Emmanuelle , de Vargas Colomban , Haramaty Liti , Falkowski Paul G. , Gattuso Jean-Pierre
Published: March 2009

The expression of genes of biogeochemical interest in calcifying and noncalcifying life stages of the coccolithophore
Emiliania huxleyi
was investigated. Transcripts potentially involved in calcification were tested through a light-dark cycle. These transcripts were more abundant in calcifying cells and were upregulated in the light. Their application as potential candidates for in situ biogeochemical proxies is also suggested.