bluuebunny/crossref_metadata_2025 huggingface dataset:
Branched fibres in old dystrophic mdx muscle are associated with mechanical weakening of the sarcolemma, abnormal Ca2+ transients and a breakdown of Ca2+ homeostasis during fatigue
Authors: Head Stewart I.
Published: February 2010

In the dystrophinopathies, skeletal muscle fibres undergo cycles of degeneration and regeneration, with regenerated fibres displaying a branched morphology. This study tests the hypothesis that regenerated branched fibres are mechanically weakened by the presence of branches and are damaged by contractions which do not affect unbranched dystrophin‐negative fibres. Experiments were carried out on single fast‐twitch fibres and whole muscle from the dystrophin‐negative mdx mouse. Fura‐2 was ionophoresed into fibres to measure intracellular Ca2+ concentration ([Ca2+]i). Single branched mdx fibres have abnormal Ca2+ kinetics, with the [Ca2+]i transient at the peak of the twitch depressed, are damaged by fatiguing activation, resulting in a breakdown of Ca2+ homeostasis, and break at branch points when submaximally activated in skinned fibre experiments. When old intact isolated mdx muscles, with >90% branched fibres, are eccentrically activated with a moderate eccentric protocol there is a 40 ± 8% reduction in maximal force. Isolated single fibres from these muscles show areas of damage at fibre branch points. This same eccentric protocol causes no force loss in either littermate control muscles or mdx muscles with <10% branched fibres. I present a two‐stage hypothesis for muscle damage in the dystrophinopathies, as follows: stage 1, the absence of dystrophin disrupts ion channel function, causing an activation of necrotizing Ca2+‐activated proteases, which results in regenerated branched fibres; and stage 2, branched fibres are mechanically damaged during contraction. These results may have implications when considering therapies for boys with Duchenne muscular dystrophy. In particular, any therapy aimed at rescuing the defective gene will presumably have to do so before the number of branched fibres has increased to a level where the muscle is mechanically compromised.
Computationally Efficient Multisensor Fusion Estimation Algorithms
Authors: Lee Seokhyoung , Shin Vladimir
Published: February 2010

This paper provides two computationally effective fusion estimation algorithms. The first algorithm is based on Cholesky factorization of a cross-covariance block matrix. This algorithm has low computational complexity and is equivalent to the standard composite fusion estimation algorithm as well. The second algorithm is based on a special approximation scheme for local cross-covariances. Such approximation is useful to compute matrix weights for fusion estimation in a multidimensional-multisensor environment. Subsequent computational analysis of the proposed fusion algorithms is presented with corresponding examples showing the low computational complexities of the new fusion estimation algorithms.
Rickettsia felisInfection in a Common Household Insect Pest,Liposcelis bostrychophila(Psocoptera: Liposcelidae)
Authors: Behar Adi , McCormick Laurie J. , Perlman Steve J.
Published: February 2010

Many species ofRickettsiaare well-known mammalian pathogens transmitted by blood-feeding arthropods. However, molecular surveys are continually uncovering novelRickettsiaspecies, often in unexpected hosts, including many arthropods that do not feed on blood. This study reports a systematic molecular characterization of aRickettsiainfecting the psocidLiposcelis bostrychophila(Psocoptera: Liposcelidae), a common and cosmopolitan household pest. Surprisingly, the psocidRickettsiais shown to beRickettsia felis, a human pathogen transmitted by fleas that causes serious morbidity and occasional mortality. The plasmid from the psocidR. feliswas sequenced and was found to be virtually identical to the one inR. felisfrom fleas. AsLiposcelisinsects are often intimately associated with humans and other vertebrates, it is speculated that they acquiredR. felisfrom fleas. Whether theR. felisin psocids causes disease in vertebrates is not known and warrants further study.
Two Types of Endosymbiotic Bacteria in the Enigmatic Marine Worm
Xenoturbella
bocki
Authors: Kjeldsen Kasper Urup , Obst Matthias , Nakano Hiroaki , Funch Peter , Schramm Andreas
Published: February 2010

Two types of endosymbiotic bacteria were identified in the gastrodermis of the marine invertebrate
Xenoturbella
bocki
(Xenoturbellida, Bilateria). While previously described
Chlamydia
-like endosymbionts were rare,
Gammaproteobacteria
distantly related to other endosymbionts and pathogens were abundant. The endosymbionts should be considered when interpreting the poorly understood ecology and evolution of
Xenoturbella
.
Detection and Quantification of Functional Genes of Cellulose- Degrading, Fermentative, and Sulfate-Reducing Bacteria and Methanogenic Archaea
Authors: Pereyra L. P. , Hiibel S. R. , Prieto Riquelme M. V. , Reardon K. F. , Pruden A.
Published: February 2010

Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria,
hydA
genes of fermentative bacteria,
dsrA
genes of sulfate-reducing bacteria, and
mcrA
genes of methanogenic archaea. Specificity was verified
in silico
and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.
Biofilm Formation by
Campylobacter jejuni
Is Increased under Aerobic Conditions
Authors: Reuter Mark , Mallett Arthur , Pearson Bruce M. , van Vliet Arnoud H. M.
Published: February 2010

The microaerophilic human pathogen
Campylobacter jejuni
is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that
C. jejuni
NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O
2
) than under microaerobic conditions (5% O
2
, 10% CO
2
), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in
C. jejuni
. Moreover, preformed biofilms shed high numbers of viable
C. jejuni
cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions,
C. jejuni
adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of
C. jejuni
.
Effects of Swine Manure on Macrolide, Lincosamide, and Streptogramin B Antimicrobial Resistance in Soils
Authors: Zhou Zhi , Raskin Lutgarde , Zilles Julie L.
Published: February 2010

Current agricultural practices involve inclusion of antimicrobials in animal feed and result in manure containing antimicrobials and antimicrobial-resistant microorganisms. This work evaluated the effects of land application of swine manure on the levels of tetracycline, macrolide, and lincosamide antimicrobials and on macrolide, lincosamide, and streptogramin B (MLS
B
) resistance in field soil samples and laboratory soil batch tests. MLS
B
and tetracycline antimicrobials were quantified after solid-phase extraction using liquid chromatography-tandem mass spectrometry. The prevalence of the ribosomal modification responsible for MLS
B
resistance in the same samples was quantified using fluorescence
in situ
hybridization. Macrolide antimicrobials were not detected in soil samples, while tetracyclines were detected, suggesting that the latter compounds persist in soil. No significant differences in ribosomal methylation or presumed MLS
B
resistance were observed when amended and unamended field soils were compared, although a transient (<20-day) increase was observed in most batch tests.
Clostridium
cluster XIVa accounted for the largest fraction of resistant bacteria identified in amended soils. Overall, this study did not detect a persistent increase in the prevalence of MLS
B
resistance due to land application of treated swine manure.
Enhancement of Enteric Adenovirus Cultivation by Viral Transactivator Proteins
Authors: Kim Misoon , Lim Mi Young , Ko GwangPyo
Published: February 2010

Human enteric adenoviruses (HAdVs; serotypes 40 and 41) are important waterborne and food-borne pathogens. However, HAdVs are fastidious, are difficult to cultivate, and do not produce a clear cytopathic effect during cell culture within a reasonable time. Thus, we examined whether the viral transactivator proteins cytomegalovirus (CMV) IE1 and hepatitis B virus (HBV) X promoted the multiplication of HAdVs. Additionally, we constructed a new 293 cell line expressing CMV IE1 protein for cultivation assays. We analyzed the nucleic acid sequences of the promoter regions of both E1A and hexon genes, which are considered to be the most important regions for HAdV replication. Expression of either HBV X or CMV IE1 protein significantly increased the promoter activities of E1A and hexon genes of HAdVs by as much as 14-fold during cell cultivation. The promotion of HAdV expression was confirmed by increased levels of both adenoviral DNA and mRNA expression. Finally, the newly developed 293 cell line expressing CMV IE1 protein showed an increase in viral DNA ranging from 574% to 619% compared with the conventional 293 cell line. These results suggest that the newly constructed cell line could be useful for efficient cultivation and research of fastidious HAdVs.
Requirement of a Mitogen-Activated Protein Kinase for Appressorium Formation and Penetration of Insect Cuticle by the Entomopathogenic Fungus
Beauveria bassiana
Authors: Zhang Yongjun , Zhang Jianqing , Jiang Xiaodong , Wang Guijiang , Luo Zhibing , Fan Yanhua , Wu Zengqiang , Pei Yan
Published: February 2010

Beauveria bassiana
is an important insect-pathogenic fungus that invades insects by direct penetration of the host cuticle. To delineate the molecular mechanisms involved in fungal infection, a mitogen-activated protein kinase (MAPK) gene, Bb
mpk1
, which encodes a YERK1 family MAPK was isolated and characterized. Targeted gene disruption of Bb
mpk1
resulted in a complete loss of virulence when applied topically to host insects but did not affect growth of the fungus when conidia were injected directly into the hemocoel. Hyphae of the mutant strain growing in the insect hemocoel were unable to penetrate the cuticle growing outwards and consequently failed to sporulate on the cadaver surface. These data suggest that BbMPK1 is essential for penetration of the insect cuticle both from the outside and from the inside-out in order to escape and disperse from the host. Inactivation of BbMPK1 also caused a significant decrease in fungal adhesion to insect cuticles and eliminated their ability to form appressoria. In order to identify downstream genes regulated by BbMPK1, a suppressive subtractive hybridization (SSH) library was generated comparing mutant and wild-type transcripts isolated during appressorium formation. Thirty-one genes screened from the SSH library were determined to be expressed in the wild-type strain but either significantly reduced or not expressed in the mutant. Ten genes showed high or medium similarity to known protein encoding genes, including proteins involved in cell surface hydrophobicity, lipid metabolism, microtubule dynamics, mitochondrial electron transport, chromatin remodeling, transcription, rRNA processing, small nucleolar RNA accumulation, oxidation of aldehydes, translation, and likely other cellular processes.
Role of Extracellular DNA during Biofilm Formation by
Listeria monocytogenes
Authors: Harmsen Morten , Lappann Martin , Knøchel Susanne , Molin Søren
Published: February 2010

Listeria monocytogenes
is a food-borne pathogen that is capable of living in harsh environments. It is believed to do this by forming biofilms, which are surface-associated multicellular structures encased in a self-produced matrix. In this paper we show that in
L. monocytogenes
extracellular DNA (eDNA) may be the only central component of the biofilm matrix and that it is necessary for both initial attachment and early biofilm formation for 41
L. monocytogenes
strains that were tested. DNase I treatment resulted in dispersal of biofilms, not only in microtiter tray assays but also in flow cell biofilm assays. However, it was also demonstrated that in a culture without eDNA, neither
Listeria
genomic DNA nor salmon sperm DNA by itself could restore the capacity to adhere. A search for additional necessary components revealed that peptidoglycan (PG), specifically
N
-acetylglucosamine (NAG), interacted with the DNA in a manner which restored adhesion. If a short DNA fragment (less than approximately 500 bp long) was added to an eDNA-free culture prior to addition of genomic or salmon sperm DNA, adhesion was prevented, indicating that high-molecular-weight DNA is required for adhesion and that the number of attachment sites on the cell surface can be saturated.
Probabilistic Model for
Listeria monocytogenes
Growth during Distribution, Retail Storage, and Domestic Storage of Pasteurized Milk
Authors: Koutsoumanis Konstantinos , Pavlis Athanasios , Nychas George-John E. , Xanthiakos Konstantinos
Published: February 2010

A survey on the time-temperature conditions of pasteurized milk in Greece during transportation to retail, retail storage, and domestic storage and handling was performed. The data derived from the survey were described with appropriate probability distributions and introduced into a growth model of
Listeria monocytogenes
in pasteurized milk which was appropriately modified for taking into account strain variability. Based on the above components, a probabilistic model was applied to evaluate the growth of
L. monocytogenes
during the chill chain of pasteurized milk using a Monte Carlo simulation. The model predicted that, in 44.8% of the milk cartons released in the market, the pathogen will grow until the time of consumption. For these products the estimated mean total growth of
L. monocytogenes
during transportation, retail storage, and domestic storage was 0.93 log CFU, with 95th and 99th percentiles of 2.68 and 4.01 log CFU, respectively. Although based on EU regulation 2073/2005 pasteurized milk produced in Greece belongs to the category of products that do not allow the growth of
L. monocytogenes
due to a shelf life (defined by law) of 5 days, the above results show that this shelf life limit cannot prevent
L. monocytogenes
from growing under the current chill chain conditions. The predicted percentage of milk cartons—initially contaminated with 1 cell/1-liter carton—in which the pathogen exceeds the safety criterion of 100 cells/ml at the time of consumption was 0.14%. The probabilistic model was used for an importance analysis of the chill chain factors, using rank order correlation, while selected intervention and shelf life increase scenarios were evaluated. The results showed that simple interventions, such as excluding the door shelf from the domestic storage of pasteurized milk, can effectively reduce the growth of the pathogen. The door shelf was found to be the warmest position in domestic refrigerators, and it was most frequently used by the consumers for domestic storage of pasteurized milk. Furthermore, the model predicted that a combination of this intervention with a decrease of the mean temperature of domestic refrigerators by 2°C may allow an extension of pasteurized milk shelf life from 5 to 7 days without affecting the current consumer exposure to
L. monocytogenes
.
Population Diversity of
Listeria monocytogenes
LO28: Phenotypic and Genotypic Characterization of Variants Resistant to High Hydrostatic Pressure
Authors: Van Boeijen Ineke K. H. , Chavaroche Anaïs A. E. , Valderrama Wladir B. , Moezelaar Roy , Zwietering Marcel H. , Abee Tjakko
Published: February 2010

A comparative phenotype analysis of 24
Listeria monocytogenes
LO28 stress-resistant variants obtained after high-pressure treatment was performed to assess their robustness and growth performance under a range of food-relevant conditions. In addition, genetic analysis was conducted to characterize the promoter regions and open reading frames of the class I and III transcriptional repressors CtsR and HrcA, which control production of specific sets of stress proteins. Analysis of stress survival capacity, motility, biofilm formation, and growth under various conditions showed all variants to be more resistant to pressure and heat than the wild type; however, differences among variants were observed in acid resistance, growth rate, motility, and biofilm-forming capacity. Genetic analysis revealed no variation in the genetic make-up of
hrcA
and its upstream region, but two variants had deletions in the upstream region of
ctsR
and seven variants had mutations in the
ctsR
gene itself. The results of the characterization were cluster analyzed to obtain insight into the diversity of variants. Ten unique variants and three clusters with specific features could be identified: one cluster consisting of seven variants having a mutation in the CtsR regulator gene, one cluster containing two variants with an aerobic biofilm formation capacity similar to that of the wild type, and a cluster composed of five immotile variants. The large population diversity of
L. monocytogenes
stress-resistant variants signifies the organism's genetic flexibility, which in turn may contribute to the survival and persistence of this human pathogen in food-processing environments.
Identification of Ciliate Grazers of Autotrophic Bacteria in Ammonia-Oxidizing Activated Sludge by RNA Stable Isotope Probing
Authors: Moreno Ana Maria , Matz Carsten , Kjelleberg Staffan , Manefield Mike
Published: February 2010

It is well understood that protozoa play a major role in controlling bacterial biomass and regulating nutrient cycling in the environment. Little is known, however, about the movement of carbon from specific reduced substrates, through functional groups of bacteria, to particular clades of protozoa. In this study we first identified the active protozoan phylotypes present in activated sludge, via the construction of an rRNA-derived eukaryote clone library. Most of the sequences identified belonged to ciliates of the subclass Peritrichia and amoebae, confirming the dominance of surface-associated protozoa in the activated sludge environment. We then demonstrated that
13
C-labeled protozoan RNA can be retrieved from activated sludge amended with
13
C-labeled protozoa or
13
C-labeled
Escherichia coli
cells by using an RNA stable isotope probing (RNA-SIP) approach. Finally, we used RNA-SIP to track carbon from bicarbonate and acetate into protozoa under ammonia-oxidizing and denitrifying conditions, respectively. RNA-SIP analysis revealed that the peritrich ciliate
Epistylis galea
dominated the acquisition of carbon from bacteria with access to CO
2
under ammonia-oxidizing conditions, while there was no evidence of specific grazing on acetate consumers under denitrifying conditions.
High-Throughput Method for Rapid Induction of Prophages from Lysogens and Its Application in the Study of Shiga Toxin-Encoding
Escherichia coli
Strains
Authors: McDonald James E. , Smith Darren L. , Fogg Paul C. M. , McCarthy Alan J. , Allison Heather E.
Published: February 2010

A high-throughput 96-well plate-based method for the rapid induction of endogenous prophages from individual bacterial strains was developed. The detection of endogenous prophages was achieved by the filtration of the culture liquor following norfloxacin induction and subsequent PCRs targeting bacteriophage-carried gene markers. The induction method was tested on 188 putative Shiga toxin (Stx)-producing
Escherichia coli
(STEC) strains and demonstrated the ability to detect both lambdoid and
stx-
carrying bacteriophages in strains for which plaques were not observed via plaque assay. Lambdoid bacteriophages were detected in 37% of the induced phage preparations via amplification of the
Q
gene, and Stx1- and Stx2-encoding phages were detected in 2 and 14% of the strains, respectively. The method therefore provided greater sensitivity for the detection of Stx and other lambdoid bacteriophage populations carried by STEC strains than that for the established method of plaque assay using bacterial indicator strains, enabling, for the first time, large-scale bacteriophage population and diversity studies.
Negative Effects of Sample Pooling on PCR-Based Estimates of Soil Microbial Richness and Community Structure
Authors: Manter Daniel K. , Weir Tiffany L. , Vivanco Jorge M.
Published: February 2010

In this study, we examined the effect of various pooling strategies on the characterization of soil microbial community composition and phylotype richness estimates. Automated ribosomal intergenic spacer analysis (ARISA) profiles were determined from soil samples that were (i) unpooled (extracted and amplified individually), (ii) pooled prior to PCR amplification, or (iii) pooled prior to DNA extraction. Regression analyses suggest that the less even the soil microbial community (i.e., low Shannon equitability,

E
H

), the greater was the impact of either pooling strategy on microbial detection (
R
2
= 0.766). For example, at a tropical rainforest site, which had the most uneven fungal (

E
H

of 0.597) and bacterial communities (

E
H

of 0.822), the unpooled procedure detected an additional 67 fungal and 115 bacterial phylotypes relative to either of the pooled procedures. Phylotype rarity, resulting in missed detection upon pooling, differed between the fungal and bacterial communities. Fungi were typified by locally abundant but spatially rare phylotypes, and the bacteria were typified by locally rare but spatially ubiquitous phylotypes. As a result, pooling differentially influenced plot comparisons, leading to an increase in similarity for the bacterial community and a decrease in the fungal community. In conclusion, although pooling reduces sample numbers and variability, it could mask a significant portion of the detectable microbial community, particularly for fungi due to their higher spatial heterogeneity.
Activated Alleles of the Schizosaccharomyces pombegpa2+Gα Gene Identify Residues Involved in GDP-GTP Exchange
Authors: Ivey F. Douglas , Taglia Francis X. , Yang Fan , Lander Matthew M. , Kelly David A. , Hoffman Charles S.
Published: February 2010

TheSchizosaccharomyces pombeglucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Gα subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activatedgpa2alleles that bypass the loss of the Git5-Git11 Gβγ dimer to repress transcription of the glucose-regulatedfbp1+gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into thegpa2+chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Gα function, thus extending our understanding of Gα protein structure-function relationships.
F-Actin Dynamics in Neurospora crassa
Authors: Berepiki Adokiye , Lichius Alexander , Shoji Jun-Ya , Tilsner Jens , Read Nick D.
Published: February 2010

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in
Neurospora crassa
and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential
Saccharomyces cerevisiae
actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in
N. crassa
without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.
Ste12 and Ste12-Like Proteins, Fungal Transcription Factors Regulating Development and Pathogenicity
Authors: Wong Sak Hoi Joanne , Dumas Bernard
Published: February 2010

Ste12 and Ste12-like proteins are transcription factors found exclusively in the fungal kingdom. In the yeast model
Saccharomyces cerevisiae
, where the first member was identified, Ste12p was shown to regulate mating and invasive/pseudohyphal growth. In recent literature, there have been several reports of Ste12-like factors in multiple fungal systems, yeasts or filamentous fungi, with saprophytic or parasitic life-styles. In all these models, Ste12 and Ste12-like factors are involved in the regulation of fungal development and pathogenicity. In this review, we discuss the features, the regulation, and the role of Ste12 and Ste12-like factors by highlighting the similarities and dissimilarities that occur within this group.
Genetic Engineering of Algae for Enhanced Biofuel Production
Authors: Radakovits Randor , Jinkerson Robert E. , Darzins Al , Posewitz Matthew C.
Published: February 2010

There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H2yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H2production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.
Genetic Modification of theSalmonellaMembrane Physical State Alters the Pattern of Heat Shock Response
Authors: Porta Amalia , Török Zsolt , Horvath Ibolya , Franceschelli Silvia , Vígh László , Maresca Bruno
Published: February 2010

It is now recognized that membranes are not simple physical barriers but represent a complex and dynamic environment that affects membrane protein structures and their functions. Recent data emphasize the role of membranes in sensing temperature changes, and it has been shown that the physical state of the plasma membrane influences the expression of a variety of genes such as heat shock genes. It has been widely shown that minor alterations in lipid membranes are critically involved in the conversion of signals from the environment to the transcriptional activation of heat shock genes. Previously, we have proposed that the composition, molecular arrangement, and physical state of lipid membranes and their organization have crucial roles in cellular responses during stress caused by physical and chemical factors as well as in pathological states. Here, we show that transformation ofSalmonella entericaserovar Typhimurium LT2 (SalmonellaTyphimurium) with a heterologous Δ12-desaturase (or with itstrans-membrane regions) causes major changes in the pathogen's membrane dynamic. In addition, this pathogen is strongly impaired in the synthesis of major stress proteins (heat shock proteins) under heat shock. These data support the hypothesis that the perception of temperature inSalmonellais strictly controlled by membrane order and by a specific membrane lipid/protein ratio that ultimately causes transcriptional activation of heat shock genes. These results represent a previously unrecognized mode of sensing temperature variation used by this pathogen at the onset of infection.
Changes in Membrane Fluid State and Heat Shock Response Cause Attenuation of Virulence
Authors: Porta Amalia , Eletto Annamaria , Török Zsolt , Franceschelli Silvia , Glatz Attila , Vígh László , Maresca Bruno
Published: February 2010

So far attenuation of pathogens has been mainly obtained by chemical or heat treatment of microbial pathogens. Recently, live attenuated strains have been produced by genetic modification. We have previously demonstrated that in several prokaryotes as well as in yeasts and mammalian cells the heat shock response is controlled by the membrane physical state (MPS). We have also shown that in
Salmonella enterica
serovar Typhimurium LT2 (
Salmonella
Typhimurium) overexpression of a Δ
12
-desaturase gene alters the MPS, inducing a sharp impairment of transcription of major heat shock genes and failure of the pathogen to grow inside macrophage (MΦ) (A. Porta et al., J. Bacteriol. 192:1988-1998, 2010). Here, we show that overexpression of a homologous Δ
9
-desaturase sequence in the highly virulent G217B strain of the human fungal pathogen
Histoplasma capsulatum
causes loss of its ability to survive and persist within murine MΦ along with the impairment of the heat shock response. When the attenuated strain of
H. capsulatum
was injected in a mouse model of infection, it did not cause disease. Further, treated mice were protected when challenged with the virulent fungal parental strain. Attenuation of virulence in MΦ of two evolutionarily distant pathogens was obtained by genetic modification of the MPS, suggesting that this is a new method that may be used to produce attenuation or loss of virulence in both other intracellular prokaryotic and eukaryotic pathogens. This new procedure to generate attenuated forms of pathogens may be used eventually to produce a novel class of vaccines based on the genetic manipulation of a pathogen's membrane fluid state and stress response.
Global Regulation of Food Supply by
P
seudomonas p
utida
DOT-T1E
Authors: Daniels Craig , Godoy Patricia , Duque Estrella , Molina-Henares M. Antonia , de la Torre Jesús , del Arco José María , Herrera Carmen , Segura Ana , Guazzaroni M. Eugenia , Ferrer Manuel , Ramos Juan Luis
Published: February 2010

Pseudomonas putida
DOT-T1E was used as a model to develop a “phenomics” platform to investigate the ability of
P. putida
to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes Δ
crc
, Δ
crp
, Δ
cyoB
, and Δ
ptsN
allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and
d
-fructose is influenced by CyoB, and
l
-
arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e.,
l
-valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides,
d
-amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine/cysteine catabolism while PtsN and Crc are needed for
N
-acetyl-
l
-cysteamine utilization. Growth of global regulator mutants was also influenced by stressors of different types (antibiotics, oxidative agents, and metals). Overall and in combination with results for growth in the presence of various stressors, these phenomics assays provide multifaceted insights into the complex decision-making process involved in nutrient supply, optimization, and survival.
Role of the DinB Homologs Rv1537 and Rv3056 in
Mycobacterium tuberculosis
Authors: Kana Bavesh D. , Abrahams Garth L. , Sung Nackmoon , Warner Digby F. , Gordhan Bhavna G. , Machowski Edith E. , Tsenova Liana , Sacchettini James C. , Stoker Neil G. , Kaplan Gilla , Mizrahi Valerie
Published: February 2010

The environment encountered by
Mycobacterium tuberculosis
during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage.
M. tuberculosis
possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both
dinB1
and
dinB2
are shown to be expressed
in vitro
in a growth phase-dependent manner, with
dinB2
levels 12- to 40-fold higher than those of
dinB1
. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of
dinB1
,
dinB2
, or both had no effect on the susceptibility of
M. tuberculosis
to compounds that form
N
2
-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring
rpoB
mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in
Mycobacterium smegmatis
. The lack of the effect of altering the complements or expression levels of
dinB1
and/or
dinB2
under conditions predicted to be phenotypically revealing suggests that the DinB homologs from
M. tuberculosis
do not behave like their counterparts from other organisms.
Effects of SequentialCampylobacter jejuni81-176 Lipooligosaccharide Core Truncations on Biofilm Formation, Stress Survival, and Pathogenesis
Authors: Naito Mizue , Frirdich Emilisa , Fields Joshua A. , Pryjma Mark , Li Jianjun , Cameron Andrew , Gilbert Michel , Thompson Stuart A. , Gaynor Erin C.
Published: February 2010

Campylobacter jejuniis a highly prevalent human pathogen for which pathogenic and stress survival strategies remain relatively poorly understood. We previously found that aC. jejunistrain 81-176 mutant defective for key virulence and stress survival attributes was also hyper-biofilm and hyperreactive to the UV fluorescent dye calcofluor white (CFW). We hypothesized that screening for CFW hyperreactive mutants would identify additional genes required forC. jejunipathogenesis properties. Surprisingly, two such mutants harbored lesions in lipooligosaccharide (LOS) genes (waaFandlgtF), indicating a complete loss of the LOS outer core region. We utilized this as an opportunity to explore the role of each LOS core-specific moiety in the pathogenesis and stress survival of this strain and thus also constructed ΔgalTand ΔcstIImutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (ΔwaaFand ΔlgtFbut not ΔgalTor ΔcstIImutants) exhibited enhanced biofilm formation. The presence of the complete outer core was also necessary for resistance to complement-mediated killing. In contrast, any LOS truncation, even that of the terminal sialic acid (ΔcstII), resulted in diminished resistance to polymyxin B. The cathelicidin LL-37 was found to be active againstC. jejuni, with the LOS mutants exhibiting modest but tiled alterations in LL-37 sensitivity. The ΔwaaFmutant but not the other LOS mutant strains also exhibited a defect in intraepithelial cell survival, an aspect ofC. jejunipathogenesis that has only recently begun to be clarified. Finally, using a mouse competition model, we now provide the first direct evidence for the importance of theC. jejuniLOS in host colonization. Collectively, this study has uncovered novel roles for theC. jejuniLOS, highlights the dynamic nature of theC. jejunicell envelope, and provides insight into the contribution of specific LOS core moieties to stress survival and pathogenesis.
Increased Expression of the Type IV Secretion System in Piliated
Neisseria gonorrhoeae
Variants
Authors: Salgado-Pabón Wilmara , Du Ying , Hackett Kathleen T. , Lyons Katelynn M. , Arvidson Cindy Grove , Dillard Joseph P.
Published: February 2010

Neisseria gonorrhoeae
produces a type IV secretion system that secretes chromosomal DNA. The secreted DNA is active in the transformation of other gonococci in the population and may act to transfer antibiotic resistance genes and variant alleles for surface antigens, as well as other genes. We observed that gonococcal variants that produced type IV pili secreted more DNA than variants that were nonpiliated, suggesting that the process may be regulated. Using microarray analysis, we found that a piliated strain showed increased expression of the gene for the putative type IV secretion coupling protein TraD, whereas a nonpiliated variant showed increased expression of genes for transcriptional and translational machinery, consistent with its higher growth rate compared to that of the piliated strain. These results suggested that type IV secretion might be controlled by either
traD
expression or growth rate. A mutant with a deletion in
traD
was found to be deficient in DNA secretion. Further mutation and complementation analysis indicated that
traD
is transcriptionally and translationally coupled to
traI
, which encodes the type IV secretion relaxase. We were able to increase DNA secretion in a nonpiliated strain by inserting a gene cassette with a strong promoter to drive the expression of the putative operon containing
traI
and
traD
. Together, these data suggest a model in which the type IV secretion system apparatus is made constitutively, while its activity is controlled through regulation of
traD
and
traI
.
Myxococcus xanthus
Viability Depends on GroEL Supplied by Either of Two Genes, but the Paralogs Have Different Functions during Heat Shock, Predation, and Development
Authors: Li Jian , Wang Yan , Zhang Cui-ying , Zhang Wen-yan , Jiang De-ming , Wu Zhi-hong , Liu Hong , Li Yue-zhong
Published: February 2010

Myxococcus xanthus
DK1622 contains two paralogous
groEL
gene loci that possess both different sequences and different organizations within the genome. Deletion of either one of these two genes alone does not affect cell viability. However, deletion of both
groEL
genes results in cell death unless a complemented
groEL1
or
groEL2
gene is present. The
groEL1
gene was determined to be essential for cell survival under heat shock conditions; a strain with mutant
groEL2
caused cells to be more sensitive than the wild-type strain to higher temperatures. Mutants with a single deletion of either
groEL1
(
MXAN_4895
) or
groEL2
(
MXAN_4467
) had a growth curve similar to that of the wild-type strain DK1622 in medium containing hydrolyzed proteins as the substrate. However, when cells were cultured on medium containing either
Escherichia coli
cells or casein as the substrate, deletion of
groEL2
, but not
groEL1
, led to a deficiency in cell predation and macromolecular feeding. Furthermore,
groEL1
was found to play an indispensable role in the development and sporulation of cells, but deletion of
groEL2
had no visible effects. Our results suggest that, although alternatively required for cell viability, the products of the two
groEL
genes have divergent functions in the multicellular social life cycle of
M. xanthus
DK1622.
Analyses of the Regulatory Mechanism and Physiological Roles of
Pseudomonas aeruginosa
OhrR, a Transcription Regulator and a Sensor of Organic Hydroperoxides
Authors: Atichartpongkul Sopapan , Fuangthong Mayuree , Vattanaviboon Paiboon , Mongkolsuk Skorn
Published: February 2010

ohrR
encodes an organic hydroperoxide sensor and a transcriptional repressor that regulates organic hydroperoxide-inducible expression of a thiol peroxidase gene,
ohr
, and itself. OhrR binds directly to the operators and represses transcription of these genes. Exposure to an organic hydroperoxide leads to oxidation of OhrR and to subsequent structural changes that result in the loss of the repressor's ability to bind to the operators that allow expression of the target genes. Differential induction of
ohrR
and
ohr
by
tert
-butyl hydroperoxide suggests that factors such as the repressor's dissociation constants for different operators and the chemical nature of the inducer contribute to OhrR-dependent organic hydroperoxide-inducible gene expression.
ohrR
and
ohr
mutants show increased and decreased resistance to organic hydroproxides, respectively, compared to a parental strain. Moreover, the
ohrR
mutant had a reduced-virulence phenotype in the
Pseudomonas aeruginosa
-
Caenorhabditis elegans
pathogenicity model.
Genome Sequence ofStreptococcus gallolyticus: Insights into Its Adaptation to the Bovine Rumen and Its Ability To Cause Endocarditis
Authors: Rusniok Christophe , Couvé Elisabeth , Da Cunha Violette , El Gana Rachida , Zidane Nora , Bouchier Christiane , Poyart Claire , Leclercq Roland , Trieu-Cuot Patrick , Glaser Philippe
Published: February 2010

Streptococcus gallolyticus(formerly known asStreptococcus bovisbiotype I) is an increasing cause of endocarditis among streptococci and frequently associated with colon cancer.S. gallolyticusis part of the rumen flora but also a cause of disease in ruminants as well as in birds. Here we report the complete nucleotide sequence of strain UCN34, responsible for endocarditis in a patient also suffering from colon cancer. Analysis of the 2,239 proteins encoded by its 2,350-kb-long genome revealed unique features among streptococci, probably related to its adaptation to the rumen environment and its capacity to cause endocarditis.S. gallolyticushas the capacity to use a broad range of carbohydrates of plant origin, in particular to degrade polysaccharides derived from the plant cell wall. Its genome encodes a large repertoire of transporters and catalytic activities, like tannase, phenolic compounds decarboxylase, and bile salt hydrolase, that should contribute to the detoxification of the gut environment. Furthermore,S. gallolyticussynthesizes all 20 amino acids and more vitamins than any other sequencedStreptococcusspecies. Many of the genes encoding these specific functions were likely acquired by lateral gene transfer from other bacterial species present in the rumen. The surface properties of strain UCN34 may also contribute to its virulence. A polysaccharide capsule might be implicated in resistance to innate immunity defenses, and glucan mucopolysaccharides, three types of pili, and collagen binding proteins may play a role in adhesion to tissues in the course of endocarditis.
TheN-Acetylmuramic Acid 6-Phosphate Etherase Gene Promotes Growth and Cell Differentiation of Cyanobacteria under Light-Limiting Conditions
Authors: Jiang Haibo , Kong Renqiu , Xu Xudong
Published: February 2010

Inactivation ofsll0861inSynechocystissp. strain PCC 6803 or the homologous genealr2432inAnabaenasp. strain PCC 7120 had no effect on the growth of these organisms at a light intensity of 30 μmol photons m−2s−1but reduced their growth at a light intensity of 5 or 10 μmol photons m−2s−1. InAnabaena, inactivation of the gene also significantly reduced the rate of heterocyst differentiation under low-light conditions. The predicted products ofsll0861andalr2432and homologs of these genes showed similarity toN-acetylmuramic acid 6-phosphate etherase (MurQ), an enzyme involved in peptidoglycan recycling, inEscherichia coli. E. coli murQand the cyanobacterial homologs could functionally substitute for each other. We hypothesize thatmurQin cyanobacteria promotes low-light adaptation through reutilization of peptidoglycan degradation products.