bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppressive Effect on Activation of Macrophages by
Lactobacillus casei
Strain Shirota Genes Determining the Synthesis of Cell Wall-Associated Polysaccharides
Authors: Yasuda Emi , Serata Masaki , Sako Tomoyuki
Published: June 2008

Although many
Lactobacillus
strains used as probiotics are believed to modulate host immune responses, the molecular natures of the components of such probiotic microorganisms directly involved in immune modulation process are largely unknown. We aimed to assess the function of polysaccharide moiety of the cell wall of
Lactobacillus casei
strain Shirota as a possible immune modulator which regulates cytokine production by macrophages. A gene survey of the genome sequence of
L. casei
Shirota hunted down a unique cluster of 10 genes, most of whose predicted amino acid sequences had similarities to various extents to known proteins involved in biosynthesis of extracellular or capsular polysaccharides from other lactic acid bacteria. Gene knockout mutants of eight genes from this cluster resulted in the loss of reactivity to
L. casei
Shirota-specific monoclonal antibody and extreme reduction of high-molecular-mass polysaccharides in the cell wall fraction, indicating that at least these genes are involved in biosynthesis of high-molecular-mass cell wall polysaccharides. By adding heat-killed mutant cells to mouse macrophage cell lines or to mouse spleen cells, the production of tumor necrosis factor alpha, interleukin-12 (IL-12), IL-10, and IL-6 was more stimulated than by wild-type cells. In addition, these mutants additively enhanced lipopolysaccharide-induced IL-6 production by RAW 264.7 mouse macrophage-like cells, while wild-type cells significantly suppressed the IL-6 production of RAW 264.7. Collectively, these results indicate that this cluster of genes of
L. casei
Shirota, which have been named
cps1A
,
cps1B
,
cps1C
,
cps1D
,
cps1E
,
cps1F
,
cps1G
, and
cps1J
, determine the synthesis of the high-molecular-mass polysaccharide moiety of the
L. casei
Shirota cell wall and that this polysaccharide moiety is the relevant immune modulator which may function to reduce excessive immune reactions during the activation of macrophages by
L. casei
Shirota.
Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State
Authors: Dimitriu Pedro A. , Pinkart Holly C. , Peyton Brent M. , Mormile Melanie R.
Published: June 2008

The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by
Gammaproteobacteria
, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the
Cytophaga-Flexibacter-Bacteroides
(CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the
Chloroflexi
and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.
Transfer of Class 1 Integron-Mediated Antibiotic Resistance Genes from Shiga Toxin-Producing
Escherichia coli
to a Susceptible
E. coli
K-12 Strain in Storm Water and Bovine Feces
Authors: Nagachinta Supakana , Chen Jinru
Published: June 2008

Transfer of class 1 integron-mediated antibiotic resistance genes has been demonstrated under laboratory conditions. However, there is no information concerning the transfer of these genes in an agricultural environment. The present study sought to determine if integron-mediated streptomycin and sulfisoxazole resistance genes could be transferred from Shiga toxin-producing
Escherichia coli
(STEC) strains 6-20 (O157:H7) and 7-63 (O111:H8) to the susceptible strain
E. coli
K-12 MG1655 in bovine feces (pH 5.5, 6.0, or 6.5) and storm water (pH 5, 6, 7, or 8) at 4, 15, and 28°C, which are average seasonal temperatures for winter, spring-fall, and summer, respectively, in the Griffin, GA, area. The results indicated that at 28°C, the integron-mediated antibiotic resistance genes were transferred from both of the STEC donors in bovine feces. Higher conjugation efficiencies were, however, observed in the conjugation experiments involving STEC strain 6-20. In storm water, the resistance genes were transferred only from STEC strain 6-20. Greater numbers of transconjugants were recovered in the conjugation experiments performed with pH 6.5 bovine feces and with pH 7 storm water. Antibiotic susceptibility tests confirmed the transfer of integron-mediated streptomycin resistance and sulfisoxazole resistance, as well as the transfer of non-integron-mediated oxytetracycline resistance and tetracycline resistance in the transconjugant cells. These results suggest that the antibiotic resistance genes in STEC could serve as a source of antibiotic resistance genes disseminated via conjugation to susceptible cells of other
E. coli
strains in an agricultural environment.
Production of an Insecticidal Crystal Protein from
Bacillus thuringiensis
by the Methylotroph
Methylobacterium extorquens
Authors: Choi Young J. , Gringorten J. Lawrence , Bélanger Louise , Morel Lyne , Bourque Denis , Masson Luke , Groleau Denis , Míguez Carlos B.
Published: June 2008

The Cry1Aa protein from
Bacillus thuringiensis
is an insecticidal protein that is highly active against several species of Lepidoptera. We cloned and expressed the
cry1Aa
gene in a plant-colonizing methylotroph,
Methylobacterium extorquens
, under the control of the strong
M. extorquens
AM1 methanol dehydrogenase promoter, P

mxaF

. Transmission electron microscopy revealed characteristic bipyramidal intracellular δ-endotoxin crystals similar to the crystalline inclusions formed by
B. thuringiensis
. Both the protoxin protein and the activated toxin were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis. In single-dose assays of the recombinant against the silkworm,
Bombyx mori
, both whole cells and cell lysates caused rapid feeding inhibition followed by mortality. The biomass and growth rate of recombinant cells in shake flask culture were similar to those of the wild-type strain, indicating a lack of fitness cost to the recombinant under controlled culture conditions. Recombinant Cry1Aa was expressed at a level of 4.5% of total
M. extorquens
cell protein. The potential benefits of modifying
M. extorquens
to deliver insecticidal Cry proteins for crop and forest protection are discussed.
Function of Heterologous
Mycobacterium tuberculosis
InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C
20
Fatty Acids to C
60
-to-C
90
Mycolic Acids, during De Novo Lipoic Acid Synthesis in
Saccharomyces cerevisiae
Authors: Gurvitz Aner , Hiltunen J. Kalervo , Kastaniotis Alexander J.
Published: June 2008

We describe the physiological function of heterologously expressed
Mycobacterium tuberculosis
InhA during de novo lipoic acid synthesis in yeast (
Saccharomyces cerevisiae
) mitochondria. InhA, representing 2-
trans
-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C
20
fatty acids to form C
60
-to-C
90
mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in
S. cerevisiae etr1
Δ cells lacking mitochondrial 2-
trans
-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast
etr1
Δ cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C
4
to C
8
) than was previously thought (>C
12
). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in
M. tuberculosis
and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.
Characterization of Verotoxin-Encoding Phages from
Escherichia coli
O103:H2 Strains of Bovine and Human Origins
Authors: Karama Musafiri , Gyles Carlton L.
Published: June 2008

The objectives of this study were to induce and characterize verotoxin-encoding phages from a collection of 91 verotoxin-producing
Escherichia coli
(VTEC) O103:H2 strains of human and bovine origins. All the strains carried the
vt1
gene, and two carried the
vt2
gene as well. The phages were induced by UV irradiation and characterized by DNA restriction fragment length polymorphism (RFLP), genome size, morphology, and
Q
and
P
genes, characteristic of lambdoid phages. A total of 32
vt
-positive phages were induced and isolated from 31 VTEC O103:H2 strains. Thirty phages were
vt1
positive, and two were
vt2
positive. Ten of the 30
vt1
-positive phages (33.3%) were from cattle strains, and 20 (66.6%) were from human strains. The two
vt2
-positive phages were from human strains. Phages belonged to 21 RFLP profiles, of which 17 were single-phage profiles and 4 were multiple-phage profiles. The estimated genome size of the phages ranged from 34 to 84 kb. Two phages that were examined by electron microscopy possessed hexagonal heads with long tails, and one had an elongated head with a long tail. The
Q
and
P
genes were amplified in all 32 phages, and the
Q
-

stxA
1

gene region yielded an amplicon in 19 phages (59.3%). It is concluded that the VTEC O103:H2 strains of human origin were more readily inducible than those of bovine origin and that the genotypic profiles of verotoxin-encoding phages were highly diverse, as revealed by their RFLP profiles.
Characterization of an
Escherichia coli
O157:H7 O-Antigen Deletion Mutant and Effect of the Deletion on Bacterial Persistence in the Mouse Intestine and Colonization at the Bovine Terminal Rectal Mucosa
Authors: Sheng Haiqing , Lim Ji Youn , Watkins Maryann K. , Minnich Scott A. , Hovde Carolyn J.
Published: June 2008

Escherichia coli
O157:H7 causes hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome in humans and transiently colonizes healthy cattle at the terminal rectal mucosa. To investigate the role of the O antigen in persistence and colonization in the animal host, we generated an
E. coli
O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (
per
) in the
rfb
cluster. The lack of O antigen was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anti-O157 antibody. The growth rate and cell membrane permeability of the Δ
per
mutant were similar to the growth rate and cell membrane permeability of the wild type. Changes in membrane and secreted proteins were observed, but the expression of intimin, EspA, and EspB, implicated in bacterial intestinal colonization, was not altered, as determined by immunoblotting and reverse transcription-PCR. Similar to other O-antigen deletion mutants, the Δ
per
mutant was pleiotropic for autoaggregation and motility (it was FliC negative as determined by immunoblotting and flagellum negative as determined by electron microscopy). The abilities of the mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa were compared. Mice fed the Δ
per
mutant shed lower numbers of bacteria (
P
< 0.05) over a shorter time than mice fed the wild-type or complemented strain. After rectal application in steers, lower numbers of the Δ
per
mutant than of the wild type colonized the rectoanal junction mucosa, and the duration of the colonization was shorter (
P
< 0.05). Our previous work showed that flagella do not influence
E. coli
O157:H7 colonization at the bovine terminal rectal mucosa, so the current findings suggest that the O antigen contributes to efficient bovine colonization.
Isolation and Characterization of Carnocyclin A, a Novel Circular Bacteriocin Produced by
Carnobacterium maltaromaticum
UAL307
Authors: Martin-Visscher Leah A. , van Belkum Marco J. , Garneau-Tsodikova Sylvie , Whittal Randy M. , Zheng Jing , McMullen Lynn M. , Vederas John C.
Published: June 2008

Carnobacterium maltaromaticum
UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous
Listeria
species. Three bacteriocins were isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and α-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by
Carnobacterium
spp.
Isolation of Microbes from Lake Vostok Accretion Ice
Authors: D'Elia Tom , Veerapaneni Ram , Rogers Scott O.
Published: June 2008

Bacteria from seven Lake Vostok accretion and two deep glacial Vostok ice core sections were characterized. The cell concentrations were low, but many of the cells were viable. From the hundreds of cultures, 18 unique bacterial rRNA gene phylotypes were determined. Lake Vostok may contain a complex microbial ecosystem.
Magnetic Resonance Imaging of Structure, Diffusivity, and Copper Immobilization in a Phototrophic Biofilm
Authors: Phoenix V. R. , Holmes W. M.
Published: June 2008

Magnetic resonance imaging (MRI) was used to spatially resolve the structure, water diffusion, and copper transport of a phototrophic biofilm and its fate. MRI was able to resolve considerable structural heterogeneity, ranging from classical laminations ∼500 μm thick to structures with no apparent ordering. Pulsed-field gradient (PFG) analysis spatially resolved water diffusion coefficients which exhibited relatively little or no attenuation (diffusion coefficients ranged from 1.7 × 10
−9
m
2
s
−1
to 2.2 × 10
−9
m
2
s
−1
). The biofilm was then reacted with a 10-mg liter
−1
Cu
2+
solution, and transverse-parameter maps were used to spatially and temporally map copper immobilization within the biofilm. Significantly, a calibration protocol similar to that used in biomedical research successfully quantified copper concentrations throughout the biofilm. Variations in Cu concentrations were controlled by the biofilm structure. Copper immobilization was most rapid (∼5 mg Cu liter
−1
h
−1
) over the first 20 to 30 h and then much slower for the remaining 60 h of the experiment. The transport of metal within the biofilm is controlled by both diffusion and immobilization. This was explored using a Bartlett and Gardner model which examined both diffusion and adsorption through a hypothetical film exhibiting properties similar to those of the phototrophic biofilm. Higher adsorption constants (
K
) resulted in longer lag times until the onset of immobilization at depth but higher actual adsorption rates. MRI and reaction transport models are versatile tools which can significantly improve our understanding of heavy metal immobilization in naturally occurring biofilms.
Effects of Transgenic Hybrid Aspen Overexpressing Polyphenol Oxidase on Rhizosphere Diversity
Authors: Oliver Kathryn L. , Hamelin Richard C. , Hintz William E.
Published: June 2008

This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the
Bacteroidetes
/
Chlorobi
group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the
Zygomycota, Chytridiomycota, Ascomycota
, and
Basidiomycota
were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.
High-Throughput, Single-Cell Analysis of Macrophage Interactions with Fluorescently LabeledBacillus anthracisSpores
Authors: Stojkovic Bojana , Torres Eric M. , Prouty Angela M. , Patel Hetal K. , Zhuang Lefan , Koehler Theresa M. , Ballard Jimmy D. , Blanke Steven R.
Published: June 2008

The engulfment ofBacillus anthracisspores by macrophages is an important step in the pathogenesis of inhalational anthrax. However, from a quantitative standpoint, the magnitude to which macrophages interact with and engulf spores remains poorly understood, in part due to inherent limitations associated with commonly used assays. To analyze phagocytosis of spores by RAW264.7 macrophage-like cells in a high-throughput, nonsubjective manner, we labeledB. anthracisSterne 7702 spores prior to infection with an Alexa Fluor 488 amine-reactive dye in a manner that did not alter their germination, growth kinetics, and heat resistance. Using flow cytometry, large numbers of cells exposed to labeled spores were screened to concurrently discriminate infected from uninfected cells and surface-associated from internalized spores. These experiments revealed that spore uptake was not uniform, but instead, highly heterogeneous and characterized by subpopulations of infected and uninfected cells, as well as considerable variation in the number of spores associated with individual cells. Flow cytometry analysis of infections demonstrated that spore uptake was independent of the presence or absence of fetal bovine serum, a germinant that, while routinely used in vitro, complicates the interpretation of the outcome of infections. Two commonly used macrophage cell lines, RAW264.7 and J774A.1 cells, were compared, revealing significant disparity between these two models in the rates of phagocytosis of labeled spores. These studies provide the experimental framework for investigating mechanisms of spore phagocytosis, as well as quantitatively evaluating strategies for interfering with macrophage binding and uptake of spores.
Molecular Identification of a SNAP-25-Like SNARE Protein inParamecium
Authors: Schilde Christina , Lutter Kaya , Kissmehl Roland , Plattner Helmut
Published: June 2008

Using database searches of the completedParamecium tetraureliamacronuclear genome with the metazoan SNAP-25 homologues, we identified a single 21-kDa Qb/c-SNARE in this ciliated protozoan, namedP. tetraureliaSNAP (PtSNAP), containing the characteristic dual heptad repeat SNARE motifs of SNAP-25. The presence of only a single Qb/c class SNARE inP. tetraureliais surprising in view of the multiple genome duplications and the high number of SNAREs found in other classes of this organism. As inferred from the subcellular localization of a green fluorescent protein (GFP) fusion construct, the protein is localized on a variety of intracellular membranes, and there is a large soluble pool of PtSNAP. Similarly, the PtSNAP that is detected with a specific antibody in fixed cells is associated with a number of intracellular membrane structures, including food vacuoles, the contractile vacuole system, and the sites of constitutive endo- and exocytosis. Surprisingly, using gene silencing, we could not assign a role to PtSNAP in the stimulated exocytosis of dense core vesicles (trichocysts), but we found an increased number of food vacuoles in PtSNAP-silenced cells. In conclusion, we identify PtSNAP as aParameciumhomologue of metazoan SNAP-25 that shows several divergent features, like resistance to cleavage by botulinum neurotoxins.
Saccharomyces cerevisiae
Afr1 Protein Is a Protein Phosphatase 1/Glc7-Targeting Subunit That Regulates the Septin Cytoskeleton during Mating
Authors: Bharucha Jennifer P. , Larson Jennifer R. , Konopka James B. , Tatchell Kelly
Published: June 2008

Glc7, the type1 serine/threonine phosphatase in the yeast
Saccharomyces cerevisiae
, is targeted by auxiliary subunits to numerous locations in the cell, where it regulates a range of physiological pathways. We show here that the accumulation of Glc7 at mating projections requires Afr1, a protein required for the formation of normal projections.
AFR1
-null mutants fail to target Glc7 to projections, and an Afr1 variant specifically defective in binding to Glc7 [Afr1(V546A F548A)] forms aberrant projections. The septin filaments in mating projections of
AFR1
mutants initiate normally but then rearrange asymmetrically as the projection develops, suggesting that the Afr1-Glc7 holoenzyme may regulate the maintenance of septin complexes during mating. These results demonstrate a previously unknown role for Afr1 in targeting Glc7 to mating projections and in regulating the septin architecture during mating.
Dolichyl-Phosphate-Glucose Is Used To Make O-Glycans on Glycoproteins of
Trichomonas vaginalis
Authors: Grabińska Kariona A. , Ghosh Sudip K. , Guan Ziqiang , Cui Jike , Raetz Christian R. H. , Robbins Phillips W. , Samuelson John
Published: June 2008

Trichomonas vaginalis
, the protist that causes vaginal itching, has a huge genome with numerous gene duplications. Recently we found that
Trichomonas
has numerous genes encoding putative dolichyl-phosphate-glucose (Dol-P-Glc) synthases (encoded by
ALG5
genes) despite the fact that
Trichomonas
lacks the glycosyltransferases (encoded by
ALG6
,
ALG8
, and
ALG10
genes) that use Dol-P-Glc to glucosylate dolichyl-PP-linked glycans. In addition,
Trichomonas
does not have a canonical
DPM1
gene, encoding a dolichyl-P-mannose (Dol-P-Man) synthase. Here we show
Trichomonas
membranes have roughly 300 times the Dol-P-Glc synthase activity of
Saccharomyces cerevisiae
membranes and about one-fifth the Dol-P-Man synthase activity of
Saccharomyces
membranes. Endogenous Dol-P-hexoses of
Trichomonas
are relatively abundant and contain 16 isoprene units. Five paralogous
Trichomonas ALG5
gene products have Dol-P-Glc synthase activity when expressed as recombinant proteins, and these
Trichomonas
Alg5s correct a carboxypeptidase N glycosylation defect in a
Saccharomyces alg5
mutant in vivo. A recombinant
Trichomonas
Dpm1, which is deeply divergent in its sequence, has Dol-P-Man synthase activity. When radiolabeled Dol-P-Glc is incubated with
Trichomonas
membranes, Glc is incorporated into reducing and nonreducing sugars of O-glycans of endogenous glycoproteins. To our knowledge, this is the first demonstration of Dol-P-Glc as a sugar donor for O-glycans on glycoproteins.
Involvement of
Saccharomyces cerevisiae
Avo3p/Tsc11p in Maintaining TOR Complex 2 Integrity and Coupling to Downstream Signaling
Authors: Ho Hsiang-Ling , Lee Hsin-Yi , Liao Hsien-Ching , Chen Mei-Yu
Published: June 2008

Target-of-rapamycin proteins (TORs) are Ser/Thr kinases serving a central role in cell growth control. TORs function in two conserved multiprotein complexes, TOR complex 1 (TORC1) and TORC2; the mechanisms underlying their actions and regulation are not fully elucidated.
Saccharomyces
TORC2, containing Tor2p, Avo1p, Avo2p, Avo3p/Tsc11p, Bit61p, and Lst8p, regulates cell integrity and actin organization. Two classes of
avo3
temperature-sensitive (
avo3
ts
) mutants that we previously identified display cell integrity and actin defects, yet one is suppressed by
AVO1
while the other is suppressed by
AVO2
or
SLM1
, defining two TORC2 downstream signaling mechanisms, one mediated by Avo1p and the other by Avo2p/Slm1p. Employing these mutants, we explored Avo3p functions in TORC2 structure and signaling. By observing binary protein interactions using coimmunoprecipitation, we discovered that the composition of TORC2 and its recruitment of the downstream effectors Slm1p and Slm2p were differentially affected in different
avo3
ts
mutants. These molecular defects can be corrected only by expressing
AVO3
, not by expressing suppressors, highlighting the role of Avo3p as a structural and signaling scaffold for TORC2. Phenotypic modifications of
avo3
ts
mutants by deletion of individual Rho1p-GTPase-activating proteins indicate that two TORC2 downstream signaling branches converge on Rho1p activation. Our results also suggest that Avo2p/Slm1p-mediated signaling, but not Avo1p-mediated signaling, links to Rho1p activation specifically through the Rho1p-guanine nucleotide exchange factor Tus1p.
Expression Quantitative Trait Locus Mapping of
Toxoplasma
Genes Reveals Multiple Mechanisms for Strain-Specific Differences in Gene Expression
Authors: Boyle Jon P. , Saeij Jeroen P. J. , Harada Scott Y. , Ajioka Jim W. , Boothroyd John C.
Published: June 2008

Toxoplasma gondii
is an intracellular parasite with a significant impact on human health, especially in cases where individuals are immunocompromised (e.g., due to human immunodeficiency virus/AIDS). In Europe and North America, only a few clonal genotypes appear to be responsible for the vast majority of
Toxoplasma
infections, and these clonotypes have been intensely studied to identify strain-specific phenotypes that may play a role in the manifestation of more-severe disease. To identify and genetically map strain-specific differences in gene expression, we have carried out expression quantitative trait locus analysis on
Toxoplasma
gene expression phenotypes by using spotted cDNA microarrays. This led to the identification of 16
Toxoplasma
genes that had significant and mappable strain-specific variation in hybridization intensity. While the analysis should identify both
cis
- and
trans
-mapping hybridization profiles, we identified only loci with strain-specific hybridization differences that are most likely due to differences in the locus itself (i.e.,
cis
mapping). Interestingly, a larger number of these
cis
-mapping genes than would be expected by chance encode either confirmed or predicted secreted proteins, many of which are known to localize to the specialized secretory organelles characteristic of members of the phylum Apicomplexa. For six of the
cis
-mapping loci, we determined if the strain-specific hybridization differences were due to true transcriptional differences or rather to strain-specific differences in hybridization efficiency because of extreme polymorphism and/or deletion, and we found examples of both scenarios.
Isolates of
Cryptococcus neoformans
from Infected Animals Reveal Genetic Exchange in Unisexual, α Mating Type Populations
Authors: Bui Tien , Lin Xiaorong , Malik Richard , Heitman Joseph , Carter Dee
Published: June 2008

Sexual reproduction and genetic exchange are important for the evolution of fungal pathogens and for producing potentially infective spores. Studies to determine whether sex occurs in the pathogenic yeast
Cryptococcus neoformans
var
. grubii
have produced enigmatic results, however: basidiospores are the most likely infective propagules, and clinical isolates are fertile and genetically diverse, consistent with a sexual species, but almost all populations examined consist of a single mating type and have little evidence for genetic recombination. The choice of population is critical when looking for recombination, particularly when significant asexual propagation is likely and when latency may complicate assessing the origin of an isolate. We therefore selected isolates from infected animals living in the region of Sydney, Australia, with the assumption that the relatively short life spans and limited travels of the animal hosts would provide a very defined population. All isolates were mating type α and were of molecular genotype VNI or VNII. A lack of linkage disequilibrium among loci suggested that genetic exchange occurred within both genotype groups. Four diploid VNII isolates that produced filaments and basidium-like structures when cultured in proximity to an a mating type strain were found. Recent studies suggest that compatible α-α unions can occur in
C. neoformans
var.
neoformans
populations and in populations of the sibling species
Cryptococcus gattii
. As a mating type strains of
C. neoformans
var.
grubii
have never been found in Australia, or in the VNII molecular type globally, the potential for α-α unions is evidence that α-α unisexual mating maintains sexual recombination and diversity in this pathogen and may produce infectious propagules.
Contribution of Galactofuranose to the Virulence of the Opportunistic Pathogen
Aspergillus fumigatus
Authors: Schmalhorst Philipp S. , Krappmann Sven , Vervecken Wouter , Rohde Manfred , Müller Meike , Braus Gerhard H. , Contreras Roland , Braun Armin , Bakker Hans , Routier Françoise H.
Published: June 2008

The filamentous fungus
Aspergillus fumigatus
is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the
A. fumigatus
cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The
glfA
deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against
A. fumigatus
. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.
Characterization of Kl
GRR1
and
SMS1
Genes, Two New Elements of the Glucose Signaling Pathway of
Kluyveromyces lactis
Authors: Hnatova Martina , Wésolowski-Louvel Micheline , Dieppois Guenaëlle , Deffaud Julien , Lemaire Marc
Published: June 2008

The expression of the major glucose transporter gene,
RAG1
, is induced by glucose in
Kluyveromyces lactis
. This regulation involves several pathways, including one that is similar to Snf3/Rgt2-ScRgt1 in
Saccharomyces cerevisiae
. We have identified missing key components of the
K. lactis
glucose signaling pathway by comparison to the same pathway of
S. cerevisiae
. We characterized a new mutation,
rag19
, which impairs
RAG1
regulation. The Rag19 protein is 43% identical to the F-box protein ScGrr1 of
S. cerevisiae
and is able to complement an Sc
grr1
mutation. In the
K. lactis
genome, we identified a single gene,
SMS1
(for
s
imilar to
M
th1 and
S
td1), that encodes a protein showing an average of 50% identity with Mth1 and Std1, regulators of the ScRgt1 repressor. The suppression of the
rag4
(glucose sensor),
rag8
(casein kinase I), and
rag19
mutations by the Δ
sms1
deletion, together with the restoration of
RAG1
transcription in the double mutants, demonstrates that Sms1 is a negative regulator of
RAG1
expression and is acting downstream of Rag4, Rag8, and Rag19 in the cascade. We report that Sms1 regulates KlRgt1 repressor activity by preventing its phosphorylation in the absence of glucose, and that
SMS1
is regulated by glucose, both at the transcriptional and the posttranslational level. Two-hybrid interactions of Sms1 with the glucose sensor and KlRgt1 repressor suggest that Sms1 mediates the glucose signal from the plasma membrane to the nucleus. All of these data demonstrated that Sms1 was the
K. lactis
homolog of
MTH1
and
STD1
of
S. cerevisiae
. Interestingly,
MTH1
and
STD1
were unable to complement a Δ
sms1
mutation.
Transfer of the First Arabinofuranose Residue to Galactan Is Essential for
Mycobacterium smegmatis
Viability
Authors: Shi Libin , Zhou Roukun , Liu Zhentong , Lowary Todd L. , Seeberger Peter H. , Stocker Bridget L. , Crick Dean C. , Khoo Kay-Hooi , Chatterjee Delphi
Published: June 2008

The mycobacterial arabinan is an elaborate component of the cell wall with multiple glycosyl linkages and no repeating units. In
Mycobacterium
spp., the Emb proteins (EmbA, EmbB, and EmbC) have been identified as putative mycobacterial arabinosyltransferases implicated in the biogenesis of the cell wall arabinan. Furthermore, it is now evident that the EmbA and EmbB proteins are involved in the assembly of the nonreducing terminal motif of arabinogalactan and EmbC is involved in transferring arabinose, perhaps in the early stage of arabinan synthesis in lipoarabinomannan. It has also been shown that the Emb proteins are a target of the antimycobacterial drug ethambutol (EMB). In the search for additional mycobacterial arabinosyltransferases in addition to the Emb proteins, we disrupted MSMEG_6386 (an orthologue of Rv3792 and a gene upstream of
embC
) in
Mycobacterium smegmatis
. Allelic exchange at the chromosomal MSMEG_6386 locus of
M. smegmatis
could only be achieved in the presence of a rescue plasmid carrying a functional copy of MSMEG_6386 or Rv3792, strongly suggesting that MSMEG_6386 is essential. An in vitro arabinosyltransferase assay using a membrane preparation from
M. smegmatis
expressing Rv3792 and synthetic β-
d
-Gal
f
-(1→5)-β-
d
-Gal
f
-(1→6)-β-
d
-Gal
f
-octyl and β-
d
-Gal
f
-(1→6)-β-
d
-Gal
f
-(1→5)-β-
d
-Gal
f
-octyl showed that Rv3792 gene product can transfer an arabinose residue to the C-5 position of the internal 6-linked galactose. The reactions were insensitive to EMB, and when α-
d
-Man
p
-(1→6)-α-
d
-Man
p
-(1→6)-α-
d
-Man
p
-octylthiomethyl was used as an acceptor, no product was formed. These observations indicate that transfer of the first arabinofuranose residue to galactan is essential for
M. smegmatis
viability.
Replacement of Adenylate Cyclase Toxin in a Lineage of
Bordetella bronchiseptica
Authors: Buboltz Anne M. , Nicholson Tracy L. , Parette Mylisa R. , Hester Sara E. , Parkhill Julian , Harvill Eric T.
Published: June 2008

Bordetella bronchiseptica
is a gram-negative respiratory pathogen that infects a wide range of hosts and causes a diverse spectrum of disease. This diversity is likely affected by multiple factors, such as host immune status, polymicrobial infection, and strain diversity. In a murine model of infection, we found that the virulence of
B. bronchiseptica
strains, as measured by the mean lethal dose, varied widely. Strain 253 was less virulent than the typically studied strain, RB50. Transcriptome analysis showed that
cyaA
, the gene encoding adenylate cyclase toxin (CyaA), was the most downregulated transcript identified in strain 253 compared to that in strain RB50. Comparative genomic hybridization and genome sequencing of strain 253 revealed that the
cya
locus, which encodes, activates, and secretes CyaA, was replaced by an operon (
ptp
) predicted to encode peptide transport proteins. Other
B. bronchiseptica
strains from the same phylogenetic lineage as that of strain 253 also lacked the
cya
locus, contained the
ptp
genes, and were less virulent than strain RB50. Although the loss of CyaA would be expected to be counterselected since it is conserved among the classical bordetellae and believed to be important to their success, our data indicate that the loss of this toxin and the gain of the
ptp
genes occurred in an ancestral strain that then expanded into a lineage. This suggests that there may be ecological niches in which CyaA is not critical for the success of
B. bronchiseptica
.
Characterization of Lipooligosaccharide-Biosynthetic Loci ofCampylobacter jejuniReveals New Lipooligosaccharide Classes: Evidence of Mosaic Organizations
Authors: Parker Craig T. , Gilbert Michel , Yuki Nobuhiro , Endtz Hubert P. , Mandrell Robert E.
Published: June 2008

The lipooligosaccharide (LOS) biosynthesis region is one of the more variable genomic regions between strains ofCampylobacter jejuni. Indeed, eight classes of LOS biosynthesis loci have been established previously based on gene content and organization. In this study, we characterize additional classes of LOS biosynthesis loci and analyze various mechanisms that result in changes to LOS structures. To gain further insights into the genomic diversity ofC. jejuniLOS biosynthesis region, we sequenced the LOS biosynthesis loci of 15 strains that possessed gene content that was distinct from the eight classes. This analysis identified 11 new classes of LOS loci that exhibited examples of deletions and insertions of genes and cassettes of genes found in other LOS classes or capsular biosynthesis loci leading to mosaic LOS loci. The sequence analysis also revealed both missense mutations leading to “allelic” glycosyltransferases and phase-variable and non-phase-variable gene inactivation by the deletion or insertion of bases. Specifically, we demonstrated that gene inactivation is an important mechanism for altering the LOS structures of strains possessing the same class of LOS biosynthesis locus. Together, these observations suggest that LOS biosynthesis region is a hotspot for genetic exchange and variability, often leading to changes in the LOS produced.
Regulation of Autolysis-Dependent Extracellular DNA Release by
Enterococcus faecalis
Extracellular Proteases Influences Biofilm Development
Authors: Thomas Vinai Chittezham , Thurlow Lance R. , Boyle Dan , Hancock Lynn E.
Published: June 2008

Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The
Enterococcus faecalis
Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by
E. faecalis
and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE
−
mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE
−
mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of
E. faecalis
biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases—GelE and SprE—is responsible for regulating autolysis and the release of high-molecular-weight eDNA, a critical component for the development of
E. faecalis
biofilms.
FtsW Is a Dispensable Cell Division Protein Required for Z-Ring Stabilization during Sporulation Septation in
Streptomyces coelicolor
Authors: Mistry Bhavesh V. , Del Sol Ricardo , Wright Chris , Findlay Kim , Dyson Paul
Published: June 2008

The conserved
rodA
and
ftsW
genes encode polytopic membrane proteins that are essential for bacterial cell elongation and division, respectively, and each gene is invariably linked with a cognate class B high-molecular-weight penicillin-binding protein (HMW PBP) gene. Filamentous differentiating
Streptomyces coelicolor
possesses four such gene pairs. Whereas
rodA
, although not its cognate HMW PBP gene, is essential in these bacteria, mutation of SCO5302 or SCO2607 (
sfr
) caused no gross changes to growth and septation. In contrast, disruption of either
ftsW
or the cognate
ftsI
gene blocked the formation of sporulation septa in aerial hyphae. The inability of spiral polymers of FtsZ to reorganize into rings in aerial hyphae of these mutants indicates an early pivotal role of an FtsW-FtsI complex in cell division. Concerted assembly of the complete divisome was unnecessary for Z-ring stabilization in aerial hyphae as
ftsQ
mutants were found to be blocked at a later stage in cell division, during septum closure. Complete cross wall formation occurred in vegetative hyphae in all three
fts
mutants, indicating that the typical bacterial divisome functions specifically during nonessential sporulation septation, providing a unique opportunity to interrogate the function and dependencies of individual components of the divisome in vivo.
RadC, a Misleading Name?
Authors: Attaiech Laetitia , Granadel Chantal , Claverys Jean-Pierre , Martin Bernard
Published: June 2008

The pfam04002 annotation describes RadC as a bacterial DNA repair protein. Although theradCgene is expressed specifically during competence for genetic transformation inStreptococcus pneumoniae, we report thatradCmutants exhibit normal uptake and processing of transforming DNA. They also display normal sensitivity to DNA-damaging agents, providing no support for the rad epithet.
Functional Characterization and Metal Ion Specificity of the Metal-Citrate Complex Transporter from
Streptomyces coelicolor
Authors: Lensbouer Joshua J. , Patel Ami , Sirianni Joseph P. , Doyle Robert P.
Published: June 2008

Secondary transporters of citrate in complex with metal ions belong to the bacterial CitMHS family, about which little is known. The transport of metal-citrate complexes in
Streptomyces coelicolor
has been investigated. The best cofactor for citrate uptake in
Streptomyces coelicolor
is Fe
3+
, but uptake was also noted for Ca
2+
, Pb
2+
, Ba
2+
, and Mn
2+
. Uptake was not observed with the Mg
2+
, Ni
2+
, or Co
2+
cofactor. The transportation of iron- and calcium-citrate makes these systems unique among the CitMHS family members reported to date. No complementary uptake akin to that observed for the CitH (Ca
2+
, Ba
2+
, Sr
2+
) and CitM (Mg
2+
, Ni
2+
, Mn
2+
, Co
2+
, Zn
2+
) systems of
Bacillus subtilis
was noted. Competitive experiments using EGTA confirmed that metal-citrate complex formation promoted citrate uptake. Uptake of free citrate was not observed. The open reading frame postulated as being responsible for the metal-citrate transport observed in
Streptomyces coelicolor
was cloned and overexpressed in
Escherichia coli
strains with the primary Fe
3+
-citrate transport system (
fecABCDE
) removed. Functional expression was successful, with uptake of Ca
2+
-citrate, Fe
3+
-citrate, and Pb
2+
-citrate observed. No free-citrate transport was observed in IPTG (isopropyl-β-
d
-thiogalactopyranoside)-induced or -uninduced
E. coli
. Metabolism of the Fe
3+
-citrate and Ca
2+
-citrate complexes, but not the Pb
2+
-citrate complex, was observed. Rationalization is based on the difference in metal-complex coordination upon binding of the metal by citrate.
Stability of the
cbb
3
-Type Cytochrome Oxidase Requires Specific CcoQ-CcoP Interactions
Authors: Peters Annette , Kulajta Carmen , Pawlik Grzegorz , Daldal Fevzi , Koch Hans-Georg
Published: June 2008

Cytochrome
cbb
3
-type oxidases are members of the heme copper oxidase superfamily and are composed of four subunits. CcoN contains the heme
b
-Cu
B
binuclear center where oxygen is reduced, while CcoP and CcoO are membrane-bound
c
-type cytochromes thought to channel electrons from the donor cytochrome into the binuclear center. Like many other bacterial members of this superfamily, the cytochrome
cbb
3
-type oxidase contains a fourth, non-cofactor-containing subunit, which is termed CcoQ. In the present study, we analyzed the role of CcoQ on the stability and activity of
Rhodobacter capsulatus cbb
3
-type oxidase. Our data showed that CcoQ is a single-spanning membrane protein with a N
out
-C
in
topology. In the absence of CcoQ,
cbb
3
-type oxidase activity is significantly reduced, irrespective of the growth conditions. Blue native polyacrylamide gel electrophoresis analyses revealed that the lack of CcoQ specifically impaired the stable recruitment of CcoP into the
cbb
3
-type oxidase complex. This suggested a specific CcoQ-CcoP interaction, which was confirmed by chemical cross-linking. Collectively, our data demonstrated that in
R. capsulatus
CcoQ was required for optimal
cbb
3
-type oxidase activity because it stabilized the interaction of CcoP with the CcoNO core complex, leading subsequently to the formation of the active 230-kDa
cbb
3
-type oxidase complex.