bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparative Characterization of the Microbial Diversities of an Artificial Microbialite Model and a Natural Stromatolite
Authors: Havemann Stephanie A. , Foster Jamie S. 
Published: October 2008

ABSTRACT

Microbialites are organosedimentary structures that result from the trapping, binding, and lithification of sediments by microbial mat communities. In this study we developed a model artificial microbialite system derived from natural stromatolites, a type of microbialite, collected from Exuma Sound, Bahamas. We demonstrated that the morphology of the artificial microbialite was consistent with that of the natural system in that there was a multilayer community with a pronounced biofilm on the surface, a concentrated layer of filamentous cyanobacteria in the top 5 mm, and a lithified layer of fused oolitic sand grains in the subsurface. The fused grain layer was comprised predominantly of the calcium carbonate polymorph aragonite, which corresponded to the composition of the Bahamian stromatolites. The microbial diversity of the artificial microbialites and that of natural stromatolites were also compared using automated ribosomal intergenic spacer analysis (ARISA) and 16S rRNA gene sequencing. The ARISA profiling indicated that the Shannon indices of the two communities were comparable and that the overall diversity was not significantly lower in the artificial microbialite model. Bacterial clone libraries generated from each of the three artificial microbialite layers and natural stromatolites indicated that the cyanobacterial and crust layers most closely resembled the ecotypes detected in the natural stromatolites and were dominated by
Proteobacteria
and
Cyanobacteria
. We propose that such model artificial microbialites can serve as experimental analogues for natural stromatolites.
Proteome-Based Comparative Analyses of Growth Stages Reveal New Cell Cycle-Dependent Functions in the Predatory Bacterium
Bdellovibrio bacteriovorus
Authors: Dori-Bachash Mally , Dassa Bareket , Pietrokovski Shmuel , Jurkevitch Edouard 
Published: October 2008

ABSTRACT

Bdellovibrio
and like organisms are obligate predators of bacteria that are ubiquitously found in the environment. Most exhibit a peculiar dimorphic life cycle during which free-swimming attack-phase (AP) cells search for and invade bacterial prey cells. The invader develops in the prey as a filamentous polynucleoid-containing cell that finally splits into progeny cells. Therapeutic and biocontrol applications of
Bdellovibrio
in human and animal health and plant health, respectively, have been proposed, but more knowledge of this peculiar cell cycle is needed to develop such applications. A proteomic approach was applied to study cell cycle-dependent expression of the
Bdellovibrio bacteriovorus
proteome in synchronous cultures of a facultative host-independent (HI) strain able to grow in the absence of prey. Results from two-dimensional gel electrophoresis, mass spectrometry, and temporal expression of selected genes in predicted operons were analyzed. In total, about 21% of the in silico predicted proteome was covered. One hundred ninety-six proteins were identified, including 63 hitherto unknown proteins and 140 life stage-dependent spots. Of those, 47 were differentially expressed, including chemotaxis, attachment, growth- and replication-related, cell wall, and regulatory proteins. Novel cell cycle-dependent adhesion, gliding, mechanosensing, signaling, and hydrolytic functions were assigned. The HI model was further studied by comparing HI and wild-type AP cells, revealing that proteins involved in DNA replication and signaling were deregulated in the former. A complementary analysis of the secreted proteome identified 59 polypeptides, including cell contact proteins and hydrolytic enzymes specific to predatory bacteria.
yKu70/yKu80 and Rif1 Regulate Silencing Differentially at Telomeres in
Candida glabrata
Authors: Rosas-Hernández Lluvia L. , Juárez-Reyes Alejandro , Arroyo-Helguera Omar E. , De Las Peñas Alejandro , Pan Shih-Jung , Cormack Brendan P. , Castaño Irene 
Published: October 2008

ABSTRACT

Candida glabrata
, a common opportunistic fungal pathogen, adheres efficiently to mammalian epithelial cells in culture. This interaction in vitro depends mainly on the adhesin Epa1, one of a large family of cell wall proteins. Most of the
EPA
genes are located in subtelomeric regions, where they are transcriptionally repressed by silencing. In order to better characterize the transcriptional regulation of the
EPA
family, we have assessed the importance of
C. glabrata
orthologues of known regulators of subtelomeric silencing in
Saccharomyces cerevisiae.
To this end, we used a series of strains containing insertions of the reporter
URA3
gene within different intergenic regions throughout four telomeres of
C. glabrata
. Using these reporter strains, we have assessed the roles of
SIR2
,
SIR3
,
SIR4
,
HDF1
(yKu70),
HDF2
(yKu80), and
RIF1
in mediating silencing at four
C. glabrata
telomeres. We found that, whereas the
SIR
proteins are absolutely required for silencing of the reporter genes and the native subtelomeric
EPA
genes, the Rif1 and the Ku proteins regulate silencing at only a subset of the analyzed telomeres. We also mapped a
cis
element adjacent to the
EPA3
locus that can silence a reporter gene when placed at a distance of 31 kb from the telomere. Our data show that silencing of the
C. glabrata
telomeres varies from telomere to telomere. In addition, recruitment of silencing proteins to the subtelomeres is likely, for certain telomeres, to depend both on the telomeric repeats and on particular discrete silencing elements.
Genetic Analysis of the
Enterococcus
Vancomycin Resistance Conjugative Plasmid pHTβ: Identification of the Region Involved in Cell Aggregation and
traB
, a Key Regulator Gene for Plasmid Transfer and Cell Aggregation
Authors: Tomita Haruyoshi , Ike Yasuyoshi 
Published: October 2008

ABSTRACT

The
Enterococcus
plasmid pHTβ (63.7 kbp) is a pheromone-independent, highly conjugative pMG1-like plasmid that carries a Tn
1546
-like transposon encoding vancomycin resistance. The transfer-related regions (Tra I, Tra II, and Tra III) containing
oriT
and a putative nickase gene (
traI
) have previously been identified in pHTβ, and in this study, we found that the plasmid conferred the ability to self-aggregate on the host strain
Enterococcus faecalis
FA2-2. A region where mutation resulted in the impairment of aggregation was identified and mapped to a point upstream of the transfer-related Tra I region. This region consisted of an approximately 6-kbp segment that contained the five open reading frames (ORFs) ORF9 to ORF13. These ORFs are considered to encode the aggregation function, although the precise mode of action of each ORF has not yet been elucidated. An in-frame deletion mutant of ORF10 resulted in reduced aggregation and decreased transfer frequency in broth mating. Transcription analysis of the aggregation region showed that the five ORFs from ORF9 to ORF13 form an operon structure, and a long transcript that started from a promoter region located upstream of ORF9 was identified. Tra II spans a 1.7-kbp region containing ORF56 and ORF57. Tn
917
-
lac
insertions into or an in-frame deletion mutant of ORF56 (187 amino acids) resulted in impaired transfer and aggregation. The cloned ORF56 complemented these functions in
trans
. The transcription levels of ORF10 and ORF13 were reduced in the in-frame mutants of ORF56, but this reduction was complemented by a cloned ORF56 in
trans
. The results indicated that ORF56 positively regulated the aggregation and plasmid transfer in the host strain, and ORF56 was designated
traB
.
Analysis ofyggXandgshAMutants Provides Insights into the Labile Iron Pool inSalmonella enterica
Authors: Thorgersen Michael P. , Downs Diana M. 
Published: October 2008

ABSTRACTStrains ofSalmonella entericalacking YggX and the cellular reductant glutathione exhibit defects similar to those resulting from iron deficiency and oxidative stress. Mutant strains are sensitive to hydrogen peroxide and superoxide, deregulate the expression of the Fur-regulated geneentB, and fail to grow on succinate medium. Suppression of someyggX gshAmutant phenotypes by the cell-permeable iron chelator deferoxamine allowed the conclusion that increased levels of cellular Fenton chemistry played a role in the growth defects. The data presented are consistent with a scenario in which glutathione acts as a physiological chelator of the labile iron pool and in which YggX acts upstream of the labile iron pool by preventing superoxide toxicity.
Interaction of the Mycobacterial Heparin-Binding Hemagglutinin with Actin, as Evidenced by Single-Molecule Force Spectroscopy
Authors: Verbelen Claire , Dupres Vincent , Raze Dominique , Bompard Coralie , Locht Camille , Dufrêne Yves F. 
Published: October 2008

ABSTRACT

Although
Mycobacterium tuberculosis
and related species are considered to be typical endosomal pathogens, recent studies have suggested that mycobacteria can be present in the cytoplasm of infected cells and cause cytoskeleton rearrangements, the mechanisms of which remain unknown. Here, we used single-molecule force spectroscopy to demonstrate that the heparin-binding hemagglutinin (HBHA), a surface adhesin from
Mycobacterium tuberculosis
displaying sequence similarities with actin-binding proteins, is able to bind to actin. Force curves recorded between actin and the coiled-coil, N-terminal domain of HBHA showed a bimodal distribution of binding forces reflecting the detection of single and double HBHA-actin interactions. Force curves obtained between actin and the lysine-rich C-terminal domain of HBHA showed a broader distribution of binding events, suggesting they originate primarily from intermolecular electrostatic bridges between cationic HBHA domains and anionic actin residues. We also explored the dynamics of the HBHA-actin interaction, showing that the binding force and binding frequency increased with the pulling speed and contact time, respectively. Taken together, our data indicate that HBHA is able to specifically bind actin, via both its N-terminal and C-terminal domains, strongly suggesting a role of the HBHA-actin interaction in the pathogenesis of mycobacterial diseases.
Streptomyces scabies87-22 Possesses a Functional Tomatinase
Authors: Seipke Ryan F. , Loria Rosemary 
Published: October 2008

ABSTRACTThe actinomyceteStreptomyces scabies87-22 is the causal agent of common scab, an economically important disease of potato and taproot crops. Sequencing of theS. scabies87-22 genome revealed the presence of a gene with high homology to the gene encoding the α-tomatine-detoxifying enzyme tomatinase found in fungal tomato pathogens. ThetomAgene fromS. scabies87-22 was cotranscribed with a putative family 1 glycosyl hydrolase gene, and purified TomA protein was active only on α-tomatine and not potato glycoalkaloids or xylans. Tomatinase-null mutants were more sensitive to α-tomatine than the wild-type strain in a disk diffusion assay. Interestingly, tomatine affected only aerial mycelium and not vegetative mycelium, suggesting that the target(s) of α-tomatine is not present during vegetative growth. Severities of disease for tomato seedlings affected byS. scabies87-22 wild-type and ΔtomA1strains were indistinguishable, suggesting that tomatinase is not important in pathogenicity on tomato plants. However, conservation oftomAon a pathogenicity island inS. acidiscabiesandS. turgidiscabiessuggests a role in plant-microbe interaction.
Regulation of Fatty Acid Metabolism by FadR Is Essential for
Vibrio vulnificus
To Cause Infection of Mice
Authors: Brown Roslyn N. , Gulig Paul A. 
Published: October 2008

ABSTRACT

The opportunistic bacterial pathogen
Vibrio vulnificus
causes severe wound infection and fatal septicemia. We used alkaline phosphatase insertion mutagenesis in a clinical isolate of
V. vulnificus
to find genes necessary for virulence, and we identified
fadR
, which encodes a regulator of fatty acid metabolism. The
fadR
::mini-Tn
5
Km2
phoA
mutant was highly attenuated in a subcutaneously inoculated iron dextran-treated mouse model of
V. vulnificus
disease, was hypersensitive to the fatty acid synthase inhibitor cerulenin, showed aberrant expression of fatty acid biosynthetic (
fab
) genes and fatty acid oxidative (
fad
) genes, produced smaller colonies on agar media, and grew slower in rich broth than did the wild-type parent. Deletion of
fadR
essentially recapitulated the phenotypes of the insertion mutant, and the Δ
fadR
mutation was complemented in
trans
with the wild-type gene. Further characterization of the Δ
fadR
mutant showed that it was not generally hypersensitive to envelope stresses but had decreased motility and showed an altered membrane lipid profile compared to that of the wild type. Supplementation of broth with the unsaturated fatty acid oleate restored wild-type growth in vitro, and infection with oleate in the inoculum increased the ability of the Δ
fadR
mutant to infect mice. We conclude that
fadR
and regulation of fatty acid metabolism are essential for
V. vulnificus
to be able to cause disease in mammalian hosts.
The
Bacillus anthracis
SleL (YaaH) Protein Is an
N
-Acetylglucosaminidase Involved in Spore Cortex Depolymerization
Authors: Lambert Emily A. , Popham David L. 
Published: October 2008

ABSTRACT

Bacillus anthracis
spores, the infectious agents of anthrax, are notoriously difficult to remove from contaminated areas because they are resistant to many eradication methods. These resistance properties are due to the spore's dehydration and dormancy and to the multiple protective layers surrounding the spore core, one of which is the cortex. In order for
B. anthracis
spores to germinate and resume growth, the cortex peptidoglycan must be depolymerized. This study reports on analyses of
sleL
(
yaaH)
, which encodes a cortex-lytic enzyme. The inactivation of
sleL
does not affect vegetative growth, spore viability, or the initial stages of germination, including dipicolinic acid release. However, mutant spores exhibit a slight delay in the loss of optical density compared to that of wild-type spores. Mutants also retain more diaminopimelic acid and
N
-acetylmuramic acid during germination than wild-type spores, suggesting that the cortex peptidoglycan is not being hydrolyzed as rapidly. This finding is supported by high-pressure liquid chromatography analysis of the peptidoglycan structure used to confirm that SleL acts as an
N
-acetylglucosaminidase. When
sleL
is inactivated, the cortex peptidoglycan is not depolymerized into small muropeptides but instead is retained within the spore as large fragments. In the absence of the
sleL
-encoded
N
-acetylglucosaminidase, other cortex-lytic enzymes break down the cortex peptidoglycan sufficiently to allow rapid germination and outgrowth.
A Unique Combination of Genetic Systems for the Synthesis of Trehalose in
Rubrobacter xylanophilus
: Properties of a Rare Actinobacterial TreT
Authors: Nobre Ana , Alarico Susana , Fernandes Chantal , Empadinhas Nuno , da Costa Milton S. 
Published: October 2008

ABSTRACT

Trehalose is the primary organic solute in
Rubrobacter xylanophilus
under all conditions tested, including those for optimal growth. We detected genes of four different pathways for trehalose synthesis in the genome of this organism, namely, the trehalose-6-phosphate synthase (Tps)/trehalose-6-phosphate phosphatase (Tpp), TreS, TreY/TreZ, and TreT pathways. Moreover,
R. xylanophilus
is the only known member of the phylum
Actinobacteria
to harbor TreT. The Tps sequence is typically bacterial, but the Tpp sequence is closely related to eukaryotic counterparts. Both the Tps/Tpp and the TreT pathways were active in vivo, while the TreS and the TreY/TreZ pathways were not active under the growth conditions tested and appear not to contribute to the levels of trehalose observed. The genes from the active pathways were functionally expressed in
Escherichia coli
, and Tps was found to be highly specific for GDP-glucose, a rare feature among these enzymes. The trehalose-6-phosphate formed was specifically dephosphorylated to trehalose by Tpp. The recombinant TreT synthesized trehalose from different nucleoside diphosphate-glucose donors and glucose, but the activity in
R. xylanophilus
cell extracts was specific for ADP-glucose. The TreT could also catalyze trehalose hydrolysis in the presence of ADP, but with a very high

K
m

. Here, we functionally characterize two systems for the synthesis of trehalose in
R. xylanophilus
, a representative of an ancient lineage of the actinobacteria, and discuss a possible scenario for the exceptional occurrence of
treT
in this extremophilic bacterium.
HetF and PatA Control Levels of HetR in
Anabaena
sp. Strain PCC 7120
Authors: Risser Douglas D. , Callahan Sean M. 
Published: October 2008

ABSTRACT

Anabaena
sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates heterocysts in response to deprivation of combined nitrogen. A
hetF
deletion strain lacked heterocysts and had aberrant cell morphology. Site-directed mutagenesis of the predicted active-site histidine and cysteine residues of this putative caspase-hemoglobinase fold protease abolished HetF function, supporting the hypothesis that HetF is a protease. Deletion of
patA
, which is necessary for the formation of most intercalary heterocysts, or
hetF
resulted in an increase in HetR protein, and extra copies of
hetF
on a plasmid functionally bypassed the deletion of
patA
. A
hetR
-
gfp
translational fusion expressed from an inducible promoter demonstrated that
hetF
-dependent downregulation of HetR levels occurs rapidly in vegetative cells, as well as developing heterocysts. “Mosaic” filaments in which only one cell of a filament had a copy of
hetR
or
hetF
indicated that
hetF
is required for differentiation only in cells that will become heterocysts.
hetF
was required for transcription from a
hetR
-dependent transcription start point of the
hetR
promoter and induction of transcription from the
patS
promoter. The inverse correlation between the level of HetR protein and transcription from
hetR
-dependent promoters suggests that the transcriptional activity of HetR is regulated by HetF and PatA.
Direct Observation of
Staphylococcus aureus
Cell Wall Digestion by Lysostaphin
Authors: Francius Grégory , Domenech Oscar , Mingeot-Leclercq Marie Paule , Dufrêne Yves F. 
Published: October 2008

ABSTRACT

The advent of
Staphylococcus aureus
strains that are resistant to virtually all antibiotics has increased the need for new antistaphylococcal agents. An example of such a potential therapeutic is lysostaphin, an enzyme that specifically cleaves the
S. aureus
peptidoglycan, thereby lysing the bacteria. Here we tracked over time the structural and physical dynamics of single
S. aureus
cells exposed to lysostaphin, using atomic force microscopy. Topographic images of native cells revealed a smooth surface morphology decorated with concentric rings attributed to newly formed peptidoglycan. Time-lapse images collected following addition of lysostaphin revealed major structural changes in the form of cell swelling, splitting of the septum, and creation of nanoscale perforations. Notably, treatment of the cells with lysostaphin was also found to decrease the bacterial spring constant and the cell wall stiffness, demonstrating that structural changes were correlated with major differences in cell wall nanomechanical properties. We interpret these modifications as resulting from the digestion of peptidoglycan by lysostaphin, eventually leading to the formation of osmotically fragile cells. This study provides new insight into the lytic activity of lysostaphin and offers promising prospects for the study of new antistaphylococcal agents.
Regulation of Cell Growth during Serum Starvation and Bacterial Survival in Macrophages by the Bifunctional Enzyme SpoT in
Helicobacter pylori
Authors: Zhou Yan Ning , Coleman William G. , Yang Zhaoxu , Yang Yi , Hodgson Nathaniel , Chen Fuxiang , Jin Ding Jun 
Published: October 2008

ABSTRACT

In
Helicobacter pylori
the stringent response is mediated solely by
spoT
. The
spoT
gene is known to encode (p)ppGpp synthetase activity and is required for
H. pylori
survival in the stationary phase. However, neither the hydrolase activity of the
H. pylori
SpoT protein nor the role of SpoT in the regulation of growth during serum starvation and intracellular survival of
H. pylori
in macrophages has been determined. In this study, we examined the effects of SpoT on these factors. Our results showed that the
H. pylori spoT
gene encodes a bifunctional enzyme with both a hydrolase activity and the previously described (p)ppGpp synthetase activity, as determined by introducing the gene into
Escherichia coli relA
and
spoT
defective strains. Also, we found that SpoT mediates a serum starvation response, which not only restricts the growth but also maintains the helical morphology of
H. pylori.
Strikingly, a
spoT
null mutant was able to grow to a higher density in serum-free medium than the wild-type strain, mimicking the “relaxed” growth phenotype of an
E. coli relA
mutant during amino acid starvation. Finally, SpoT was found to be important for intracellular survival in macrophages during phagocytosis. The unique role of (p)ppGpp in cell growth during serum starvation, in the stress response, and in the persistence of
H. pylori
is discussed.
Genetic Dissection of the
Francisella novicida
Restriction Barrier
Authors: Gallagher Larry A. , McKevitt Matthew , Ramage Elizabeth R. , Manoil Colin 
Published: October 2008

ABSTRACT

Francisella tularensis
is the causative agent of tularemia and is a category A select agent.
Francisella novicida
, considered by some to be one of four subspecies of
F. tularensis
, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans.
F
.
novicida
exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 10
6
-fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the
F. novicida
genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the
F. tularensis
subspecies are pseudogenes, explaining the unusually strong restriction barrier in
F. novicida
and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into
F. novicida
efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes.