bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Structure and Aggressiveness of
Erysiphe necator
Populations during Grapevine Powdery Mildew Epidemics
Authors: Montarry Josselin , Cartolaro Philippe , Delmotte François , Jolivet Jérôme , Willocquet Laetitia
Published: August 2008

Isolates of the causal ascomycete of grapevine powdery mildew,
Erysiphe necator
, correspond to two genetically differentiated groups (A and B) that coexist on the same host. This coexistence was analyzed by investigating temporal changes in the genetic and phenotypic structures of
E. necator
populations during three epidemics. Group A was present only at the start of the growing season, whereas group B was present throughout all three epidemics. Group A was less aggressive in terms of germination and infection efficiency but was more aggressive than group B in terms of the latency period, lesion diameter, and spore production. Our results are consistent with a temporal differentiation of niches, preventing recombination, and suggest an association between the disease level and the frequencies of genetic groups.
Genetic Tools for Studying
Capnocytophaga canimorsus
Authors: Mally Manuela , Cornelis Guy R.
Published: August 2008

Capnocytophaga canimorsus
, a commensal bacterium from canine oral flora, has been isolated throughout the world from severe human infections caused by dog bites. Due to the low level of evolutionary relationship to
Proteobacteria
, genetic methods suitable for the genus
Capnocytophaga
needed to be established. Here, we show that Tn
4351
, derived from
Bacteroides fragilis
, could be introduced by conjugation into
C. canimorsus
and conferred resistance to erythromycin. By mapping and sequencing a naturally occurring plasmid isolated from a clinical isolate of
C. canimorsus
, we identified a
repA
gene that allowed us to construct
Escherichia coli-Capnocytophaga
shuttle vectors. Most commonly used antibiotic markers were not functional in
C. canimorsus
, but cefoxitin (
cfxA
), tetracycline (
tetQ
), and erythromycin (
ermF
) resistances could be used as markers for plasmid maintenance in
C. canimorsus
and even in some other
Capnocytophaga
spp. Shuttle vectors were introduced into
C. canimorsus
either by conjugation using the origin of transfer (
oriT
) of RP4 or by electrotransformation. Taking advantage of the promoter of
ermF
, an expression vector was constructed. Finally, a method that allows site-directed mutagenesis is described. All these genetic tools pave the way, not only for molecular studies of the pathogenesis of
C. canimorsus
, but also for studies of other oral
Capnocytophaga
species.
The Deep-Sea Bacterium
Photobacterium profundum
SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions
Authors: Eloe Emiley A. , Lauro Federico M. , Vogel Rudi F. , Bartlett Douglas H.
Published: August 2008

Motility is a critical function needed for nutrient acquisition, biofilm formation, and the avoidance of harmful chemicals and predators. Flagellar motility is one of the most pressure-sensitive cellular processes in mesophilic bacteria; therefore, it is ecologically relevant to determine how deep-sea microbes have adapted their motility systems for functionality at depth. In this study, the motility of the deep-sea piezophilic bacterium
Photobacterium profundum
SS9 was investigated and compared with that of the related shallow-water piezosensitive strain
Photobacterium profundum
3TCK, as well as that of the well-studied piezosensitive bacterium
Escherichia coli
. The SS9 genome contains two flagellar gene clusters: a polar flagellum gene cluster (PF) and a putative lateral flagellum gene cluster (LF). In-frame deletions were constructed in the two flagellin genes located within the PF cluster (
flaA
and
flaC
), the one flagellin gene located within the LF cluster (
flaB
), a component of a putative sodium-driven flagellar motor (
motA2
), and a component of a putative proton-driven flagellar motor (
motA1
). SS9 PF
flaA
,
flaC
, and
motA2
mutants were defective in motility under all conditions tested. In contrast, the
flaB
and
motA1
mutants were defective only under conditions of high pressure and high viscosity.
flaB
and
motA1
gene expression was strongly induced by elevated pressure plus increased viscosity. Direct swimming velocity measurements were obtained using a high-pressure microscopic chamber, where increases in pressure resulted in a striking decrease in swimming velocity for
E. coli
and a gradual reduction for 3TCK which proceeded up to 120 MPa, while SS9 increased swimming velocity at 30 MPa and maintained motility up to a maximum pressure of 150 MPa. Our results indicate that
P. profundum
SS9 possesses two distinct flagellar systems, both of which have acquired dramatic adaptations for optimal functionality under high-pressure conditions.
Bacteriophages Reduce Experimental Contamination of Hard Surfaces, Tomato, Spinach, Broccoli, and Ground Beef by
Escherichia coli
O157:H7
Authors: Abuladze Tamar , Li Manrong , Menetrez Marc Y. , Dean Timothy , Senecal Andre , Sulakvelidze Alexander
Published: August 2008

A bacteriophage cocktail (designated ECP-100) containing three
Myoviridae
phages lytic for
Escherichia coli
O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three
E. coli
O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable
E. coli
organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (10
10
, 10
9
, and 10
8
PFU/ml) resulted in statistically significant reductions (
P =
<0.05) of 99.99%, 98%, and 94%, respectively, in the number of
E. coli
O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of
E. coli
O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 10
9
PFU/ml) also significantly reduced the number of viable
E. coli
O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 ± 4 h posttreatment of tomato samples) to 100% (at 24 ± 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by
E. coli
O157:H7.
TOR1 and TOR2 Have Distinct Locations in Live Cells
Authors: Sturgill Thomas W. , Cohen Adiel , Diefenbacher Melanie , Trautwein Mark , Martin Dietmar E. , Hall Michael N.
Published: August 2008

TOR is a structurally and functionally conserved Ser/Thr kinase found in two multiprotein complexes that regulate many cellular processes to control cell growth. Although extensively studied, the localization of TOR is still ambiguous, possibly because endogenous TOR in live cells has not been examined. Here, we examined the localization of green fluorescent protein (GFP) tagged, endogenous TOR1 and TOR2 in live
S. cerevisiae
cells. A DNA cassette encoding three copies of green fluorescent protein (3XGFP) was inserted in the
TOR1
gene (at codon D330) or the
TOR2
gene (at codon N321). The TORs were tagged internally because TOR1 or TOR2 tagged at the N or C terminus was not functional. The
TOR1
D330-3XGFP
strain was not hypersensitive to rapamycin, was not cold sensitive, and was not resistant to manganese toxicity caused by the loss of Pmr1, all indications that TOR1-3XGFP was expressed and functional. TOR2-3XGFP was functional, as
TOR2
is an essential gene and
TOR2
N321-3XGFP
haploid cells were viable. Thus, TOR1 and TOR2 retain function after the insertion of 748 amino acids in a variable region of their noncatalytic domain. The localization patterns of TOR1-3XGFP and TOR2-3XGFP were documented by imaging of live cells. TOR1-3XGFP was diffusely cytoplasmic and concentrated near the vacuolar membrane. The TOR2-3XGFP signal was cytoplasmic but predominately in dots at the plasma membrane. Thus, TOR1 and TOR2 have distinct localization patterns, consistent with the regulation of cellular processes as part of two different complexes.
Copper Distributed by Atx1 Is Available to Copper Amine Oxidase 1 in
Schizosaccharomyces pombe
Authors: Peter Chardeen , Laliberté Julie , Beaudoin Jude , Labbé Simon
Published: August 2008

Copper amine oxidases (CAOs) have been proposed to be involved in the metabolism of xenobiotic and biogenic amines. The requirement for copper is absolute for their activity. In the fission yeast
Schizosaccharomyces pombe
,
cao1

+

and
cao2

+

genes are predicted to encode members of the CAO family. While both genes are expressed in wild-type cells, we determined that the expression of only
cao1

+

but not
cao2

+

results in the production of an active enzyme. Site-directed mutagenesis identified three histidine residues within the C-terminal region of Cao1 that are necessary for amine oxidase activity. By use of a
cao1

+

-
GFP
allele that retained wild-type function, Cao1-GFP was localized in the cytosol (GFP is green fluorescent protein). Under copper-limiting conditions, disruption of
ctr4

+

,
ctr5

+

, and
cuf1

+

produced a defect in amine oxidase activity, indicating that a functionally active Cao1 requires Ctr4/5-mediated copper transport and the transcription factor Cuf1. Likewise,
atx1
null cells exhibited substantially decreased levels of amine oxidase activity. In contrast, deletion of
ccc2
,
cox17
, and
pccs
had no significant effect on Cao1 activity. Residual amine oxidase activity in cells lacking
atx1

+

can be restored to normal levels by returning an
atx1

+

allele, underscoring the critical importance of the presence of Atx1 in cells. Using two-hybrid analysis, we demonstrated that Cao1 physically interacts with Atx1 and that this association is comparable to that of Atx1 with the N-terminal region of Ccc2. Collectively, these results describe the first example of the ability of Atx1 to act as a copper carrier for a molecule other than Ccc2 and its critical role in delivering copper to Cao1.
Trade-Off between Iron Uptake and Protection against Oxidative Stress: Deletion ofcueOPromotes UropathogenicEscherichia coliVirulence in a Mouse Model of Urinary Tract Infection
Authors: Tree Jai J. , Ulett Glen C. , Ong Cheryl-Lynn Y. , Trott Darren J. , McEwan Alastair G. , Schembri Mark A.
Published: August 2008

The periplasmic multicopper oxidase (CueO) is involved in copper homeostasis and protection against oxidative stress. Here, we show that the deletion ofcueOin uropathogenicEscherichia coliincreases its colonization of the urinary tract despite its increased sensitivity to hydrogen peroxide. ThecueOdeletion mutant accumulated iron with increased efficiency compared to its parent strain; this may account for its advantage in the iron-limited environment of the urinary tract.
ESAT-6-Like Protein Secretion inBacillus anthracis
Authors: Garufi Gabriella , Butler Emily , Missiakas Dominique
Published: August 2008

Proteins of the WXG100 family represent the prototypical substrates of bacterial type VII secretion systems that typically encompass 100 residues, lack canonical signal peptides, and form helix-turn-helix hairpin structures with WXG positioned in the turn element.Bacillus anthracisencodes six WXG100 proteins, herein referred to as EsxB, EsxL, EsxP, EsxQ, EsxV, and EsxW. With the exception of EsxB,B. anthracisproteins harbor C-terminal extensions that are appended to canonical WXG domains. When cultured in liquid broth,B. anthracissecretes two substrates, EsxB and EsxW, into the extracellular environment. EsxB is required for the stability and secretion of EsxW; however, EsxW is dispensable for EsxB secretion. In agreement with the hypothesis that EsxB binding to substrates promotes recognition and secretion by the type VII pathway, EsxB is reported to interact with EsxB and EsxW. Unlike deletions in mycobacterial EsxB, deletion of five N- or C-terminal residues does not affect the ability of mutantB. anthracisEsxB to travel the type VII pathway and initiate secretion of EsxW. Translational fusion of ubiquitin to the N or C terminus of EsxB also had no effect, while ubiquitin insertion into the center turn abrogated secretion. Anthrax-infected guinea pigs mounted humoral immune responses to EsxB, EsxP, and EsxW, which suggests thatB. anthracisactivates the type VII secretion pathway during infection.
Suppressor Analysis of the MotB(D33E) Mutation To Probe Bacterial Flagellar Motor Dynamics Coupled with Proton Translocation
Authors: Che Yong-Suk , Nakamura Shuichi , Kojima Seiji , Kami-ike Nobunori , Namba Keiichi , Minamino Tohru
Published: August 2008

MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of
Salmonella enterica
serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile
motB
(
D33E
) mutant and its pseudorevertants. We first confirmed that the poor motility of the
motB
(
D33E
) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the
motB
(
D33E
) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/deprotonation of Glu-33 but not maximum proton conductivity.
Reexamination of the Role of the Amino Terminus of SecA in Promoting Its Dimerization and Functional State
Authors: Das Sanchaita , Stivison Elizabeth , Folta-Stogniew Ewa , Oliver Donald
Published: August 2008

The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a monomer or dimer during this dynamic process. Here we reexamined the roles of the amino and carboxyl termini of SecA in promoting its dimerization and functional state by examining three
secA
mutants and the corresponding proteins: SecAΔ8 lacking residues 2 to 8, SecAΔ11 lacking residues 2 to 11, and SecAΔ11/N95 lacking both residues 2 to 11 and the carboxyl-terminal 70 residues. We demonstrated that whether SecAΔ11 or SecAΔ11/N95 was functional for promoting cell growth depended solely on the vivo level of the protein, which appeared to govern residual dimerization. All three SecA mutant proteins were defective for promoting cell growth unless they were highly overproduced. Cell fractionation revealed that SecAΔ11 and SecAΔ11/N95 were proficient in membrane association, although the formation of integral membrane SecA was reduced. The presence of a modestly higher level of SecAΔ11/N95 in the membrane and the ability of this protein to form dimers, as detected by chemical cross-linking, were consistent with the higher level of
secA
expression and better growth of the SecAΔ11/N95 mutant than of the SecAΔ11 mutant. Biochemical studies showed that SecAΔ11 and SecAΔ11/N95 had identical dimerization defects, while SecAΔ8 was intermediate between these proteins and wild-type SecA in terms of dimer formation. Furthermore, both SecAΔ11 and SecAΔ11/N95 were equally defective in translocation ATPase specific activity. Our studies showed that the nonessential carboxyl-terminal 70 residues of SecA play no role in its dimerization, while increasing the truncation of the amino-terminal region of SecA from 8 to 11 residues results in increased defects in SecA dimerization and poor in vivo function unless the protein is highly overexpressed. They also clarified a number of conflicting previous reports and support the essential nature of the SecA dimer.
NorB, an Efflux Pump in
Staphylococcus aureus
Strain MW2, Contributes to Bacterial Fitness in Abscesses
Authors: Ding Yanpeng , Onodera Yoshikuni , Lee Jean C. , Hooper David C.
Published: August 2008

While remaining a major problem in hospitals,
Staphylococcus aureus
is now spreading in communities. Strain MW2 (USA400 lineage) and other community methicillin-resistant
S. aureus
strains most commonly cause skin infections with abscess formation. Multidrug resistance (MDR) efflux pumps contribute to antimicrobial resistance but may also contribute to bacterial survival by removal of environmental toxins. In
S. aureus
, NorA, NorB, NorC, and Tet38 are chromosomally encoded efflux pumps whose overexpression can confer MDR to quinolones and other compounds (Nor pumps) or tetracyclines alone (Tet38), but the natural substrates of these pumps are not known. To determine the role of these efflux pumps in a natural environment in the absence of antibiotics, we used strain MW2 in a mouse subcutaneous abscess model and compared pump gene expression as determined by reverse transcription-PCR in the abscesses and in vitro.
norB
and
tet38
were selectively upregulated in vivo more than 171- and 24-fold, respectively, whereas
norA
and
norC
were downregulated. These changes were associated with an increase in expression of
mgrA
, which encodes a transcriptional regulator known to affect pump gene expression. In competition experiments using equal inocula of a
norB
or
tet38
mutant and parent strain MW2, each mutant exhibited growth defects of about two- to threefold in vivo. In complementation experiments, a single-copy insertion of
norB
(but not a single-copy insertion of
tet38
) in the
attB
site within
geh
restored the growth fitness of the
norB
mutant in vivo. Our findings indicate that some MDR pumps, like NorB, can facilitate bacterial survival when they are overexpressed in a staphylococcal abscess and may contribute to the relative resistance of abscesses to antimicrobial therapy, thus linking bacterial fitness and resistance in vivo.
Purification and Characterization of the Bacterial UDP-GlcNAc:Undecaprenyl-Phosphate GlcNAc-1-Phosphate Transferase WecA
Authors: Al-Dabbagh Bayan , Mengin-Lecreulx Dominique , Bouhss Ahmed
Published: August 2008

To date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate
N
-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. In the present work and for the first time, the integral membrane protein WecA that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl-GlcNAc, the lipid intermediate involved in the synthesis of various bacterial cell envelope components, was overproduced and purified to near homogeneity in milligram quantities. An enzymatic assay was developed, and the kinetic parameters of WecA as well as the effects of pH, salts, cations, detergents, and temperature on the enzyme activity were determined. A minimal length of 35 carbons was required for the lipid substrate, and tunicamycin was shown to inhibit the enzyme at submicromolar concentrations.
SpoT Regulates DnaA Stability and Initiation of DNA Replication in Carbon-StarvedCaulobacter crescentus
Authors: Lesley Joseph A. , Shapiro Lucy
Published: August 2008

Cell cycle progression and polar differentiation are temporally coordinated inCaulobacter crescentus. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. We analyzed the adaptive response ofC. crescentusswarmer cells to carbon starvation and found that there was a block in both the swarmer-to-stalked cell polar differentiation program and the initiation of DNA replication. SpoT is a bifunctional synthase/hydrolase that controls the steady-state level of the stress-signaling nucleotide (p)ppGpp, and carbon starvation caused a SpoT-dependent increase in (p)ppGpp concentration. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. Microbiol.55:1233-1245, 2005). We observed that SpoT is required for this phenomenon in swarmer cells, and in the absence of SpoT, carbon-starved swarmer cells inappropriately initiated DNA replication. Since SpoT controls (p)ppGpp abundance, we propose that this nucleotide relays carbon starvation signals to the cellular factors responsible for activating DnaA proteolysis, thereby inhibiting the initiation of DNA replication. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. Thus, swarmer cells utilize at least two independent signaling pathways to relay carbon starvation signals: a SpoT-dependent pathway mediating the inhibition of DNA replication initiation, and a SpoT-independent pathway(s) that blocks morphological differentiation.
Genome Sequence of the Fish Pathogen
Renibacterium salmoninarum
Suggests Reductive Evolution away from an Environmental
Arthrobacter
Ancestor
Authors: Wiens Gregory D. , Rockey Daniel D. , Wu Zaining , Chang Jean , Levy Ruth , Crane Samuel , Chen Donald S. , Capri Gina R. , Burnett Jeffrey R. , Sudheesh Ponnerassery S. , Schipma Matthew J. , Burd Henry , Bhattacharyya Anamitra , Rhodes Linda D. , Kaul Rajinder , Strom Mark S.
Published: August 2008

Renibacterium salmoninarum
is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of
R. salmoninarum
ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The
R. salmoninarum
genome has extended regions of synteny to the
Arthrobacter
sp. strain FB24 and
Arthrobacter aurescens
TC1 genomes, but it is approximately 1.9 Mb smaller than both
Arthrobacter
genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding β-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into
R. salmoninarum
evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.
The Origins of 168, W23, and Other
Bacillus subtilis
Legacy Strains
Authors: Zeigler Daniel R. , Prágai Zoltán , Rodriguez Sabrina , Chevreux Bastien , Muffler Andrea , Albert Thomas , Bai Renyuan , Wyss Markus , Perkins John B.
Published: August 2008

Bacillus subtilis
is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of
B. subtilis
genetics. For three—NCIB 3610
T
, PY79, and SMY—we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more “W23 islands” in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its
trp
and
sac
loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these
B. subtilis
legacy strains.
Stygiolobus
Rod-Shaped Virus and the Interplay of Crenarchaeal Rudiviruses with the CRISPR Antiviral System
Authors: Vestergaard Gisle , Shah Shiraz A. , Bize Ariane , Reitberger Werner , Reuter Monika , Phan Hien , Briegel Ariane , Rachel Reinhard , Garrett Roger A. , Prangishvili David
Published: August 2008

A newly characterized archaeal rudivirus
Stygiolobus
rod-shaped virus (SRV), which infects a hyperthermophilic
Stygiolobus
species, was isolated from a hot spring in the Azores, Portugal. Its virions are rod-shaped, 702 (± 50) by 22 (± 3) nm in size, and nonenveloped and carry three tail fibers at each terminus. The linear double-stranded DNA genome contains 28,096 bp and an inverted terminal repeat of 1,030 bp. The SRV shows morphological and genomic similarities to the other characterized rudiviruses
Sulfolobus
rod-shaped virus 1 (SIRV1), SIRV2, and
Acidianus
rod-shaped virus 1, isolated from hot acidic springs of Iceland and Italy. The single major rudiviral structural protein is shown to generate long tubular structures in vitro of similar dimensions to those of the virion, and we estimate that the virion constitutes a single, superhelical, double-stranded DNA embedded into such a protein structure. Three additional minor conserved structural proteins are also identified. Ubiquitous rudiviral proteins with assigned functions include glycosyl transferases and a
S
-adenosylmethionine-dependent methyltransferase, as well as a Holliday junction resolvase, a transcriptionally coupled helicase and nuclease implicated in DNA replication. Analysis of matches between known crenarchaeal chromosomal CRISPR spacer sequences, implicated in a viral defense system, and rudiviral genomes revealed that about 10% of the 3,042 unique acidothermophile spacers yield significant matches to rudiviral genomes, with a bias to highly conserved protein genes, consistent with the widespread presence of rudiviruses in hot acidophilic environments. We propose that the 12-bp indels which are commonly found in conserved rudiviral protein genes may be generated as a reaction to the presence of the host CRISPR defense system.
The Carboxyl-Terminal Domain of TraR, a
Streptomyces
HutC Family Repressor, Functions in Oligomerization
Authors: Kataoka Masakazu , Tanaka Takeshi , Kohno Toshiyuki , Kajiyama Yusuke
Published: August 2008

Efficient conjugative transfer of the
Streptomyces
plasmid pSN22 is accomplished by regulated expression of the
tra
operon genes,
traA, traB
, and
spdB
. The TraR protein is the central transcriptional repressor regulating the expression of the
tra
operon and itself and is classified as a member of the HutC subfamily in the helix-turn-helix (HTH) GntR protein family. Sequence information predicts that the N-terminal domain (NTD) of TraR, containing an HTH motif, functions in binding of DNA to the
cis
element; however, the function of the C-terminal region remains obscure, like that for many other GntR family proteins. Here we demonstrate the domain structure of the TraR protein and explain the role of the C-terminal domain (CTD). The TraR protein can be divided into two structural domains, the NTD of M1 to R95 and the CTD of Y96 to E246, revealed by limited proteolysis. Domain expression experiments revealed that both domains retained their function. An in vitro pull-down assay using recombinant TraR proteins revealed that TraR oligomerization depended on the CTD. A bacterial two-hybrid system interaction assay revealed that the minimum region necessary for this binding is R95 to P151. A mutant TraR protein in which Leu121 was replaced by His exhibited a loss of both oligomerization ability and repressor function. An in vitro cross-linking assay revealed preferential tetramer formation by TraR and the minimum CTD. These results indicate that the C-terminal R95-to-P151 region of TraR functions to form an oligomer, preferentially a tetramer, that is essential for the repressor function of TraR.
Use of CDP-Glycerol as an Alternate Acceptor for the Teichoic Acid Polymerase Reveals that Membrane Association Regulates Polymer Length
Authors: Schertzer Jeffrey W. , Brown Eric D.
Published: August 2008

The study of bacterial extracellular polysaccharide biosynthesis is hampered by the fact that these molecules are synthesized on membrane-resident carrier lipids. To get around this problem, a practical solution has been to synthesize soluble lipid analogs and study the biosynthetic enzymes using a soluble system. This has been done for the
Bacillus subtilis
teichoic acid polymerase, TagF, although several aspects of catalysis were inconsistent with the results obtained with reconstituted membrane systems or physiological observations. In this work we explored the acceptor substrate promiscuity and polymer length disregulation that appear to be characteristic of TagF activity away from biological membranes. Using isotope labeling, steady-state kinetics, and chemical lability studies, we demonstrated that the enzyme can synthesize poly(glycerol phosphate) teichoic acid using the elongation substrate CDP-glycerol as an acceptor. This suggests that substrate specificity is relaxed in the region distal to the glycerol phosphate moiety in the acceptor molecule under these conditions. Polymer synthesis proceeded at a rate (27 min
−1
) comparable to that in the reconstituted membrane system after a distinct lag period which likely represented slower initiation on the unnatural CDP-glycerol acceptor. We confirmed that polymer length became disregulated in the soluble system as the polymers synthesized on CDP-glycerol acceptors were much larger than the polymers synthesized on the membrane or previously found attached to bacterial cell walls. Finally, polymer synthesis on protease-treated membranes suggested that proper length regulation is retained in the absence of accessory proteins and provided evidence that such regulation is conferred through proper association of the polymerase with the membrane.
Functional Definition and Global Regulation of Zur, a Zinc Uptake Regulator in a
Streptococcus suis
Serotype 2 Strain Causing Streptococcal Toxic Shock Syndrome
Authors: Feng Youjun , Li Ming , Zhang Huimin , Zheng Beiwen , Han Huiming , Wang Changjun , Yan Jinghua , Tang Jiaqi , Gao George F.
Published: August 2008

Zinc is an essential trace element for all living organisms and plays pivotal roles in various cellular processes. However, an excess of zinc is extremely deleterious to cells. Bacteria have evolved complex machineries (such as efflux/influx systems) to control the concentration at levels appropriate for the maintenance of zinc homeostasis in cells and adaptation to the environment. The Zur (zinc uptake regulator) protein is one of these functional members involved in the precise control of zinc homeostasis. Here we identified a
zur
homologue designated 310 from
Streptococcus suis
serotype 2, strain 05ZYH33, a highly invasive isolate causing streptococcal toxic shock syndrome. Biochemical analysis revealed that the protein product of gene 310 exists as a dimer form and carries zinc ions. An isogenic gene replacement mutant of gene 310, the Δ310 mutant, was obtained by homologous recombination. Physiological tests demonstrated that the Δ310 mutant is specifically sensitive to Zn
2+
, while functional complementation of the Δ310 mutant can restore its duration capability, suggesting that 310 is a functional member of the Zur family. Two-dimensional electrophoresis indicated that nine proteins in the Δ310 mutant are overexpressed in comparison with those in the wild type. DNA microarray analyses suggested that 121 genes in the Δ310 mutant are affected, of which 72 genes are upregulated and 49 are downregulated. The transcriptome of
S. suis
serotype 2 with high Zn
2+
concentrations also showed 117 differentially expressed genes, with 71 upregulated and 46 downregulated. Surprisingly, more than 70% of the genes differentially expressed in the Δ310 mutant were the same as those in
S. suis
serotype 2 that were differentially expressed in response to high Zn
2+
concentration, consistent with the notion that 310 is involved in zinc homeostasis. We thus report for the first time a novel zinc-responsive regulator, Zur, from
Streptococcus suis
serotype 2.
TheACTN3R577X polymorphism in Russian endurance athletes
Authors: Ahmetov I I , Druzhevskaya A M , Astratenkova I V , Popov D V , Vinogradova O L , Rogozkin V A
Published: August 2008

Objective

The functional 577R allele of the α-actinin-3 (ACTN3) gene has been reported to be associated with elite power athlete status, while the nonfunctional 577XX genotype (predicts an α-actinin-3 deficient phenotype) has been hypothesised as providing some sort of advantage for endurance athletes. In the present study, the distribution ofACTN3genotypes and alleles in Russian endurance-oriented athletes were examined and association betweenACTN3genotypes and the competition results of rowers were sought.

Methods

456 Russian endurance-oriented athletes of regional or national competitive standard were involved in the study.ACTN3genotype and allele frequencies were compared with 1211 controls. The data from the Russian Cup Rowing Tournament were used to search for possible association between theACTN3genotype and the long-distance (∼6 km) rowing results of 54 athletes. DNA was extracted from mouthwash samples. Genotyping for the R577X variant was performed by PCR and restriction enzyme digestion.

Results

The frequencies of theACTN3577XX genotype (5.7% vs 14.5%; p<0.0001) and 577X allele (33.2% vs 39.0%; p = 0.0025) were significantly lower in endurance-oriented athletes compared with the controls, and none of the highly elite athletes had the 577XX genotype. Furthermore, male rowers withACTN3577RR genotype showed better results (1339 (11) s) in long-distance rowing than carriers of 577RX (1386 (12) s) or 577XX (1402 (10) s) genotypes (p = 0.016).

Conclusion

Our data show that theACTN3577X allele is under-represented in Russian endurance athletes and is associated with the rowers’ competition results.
How do I measure physical activity in my patients? Questionnaires and objective methods
Authors: Ainsworth B E
Published: August 2008

How do healthcare providers know if their patients are getting enough physical activity to promote good health and to reduce their risks of chronic diseases and injury? The first step is to identify the patient’s current level of physical activity using questionnaires and/or motion sensors. Questionnaires assess activity levels by having patients answer a set of questions about the types and amounts of activity performed at some time in the past. Motion sensors assess physical activity by patients wearing a small monitoring device that records their body movement as it occurs. If a provider is interested in determining a patient’s caloric energy expenditure, he/she can apply statistical regression models to the questionnaire and motion sensor data to estimate kilocalories. If more precise measures of energy expenditure are desired, a provider can use the isotopic doubly labelled water method to estimate kilocalories; however, this method is costly and is impractical in non-research clinical settings.
Does tobacco industry marketing excessively impact lesbian, gay and bisexual communities?
Authors: Dilley J A , Spigner C , Boysun M J , Dent C W , Pizacani B A
Published: August 2008

Background:

Tobacco industry documents have revealed marketing plans specifically to reach lesbian, gay and bisexual (LGB) populations. Research supports a causal linkage between receptivity and exposure to tobacco industry marketing and tobacco use uptake among adolescents. Pro-tobacco messages may diminish the effectiveness of tobacco control activities and contribute to the high smoking prevalence among LGB populations.

Objective:

To compare receptivity and exposure to tobacco industry marketing between LGB and heterosexual populations.

Methods:

Nearly 400 gay or bisexual men and more than 600 lesbian or bisexual women were identified in the 2003–2006 Washington State Behavioral Risk Factor Surveillance System (BRFSS), a state-wide, population-based telephone survey of adults. The BRFSS included questions measuring receptivity and exposure to tobacco industry marketing. Multiple logistic regression models stratified by gender were used to assess differences for lesbians, gays and bisexuals separately, in comparison to their heterosexual counterparts.

Results:

As expected, smoking prevalence was higher among LGB populations than among heterosexuals. After adjustment for demographic differences and smoking status, gay and bisexual men reported more exposure to tobacco industry marketing (free sample distribution) than straight men, but were equally receptive to it. Lesbian and bisexual women were more receptive to and reported more exposure to tobacco industry marketing than straight women.

Conclusion:

LGB communities, especially lesbian and bisexual women, appear to be effectively targeted by tobacco industry marketing activities. Strategies to limit tobacco industry marketing, and increase individuals’ resistance to marketing, may be critical to reducing smoking among LGB populations.
PRODUCTION OF CYTASES ACTIVE ON BARLEY GUM BY BACTERIA OF THE GENUS BACILLUS
Authors: Blackwood A. C.
Published: August 2008

One hundred and fourteen bacterial cultures representing most of the species in the Bacillus genus were tested for the production of extracellular barley gum cytase. Assays were made on shake-flask cultures grown on a medium containing glucose and yeast extract. Although all the organisms had some enzymatic activity, certain strains of Bacillus subtilis gave the best yields of cytase. On a medium with asparagine as the sole nitrogen source even higher yields were obtained. The crude cytase preparations were stable and after freeze-drying most of the original activity remained.
FURTHER OBSERVATIONS ON THE REDUCTION OF FUSIFORM CAMBIAL CELLS IN THUJA OCCIDENTALIS L.
Authors: Bannan M. W.
Published: August 2008

The multiplication of fusiform initials in the cambium is accompanied by extensive loss or transformation of these cells. A few of the failing cambial cells lapse into maturation quickly, but the majority are transversely subdivided with varying proportions of the segments surviving and undergoing ultimate conversion to ray initials. The loss or conversion is attended with reduction in cell size. Increase in cell volume lags behind cell division during the periclinal divisions of the transitional period. The tangential dimensions of the successively formed cells are continuously reduced, and sometimes radial expansion is also retarded, especially toward the cell tips. Simultaneous shortening of the cells is due to alteration in cell shape combined with asymmetry in periclinal division such that daughter cells of unequal lengths are produced. Repetition of the process, the smaller cell functioning as the initiating cambial cell in each instance, results in continued shortening.
PHYSIOLOGICAL AND BIOCHEMICAL STUDIES IN PLANT METABOLISM: VII. THE INVERTASE OF THE SEEDLING WHEAT LEAF
Authors: Ward G. M.
Published: August 2008

The press juice from mature seedling wheat leaves possesses a high thermo-labile activity in the inversion of sucrose. It is concluded that an invertase is present. The active material is not associated with the coarse particles of the protoplasm but is soluble in the sap and in that condition is very stable in the cold. It is, however, sensitive to drying and precipitation. Some kinetic characteristics of this enzyme resemble those of invertases from other sources. But the Michaelis constant is small by comparison with that of yeast invertase and therefore a relatively high affinity of the leaf enzyme for its substrate is indicated.
STUDIES ON MICROORGANISMS IN ARCTIC AIR DURING 1949 AND 1950
Authors: Pady S. M. , Kelly C. D.
Published: August 2008

The numbers of bacteria and fungi in arctic air were determined by daily sampling at Churchill, Man., during July and August 1950, and in three flights, one to Baker Lake, N.W.T., and the remainder to Resolute Bay, N.W.T. Of the three samplers which were used simultaneously the G.E. Bacterial Air Sampler gave slightly higher readings than the Bourdillon Slit Sampler, while the filter gave low readings throughout.Daily averages of bacteria at ground level ranged from 0.9 to 30.1 per cu. ft., and in the flight to Resolute Bay from 0.3 to 0.9 per cu. ft. while the comparable readings of the fungi were 0.5 to 4.4 and 0.01 to 0.7 per cu. ft. with the slit sampler. Considerable variation occurred in ground level samples, not only from day to day but throughout the day. Silicone slide studies revealed high numbers, up to 115 per cu. ft., which includes a high proportion of nonviable fungus spores. Most of the organisms are soil inhabiting forms but some fungus parasites were present, chiefly as smut (Ustilago) spores. Evidence indicates that winds originating in the south carry large numbers of organisms northward, many of which are nonviable when they reach the arctic, while north winds of polar origin contain very low numbers even in the summer.
STUDIES IN FOREST PATHOLOGY: X. DECAY OF WHITE PINE IN THE TIMAGAMI LAKE AND OTTAWA VALLEY AREAS
Authors: White L. T.
Published: August 2008

The study of decay and decay relationships of white pine was undertaken to provide forest authorities in Ontario with information essential to the management of even-aged stands. Of 1012 white pine trees examined in 10 1-acre plots, located in even-aged white pine stands, 52% contained decay. A well-defined relationship existed between age and decay. The proportion of trees with decay increased gradually with age from 40% in the 60-year age class to 100% in the 220-year age class. The loss in merchantable volume increased from 4% at 60 years to 40% at 200 years. No consistent relationship between percentage of decay and diameter was found. The incidence of decay in vigorous and non-vigorous trees was about the same but the percentage of volume lost through decay was considerably higher in slow-growing, less vigorous trees. The pathological rotation, as indicated by the maximum periodic increment, was 160 to 170 years. A number of methods of estimating the volume of decay in logs are compared. Regression lines, which define the percentage of cull allowances at all ages, are presented. Thirteen wood-destroying fungi were found to be associated with the decay of living white pine in Ontario. Fomes pini (Thore) Lloyd was responsible for 90% of all loss in trunks and butts of living trees. External signs of decay were rare, except in cases of advanced white pocket rot caused by F. pini.
NITROGEN AND VITAMIN REQUIREMENTS OF PSEUDOMONAS HYDROPHILA
Authors: Tamboline Florence R.
Published: August 2008

Yeast extract and vitamin-free casamino acids were found to be equivalent as sources of nitrogen for the growth of Pseudomonas hydrophila in a glucose – mineral salts medium. The addition of a mixture of thiamine, riboflavin, pantothenic acid, pyridoxine, nicotinic acid, para-aminobenzoic acid, biotin, and folic acid to the medium containing vitamin-free casamino acids did not stimulate growth. About 67% as much growth was obtained with a mixture of 20 amino acids and asparagine as with the vitamin-free casamino acids and the mixture could be replaced by any one of asparagine, aspartic acid, serine, and alanine. Of the 11 simple nitrogen compounds tested, including urea, nitrates, and ammonium salts, only ammonium citrate and dibasic ammonium phosphate were utilized appreciably. A simple synthetic medium consisting of ammonium citrate, glucose, and mineral salts was found to give approximately the same amount of growth as the more complex yeast extract – glucose – mineral salts medium.
FACTORS INFLUENCING THE GROWTH AND SURVIVAL OF RHIZOBIA IN HUMUS AND SOIL CULTURES.II
Authors: Spencer J. F. T. , Newton J. D.
Published: August 2008

A study was made of several factors influencing the growth and survival of rhizobia in humus and soil cultures. Screw-capped glass jars were more satisfactory than the foil-lined cardboard cartons used commercially for packaging of seed inoculation cultures because contaminants were excluded and moisture retained more effectively. Refrigeration of the cultures reduced water losses and proved to be an excellent method of maintaining large numbers of living bacteria for a long time. The addition of sucrose to the humus cultures ordinarily produced large increases in numbers of rhizobia, as determined by plate counts, and the addition of various inorganic nutrients generally produced smaller increases. In general, any particular element stimulated growth only if the level of that element in the soil used as a base was below optimum. Nitrate and sucrose stimulated growth when either was used alone, but together they greatly depressed growth.
NEW INFORMATION CONCERNING BALSAM FIR DECAYS IN EASTERN NORTH AMERICA
Authors: Basham J. T. , Mook P. V. , Davidson A. G.
Published: August 2008

Four separate investigations of decay in balsam fir (Abies balsamea (L.) Mill.) have been carried out in recent years by forest pathologists in Eastern North America. Early in these studies it became apparent that the existing ideas concerning the identity of the fungi responsible for decay in living balsam fir trees required considerable revision. Trunk rot was confirmed as being caused mainly by Stereum sanguinolentum Alb. and Schw. ex Fries; however, five fungi, headed by Corticium galactinum (Fries) Burt, were found associated with most of the white stringy butt rots, and two fungi, Coniophora puteana (Schum. ex Fries) Karst. and Polyporus balsameus Peck, were associated with most of the brown cubical butt rots. Hitherto Poria subacida (Peck) Sacc. and P. balsameus were believed to cause practically all white and brown butt rots, respectively, in this species. The effect of site on the decay of living balsam fir is discussed, as is the extent and kind of decay with which each of the nine predominant heart-rot fungi was found associated. A brief outline is presented concerning the fungi found associated with the deterioration of dead balsam fir.