bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitrogen starvation induces extensive changes in the redox proteome of Prochlorococcus sp. strain SS120
Authors: McDonagh Brian , Domínguez‐Martín Ma Agustina , Gómez‐Baena Guadalupe , López‐Lozano Antonio , Diez Jesús , Bárcena Jose A. , García Fernández Jose M.
Published: February 2012

Very low nitrogen concentration is a critical limitation in the oligotrophic oceans inhabited by the cyanobacterium Prochlorococccus, one of the main primary producers on Earth. It is well known that nitrogen starvation affects redox homeostasis in cells. We have studied the effect of nitrogen starvation on the thiol redox proteome in the Prochlorococcus sp. SS120 strain, by using shotgun proteomic techniques to map the cysteine modified in each case and to quantify the ratio of reversibly oxidized/reduced species. We identified a number of proteins showing modified cysteines only under either control or N‐starvation, including isocitrate dehydrogenase and ribulose phosphate 3‐epimerase. We detected other key enzymes, such as glutamine synthetase, transporters and transaminases, showing that nitrogen‐related pathways were deeply affected by nitrogen starvation. Reversibly oxidized cysteines were also detected in proteins of other important metabolic pathways, such as photosynthesis, phosphorus metabolism, ATP synthesis and nucleic acids metabolism. Our results demonstrate a wide effect of nitrogen limitation on the redox status of the Prochlorococcus proteome, suggesting that besides previously reported transcriptional changes, this cyanobacterium responds with post‐translational redox changes to the lack of nitrogen in its environment.
The Effect of the Resources Boom on the Population and Labour Market of Western Australia
Authors: Garnett Anne M.
Published: February 2012

During the 2000s, Australia experienced a rapid and substantial resources boom. The impacts from this boom were particularly felt in Western Australia, which has the largest share of mining revenue as a proportion of both gross state product and exports. Some states have reported that although mining export earnings have risen due to strong commodity prices, there was less impact on the populations, labour markets and regional development than may have been expected. This paper will investigate the impact of the mining boom on the population and labour markets of Western Australia. Of particular policy relevance are skilled labour shortages and the effects that any changes in population and employment have on the remote communities where the majority of the mining activities are located.
Principles and popularity: The interplay of moral norms and descriptive norms in the context of volunteerism
Authors: Smith Megan K. , Masser Barbara M.
Published: February 2012

Can a moral norm be manipulated and if so, how does a manipulated moral norm interact with a descriptive norm to impact on behavioural intentions? The present research addressed these questions in three studies. Study 1a and 1b demonstrated the validity of a manipulation designed to activate a moral norm for volunteer activity. In Study 2, activating a moral norm for volunteering strengthened intentions to volunteer and inhibited intentions to conform to a descriptive norm. Conceptual and applied implications of activating a moral norm are discussed.
Topology Optimization for Structures With Nonlinear Behavior Using the Equivalent Static Loads Method
Authors: Lee Hyun-Ah , Park Gyung-Jin
Published: February 2012

Many structures in the real world show nonlinear responses. The nonlinearity may be due to some reasons, such as nonlinear material (material nonlinearity), large deformation of the structures (geometric nonlinearity), or contact between the parts (contact nonlinearity). Conventional optimization algorithms considering the nonlinearities are fairly difficult and expensive because many nonlinear analyses are required. It is quite difficult to perform topology optimization considering nonlinear static behavior because of the many design variables. In the current element density based topology optimization considering nonlinear behavior, low-density finite elements cause serious numerical problems due to excessive mesh distortion. Updating the material of the finite elements based on the density is considerably complicated because of the relationship between the element density and structural material. The equivalent static loads method for nonlinear static response structural optimization (ESLSO) has been proposed for size and shape optimization. The equivalent static loads (ESLs) are defined as the linear static load sets which generate the same displacement field from nonlinear static analysis. In this research, a new algorithm is proposed for topology optimization considering all kinds of nonlinearities by modifying the existing ESLSO. The new ESLSO can overcome the difficulties which may occur in topology optimization with nonlinear static behavior. A nonlinear static response optimization problem is converted to cyclic use of linear static response optimization with ESLs. Therefore, the new ESLSO can generate results of nonlinear static response topology optimization by using well established nonlinear static analysis and linear static response topology optimization methods. Four structural examples are demonstrated using the finite element method. Different kinds of nonlinearities are involved in each example.
A Backwards Design Method for Mechanical Conceptual Design
Authors: Burgess Stuart C.
Published: February 2012

This paper presents a backwards design method for mechanical conceptual design. The method involves starting with a very idealistic semiworking solution and then systematically solving the unworkable parts of the solution until a complete solution is found. The method can work in conjunction with other methods such as Theory of Inventive Problem Solving (TRIZ) or brainstorming. The method tries to exploit the principle that it is easier to critique and modify a design than it is to create a fully working solution in one go. The visual nature of the method means that it is suited to design problems where geometry is important such as mechanism design and machine design. Three case studies of conceptual design are presented. The case studies involve a novel clutch, a novel rotary damping mechanism and a novel worm gearbox. Each of these designs won at least one national design competition in the UK. A simple design experiment has been carried out which indicated that the backwards design method was at least as effective as the morphological chart method for a simple machine design exercise.
Employing a Recombinant Strain of Advenella mimigardefordensis for Biotechnical Production of Homopolythioesters from 3,3′-Dithiodipropionic Acid
Authors: Xia Yongzhen , Wübbeler Jan Hendrik , Qi Qingsheng , Steinbüchel Alexander
Published: February 2012

Advenella mimigardefordensis
strain DPN7
T
was genetically modified to produce poly(3-mercaptopropionic acid) (PMP) homopolymer by exploiting the recently unraveled process of 3,3′-dithiodipropionic acid (DTDP) catabolism. Production was achieved by systematically engineering the metabolism of this strain as follows: (i) deletion of its inherent 3MP dioxygenase-encoding gene (
mdo
), (ii) introduction of the
buk-ptb
operon (genes encoding the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, from
Clostridium acetobutylicum
), and (iii) overexpression of its own polyhydroxyalkanoate synthase (

phaC
Am

). These measures yielded the potent PMP production strain
A. mimigardefordensis
strain SHX22. The deletion of
mdo
was required for adequate synthesis of PMP due to the resulting accumulation of 3MP during utilization of DTDP. Overexpression of the plasmid-borne
buk-ptb
operon caused a severe growth repression. This effect was overcome by inserting this operon into the genome. Polyhydroxyalkanoate (PHA) synthases from different origins were compared. The native PHA synthase of
A. mimigardefordensis
(

phaC
Am

) was obviously the best choice to establish homopolythioester production in this strain. In addition, the cultivation conditions, including an appropriate provision of the carbon source, were further optimized to enhance PMP production. The engineered strain accumulated PMP up to approximately 25% (wt/wt) of the cell dry weight when cultivated in mineral salts medium containing glycerol as the carbon source in addition to DTDP as the sulfur-providing precursor. According to our knowledge, this is the first report of PMP homopolymer production by a metabolically engineered bacterium using DTDP, which is nontoxic, as the precursor substrate.
Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972
Authors: Rodríguez-Rubio Lorena , Gutiérrez Dolores , Martínez Beatriz , Rodríguez Ana , García Pilar
Published: February 2012

Bacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded by
Staphylococcus aureus
phage vB_SauS-phi-IPLA88, was expressed and secreted in
Lactococcus lactis
using the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts.
Emergence and Stability of High-Pressure Resistance in Different Food-Borne Pathogens
Authors: Vanlint Dietrich , Rutten Nele , Michiels Chris W. , Aertsen Abram
Published: February 2012

High hydrostatic pressure (HHP) processing is becoming a valuable nonthermal food pasteurization technique, although there is reasonable concern that bacterial HHP resistance could compromise the safety and stability of HHP-processed foods. While the degree of natural HHP resistance has already been shown to vary greatly among and within bacterial species, a still unresolved question remains as to what extent different food-borne pathogens can actually develop HHP resistance. In this study, we therefore examined and compared the intrinsic potentials for HHP resistance development among strains of
Escherichia coli
,
Shigella flexneri
,
Salmonella enterica
serovars Typhimurium and Enteritidis,
Yersinia enterocolitica
,
Aeromonas hydrophila
,
Pseudomonas aeruginosa
, and
Listeria innocua
using a selective enrichment approach. Interestingly, of all strains examined, the acquisition of extreme HHP resistance could be detected in only some of the
E. coli
strains, indicating that a specific genetic predisposition might be required for resistance development. Furthermore, once acquired, HHP resistance proved to be a very stable trait that was maintained for >80 generations in the absence of HHP exposure. Finally, at the mechanistic level, HHP resistance was not necessarily linked to derepression of the heat shock genes and was not related to the phenomenon of persistence.
Establishment of a Tractable Genetic Transformation System in Veillonella spp
Authors: Liu Jinman , Xie Zhoujie , Merritt Justin , Qi Fengxia
Published: February 2012

We have constructed the first
Escherichia coli-Veillonella
shuttle vector based on an endogenous plasmid (pVJL1) isolated from a clinical
Veillonella
strain. A highly transformable
Veillonella
strain was also identified. Both the shuttle vector and the transformable strain should be valuable tools for future
Veillonella
genetic studies.
Ecological Success of a Group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii Hybrids in the Northern European Wine-Making Environment
Authors: Erny C. , Raoult P. , Alais A. , Butterlin G. , Delobel P. , Matei-Radoi F. , Casaregola S. , Legras J. L.
Published: February 2012

The hybrid nature of lager-brewing yeast strains has been known for 25 years; however, yeast hybrids have only recently been described in cider and wine fermentations. In this study, we characterized the hybrid genomes and the relatedness of the Eg8 industrial yeast strain and of 24
Saccharomyces cerevisiae/Saccharomyces kudriavzevii
hybrid yeast strains used for wine making in France (Alsace), Germany, Hungary, and the United States. An array-based comparative genome hybridization (aCGH) profile of the Eg8 genome revealed a typical chimeric profile. Measurement of hybrids DNA content per cell by flow cytometry revealed multiple ploidy levels (2n, 3n, or 4n), and restriction fragment length polymorphism analysis of 22 genes indicated variable amounts of
S. kudriavzevii
genetic content in three representative strains. We developed microsatellite markers for
S. kudriavzevii
and used them to analyze the diversity of a population isolated from oaks in Ardèche (France). This analysis revealed new insights into the diversity of this species. We then analyzed the diversity of the wine hybrids for 12
S. cerevisiae
and 7
S. kudriavzevii
microsatellite loci and found that these strains are the products of multiple hybridization events between several
S. cerevisiae
wine yeast isolates and various
S. kudriavzevii
strains. The Eg8 lineage appeared remarkable, since it harbors strains found over a wide geographic area, and the interstrain divergence measured with a (δμ)
2
genetic distance indicates an ancient origin. These findings reflect the specific adaptations made by
S. cerevisiae/S. kudriavzevii
cryophilic hybrids to winery environments in cool climates.
Distribution Patterns and Phylogeny of Marine Stramenopiles in the North Pacific Ocean
Authors: Lin Yun-Chi , Campbell Tracy , Chung Chih-Ching , Gong Gwo-Ching , Chiang Kuo-Ping , Worden Alexandra Z.
Published: February 2012

Marine stramenopiles (MASTs) are a diverse suite of eukaryotic microbes found in marine environments. Several MAST lineages are thought to contain heterotrophic nanoflagellates. However, MASTs remain uncultured and data on distributions and trophic modes are limited. We investigated MASTs in provinces on the west and east sides of the North Pacific Subtropical Gyre, specifically the East China Sea (ECS) and the California Current system (CALC). For each province, DNA was sampled from three zones: coastal, mesotrophic transitional, and more oligotrophic euphotic waters. Along with diatoms, chrysophytes, and other stramenopiles, sequences were recovered from nine MAST lineages in the six ECS and four CALC 18S rRNA gene clone libraries. All but one of these libraries were from surface samples. MAST clusters 1, 3, 7, 8, and 11 were identified in both provinces, with MAST cluster 3 (MAST-3) being found the most frequently. Additionally, MAST-2 was detected in the ECS and MAST-4, -9, and -12 were detected in the CALC. Phylogenetic analysis indicated that some subclades within these lineages differ along latitudinal gradients. MAST-1A, -1B, and -1C and MAST-4 size and abundance estimates obtained using fluorescence
in situ
hybridization on 79 spring and summer ECS samples showed a negative correlation between size of MAST-1B and MAST-4 cells and temperature. MAST-1A was rarely detected, but MAST-1B and -1C and MAST-4 were abundant in summer and MAST-1C and MAST-4 were more so at the coast, with maximum abundances of 543 and 1,896 cells ml
−1
, respectively. MAST-4 and
Synechococcus
abundances were correlated, and experimental work showed that MAST-4 ingests
Synechococcus
. Together with previous studies, this study helps refine hypotheses on distribution and trophic modes of MAST lineages.
Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL
Authors: Ko Hyeok-Jin , Park Eunhye , Song Joseph , Yang Taek Ho , Lee Hee Jong , Kim Kyoung Heon , Choi In-Geol
Published: February 2012

Autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of
Escherichia coli
for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from
Saccharophagus degradans
2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 10
4
molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing
E. coli
.
Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids
Authors: Sakaguchi Keishi , Matsuda Takanori , Kobayashi Takumi , Ohara Jun-ichiro , Hamaguchi Rie , Abe Eriko , Nagano Naoki , Hayashi Masahiro , Ueda Mayumi , Honda Daiske , Okita Yuji , Taoka Yousuke , Sugimoto Shinichi , Okino Nozomu , Ito Makoto
Published: February 2012

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (
neo
r
), driven with an ubiquitin or an EF-1α promoter-terminator from
Thraustochytrium aureum
ATCC 34304, was introduced into representatives of two thraustochytrid genera,
Aurantiochytrium
and
Thraustochytrium
. The
neo
r
marker was integrated into the chromosomal DNA by random recombination and then functionally translated into
neo
r
mRNA. Additionally, we confirmed that another two genera,
Parietichytrium
and
Schizochytrium
, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile,
T. aureum
ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in
T. aureum
ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C
20:3n-6
) and eicosatetraenoic acid (C
20:4n-3
), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C
20:4n-6
) and eicosapentaenoic acid (C
20:5n-3
), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.
Transcriptome Mapping of pAR060302, a
bla
CMY-2
-Positive Broad-Host-Range IncA/C Plasmid
Authors: Lang Kevin S. , Danzeisen Jessica L. , Xu Wayne , Johnson Timothy J.
Published: February 2012

The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among
Escherichia coli
and
Salmonella enterica
strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using
E. coli
strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes
floR
,
bla
CMY-2
,
aadA
, and
aacA
. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of
floR
and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators.
Antimicrobial Mechanism of Monocaprylate
Authors: Hyldgaard Morten , Sutherland Duncan S. , Sundh Maria , Mygind Tina , Meyer Rikke Louise
Published: February 2012

Monoglyceride esters of fatty acids occur naturally and encompass a broad spectrum of antimicrobial activity. Monocaprylate is generally regarded as safe (GRAS) and can function both as an emulsifier and as a preservative in food. However, knowledge about its mode of action is lacking. The aim of this study was therefore to elucidate the mechanism behind monocaprylate's antimicrobial effect. The cause of cell death in
Escherichia coli
,
Staphylococcus xylosus
, and
Zygosaccharomyces bailii
was investigated by examining monocaprylate's effect on cell structure, membrane integrity, and its interaction with model membranes. Changes in cell structure were visible by atomic force microscopy (AFM), and propidium iodide staining showed membrane disruption, indicating the membrane as a site of action. This indication was confirmed by measuring calcein leakage from membrane vesicles exposed to monocaprylate. AFM imaging of supported lipid bilayers visualized the integration of monocaprylate into the liquid disordered, and not the solid ordered, phase of the membrane. The integration of monocaprylate was confirmed by quartz crystal microbalance measurements, showing an abrupt increase in mass and hydration of the membrane after exposure to monocaprylate above a threshold concentration. We hypothesize that monocaprylate destabilizes membranes by increasing membrane fluidity and the number of phase boundary defects. The sensitivity of cells to monocaprylate will therefore depend on the lipid composition, fluidity, and curvature of the membrane.
Leptospire Genomic Diversity Revealed by Microarray-Based Comparative Genomic Hybridization
Authors: Eribo Broderick , Mingmongkolchai Sirima , Yan Tingfen , Dubbs Padunsri , Nelson Karen E.
Published: February 2012

Comparative genomic hybridization was used to compare genetic diversity of five strains of
Leptospira
(
Leptospira interrogans
serovars Bratislava, Canicola, and Hebdomadis and
Leptospira kirschneri
serovars Cynopteri and Grippotyphosa). The array was designed based on two available sequenced
Leptospira
reference genomes, those of
L. interrogans
serovar Copenhageni and
L. interrogans
serovar Lai. A comparison of genetic contents showed that
L. interrogans
serovar Bratislava was closest to the reference genomes while
L. kirschneri
serovar Grippotyphosa had the least similarity to the reference genomes. Cluster analysis indicated that
L. interrogans
serovars Bratislava and Hebdomadis clustered together first, followed by
L. interrogans
serovar Canicola, before the two
L. kirschneri
strains. Confirmed/potential virulence factors identified in previous research were also detected in the tested strains.
Aceticlastic and NaCl-Requiring Methanogen “Methanosaeta pelagica” sp. nov., Isolated from Marine Tidal Flat Sediment
Authors: Mori Koji , Iino Takao , Suzuki Ken-Ichiro , Yamaguchi Kaoru , Kamagata Yoichi
Published: February 2012

Among methanogens, only 2 genera,
Methanosaeta
and
Methanosarcina
, are known to contribute to methanogenesis from acetate, and
Methanosaeta
is a specialist that uses acetate specifically. However,
Methanosaeta
strains so far have mainly been isolated from anaerobic digesters, despite the fact that it is widespread, not only in anaerobic methanogenic reactors and freshwater environments, but also in marine environments, based upon extensive 16S rRNA gene-cloning analyses. In this study, we isolated an aceticlastic methanogen, designated strain 03d30q
T
, from a tidal flat sediment. Phylogenetic analyses based on 16S rRNA and
mcrA
genes revealed that the isolate belongs to the genus
Methanosaeta
. Unlike the other known
Methanosaeta
species, this isolate grows at Na
+
concentrations of 0.20 to 0.80 M, with an optimum concentration of 0.28 M. Quantitative estimation using real-time PCR detected the 16S rRNA gene of the genus
Methanosaeta
in the marine sediment, and relative abundance ranged from 3.9% to 11.8% of the total archaeal 16S rRNA genes. In addition, the number of
Methanosaeta
organisms increased with increasing depth and was much higher than that of
Methanosarcina
organisms, suggesting that aceticlastic methanogens contribute to acetate metabolism to a greater extent than previously thought in marine environments, where sulfate-reducing acetate oxidation prevails. This is the first report on marine
Methanosaeta
species, and based on phylogenetic and characteristic studies, the name “
Methanosaeta pelagica
” sp. nov. is proposed for this novel species, with type strain 03d30q.
Impact of Xynthia Tempest on Viral Contamination of Shellfish
Authors: Grodzki Marco , Ollivier Joanna , Le Saux Jean-Claude , Piquet Jean-Côme , Noyer Mathilde , Le Guyader Françoise S.
Published: February 2012

Viral contamination in oyster and mussel samples was evaluated after a massive storm with hurricane wind named “Xynthia tempest” destroyed a number of sewage treatment plants in an area harboring many shellfish farms. Although up to 90% of samples were found to be contaminated 2 days after the disaster, detected viral concentrations were low. A 1-month follow-up showed a rapid decrease in the number of positive samples, even for norovirus.
One-Step Triplex High-Resolution Melting Analysis for Rapid Identification and Simultaneous Subtyping of Frequently Isolated Salmonella Serovars
Authors: Zeinzinger Josef , Pietzka Ariane T. , Stöger Anna , Kornschober Christian , Kunert Renate , Allerberger Franz , Mach Robert , Ruppitsch Werner
Published: February 2012

Salmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping of
Salmonella enterica
isolates based on specific single-nucleotide polymorphisms in fragments of
fljB
,
gyrB
, and
ycfQ
. Simultaneous gene scanning of
fljB
,
gyrB
, and
ycfQ
by high-resolution melting-curve analysis of 417
Salmonella
isolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases from
Salmonella enterica
serotype Enteritidis and
Salmonella enterica
serotype Dublin, in all cases from
Salmonella enterica
serotype Ohio and
Salmonella enterica
serotype Rissen, from
Salmonella enterica
serotype Mbandaka and
Salmonella enterica
serotype Kentucky, and from
Salmonella enterica
serotype Bredeney,
Salmonella enterica
serotype Give, and
Salmonella enterica
serotype Schwarzengrund. To differentiate the most frequent
Salmonella
serotype, Enteritidis, from some
S
. Dublin isolates, an additional single PCR assay was developed for specific identification of
S
. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with an
S
. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39
Salmonella
serotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhuman
Salmonella
isolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.
Induction of Holomycin Production and Complex Metabolic Changes by the
argR
Mutation in Streptomyces clavuligerus NP1
Authors: Yin Hua , Xiang Sihai , Zheng Jianting , Fan Keqiang , Yu Tingting , Yang Xu , Peng Yanfeng , Wang Haibin , Feng Deqin , Luo Yuanming , Bai Hua , Yang Keqian
Published: February 2012

In bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption of
argR
in
Streptomyces clavuligerus
NP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and its
argR
disruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetyl-CoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin in
S. clavuligerus
.
Increased Competitive Fitness of Bacillus subtilis under Nonsporulating Conditions via Inactivation of Pleiotropic Regulators AlsR, SigD, and SigW
Authors: Nicholson Wayne L.
Published: February 2012

Previous studies implicated loss of motility and mutations of the
alsR
and
sigW
regulatory genes in enhanced fitness of the
Bacillus subtilis
evolved strain WN716 over that of its ancestral strain WN624. The fitness of strains carrying knockout mutations
alsR
::
spc
,
sigD
::
kan
, and/or
sigW
::
erm
was measured and compared to that of the congenic ancestral strain by competition experiments.
Corals Form Characteristic Associations with Symbiotic Nitrogen-Fixing Bacteria
Authors: Lema Kimberley A. , Willis Bette L. , Bourne David G.
Published: February 2012

The complex symbiotic relationship between corals and their dinoflagellate partner
Symbiodinium
is believed to be sustained through close associations with mutualistic bacterial communities, though little is known about coral associations with bacterial groups able to fix nitrogen (diazotrophs). In this study, we investigated the diversity of diazotrophic bacterial communities associated with three common coral species (
Acropora millepora
,
Acropora muricata
, and
Pocillopora damicormis
) from three midshelf locations of the Great Barrier Reef (GBR) by profiling the conserved subunit of the
nifH
gene, which encodes the dinitrogenase iron protein. Comparisons of diazotrophic community diversity among coral tissue and mucus microenvironments and the surrounding seawater revealed that corals harbor diverse
nifH
phylotypes that differ between tissue and mucus microhabitats. Coral mucus
nifH
sequences displayed high heterogeneity, and many bacterial groups overlapped with those found in seawater. Moreover, coral mucus diazotrophs were specific neither to coral species nor to reef location, reflecting the ephemeral nature of coral mucus. In contrast, the dominant diazotrophic bacteria in tissue samples differed among coral species, with differences remaining consistent at all three reefs, indicating that coral-diazotroph associations are species specific. Notably, dominant diazotrophs for all coral species were closely related to the bacterial group rhizobia, which represented 71% of the total sequences retrieved from tissue samples. The species specificity of coral-diazotroph associations further supports the coral holobiont model that bacterial groups associated with corals are conserved. Our results suggest that, as in terrestrial plants, rhizobia have developed a mutualistic relationship with corals and may contribute fixed nitrogen to
Symbiodinium
.
Emission of Methane by Eudrilus eugeniae and Other Earthworms from Brazil
Authors: Depkat-Jakob Peter S. , Hunger Sindy , Schulz Kristin , Brown George G. , Tsai Siu M. , Drake Harold L.
Published: February 2012

Earthworms emit denitrification-derived nitrous oxide and fermentation-derived molecular hydrogen. The present study demonstrated that the earthworm
Eudrilus eugeniae
, obtained in Brazil, emitted methane. Other worms displayed a lesser or no capacity to emit methane. Gene and transcript analyses of
mcrA
(encoding the alpha subunit of methyl-CoM reductase) in gut contents of
E. eugeniae
suggested that
Methanosarcinaceae
,
Methanobacteriaceae
, and
Methanomicrobiaceae
might be associated with this emission.
Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus
Authors: Dinamarco Taísa Magnani , Almeida Ricardo S. , Alves de Castro Patrícia , Brown Neil Andrew , dos Reis Thaila Fernanda , Zambelli Ramalho Leandra Naira , Savoldi Marcela , Goldman Maria Helena S. , Goldman Gustavo Henrique
Published: February 2012

Aspergillus fumigatus
is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for
A. fumigatus
virulence and survival in the mammalian host. This study characterized the
A. fumigatus
SebA transcription factor, which is the putative homologue of the factor encoded by
Trichoderma atroviride
seb1
. The Δ
sebA
mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl
2
, and poor nutritional conditions, while viability associated with
sebA
was also affected by heat shock exposure. Accordingly, SebA::GFP (SebA::green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the Δ
sebA
mutant. The
A. fumigatus
Δ
sebA
strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the Δ
sebA
mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain.
A. fumigatus
SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses.
Deciphering Transcriptional Regulatory Mechanisms Associated with Hemicellulose Degradation in Neurospora crassa
Authors: Sun Jianping , Tian Chaoguang , Diamond Spencer , Glass N. Louise
Published: February 2012

Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential substrate for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs).
Neurospora crassa
, a model filamentous fungus, expresses and secretes enzymes required for plant cell wall deconstruction. To better understand genes specifically associated with degradation of hemicellulose, we applied secretome and transcriptome analysis to
N. crassa
grown on beechwood xylan. We identified 34 secreted proteins and 353 genes with elevated transcription on xylan. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of the wild type was assessed, revealing functions for known and unknown proteins associated with hemicellulose degradation. By evaluating phenotypes of strains containing deletions of predicted TF genes in
N. crassa
, we identified a TF (XLR-1;
x
y
l
an degradation
r
egulator
1
) essential for hemicellulose degradation that is an ortholog to XlnR/XYR1 in
Aspergillus
and
Trichoderma
species, respectively, a major transcriptional regulator of genes encoding both cellulases and hemicellulases. Deletion of
xlr-1
in
N. crassa
abolished growth on xylan and xylose, but growth on cellulose and cellulolytic activity were only slightly affected. To determine the regulatory mechanisms for hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose, xylanolytic, and cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes in
N. crassa
and was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. This systematic analysis illustrates the similarities and differences in regulation of hemicellulose degradation among filamentous fungi.
Transcriptional Organization and Physiological Contributions of the
relQ
Operon of Streptococcus mutans
Authors: Kim Jeong Nam , Ahn Sang-Joon , Seaton Kinda , Garrett Steven , Burne Robert A.
Published: February 2012

The molecular alarmone (p)ppGpp functions as a global regulator of gene expression in bacteria. In
Streptococcus mutans
, (p)ppGpp synthesis is catalyzed by three gene products: RelA, RelP, and RelQ. RelA is responsible for (p)ppGpp production during a stringent response, and RelP is the primary source of (p)ppGpp during exponential growth, but the role of RelQ has not been thoroughly investigated. In this study, we analyzed the four-gene
relQ
operon to establish how these gene products may affect homeostasis and stress tolerance in the dental caries pathogen
S. mutans
. Northern blotting and reverse transcriptase PCR demonstrated that
relQ
is cotranscribed with the downstream genes
ppnK
(NAD kinase),
rluE
(pseudouridine synthase), and
pta
(phosphotransacetylase). In addition, a promoter located within the
rluE
gene was shown to drive transcription of
pta
. Inactivation of
relQ
,
ppnK
, or
rluE
did not significantly affect growth of or stress tolerance by
S. mutans
, whereas strains lacking
pta
were more sensitive to acid and oxidative stresses. Interestingly, introduction of an
rluE
deletion into the
pta
mutant reversed the deleterious effects of the
pta
mutation on growth and stress tolerance. Accumulation of (p)ppGpp was also decreased in a
pta
mutant strain, whereas inactivation of
relQ
caused enhanced (p)ppGpp synthesis in exponential-phase cells. The results reveal an important role for the
relQ
operon in the expression of traits that are essential for persistence and pathogenesis by
S. mutans
and provide evidence for a molecular connection of acetate and (p)ppGpp metabolism with tolerance of acid and oxidative stresses.
Identification of the
cglC
,
cglD
,
cglE
, and
cglF
Genes and Their Role in Cell Contact-Dependent Gliding Motility in Myxococcus xanthus
Authors: Pathak Darshankumar T. , Wall Daniel
Published: February 2012

Within
Myxococcus xanthus
biofilms, cells actively move and exchange their outer membrane (OM) lipoproteins and lipids. Between genetically distinct strains, OM exchange can regulate recipient cell behaviors, including gliding motility and development. Although many different proteins are thought to be exchanged, to date, only two endogenous OM lipoproteins, CglB and Tgl, are known to be transferred. Protein exchange requires the TraAB proteins in recipient and donor cells, where they are hypothesized to facilitate OM fusion for transfer. To better understand the types of proteins exchanged, we identified the genes for the remaining set of
cgl
gliding motility mutants. These mutants are unique because their motility defect can be transiently restored by physical contact with donor cells that encode the corresponding wild-type protein, a process called stimulation. Similar to CglB and Tgl, the
cglC
and
cglD
genes encode type II signal sequences, suggesting that they are also lipoproteins. Surprisingly, the
cglE
and
cglF
genes instead encode type I signal sequences, suggesting that nonlipoproteins are also exchanged. Consistent with this idea, the addition of exogenous synthetic CglF protein (71 amino acids) to a
cglF
mutant rescued its motility defect. In contrast to a live donor cell, stimulation with purified CglF protein occurred independently of TraA. These results also indicate that CglF may localize to the cell surface. The implications of our findings on OM exchange are discussed.
Novel Colicin F
Y
of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation
Authors: Bosák Juraj , Laiblová Petra , Šmarda Jan , Dědičová Daniela , Šmajs David
Published: February 2012

A novel colicin type, designated colicin F
Y
, was found to be encoded and produced by the strain
Yersinia frederiksenii
Y27601. Colicin F
Y
was active against both pathogenic and nonpathogenic strains of the genus
Yersinia
. Plasmid YF27601 (5,574 bp) of
Y. frederiksenii
Y27601 was completely sequenced. The colicin F
Y
activity gene (
cfyA
) and the colicin F
Y
immunity gene (
cfyI
) were identified. The deduced amino acid sequence of colicin F
Y
was very similar in its C-terminal pore-forming domain to colicin Ib (69% identity in the last 178 amino acid residues), indicating pore forming as its lethal mode of action. Transposon mutagenesis of the colicin F
Y
-susceptible strain
Yersinia kristensenii
Y276 revealed the
yiuR
gene (ykris001_4440), which encodes the YiuR outer membrane protein with unknown function, as the colicin F
Y
receptor molecule. Introduction of the
yiuR
gene into the colicin F
Y
-resistant strain
Y. kristensenii
Y104 restored its susceptibility to colicin F
Y
. In contrast, the colicin F
Y
-resistant strain
Escherichia coli
TOP10F′ acquired susceptibility to colicin F
Y
only when both the
yiuR
and
tonB
genes from
Y. kristensenii
Y276 were introduced. Similarities between colicins F
Y
and Ib, similarities between the Cir and YiuR receptors, and the detected partial cross-immunity of colicin F
Y
and colicin Ib producers suggest a common evolutionary origin of the colicin F
Y
-YiuR and colicin Ib-Cir systems.
Characterization of Campylobacter jejuni RacRS Reveals Roles in the Heat Shock Response, Motility, and Maintenance of Cell Length Homogeneity
Authors: Apel Dmitry , Ellermeier Jeremy , Pryjma Mark , DiRita Victor J. , Gaynor Erin C.
Published: February 2012

Campylobacter jejuni
commensally colonizes the cecum of birds. The RacR (
r
educed
a
bility to
c
olonize) response
r
egulator was previously shown to be important in avian colonization. To explore the means by which RacR and its cognate sensor kinase RacS may modulate
C. jejuni
physiology and colonization, Δ
racR
and Δ
racS
mutations were constructed in the invasive, virulent strain 81-176, and extensive phenotypic analyses were undertaken. Both the Δ
racR
and Δ
racS
mutants exhibited a ∼100-fold defect in chick colonization despite no (Δ
racS
) or minimal (Δ
racR
) growth defects at 42°C, the avian body temperature. Each mutant was defective for colony formation at 44°C and in the presence of 0.8% NaCl, both of which are stresses associated with the heat shock response. Promoter-reporter and real-time quantitative PCR (RT-qPCR) analyses revealed that RacR activates
racRS
and represses
dnaJ
. Although disregulation of several other heat shock genes was not observed at 38°C, the Δ
racR
and Δ
racS
mutants exhibited diminished upregulation of these genes upon a rapid temperature upshift. Furthermore, the Δ
racR
and Δ
racS
mutants displayed increased length heterogeneity during exponential growth, with a high proportion of filamented bacteria. Filamented bacteria had reduced swimming speed and were defective for invasion of Caco-2 epithelial cells. Soft-agar studies also revealed that the loss of
racR
or
racS
resulted in whole-population motility defects in viscous medium. These findings reveal new roles for RacRS in
C. jejuni
physiology, each of which is likely important during colonization of the avian host.
Transcriptional Regulation of the
assT
-
dsbL
-
dsbI
Gene Cluster in Salmonella enterica Serovar Typhi IMSS-1 Depends on LeuO, H-NS, and Specific Growth Conditions
Authors: Gallego-Hernández A. L. , Hernández-Lucas I. , De la Cruz M. A. , Olvera L. , Morett E. , Medina-Aparicio L. , Ramírez-Trujillo J. A. , Vázquez A. , Fernández-Mora M. , Calva E.
Published: February 2012

The
assT
gene encodes an arylsulfate sulfotransferase, an enzyme that catalyzes sulfuryl transfer from phenolic sulfate to a phenolic acceptor. In
Salmonella enterica
serovar Typhi IMSS-1, the
assT
gene is located upstream of the
dsbL
and
dsbI
genes, which are involved in a disulfide bond formation required for its activation. The
assT-dsbL-dsbI
gene cluster forms an operon transcribed by a LeuO-dependent promoter, in rich medium A (MA). Interestingly, in the absence of cloned
leuO
and in a Δ
leuO
background, two transcription start sites were detected for
assT
and two for
dsbL-dsbI
in minimal medium. The H-NS nucleoid protein repressed the expression of the
assT-dsbL-dsbI
LeuO-dependent operon, as well as of the
assT
transcriptional units. Thus, the expression of the
assT-dsbL-dsbI
gene cluster depends on the global regulatory proteins LeuO and H-NS, as well as on specific growth conditions.
The
rcbA
Gene Product Reduces Spontaneous and Induced Chromosome Breaks in Escherichia coli
Authors: Felczak Magdalena M. , Kaguni Jon M.
Published: February 2012

Elevated levels of DnaA cause excessive initiation, which leads to an increased level of double-strand breaks that are proposed to arise when newly formed replication forks collide from behind with stalled or collapsed forks. These double-strand breaks are toxic in mutants that are unable to repair them. Using a multicopy suppressor assay to identify genes that suppress this toxicity, we isolated a plasmid carrying a gene whose function had been unknown. This gene, carried by the cryptic
rac
prophage, has been named
rcbA
for its ability to reduce the frequency of chromosome breaks. Our study shows that the colony formation of strains bearing mutations in
rep
,
recG
, and
rcbA
, like
recA
and
recB
mutants, is inhibited by an oversupply of DnaA and that a multicopy plasmid carrying
rcbA
neutralizes this inhibition. These and other results suggest that
rcbA
helps to maintain the integrity of the bacterial chromosome by lowering the steady-state level of double-strand breaks.