bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptation in a Mouse Colony Monoassociated with
Escherichia coli
K-12 for More than 1,000 Days
Authors: Lee Sean M. , Wyse Aaron , Lesher Aaron , Everett Mary Lou , Lou Linda , Holzknecht Zoie E. , Whitesides John F. , Spears Patricia A. , Bowles Dawn E. , Lin Shu S. , Tonkonogy Susan L. , Orndorff Paul E. , Bollinger R. Randal , Parker William
Published: May 2010

Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with
Escherichia coli
K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of
E. coli
was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent
E. coli
. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of
E. coli
, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis
in vivo
, and an increased tendency for spontaneous lysis
in vitro
. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.
Natural Competence in
Thermoanaerobacter
and
Thermoanaerobacterium
Species
Authors: Shaw A. Joe , Hogsett David A. , Lynd Lee R.
Published: May 2010

Low-G+C thermophilic obligate anaerobes in the class
Clostridia
are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13
Thermoanaerobacter
and
Thermoanaerobacterium
strains previously believed to be difficult to transform or genetically recalcitrant. In
Thermoanaerobacterium saccharolyticum
JW/SL-YS485, natural competence-mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In
T. saccharolyticum
, disruptions of genes similar to
comEA
,
comEC
, and a type IV pilus (T4P) gene operon result in strains unable to incorporate further DNA, suggesting that natural competence occurs via a conserved Gram-positive mechanism. The relative ease of employing natural competence for gene transfer should foster genetic engineering in these industrially relevant organisms, and understanding the mechanisms underlying natural competence may be useful in increasing the applicability of genetic tools to difficult-to-transform organisms.
Influence of Low Oxygen Tensions and Sorption to Sediment Black Carbon on Biodegradation of Pyrene
Authors: Ortega-Calvo José-Julio , Gschwend Philip M.
Published: May 2010

Sorption to sediment black carbon (BC) may limit the aerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) in resuspension events and intact sediment beds. We examined this hypothesis experimentally under conditions that were realistic in terms of oxygen concentrations and BC content. A new method, based on synchronous fluorescence observations of
14
C-pyrene, was developed for continuously measuring the uptake of dissolved pyrene by
Mycobacterium gilvum
VM552, a representative degrader of PAHs. The effect of oxygen and pyrene concentrations on pyrene uptake followed Michaelis-Menten kinetics, resulting in a dissolved oxygen half-saturation constant (
K
om
) of 14.1 μM and a dissolved pyrene half-saturation constant (
K
pm
) of 6 nM. The fluorescence of
14
C-pyrene in air-saturated suspensions of sediments and induced cells followed time courses that reflected simultaneous desorption and biodegradation of pyrene, ultimately causing a quasi-steady-state concentration of dissolved pyrene balancing desorptive inputs and biodegradation removals. The increasing concentrations of
14
CO
2
in these suspensions, as determined with liquid scintillation, evidenced the strong impact of sorption to BC-rich sediments on the biodegradation rate. Using the best-fit parameter values, we integrated oxygen and sorption effects and showed that oxygen tensions far below saturation levels in water are sufficient to enable significant decreases in the steady-state concentrations of aqueous-phase pyrene. These findings may be relevant for bioaccumulation scenarios that consider the effect of sediment resuspension events on exposure to water column and sediment pore water, as well as the direct uptake of PAHs from sediments.
Escherichia coli
Strains Engineered for Homofermentative Production of
d
-Lactic Acid from Glycerol
Authors: Mazumdar Suman , Clomburg James M. , Gonzalez Ramon
Published: May 2010

Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in
Escherichia coli
under anaerobic and microaerobic conditions. In this work, we engineer
E. coli
for the efficient conversion of glycerol to
d
-lactic acid (
d
-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for
d
-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of
d
-lactate from pyruvate (
d
-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (Δ
pflB
) and fumarate reductase (Δ
frdA
) (strain LA01) and (ii) inactivation of fumarate reductase (Δ
frdA
), phosphate acetyltransferase (Δ
pta
), and alcohol/acetaldehyde dehydrogenase (Δ
adhE
) (strain LA02). A mutation that blocked the aerobic
d
-lactate dehydrogenase (Δ
dld
) also was introduced in both LA01 and LA02 to prevent the utilization of
d
-lactate. The most efficient strain (LA02Δ
dld
, with GlpK-GlpD overexpressed) produced 32 g/liter of
d
-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for
d
-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of
d
-lactate and is redox balanced, thus representing a viable metabolic pathway.
Genetic and Metabolic Divergence within aRhizobium leguminosarumbv.trifoliiPopulation Recovered from Clover Nodules
Authors: Wielbo Jerzy , Marek-Kozaczuk Monika , Mazur Andrzej , Kubik-Komar Agnieszka , Skorupska Anna
Published: May 2010

Rhizobia are able to establish symbiosis with leguminous plants and usually occupy highly complex soil habitats. The large size and complexity of their genomes are considered advantageous, possibly enhancing their metabolic and adaptive potential and, in consequence, their competitiveness. A population ofRhizobium leguminosarumbv.trifoliiorganisms recovered from nodules of several clover plants growing in each other's vicinity in the soil was examined regarding possible relationships between their metabolic-physiological properties and their prevalence in such a local population. Genetic and metabolic variability within theR. leguminosarumbv.trifoliistrains occupying nodules of several plants was of special interest, and both types were found to be considerable. Moreover, a prevalence of metabolically versatile strains, i.e., those not specializing in utilization of any group of substrates, was observed by combining statistical analyses of Biolog test results with the frequency of occurrence of genetically distinct strains. Metabolic versatility with regard to nutritional requirements was not directly advantageous for effectiveness in the symbiotic interaction with clover: rhizobia with specialized metabolism were more effective in symbiosis but rarely occurred in the population. The significance of genetic and, especially, metabolic complexity of bacteria constituting a nodule population is discussed in the context of strategies employed by bacteria in competition.
Application of the
Saccharomyces cerevisiae
FLP/
FRT
Recombination System in Filamentous Fungi for Marker Recycling and Construction of Knockout Strains Devoid of Heterologous Genes
Authors: Kopke Katarina , Hoff Birgit , Kück Ulrich
Published: May 2010

To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/
FRT
recombination system from the yeast
Saccharomyces cerevisiae
for marker recycling. We tested this system in the penicillin producer
Penicillium chrysogenum
using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the
FRT
sequences in direct repeat orientation (
FRT
-
nat1
cassette) into a
P. chrysogenum
recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized
P. chrysogenum flp
(
Pcflp
) recombinase gene, were transferred into the
P. chrysogenum
strain carrying the
FRT
-
nat1
cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional recombination system be generated in
P. chrysogenum
. As a proof of application of this system, we constructed a ΔPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/
FRT
system, we produced a flipper cassette carrying the
FRT
sites as well as the
Pcflp
gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/
FRT
recombination system in other fungi was further demonstrated by marker recycling in the ascomycete
Sordaria macrospora
. Here, we discuss the application of the optimized FLP/
FRT
recombination system as a molecular tool for the genetic manipulation of filamentous fungi.
γ-Glutamylmethylamide Is an Essential Intermediate in the Metabolism of Methylamine by
Methylocella silvestris
Authors: Chen Yin , Scanlan Julie , Song Lijiang , Crombie Andrew , Rahman M. Tanvir , Schäfer Hendrik , Murrell J. Colin
Published: May 2010

Methylocella silvestris
BL2, a facultative methane utilizer, can grow on monomethylamine (MMA) as a sole carbon and nitrogen source. No activity of MMA dehydrogenase was detectable. Instead, this bacterium utilizes a methylated amino acid pathway (γ-glutamylmethylamide [GMA] and
N
-methylglutamate [NMG]) for MMA metabolism. The activities of the two key enzymes in this pathway, GMA synthetase and NMG dehydrogenase, were found when the bacterium was grown on MMA. GMA was detected by high-performance liquid chromatography-mass spectrometry only when the bacterium was grown on MMA but not when it was grown on methanol. Proteomic analysis of soluble and membrane fractions of the proteome from MMA- and methanol-grown cultures revealed that an eight-gene cluster (Msil2632 to Msil2639) was induced by MMA and cotranscribed as an operon, as shown by reverse transcription-PCR. GMA-dissimilating enzyme activity was also detected when it was grown on MMA. Formaldehyde and ammonium production from GMA was dependent on glutamate but not on α-ketoglutarate. Marker exchange mutagenesis of a putative GMAS gene homologue (
gmas
, Msil2635) within this eight-gene cluster, with a kanamycin gene cassette, abolished growth of
M. silvestris
on MMA as either a sole carbon or a sole nitrogen source. Overall, our results suggest that
gmas
is essential in MMA metabolism by
M. silvestris
.
Expression of
Xylella fastidiosa
Fimbrial and Afimbrial Proteins during Biofilm Formation
Authors: Caserta R. , Takita M. A. , Targon M. L. , Rosselli-Murai L. K. , de Souza A. P. , Peroni L. , Stach-Machado D. R. , Andrade A. , Labate C. A. , Kitajima E. W. , Machado M. A. , de Souza A. A.
Published: May 2010

Complete sequencing of the
Xylella fastidiosa
genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of
X. fastidiosa
: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in
X. fastidiosa
biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants.
Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures
Authors: Hyka Petr , Züllig Thomas , Ruth Claudia , Looser Verena , Meier Christian , Klein Joachim , Melzoch Karel , Meyer Hans-Peter , Glieder Anton , Kovar Karin
Published: May 2010

Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinantPichia pastorisMut+(methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.
Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples
Authors: Parshionikar Sandhya , Laseke Ian , Fout G. Shay
Published: May 2010

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.
Burkholderia phymatum
Strains Capable of Nodulating
Phaseolus vulgaris
Are Present in Moroccan Soils
Authors: Talbi C. , Delgado M. J. , Girard L. , Ramírez-Trujillo A. , Caballero-Mellado J. , Bedmar E. J.
Published: May 2010

Phylogenetic analysis of 16S rRNA,
nodC
, and
nifH
genes of four bacterial strains isolated from root nodules of
Phaseolus vulgaris
grown in Morocco soils were identified as
Burkholderia phymatum
. All four strains formed N
2
-fixing nodules on
P. vulgaris
and
Mimosa
,
Acacia
, and
Prosopis
species and reduced acetylene to ethylene when cultured
ex planta
.
Biosynthetic Potential of Phylogenetically Unique Endophytic Actinomycetes from Tropical Plants
Authors: Janso Jeffrey E. , Carter Guy T.
Published: May 2010

The culturable diversity of endophytic actinomycetes associated with tropical, native plants is essentially unexplored. In this study, 123 endophytic actinomycetes were isolated from tropical plants collected from several locations in Papua New Guinea and Mborokua Island, Solomon Islands. Isolates were found to be prevalent in roots but uncommon in leaves. Initially, isolates were dereplicated to the strain level by ribotyping. Subsequent characterization of 105 unique strains by 16S rRNA gene sequence analysis revealed that 17 different genera were represented, and rare genera, such as
Sphaerisporangium
and
Planotetraspora
, which have never been previously reported to be endophytic, were quite prevalent. Phylogenetic analyses grouped many of the strains into clades distinct from known genera within
Thermomonosporaceae
and
Micromonosporaceae
, indicating that they may be unique genera. Bioactivity testing and liquid chromatography-mass spectrometry (LC-MS) profiling of crude fermentation extracts were performed on 91 strains. About 60% of the extracts exhibited bioactivity or displayed LC-MS profiles with spectra indicative of secondary metabolites. The biosynthetic potential of 29 nonproductive strains was further investigated by the detection of putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes. Despite their lack of detectable secondary metabolite production in fermentation, most were positive for type I (66%) and type II (79%) PKS genes, and all were positive for NRPS genes. These results suggest that tropical plants from New Guinea and the adjacent archipelago are hosts to unique endophytic actinomycetes that possess significant biosynthetic potential.
Pork Contaminated with
Salmonella
enterica
Serovar 4,[5],12:i:−, an Emerging Health Risk for Humans
Authors: Hauser Elisabeth , Tietze Erhard , Helmuth Reiner , Junker Ernst , Blank Kathrin , Prager Rita , Rabsch Wolfgang , Appel Bernd , Fruth Angelika , Malorny Burkhard
Published: May 2010

Salmonella enterica
subsp.
enterica
serovar 4,[5],12:i:− is a monophasic variant of
S
.
enterica
serovar Typhimurium (antigenic formula 4,[5],12:i:1,2). Worldwide, especially in several European countries and the United States, it has been reported among the 10 most frequently isolated serovars in pigs and humans. In the study reported here, 148 strains of the monophasic serovar isolated from pigs, pork, and humans in 2006 and 2007 in Germany were characterized by various phenotypic and genotypic methods. This characterization was done in order to investigate their clonality, the prevalence of identical subtypes in pigs, pork, and humans, and the genetic relatedness to other
S. enterica
serovar Typhimurium subtypes in respect to the pathogenic and resistance gene repertoire. Two major clonal lineages of the monophasic serovar were detected which can be differentiated by their phage types and pulsed-field gel electrophoresis (PFGE) profiles. Seventy percent of the strains tested belonged to definite phage type DT193, and those strains were mainly assigned to PFGE cluster B. Nineteen percent of the strains were typed to phage type DT120 and of these 86% belonged to PFGE cluster A. Sixty-five percent of the isolates of both lineages carried core multiresistance to ampicillin, streptomycin, tetracycline, and sulfamethoxazole encoded by the genes
bla
TEM1-like
,
strA
-
str
B
,
tet
(B), and
sul2.
No correlation to the source of isolation was observed in either lineage. Microarray analysis of 61
S. enterica
serovar 4,[5],12:i:− and 20
S. enterica
serovar Typhimurium isolates tested determining the presence or absence of 102 representative pathogenicity genes in
Salmonella
revealed no differences except minor variations in single strains within and between the serovars, e.g., by presence of the virulence plasmid in four strains. Overall the study indicates that in Germany
S. enterica
serovar 4,[5],12:i:− strains isolated from pig, pork, and human are highly related, showing their transmission along the food chain. Since the pathogenicity gene repertoire is highly similar to that of
S. enterica
serovar Typhimurium, it is essential that interventions are introduced at the farm level in order to limit human infection.
Diversity of Dissimilatory Sulfite Reductase Genes (
dsrAB
) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage
Authors: Moreau John W. , Zierenberg Robert A. , Banfield Jillian F.
Published: May 2010

Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered
dsrAB
sequences from three sites sampled along an AMD flow path indicated the dominance of a single
Desulfovibrio
species. Other major sequence clades were related most closely to
Desulfosarcina
,
Desulfococcus
,
Desulfobulbus
, and
Desulfosporosinus
species. The presence of metal sulfides with low δ
34
S values relative to δ
34
S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ
34
S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.
Cometabolic Degradation of Trichloroethene by
Rhodococcus
sp. Strain L4 Immobilized on Plant Materials Rich in Essential Oils
Authors: Suttinun Oramas , Müller Rudolf , Luepromchai Ekawan
Published: May 2010

The cometabolic degradation of trichloroethene (TCE) by
Rhodococcus
sp. L4 was limited by the loss of enzyme activity during TCE transformation. This problem was overcome by repeated addition of inducing substrates, such as cumene, limonene, or cumin aldehyde, to the cells. Alternatively,
Rhodococcus
sp. L4 was immobilized on plant materials which contain those inducers in their essential oils. Cumin seeds were the most suitable immobilizing material, and the immobilized cells tolerated up to 68 μM TCE and degraded TCE continuously. The activity of immobilized cells, which had been inactivated partially during TCE degradation, could be reactivated by incubation in mineral salts medium without TCE. These findings demonstrate that immobilization of
Rhodococcus
sp. L4 on plant materials rich in essential oils is a promising method for efficient cometabolic degradation of TCE.
Exopolysaccharide Synthesized byLactobacillus reuteriDecreases the Ability of EnterotoxigenicEscherichia coliTo Bind to Porcine Erythrocytes
Authors: Wang Yvonne , Gänzle Michael G. , Schwab Clarissa
Published: May 2010

This study investigated the therapeutic potential of bacterial polysaccharides by employing a model system based on enteroxigenicEscherichia coli(ETEC)-induced hemagglutination of erythrocytes. Exopolysaccharides produced by strains ofLactobacillus reuteriinhibited ETEC-induced hemagglutination of porcine erythrocytes. No effect was observed for dextran produced fromWeissella cibariaand commercially available oligo- and polysaccharides.
Microtubule-Dependent mRNA Transport in Fungi
Authors: Zarnack Kathi , Feldbrügge Michael
Published: May 2010

The localization and local translation of mRNAs constitute an important mechanism to promote the correct subcellular targeting of proteins. mRNA localization is mediated by the active transport of mRNPs, large assemblies consisting of mRNAs and associated factors such as RNA-binding proteins. Molecular motors move mRNPs along the actin or microtubule cytoskeleton for short-distance or long-distance trafficking, respectively. In filamentous fungi, microtubule-based long-distance transport of vesicles, which are involved in membrane and cell wall expansion, supports efficient hyphal growth. Recently, we discovered that the microtubule-mediated transport of mRNAs is essential for the fast polar growth of infectious filaments in the corn pathogen
Ustilago maydis
. Combining
in vivo
UV cross-linking and RNA live imaging revealed that the RNA-binding protein Rrm4, which constitutes an integral part of the mRNP transport machinery, mediates the transport of distinct mRNAs encoding polarity factors, protein synthesis factors, and mitochondrial proteins. Moreover, our results indicate that microtubule-dependent mRNA transport is evolutionarily conserved from fungi to higher eukaryotes. This raises the exciting possibility of
U. maydis
as a model system to uncover basic concepts of long-distance mRNA transport.
Systematic Genetic Analysis of the Plasmodium falciparum MSP7-Like Family Reveals Differences in Protein Expression, Location, and Importance in Asexual Growth of the Blood-Stage Parasite
Authors: Kadekoppala Madhusudan , Ogun Solabomi A. , Howell Steven , Gunaratne Ruwani S. , Holder Anthony A.
Published: May 2010

Proteins located on
Plasmodium falciparum
merozoites, the invasive form of the parasite's asexual blood stage, are of considerable interest in vaccine research. Merozoite surface protein 7 (MSP7) forms a complex with MSP1 and is encoded by a member of a multigene family located on chromosome 13. The family codes for MSP7 and five MSP7-related proteins (MSRPs). In the present study, we have investigated the expression and the effect of
msrp
gene deletion at the asexual blood stage. In addition to
msp7
,
msrp2
,
msrp3
, and
msrp5
are transcribed, and mRNA was easily detected by hybridization analysis, whereas mRNA for
msrp1
and
msrp4
could be detected only by reverse transcription (RT)-PCR. Notwithstanding evidence of transcription, antibodies to recombinant MSRPs failed to detect specific proteins, except for antibodies to MSRP2. Sequential proteolytic cleavages of MSRP2 resulted in 28- and 25-kDa forms. However, MSRP2 was absent from merozoites; the 25-kDa MSRP2 protein (MSRP2
25
) was soluble and secreted upon merozoite egress. The
msrp
genes were deleted by targeted disruption in the 3D7 line, leading to ablation of full-length transcripts. MSRP deletion mutants had no detectable phenotype, with growth and invasion characteristics comparable to those of the parental parasite; only the deletion of MSP7 led to a detectable growth phenotype. Thus, within this family some of the genes are transcribed at a significant level in asexual blood stages, but the corresponding proteins may or may not be detectable. Interactions of the expressed proteins with the merozoite also differ. These results highlight the potential for unexpected differences of protein expression levels within gene families.
MoTea4-Mediated Polarized Growth Is Essential for Proper Asexual Development and Pathogenesis in Magnaporthe oryzae
Authors: Patkar Rajesh N. , Suresh Angayarkanni , Naqvi Naweed I.
Published: May 2010

Polarized growth is essential for cellular development and function and requires coordinated organization of the cytoskeletal elements. Tea4, an important polarity determinant, regulates localized F-actin assembly and bipolar growth in fission yeast and directional mycelial growth in
Aspergillus
. Here, we characterize Tea4 in the rice blast fungus
Magnaporthe oryzae
(MoTea4). Similar to its orthologs, MoTea4-green fluorescent protein (MoTea4-GFP) showed punctate distribution confined to growth zones, particularly in the mycelial tips, aerial hyphae, conidiophores, conidia, and infection structures (appressoria) in
Magnaporthe
. MoTea4 was dispensable for vegetative growth in
Magnaporthe
. However, loss of MoTea4 led to a zigzag morphology in the aerial hyphae and a huge reduction in conidiation. The majority of the
tea4
Δ conidia were two celled, as opposed to the tricellular conidia in the wild type. Structure-function analysis indicated that the SH3 and coiled-coil domains of MoTea4 are necessary for proper conidiation in
Magnaporthe
. The
tea4
Δ conidia failed to produce proper appressoria and consequently failed to infect the host plants. The
tea4
Δ conidia and germ tubes showed disorganized F-actin structures with significantly reduced numbers of cortical actin patches. Compared to the wild-type conidia, the
tea4
Δ conidia showed aberrant germination, poor cytoplasmic streaming, and persistent accumulation of lipid droplets, likely due to the impaired F-actin cytoskeleton. Latrunculin A treatment of germinating wild-type conidia showed that an intact F-actin cytoskeleton is indeed essential for appressorial development in
Magnaporthe
. We show that MoTea4 plays an important role in organizing the F-actin cytoskeleton and is essentially required for polarized growth and morphogenesis during asexual and pathogenic development in
Magnaporthe
.
Unconventional Secretion of AcbA in Dictyostelium discoideum through a Vesicular Intermediate
Authors: Cabral Matthew , Anjard Christophe , Malhotra Vivek , Loomis William F. , Kuspa Adam
Published: May 2010

The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in
Dictyostelium discoideum
. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires
N
-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.
Sensor Kinases RetS and LadS RegulatePseudomonas syringaeType VI Secretion and Virulence Factors
Authors: Records Angela R. , Gross Dennis C.
Published: May 2010

Pseudomonas syringaepv. syringae B728a is a resident on leaves of common bean, where it utilizes several well-studied virulence factors, including secreted effectors and toxins, to develop a pathogenic interaction with its host. The B728a genome was recently sequenced, revealing the presence of 1,297 genes with unknown function. This study demonstrates that a 29.9-kb cluster of genes in the B728a genome shares homology to the novel type VI secretion system (T6SS) locus recently described for other Gram-negative bacteria. Western blot analyses showed that B728a secretes Hcp, a T6SS protein, in culture and that this secretion is dependent onclpV, a gene that likely encodes an AAA+ATPase. In addition, we have identified two B728a sensor kinases that have homology to theP. aeruginosaproteins RetS and LadS. We demonstrate that B728a RetS and LadS reciprocally regulate the T6SS and collectively modulate several virulence-related activities. Quantitative PCR analyses indicated that RetS and LadS regulate genes associated with the type III secretion system and that LadS controls the expression of genes involved in the production of the exopolysaccharides alginate and levan. These analyses also revealed that LadS and the hybrid sensor kinase GacS positively regulate the expression of a putative novel exopolysaccharide called Psl. Plate assays demonstrated that RetS negatively controls mucoidy, while LadS negatively regulates swarming motility. A mutation inretSaffected B728a population levels on the surfaces of bean leaves. A model for the LadS and RetS control of B728a virulence activities is proposed.
Variation among Genome Sequences of H37Rv Strains of
Mycobacterium tuberculosis
from Multiple Laboratories
Authors: Ioerger Thomas R. , Feng Yicheng , Ganesula Krishna , Chen Xiaohua , Dobos Karen M. , Fortune Sarah , Jacobs William R. , Mizrahi Valerie , Parish Tanya , Rubin Eric , Sassetti Chris , Sacchettini James C.
Published: May 2010

The publication of the complete genome sequence for
Mycobacterium tuberculosis
H37Rv in 1998 has had a great impact on the research community. Nonetheless, it is suspected that genetic differences have arisen in stocks of H37Rv that are maintained in different laboratories. In order to assess the consistency of the genome sequences among H37Rv strains in use and the extent to which they have diverged from the original strain sequenced, we carried out whole-genome sequencing on six strains of H37Rv from different laboratories. Polymorphisms at 73 sites were observed, which were shared among the lab strains, though 72 of these were also shared with H37Ra and are likely to be due to sequencing errors in the original H37Rv reference sequence. An updated H37Rv genome sequence should be valuable to the tuberculosis research community as well as the broader microbial research community. In addition, several polymorphisms unique to individual strains and several shared polymorphisms were identified and shown to be consistent with the known provenance of these strains. Aside from nucleotide substitutions and insertion/deletions, multiple IS
6110
transposition events were observed, supporting the theory that they play a significant role in plasticity of the
M. tuberculosis
genome. This genome-wide catalog of genetic differences can help explain any phenotypic differences that might be found, including a frameshift mutation in the mycocerosic acid synthase gene which causes two of the strains to be deficient in biosynthesis of the surface glycolipid phthiocerol dimycocerosate (PDIM). The resequencing of these six lab strains represents a fortuitous “
in vitro
evolution” experiment that demonstrates how the
M. tuberculosis
genome continues to evolve even in a controlled environment.
Down-Regulation of theEscherichia coliK-12nrfPromoter by Binding of the NsrR Nitric Oxide-Sensing Transcription Repressor to an Upstream Site
Authors: Browning Douglas F. , Lee David J. , Spiro Stephen , Busby Stephen J. W.
Published: May 2010

FNR-dependent activation of theEscherichia coliK-12nrfpromoter is downregulated by the nitric oxide-sensitive NsrR protein together with the nucleoid-associated protein IHF, which bind to overlapping targets adjacent to the DNA site for FNR. The NsrR target is inactivated by mutation at theSalmonella entericaserovar Typhimuriumnrfpromoter.
The Genetic Basis of Laboratory Adaptation in
Caulobacter crescentus
Authors: Marks Melissa E. , Castro-Rojas Cyd Marie , Teiling Clotilde , Du Lei , Kapatral Vinayak , Walunas Theresa L. , Crosson Sean
Published: May 2010

The dimorphic bacterium
Caulobacter crescentus
has evolved marked phenotypic changes during its 50-year history of culture in the laboratory environment, providing an excellent system for the study of natural selection and phenotypic microevolution in prokaryotes. Combining whole-genome sequencing with classical molecular genetic tools, we have comprehensively mapped a set of polymorphisms underlying multiple derived phenotypes, several of which arose independently in separate strain lineages. The genetic basis of phenotypic differences in growth rate, mucoidy, adhesion, sedimentation, phage susceptibility, and stationary-phase survival between
C. crescentus
strain CB15 and its derivative NA1000 is determined by coding, regulatory, and insertion/deletion polymorphisms at five chromosomal loci. This study evidences multiple genetic mechanisms of bacterial evolution as driven by selection for growth and survival in a new selective environment and identifies a common polymorphic locus,
zwf
, between lab-adapted
C. crescentus
and clinical isolates of
Pseudomonas aeruginosa
that have adapted to a human host during chronic infection.
Structure-Specific Nuclease Activities of
Pyrococcus abyssi
RNase HII
Authors: Le Laz Sébastien , Le Goaziou Audrey , Henneke Ghislaine
Published: May 2010

Faithful DNA replication involves the removal of RNA residues from genomic DNA prior to the ligation of nascent DNA fragments in all living organisms. Because the physiological roles of archaeal type 2 RNase H are not fully understood, the substrate structure requirements for the detection of RNase H activity need further clarification. Biochemical characterization of a single RNase H detected within the genome of
Pyrococcus abyssi
showed that this type 2 RNase H is an Mg- and alkaline pH-dependent enzyme.
Pab
RNase HII showed RNase activity and acted as a specific endonuclease on RNA-DNA/DNA duplexes. This specific cleavage, 1 nucleotide upstream of the RNA-DNA junction, occurred on a substrate in which RNA initiators had to be fully annealed to the cDNA template. On the other hand, a 5′ RNA flap Okazaki fragment intermediate impaired
Pab
RNase HII endonuclease activity. Furthermore, introduction of mismatches into the RNA portion near the RNA-DNA junction decreased both the specificity and the efficiency of cleavage by
Pab
RNase HII. Additionally,
Pab
RNase HII could cleave a single ribonucleotide embedded in a double-stranded DNA. Our data revealed
Pab
RNase HII as a dual-function enzyme likely required for the completion of DNA replication and DNA repair.
Frequency and Rate of Pilin Antigenic Variation of
Neisseria meningitidis
Authors: Helm R. Allen , Seifert H. Steven
Published: May 2010

The rates of pilin antigenic variation (Av) of two strains of
Neisseria meningitidis
were determined using an unbiased DNA sequencing assay. Strain MC58 underwent pilin Av at a rate similar to that of
N. gonorrhoeae
strain MS11 but lower than that of
N. gonorrhoeae
strain FA1090. Pilin Av was undetectable in strain FAM18.
The Response Regulator SprE (RssB) Is Required for Maintaining Poly(A) Polymerase I-Degradosome Association during Stationary Phase
Authors: Carabetta Valerie J. , Silhavy Thomas J. , Cristea Ileana M.
Published: May 2010

Poly(A) polymerase I (PAP I) is the enzyme responsible for the addition of poly(A) tails onto RNA molecules inEscherichia coli. Polyadenylation is believed to facilitate the destruction of such RNAs by the mRNA degradosome. Recently, it was discovered that the stationary-phase regulatory protein SprE (RssB) has a second function in the control of polyadenylation that is distinct from its known function in the regulated proteolysis of RpoS. In the work presented herein, we used a targeted proteomic approach to further investigate SprE's involvement in the polyadenylation pathway. Specifically, we used cryogenic cell lysis, immunopurifications on magnetic beads, and mass spectrometry to identify interacting partners of PAP I-green fluorescent protein. We provide the firstin vivoevidence that PAP I interacts with the mRNA degradosome during both exponential and stationary phases and find that the degradosome can contain up to 10 different proteins under certain conditions. Moreover, we demonstrate that the majority of these PAP I interactions are formed via protein-protein interactions and that SprE plays an important role in the maintenance of the PAP I-degradosome association during stationary phase.
Redundant Function ofcmaA2andmmaA2inMycobacterium tuberculosis cisCyclopropanation of Oxygenated Mycolates
Authors: Barkan Daniel , Rao Vivek , Sukenick George D. , Glickman Michael S.
Published: May 2010

TheMycobacterium tuberculosiscell envelope contains a wide variety of lipids and glycolipids, including mycolic acids, long-chain branched fatty acids that are decorated by cyclopropane rings. Genetic analysis of the mycolate methyltransferase family has been a powerful approach to assign functions to each of these enzymes but has failed to reveal the origin ofciscyclopropanation of the oxygenated mycolates. Here we examine potential redundancy between mycolic acid methyltransferases by generating and analyzingM. tuberculosisstrains lackingmmaA2andcmaA2,mmaA2andcmaA1, ormmaA1alone.M. tuberculosislacking bothcmaA2andmmaA2cannotciscyclopropanate methoxymycolates or ketomycolates, phenotypes not shared by themmaA2andcmaA2single mutants. In contrast, a combined loss ofcmaA1andmmaA2had no effect on mycolic acid modification compared to results with a loss ofmmaA2alone. Deletion ofmmaA1fromM. tuberculosisabolishestranscyclopropanation without accumulation oftrans-unsaturated oxygenated mycolates, placing MmaA1 in the biosynthetic pathway fortrans-cyclopropanated oxygenated mycolates before CmaA2. These results define new functions for the mycolic acid methyltransferases ofM. tuberculosisand indicate a substantial redundancy of function for MmaA2 and CmaA2, the latter of which can function as both acisandtranscyclopropane synthase for the oxygenated mycolates.
The
Yersinia pestis
Chromosome Encodes Active Addiction Toxins
Authors: Goulard Céline , Langrand Sophie , Carniel Elisabeth , Chauvaux Sylvie
Published: May 2010

Toxin-antitoxin (TA) loci consist of two genes in an operon, encoding a stable toxin and an unstable antitoxin. The expression of toxin leads to cell growth arrest and sometimes bacterial death, while the antitoxin prevents the cytotoxic activity of the toxin. In this study, we show that the chromosome of
Yersinia pestis
, the causative agent of plague, carries 10 putative TA modules and two solitary antitoxins that belong to five different TA families (HigBA, HicAB, RelEB, Phd/Doc, and MqsRA). Two of these toxin genes (
higB2
and
hicA1
) could not be cloned in
Escherichia coli
unless they were coexpressed with their cognate antitoxin gene, indicating that they are highly toxic for this species. One of these toxin genes (
higB2
) could, however, be cloned directly and expressed in
Y. pestis
, where it was highly toxic, while the other one (
hicA1
) could not, probably because of its extreme toxicity. All eight other toxin genes were successfully cloned into the expression vector pBAD-TOPO. For five of them (
higB1
,
higB3
,
higB5
,
hicA2
, and
tox
), no toxic activity was detected in either
E. coli
or
Y. pestis
despite their overexpression. The three remaining toxin genes (
relE1
,
higB4
, and
doc
) were toxic for
E. coli
, and this toxic activity was abolished when the cognate antitoxin was coexpressed, showing that these three TA modules are functional in
E. coli
. Curiously, only one of these three toxins (RelE1) was active in
Y. pestis
. Cross-interaction between modules of the same family was observed but occurred only when the antitoxins were almost identical. Therefore, our study demonstrates that of the 10 predicted TA modules encoded by the
Y. pestis
chromosome, at least 5 are functional in
E. coli
and/or in
Y. pestis
. This is the first demonstration of active addiction toxins produced by the plague agent.