bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling of Spatially Referenced Environmental and Meteorological Factors Influencing the Probability ofListeriaSpecies Isolation from Natural Environments
Authors: Ivanek R. , Gröhn Y. T. , Wells M. T. , Lembo A. J. , Sauders B. D. , Wiedmann M.
Published: August 2009

Many pathogens have the ability to survive and multiply in abiotic environments, representing a possible reservoir and source of human and animal exposure. Our objective was to develop a methodological framework to study spatially explicit environmental and meteorological factors affecting the probability of pathogen isolation from a location. Isolation ofListeriaspp. from the natural environment was used as a model system. Logistic regression and classification tree methods were applied, and their predictive performances were compared. Analyses revealed that precipitation and occurrence of alternating freezing and thawing temperatures prior to sample collection, loam soil, water storage to a soil depth of 50 cm, slope gradient, and cardinal direction to the north are key predictors for isolation ofListeriaspp. from a spatial location. Different combinations of factors affected the probability of isolation ofListeriaspp. from the soil, vegetation, and water layers of a location, indicating that the three layers represent different ecological niches forListeriaspp. The predictive power of classification trees was comparable to that of logistic regression. However, the former were easier to interpret, making them more appealing for field applications. Our study demonstrates how the analysis of a pathogen's spatial distribution improves understanding of the predictors of the pathogen's presence in a particular location and could be used to propose novel control strategies to reduce human and animal environmental exposure.
Protein
O
-Mannosyltransferases B and C Support Hyphal Development and Differentiation in
Aspergillus nidulans
Authors: Goto Masatoshi , Harada Yuka , Oka Takuji , Matsumoto Sho , Takegawa Kaoru , Furukawa Kensuke
Published: August 2009

Aspergillus nidulans
possesses three
pmt
genes encoding protein
O
-
d
-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology
150:
1973-1982, 2004). In the present paper, we describe the characterization of the
pmtA
paralogues
pmtB
and
pmtC
. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A
pmtB
disruptant showed wild-type (wt) colony formation at 30°C but slightly repressed growth at 42°C. Conidiation of the
pmtB
disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. A
pmtA
and
pmtB
double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single
pmt
mutants, the
pmtC
disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42°C further enabled the
pmtC
disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three
pmt
disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth.
Flagellum-Mediated Biofilm Defense Mechanisms of
Pseudomonas aeruginosa
against Host-Derived Lactoferrin
Authors: Leid Jeff G. , Kerr Mathias , Selgado Candice , Johnson Chelsa , Moreno Gabriel , Smith Alyssa , Shirtliff Mark E. , O'Toole George A. , Cope Emily K.
Published: August 2009

Chronic infection with the gram-negative organism
Pseudomonas aeruginosa
is a leading cause of morbidity and mortality in human patients, despite high doses of antibiotics used to treat the various diseases this organism causes. These infections are chronic because
P. aeruginosa
readily forms biofilms, which are inherently resistant to antibiotics as well as the host's immune system. Our laboratory has been investigating specific mutations in
P. aeruginosa
that regulate biofilm bacterial susceptibility to the host. To continue our investigation of the role of genetics in bacterial biofilm host resistance, we examined
P. aeruginosa
biofilms that lack the
flgK
gene. This mutant lacks flagella, which results in defects in early biofilm development (up to 36 h). For these experiments, the
flgK
-disrupted strain and the parental strain (PA14) were used in a modified version of the 96-well plate microtiter assay. Biofilms were challenged with freshly isolated human leukocytes for 4 to 6 h and viable bacteria enumerated by CFU. Subsequent to the challenge, both mononuclear cells (monocytes and lymphocytes) and neutrophils, along with tumor necrosis factor alpha (TNF-α), were required for optimal killing of the
flgK
biofilm bacteria. We identified a cytokine cross talk network between mononuclear cells and neutrophils that was essential to the production of lactoferrin and bacterial killing. Our data suggest that TNF-α is secreted from mononuclear cells, causing neutrophil activation, resulting in the secretion of bactericidal concentrations of lactoferrin. These results extend previous studies of the importance of lactoferrin in the innate immune defense against bacterial biofilms.
Powerful Induction of Divergent
tgs1-
Rv3131 Genes in
Mycobacterium tuberculosis
Is Mediated by DevR Interaction with a High-Affinity Site and an Adjacent Cryptic Low-Affinity Site
Authors: Chauhan Santosh , Tyagi Jaya Sivaswami
Published: August 2009

DevR activates the transcription of ∼48 genes in response to hypoxia and other stresses and triggers metabolic downshift and dormancy development in
Mycobacterium tuberculosis. tgs1
and Rv3131 encode triacylglycerol synthase and a putative nitroreductase, respectively, and both are members of the DevR regulon. This study aimed to understand how a single putative DevR binding site identified previously could sustain powerful induction of divergent
tgs1-
Rv3131 genes. DNase I footprinting revealed that phosphorylated DevR in fact binds to two sites symmetrically located at −42.5 and −63.5 bp from transcription start points of both genes. DevR first bound to the high-affinity site, P, and cooperatively recruited another DevR molecule to the secondary low-affinity site, S, to activate
tgs1
-Rv3131 transcription by ∼210- and ∼110-fold, respectively. The presence of a single P site significantly reduced activation of
tgs1
expression and abolished Rv3131 activity, reinforcing the requirement of two binding sites for robust expression in both directions. P site inversion abolished
tgs1
but not Rv3131 transcription despite DevR occupancy at both sites. The lack of
tgs1
expression is most likely due to disruption of its −35 promoter element rather than inversion of the binding site per se. We conclude that (i) an overlap of a DevR binding site and −35 sequence is indispensable for promoter activation, (ii) DevR interaction with two binding sites is obligatory for synergistic activation of
tgs1
-Rv3131 promoters, and (iii) DevR interaction with binding sites of different affinities offers scope for temporal and differential expression of target genes.
The Role of PerR in O2-Affected Gene Expression ofClostridium acetobutylicum
Authors: Hillmann Falk , Döring Christina , Riebe Oliver , Ehrenreich Armin , Fischer Ralf-Jörg , Bahl Hubert
Published: August 2009

In the strict anaerobeClostridium acetobutylicum, a PerR-homologous protein has recently been identified as being a key repressor of a reductive machinery for the scavenging of reactive oxygen species and molecular O2. In the absence of PerR, the full derepression of its regulon resulted in increased resistance to oxidative stress and nearly full tolerance of an aerobic environment. In the present study, the complementation of aBacillus subtilisPerR mutant confirmed that the homologous protein fromC. acetobutylicumacts as a functional peroxide sensor in vivo. Furthermore, we used a transcriptomic approach to analyze gene expression in the aerotolerant PerR mutant strain and compared it to the O2stimulon of wild-typeC. acetobutylicum. The genes encoding the components of the alternative detoxification system were PerR regulated. Only few other targets of direct PerR regulation were identified, including two highly expressed genes encoding enzymes that are putatively involved in the central energy metabolism. All of them were highly induced when wild-type cells were exposed to sublethal levels of O2. Under these conditions,C. acetobutylicumalso activated the repair and biogenesis of DNA and Fe-S clusters as well as the transcription of a gene encoding an unknown CO dehydrogenase-like enzyme. Surprisingly few genes were downregulated when exposed to O2, including those involved in butyrate formation. In summary, these results show that the defense of this strict anaerobe against oxidative stress is robust and by far not limited to the removal of O2and its reactive derivatives.
Integrative and Sequence Characteristics of a Novel Genetic Element, ICE
6013
, in
Staphylococcus aureus
Authors: Smyth Davida S. , Robinson D. Ashley
Published: August 2009

A survey of chromosomal variation in the ST239 clonal group of methicillin-resistant
Staphylococcus aureus
(MRSA) revealed a novel genetic element, ICE
6013
. The element is 13,354 bp in length, excluding a 6,551-bp Tn
552
insertion. ICE
6013
is flanked by 3-bp direct repeats and is demarcated by 8-bp imperfect inverted repeats. The element was present in 6 of 15 genome-sequenced
S. aureus
strains, and it was detected using genetic markers in 19 of 44 diverse MRSA and methicillin-susceptible strains and in all 111 ST239 strains tested. Low integration site specificity was discerned. Multiple chromosomal copies and the presence of extrachromosomal circular forms of ICE
6013
were detected in various strains. The circular forms included 3-bp coupling sequences, located between the 8-bp ends of the element, that corresponded to the 3-bp direct repeats flanking the chromosomal forms. ICE
6013
is predicted to encode 15 open reading frames, including an IS
30
-like DDE transposase in place of a Tyr/Ser recombinase and homologs of gram-positive bacterial conjugation components. Further sequence analyses indicated that ICE
6013
is more closely related to ICE
Bs1
from
Bacillus subtilis
than to the only other potential integrative conjugative element known from
S. aureus
, Tn
5801
. Evidence of recombination between ICE
6013
elements is also presented. In summary, ICE
6013
is the first member of a new family of active, integrative genetic elements that are widely dispersed within
S. aureus
strains.
MrdH, a Novel Metal Resistance Determinant of
Pseudomonas putida
KT2440, Is Flanked by Metal-Inducible Mobile Genetic Elements
Authors: Haritha Adhikarla , Sagar Korripally Prem , Tiwari Anand , Kiranmayi Patnala , Rodrigue Agnes , Mohan Pamarthi Maruthi , Singh Surya Satyanarayana
Published: August 2009

We report here the identification and characterization of
mrdH
, a novel chromosomal metal resistance determinant, located in the genomic island 55 of
Pseudomonas putida
KT2440. It encodes for MrdH, a predicted protein of ∼40 kDa with a chimeric domain organization derived from the RcnA and RND (for resistance-nodulation-cell division) metal efflux proteins. The metal resistance function of
mrdH
was identified by the ability to confer nickel resistance upon its complementation into
rcnA
mutant (a nickel- and cobalt-sensitive mutant) of
Escherichia coli
. However, the disruption of
mrdH
in
P. putida
resulted in an increased sensitivity to cadmium and zinc apart from nickel. Expression studies using quantitative reverse transcription-PCR showed the induction of
mrdH
by cadmium, nickel, zinc, and cobalt. In association with
mrdH
, we also identified a conserved hypothetical gene
mreA
whose encoded protein showed significant homology to NreA and NreA-like proteins. Expression of the
mreA
gene in
rcnA
mutant of
E. coli
enhanced its cadmium and nickel resistance. Transcriptional studies showed that both
mrdH
and
mreA
underwent parallel changes in gene expression. The mobile genetic elements Tn
4652
and IS
1246
, flanking
mrdH
and
mreA
were found to be induced by cadmium, nickel, and zinc, but not by cobalt. This study is the first report of a single-component metal efflux transporter,
mrdH
, showing chimeric domain organization, a broad substrate spectrum, and a location amid metal-inducible mobile genetic elements.
Identification of the [FeFe]-Hydrogenase Responsible for Hydrogen Generation inThermoanaerobacterium saccharolyticumand Demonstration of Increased Ethanol Yield via Hydrogenase Knockout
Authors: Shaw A. Joe , Hogsett David A. , Lynd Lee R.
Published: August 2009

Three putative hydrogenase enzyme systems inThermoanaerobacterium saccharolyticumwere investigated at the genetic, mRNA, enzymatic, and phenotypic levels. A four-gene operon containing two [FeFe]-hydrogenase genes, provisionally termedhfs(hydrogenase-Fe-S), was found to be the main enzymatic catalyst of hydrogen production.hfsB, perhaps the most interesting gene of the operon, contains an [FeFe]-hydrogenase and a PAS sensory domain and has several conserved homologues among clostridial saccharolytic, cellulolytic, and pathogenic bacteria. A second hydrogenase gene cluster,hyd, exhibited methyl viologen-linked hydrogenase enzymatic activity, buthydgene knockouts did not influence the hydrogen yield of cultures grown in closed-system batch fermentations. This result, combined with the observation thathydBcontains NAD(P)+ and FMN binding sites, suggests that thehydgenes are specific to the transfer of electrons from NAD(P)H to hydrogen ions. A third gene cluster, a putative [NiFe]-hydrogenase with homology to theechgenes, did not exhibit hydrogenase activity under any of the conditions tested. Deletion of thehfsandhydAgenes resulted in a loss of detectable methyl viologen-linked hydrogenase activity. Strains with a deletion of thehfsgenes exhibited a 95% reduction in hydrogen and acetic acid production. A strain withhfsandldhdeletions exhibited an increased ethanol yield from consumed carbohydrates and represents a new strategy for engineering increased ethanol yields inT. saccharolyticum.
Mutational Analysis ofBurkholderia thailandensisQuorum Sensing and Self-Aggregation
Authors: Chandler Josephine R. , Duerkop Breck A. , Hinz Aaron , West T. Eoin , Herman Jake P. , Churchill Mair E. A. , Skerrett Shawn J. , Greenberg E. Peter
Published: August 2009

Acyl-homoserine lactone (acyl-HSL) quorum-sensing signaling is common to manyProteobacteria. Acyl-HSLs are synthesized by the LuxI family of synthases, and the signal response is mediated by members of the LuxR family of transcriptional regulators.Burkholderia thailandensisis a member of a closely related cluster of three species, including the animal pathogensBurkholderia malleiandBurkholderia pseudomallei. Members of this group have similarluxIandluxRhomologs, and these genes contribute toB. pseudomalleiandB. malleivirulence.B. thailandensispossesses three pairs ofluxI-luxRhomologs. One of these pairs, BtaI2-BtaR2, has been shown to produce and respond to 3OHC10-HSL and to control the synthesis of an antibiotic. By using a markerless-exhange method, we constructed an assortment ofB. thailandensisquorum-sensing mutants, and we used these mutants to show that BtaI1 is responsible for C8-HSL production and BtaI3 is responsible for 3OHC8-HSL production. We also show that a strain incapable of acyl-HSL production is capable of growth on the same assortment of carbon and nitrogen sources as the wild type. Furthermore, this mutant shows no loss of virulence compared to the wild type in mice. However, the wild type self-aggregates in minimal medium, whereas the quorum-sensing mutant does not. The wild-type aggregation phenotype is recovered by addition of the BtaI1-R1 HSL signal C8-HSL. We propose that the key function of the BtaR1-BtaI1 quorum-sensing system is to cause cells to gather into aggregates once a sufficient population has been established.
A Unique β-1,2-Mannosyltransferase of
Thermotoga maritima
That Uses Di-
myo
-Inositol Phosphate as the Mannosyl Acceptor
Authors: Rodrigues Marta V. , Borges Nuno , Almeida Carla P. , Lamosa Pedro , Santos Helena
Published: August 2009

In addition to di-
myo
-inositol-1,3′-phosphate (DIP), a compatible solute widespread in hyperthermophiles, the organic solute pool of
Thermotoga maritima
comprises 2-(
O
-β-
d-
mannosyl)-di-
myo
-inositol-1,3′-phosphate (MDIP) and 2-(
O
-β-
d-
mannosyl-1,2-
O
-β-
d-
mannosyl)-di-
myo
-inositol-1,3′-phosphate (MMDIP), two newly identified β-1,2-mannosides. In cells grown under heat stress, MDIP was the major solute, accounting for 43% of the total pool; MMDIP and DIP accumulated to similar levels, each corresponding to 11.5% of the total pool. The synthesis of MDIP involved the transfer of the mannosyl group from GDP-mannose to DIP in a single-step reaction catalyzed by MDIP synthase. This enzyme used MDIP as an acceptor of a second mannose residue, yielding the di-mannosylated compound. Minor amounts of the tri-mannosylated form were also detected. With a genomic approach, putative genes for MDIP synthase were identified in the genome of
T. maritima
, and the assignment was confirmed by functional expression in
Escherichia coli
. Genes with significant sequence identity were found only in the genomes of
Thermotoga
spp.,
Aquifex aeolicus
, and
Archaeoglobus profundus
. MDIP synthase of
T. maritima
had maximal activity at 95°C and apparent

K
m

values of 16 mM and 0.7 mM for DIP and GDP-mannose, respectively. The stereochemistry of MDIP was characterized by isotopic labeling and nuclear magnetic resonance (NMR): DIP selectively labeled with carbon 13 at position C1 of the
l-
inositol moiety was synthesized and used as a substrate for MDIP synthase. This β-1,2-mannosyltransferase is unrelated to known glycosyltransferases, and within the domain
Bacteria
, it is restricted to members of the two deepest lineages, i.e., the
Thermotogales
and the
Aquificales
. To our knowledge, this is the first β-1,2-mannosyltransferase characterized thus far.
Flagellar Formation in C-Ring-Defective Mutants by Overproduction of FliI, the ATPase Specific for Flagellar Type III Secretion
Authors: Konishi Manabu , Kanbe Masaomi , McMurry Jonathan L. , Aizawa Shin-Ichi
Published: August 2009

The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the
fliG
,
fliM
, and
fliN
genes results in a Fla
−
phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an intact C ring is essential for motor function. In FliN- or FliM-deficient mutants, flagellum production was about 10% of the wild-type level, while it was only a few percent in FliG-deficient mutants, suggesting that the size of partial C rings affects the extent of flagellation. For flagella made in C-ring mutants, the hook length varied considerably, with many being markedly shorter or longer than that of the wild type. The broad distribution of hook lengths suggests that defective C rings cannot control the hook length as tightly as the wild type even though FliK and FlhB are both intact.
Homologues of the
Bacillus subtilis
SpoVB Protein Are Involved in Cell Wall Metabolism
Authors: Vasudevan Pradeep , McElligott Jessica , Attkisson Christa , Betteken Michael , Popham David L.
Published: August 2009

Members of the COG2244 protein family are integral membrane proteins involved in synthesis of a variety of extracellular polymers. In several cases, these proteins have been suggested to move lipid-linked oligomers across the membrane or, in the case of
Escherichia coli
MviN, to flip the lipid II peptidoglycan precursor.
Bacillus subtilis
SpoVB was the first member of this family implicated in peptidoglycan synthesis and is required for spore cortex polymerization. Three other COG2244 members with high similarity to SpoVB are encoded within the
B. subtilis
genome. Mutant strains lacking any or all of these genes (
yabM
,
ykvU
, and
ytgP
) in addition to
spoVB
are viable and produce apparently normal peptidoglycan, indicating that their function is not essential in
B. subtilis
. Phenotypic changes associated with loss of two of these genes suggest that they function in peptidoglycan synthesis. Mutants lacking YtgP produce long cells and chains of cells, suggesting a role in cell division. Mutants lacking YabM exhibit sensitivity to moenomycin, an antibiotic that blocks peptidoglycan polymerization by class A penicillin-binding proteins. This result suggests that YabM may function in a previously observed alternate pathway for peptidoglycan strand synthesis.
Homotaurine Metabolized to 3-Sulfopropanoate in
Cupriavidus necator
H16: Enzymes and Genes in a Patchwork Pathway
Authors: Mayer Jutta , Cook Alasdair M.
Published: August 2009

Homotaurine (3-aminopropanesulfonate), a natural product and an analogue of GABA (4-aminobutyrate), was found to be a sole source of nitrogen for
Cupriavidus necator
(
Ralstonia eutropha
) H16, whose genome sequence is known. Homotaurine nitrogen was assimilated into cell material, and the quantitative fate of the organosulfonate was sulfopropanoate, which was recovered in the growth medium. The first scalar reaction was shown to be inducible homotaurine:2-oxoglutarate aminotransferase, which released 3-sulfopropanal from homotaurine. This aminotransferase was purified to homogeneity and characterized. Peptide mass fingerprinting yielded locus tag H16_B0981, which was annotated
gabT
, for GABA transaminase (EC 2.6.1.19). Inducible, NAD(P)
+
-coupled 3-sulfopropanal dehydrogenase, which yielded 3-sulfopropanoate from 3-sulfopropanal, was also purified and characterized. Peptide mass fingerprinting yielded locus tag H16_B0982, which was annotated
gabD1
, for succinate-semialdehyde dehydrogenase (EC 1.2.1.16). GabT and GabD1 were each induced during growth with GABA, and cotranscription of
gabTD
was observed. In other organisms, regulator GabC or GabR is encoded contiguous with
gabTD
: candidate GabR′ was found in strain H16 and in many other organisms. An orthologue of the GABA permease (GabP), established in
Escherichia coli
, is present at H16_B1890, and it was transcribed constitutively. We presume that GabR′PTD are responsible for the inducible metabolism of homotaurine to intracellular 3-sulfopropanoate. The nature of the exporter of this highly charged compound was unclear until we realized from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data that sulfoacetaldehyde acetyltransferase (EC 2.3.3.15; H16_B1872) was strongly induced during growth with homotaurine and inferred that the sulfite exporter encoded at the end of the gene cluster (H16_B1874) has a broad substrate range that includes 3-sulfopropanoate.
Identification of the Site-Specific DNA Invertase Responsible for the Phase Variation of SusC/SusD Family Outer Membrane Proteins in
Bacteroides fragilis
Authors: Nakayama-Imaohji Haruyuki , Hirakawa Hideki , Ichimura Minoru , Wakimoto Shin , Kuhara Satoru , Hayashi Tetsuya , Kuwahara Tomomi
Published: August 2009

The human gut microbe
Bacteroides fragilis
can alter the expression of its surface molecules, such as capsular polysaccharides and SusC/SusD family outer membrane proteins, through reversible DNA inversions. We demonstrate here that DNA inversions at 12 invertible regions, including three gene clusters for SusC/SusD family proteins, were controlled by a single tyrosine site-specific recombinase (Tsr0667) encoded by BF0667 in
B. fragilis
strain YCH46. Genetic disruption of BF0667 diminished or attenuated shufflon-type DNA inversions at all three
susC
/
susD
genes clusters, as well as simple DNA inversions at nine other loci, most of which colocalized with
susC/susD
family genes. The inverted repeat sequences found within the Tsr0667-regulated invertible regions shared the consensus motif sequence AGTYYYN
4
GDACT. Tsr0667 specifically mediated the DNA inversions of 10 of the 12 regions, even under an
Escherichia coli
background when the invertible regions were exposed to BF0667 in
E. coli
cells. Thus, Tsr0667 is an additional globally acting DNA invertase in
B. fragilis
, which probably involves the selective expression of SusC/SusD family outer membrane proteins.
CebR as a Master Regulator for Cellulose/Cellooligosaccharide Catabolism Affects Morphological Development in
Streptomyces griseus
Authors: Marushima Kazuya , Ohnishi Yasuo , Horinouchi Sueharu
Published: August 2009

Streptomyces griseus
mutants exhibiting deficient glucose repression of β-galactosidase activity on lactose-containing minimal medium supplemented with a high concentration of glucose were isolated. One of these mutants had a 12-bp deletion in
cebR
, which encodes a LacI/GalR family regulator. Disruption of
cebR
in the wild-type strain caused the same phenotype as the mutant, indicating that CebR is required for glucose repression of β-galactosidase activity. Recombinant CebR protein bound to a 14-bp inverted-repeat sequence (designated the CebR box) present in the promoter regions of
cebR
and the putative cellobiose utilization operon,
cebEFG-bglC
. The DNA-binding activity of CebR was impaired by cellooligosaccharides, including cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose. In agreement with this observation, transcription from the
cebE
and
cebR
promoters was greatly enhanced by the addition of cellobiose to the medium. Seven other genes containing one or two CebR boxes in their upstream regions were found in the
S. griseus
genome. Five of these genes encode putative secreted proteins: two cellulases, a cellulose-binding protein, a pectate lyase, and a protein of unknown function. These five genes and
cebEFG-bglC
were transcribed at levels 4 to 130 times higher in the Δ
cebR
mutant than in the wild-type strain, as determined by quantitative reverse transcription-PCR. These findings indicate that CebR is a master regulator of cellulose/cellooligosaccharide catabolism. Unexpectedly, the Δ
cebR
mutant formed very few aerial hyphae on lactose-containing medium, demonstrating a link between carbon source utilization and morphological development.
Characterization of the FtsZ-Interacting Septal Proteins SepF and Ftn6 in the Spherical-Celled Cyanobacterium
Synechocystis
Strain PCC 6803
Authors: Marbouty Martial , Saguez Cyril , Cassier-Chauvat Corinne , Chauvat Franck
Published: August 2009

Assembly of the tubulin-like cytoskeletal protein FtsZ into a ring structure at midcell establishes the location of the nascent division sites in prokaryotes. However, it is not yet known how the assembly and contraction of the Z ring are regulated, especially in cyanobacteria, the environmentally crucial organisms for which only one FtsZ partner protein, ZipN, has been described so far. Here, we characterized SepF and Ftn6, two novel septal proteins, in the spherical-celled strain
Synechocystis
PCC 6803. Both proteins were found to be indispensable to
Synechocystis
sp. strain PCC 6803. The depletion of both SepF and Ftn6 resulted in delayed cytokinesis and the generation of giant cells but did not prevent FtsZ polymerization, as shown by the visualization of green fluorescent protein (GFP)-tagged FtsZ polymers. These GFP-tagged Z-ring-like structures often appeared to be abnormal, because these reporter cells respond to the depletion of either SepF or Ftn6 with an increased abundance of total, natural, and GFP-tagged FtsZ proteins. In agreement with their septal localization, we found that both SepF and Ftn6 interact physically with FtsZ. Finally, we showed that SepF, but not Ftn6, stimulates the formation and/or stability of FtsZ polymers in vitro.
Stress-Induced β-Lactam Antibiotic Resistance Mutation and Sequences of Stationary-Phase Mutations in the
Escherichia coli
Chromosome
Authors: Petrosino Joseph F. , Galhardo Rodrigo S. , Morales Liza D. , Rosenberg Susan M.
Published: August 2009

In some enterobacterial pathogens, but not in
Escherichia coli
, loss-of-function mutations are a common route to clinically relevant β-lactam antibiotic resistance. We previously constructed an assay system for studying enterobacterial β-lactam resistance mutations using the well-developed genetics of
E. coli
by integrating enterobacterial
ampRC
genes into the
E. coli
chromosome. Like the cells of other enterobacteria,
E. coli
cells acquire β-lactam resistance by
ampD
mutation. Here we show that starvation and stress responses provoke
ampD
β-lactam resistance mutagenesis. When starved on lactose medium, Lac
−
strains used in mutagenesis studies accumulate
ampD
β-lactam resistance mutations independent of Lac reversion. DNA double-strand break repair (DSBR) proteins and the SOS and RpoS stress responses are required for this mutagenesis, in agreement with the results obtained for
lac
reversion in these cells. Surprisingly, the stress-induced
ampD
mutations require DinB (DNA polymerase IV) and partially require error-prone DNA polymerase V, unlike
lac
mutagenesis, which requires only DinB. This assay demonstrates that real-world stressors, such as starvation, can induce clinically relevant resistance mutations. Finally, we used the
ampD
system to observe the true forward-mutation sequence spectrum of DSBR-associated stress-induced mutagenesis, for which previously only frameshift reversions were studied. We found that base substitutions outnumber frameshift mutations, as seen in other experimental systems showing stress-induced mutagenesis. The important evolutionary implication is that not only loss-of-function mutations but also change-of-function mutations can be generated by this mechanism.
NtrC-Dependent Regulatory Network for Nitrogen Assimilation in
Pseudomonas putida
Authors: Hervás Ana B. , Canosa Inés , Little Richard , Dixon Ray , Santero Eduardo
Published: August 2009

Pseudomonas putida
KT2440 is a model strain for studying bacterial biodegradation processes. However, very little is known about nitrogen regulation in this strain. Here, we show that the nitrogen regulatory NtrC proteins from
P. putida
and
Escherichia coli
are functionally equivalent and that substitutions leading to partially active forms of enterobacterial NtrC provoke the same phenotypes in
P. putida
NtrC.
P. putida
has only a single P
II
-like protein, encoded by
glnK
, whose expression is nitrogen regulated. Two contiguous NtrC binding sites located upstream of the σ
N
-dependent
glnK
promoter have been identified by footprinting analysis. In vitro experiments with purified proteins demonstrated that
glnK
transcription was directly activated by NtrC and that open complex formation at this promoter required integration host factor. Transcription of genes orthologous to enterobacterial
codB
,
dppA
, and
ureD
genes, whose transcription is dependent on σ
70
and which are activated by Nac in
E. coli
, has also been analyzed for
P. putida
. Whereas
dppA
does not appear to be regulated by nitrogen via NtrC, the
codB
and
ureD
genes have σ
N
-dependent promoters and their nitrogen regulation was exerted directly by NtrC, thus avoiding the need for Nac, which is missing in this bacterial species. Based upon these results, we propose a simplified nitrogen regulatory network in
P. putida
(compared to that in enterobacteria), which involves an indirect-feedback autoregulation of
glnK
using NtrC as an intermediary.
mosR
, a Novel Transcriptional Regulator of Hypoxia and Virulence in
Mycobacterium tuberculosis
Authors: Abomoelak Bassam , Hoye Elizabeth A. , Chi Jing , Marcus Sarah A. , Laval Francoise , Bannantine John P. , Ward Sarah K. , Daffé Mamadou , Liu Hong Di , Talaat Adel M.
Published: August 2009

Latent tuberculosis represents a high-risk burden for one-third of the world population. Previous analysis of murine tuberculosis identified a novel transcriptional regulator encoded by Rv0348 that could control the establishment of persistent tuberculosis. Disruption of the Rv0348 gene from the genome of the virulent H37Rv strain of
Mycobacterium tuberculosis
revealed a global impact on the transcriptional profiles of 163 genes, including induction of the mammalian cell entry (
mce1
) operon and the repression of a significant number of genes involved in hypoxia and starvation responses. Nonetheless, gel shift assays did not reveal direct binding between Rv0348 and a set of regulated promoters, suggesting an indirect regulatory role. However, when expressed in
Mycobacterium smegmatis
, the Rv0348 transcripts were significantly responsive to different levels of hypoxia and the encoded protein was shown to regulate genes involved in hypoxia [e.g., Rv3130c (
tgs1
)] and intracellular survival (e.g.,
mce1
), among other genes. Interestingly, the colonization level of the Δ
mosR
mutant strain was significantly lower than that of the wild-type strain of
M. tuberculosis
, suggesting its attenuation in the murine model of tuberculosis. Taken together, our analyses indicated that the Rv0348 gene encodes a novel transcriptional factor that regulates several operons involved in mycobacterial survival, especially during hypoxia; hence, we propose that Rv0348 be renamed
mosR
for
r
egulator of
m
ycobacterial
o
perons of
s
urvival.
Contributions of Zur-Controlled Ribosomal Proteins to Growth under Zinc Starvation Conditions
Authors: Gabriel Scott E. , Helmann John D.
Published: August 2009

Maintaining intracellular zinc levels is critical, because zinc serves as a cofactor for many required enzymes and is toxic in excess.Bacillus subtilisZur, a Fur family repressor, controls the zinc starvation response including two ribosomal proteins (r-proteins) paralogous to L31 and S14. Biochemical analyses suggest that Zur-controlled r-proteins (which lack the two CXXC metal-binding motifs) may functionally replace their cognate zinc-requiring proteins during zinc limitation. We demonstrate here that Zur regulates the expression of an additional r-protein paralog, RpmGC (L33c), and, using strains defective in zinc uptake, we investigate the physiological contributions of all three Zur-regulated r-proteins. In the 168 lineage,rpmGCis a pseudogene containing a frameshift mutation. Correction of this mutation allows expression of a functional L33c that can suppress the poor growth phenotype of anrpmGA rpmGB(encoding L33a, L33b) double mutant. Similarly, we provide physiological evidence in support of the “failsafe” model (Y. Natori et al., Mol. Microbiol. 63:294-307, 2007) in which the Zur-regulated S14 paralog YhzA allows continued ribosome synthesis when there is insufficient zinc to support S14 function. The L31 paralog YtiA can replace L31 and complement the growth defect of anrpmEmutant (Nanamiya et al., Mol. Microbiol. 52:273-283). We show that, under zinc starvation conditions, derepression of YtiA significantly increases the growth of cells in which preexisting ribosomes carry, as the sole L31 protein, RpmE (containing zinc), but not if they carry YtiA (which lacks zinc). These results support a direct and physiologically relevant role for YtiA in mobilizing zinc from ribosomes.
Involvement of NADH:Acceptor Oxidoreductase and Butyryl Coenzyme A Dehydrogenase in Reversed Electron Transport during Syntrophic Butyrate Oxidation by
Syntrophomonas wolfei
Authors: Müller Nicolai , Schleheck David , Schink Bernhard
Published: August 2009

Methanogenic oxidation of butyrate to acetate requires a tight cooperation between the syntrophically fermenting
Syntrophomonas wolfei
and the methanogen
Methanospirillum hungatei
, and a reversed electron transport system in
S. wolfei
was postulated to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH for H
2
generation. The metabolic activity of butyrate-oxidizing
S. wolfei
cells was measured via production of formazan and acetate from butyrate, with 2,3,5-triphenyltetrazolium chloride as electron acceptor. This activity was inhibited by trifluoperazine (TPZ), an antitubercular agent known to inhibit NADH:menaquinone oxidoreductase. In cell extracts of
S. wolfei
, the oxidation of NADH could be measured with quinones, viologens, and tetrazolium dyes as electron acceptors, and also this activity was inhibited by TPZ. The TPZ-sensitive NADH:acceptor oxidoreductase activity appeared to be membrane associated but could be dissociated from the membrane as a soluble protein and was semipurified by anion-exchange chromatography. Recovered proteins were identified by peptide mass fingerprinting, which indicated the presence of an NADH:acceptor oxidoreductase as part of a three-component [FeFe] hydrogenase complex and a selenocysteine-containing formate dehydrogenase. Furthermore, purification of butyryl-CoA dehydrogenase (Bcd) activity and peptide mass fingerprinting revealed two Bcd proteins different from the Bcd subunit of the Bcd/electron-transfer flavoprotein complex (Bcd/EtfAB) predicted from the genome sequence of
S. wolfei
. The results suggest that syntrophic oxidation of butyrate in
S. wolfei
involves a membrane-associated TPZ-sensitive NADH:acceptor oxidoreductase as part of a hydrogenase complex similar to the recently discovered “bifurcating” hydrogenase in
Thermotoga maritima
and butyryl-CoA dehydrogenases that are different from Bcd of the Bcd/EtfAB complex.
Trichosporon mycotoxinivorans
, a Novel Respiratory Pathogen in Patients with Cystic Fibrosis
Authors: Hickey Patrick W. , Sutton Deanna A. , Fothergill Annette W. , Rinaldi Michael G. , Wickes Brian L. , Schmidt Howard J. , Walsh Thomas J.
Published: August 2009

This report describes the molecular epidemiology, in vitro susceptibility, colonial and microscopic morphologies, and biochemical features of
Trichosporon mycotoxinivorans
, a newly recognized pathogen that appears to have a propensity for patients with cystic fibrosis. The index patient died with histologically documented
Trichosporon
pneumonia complicating cystic fibrosis. This is also the first report of disease caused by a
Trichosporon
species in a nontransplant patient with cystic fibrosis. As
T. mycotoxinivorans
has not previously been recognized as a respiratory pathogen, the significance of its recovery from sputum samples was not initially appreciated. Genetic analysis of archived clinical samples found three additional cases of
T. mycotoxinivorans
infection which had previously been identified as other members of the genus. An additional isolate of
T. mycotoxinivorans
was identified from a clinical sample on initial testing. Three of these four cases were also patients with cystic fibrosis. All isolates had MICs at 48 h of amphotericin B of ≥1 μg/ml and of echinocandins of ≥16 μg/ml, but they displayed various susceptibilities to the triazoles. In summary,
Trichosporon mycotoxinivorans
is a newly recognized human pathogen that is associated with cystic fibrosis.
Comparative Evaluation of the ExaVir Load Version 3 Reverse Transcriptase Assay for Measurement of Human Immunodeficiency Virus Type 1 Plasma Load
Authors: Labbett Wendy , Garcia-Diaz Ana , Fox Zoe , Clewley Gillian S. , Fernandez Thomas , Johnson Margaret , Geretti Anna Maria
Published: August 2009

In resource-limited settings, the virological monitoring of antiretroviral therapy is limited by high cost and the lack of infrastructure. The Cavidi ExaVir Load assay employs a simple and inexpensive enzyme-linked immunosorbent assay format to measure human immunodeficiency virus (HIV) reverse transcriptase activity, which correlates with plasma RNA load. The version 3 assay has been described as having improved precision and sensitivity. There are limited data on its performance relative to those of current real-time assays. Our objective was to compare HIV type 1 (HIV-1) RNA load measurement in plasma by ExaVir Load version 3 (designated ExaVir), Abbott M2000sp/M2000rt RealTime HIV-1 assay (designated RealTime), and Roche COBAS Ampliprep/COBAS TaqMan HIV-1 version 1 assay (designated TaqMan). Plasma from 119 patients (34 with subtype B infection, 85 with non-subtype B infection [A-H, CRF01, CRF02, CRF06, CRF12, CRF14, and complex]; 48 subjects were treatment experienced, 71 were naive) and serial dilutions of the second international standard (IS) were tested. Assay relationship and agreement were determined by linear regression, correlation analysis, and the Bland-Altman method. The ExaVir assay quantified 77/83 (92.8%) samples with viral loads of >2.3 log
10
copies/ml by the molecular assays. Results were linearly associated and strongly correlated with RealTime and TaqMan measurements (
R
of 0.94 and 0.92, respectively) for both subtype B (
R
of 0.97 and 0.95, respectively) and non-subtype B (
R
of 0.93 and 0.91, respectively) samples. Mean differences were 0.28 and 0.18 log
10
copies/ml in favor of the two molecular assays; 7/119 (5.9%) and 5/119 (4.2%) samples were outside the 95% level of agreement. ExaVir underquantified the IS by a mean of 0.2 (range, 0.0 to 0.5) log
10
copies/ml. The ExaVir assay showed excellent concordance with real-time molecular assays, offering a suitable option for virological monitoring in settings with limited infrastructure.
Community-Associated Methicillin-Resistant
Staphylococcus aureus
Mediastinitis
Authors: Calvano Tatjana P. , Ferraro David M. , Prakash Vidhya , Mende Katrin , Hospenthal Duane R.
Published: August 2009

Community-associated methicillin (meticillin)-resistant
Staphylococcus aureus
(CA-MRSA) continues to emerge as a cause of serious infections, chiefly of the skin and soft tissues. We present the first documented case of CA-MRSA mediastinitis in an adult. Blood and mediastinal isolates were characterized as CA-MRSA by pulsed-field gel electrophoresis and susceptibility testing.
Open Reading Frame 33 of a Gammaherpesvirus Encodes a Tegument Protein Essential for Virion Morphogenesis and Egress
Authors: Guo Haitao , Wang Lili , Peng Li , Zhou Z. Hong , Deng Hongyu
Published: August 2009

Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among
Alpha-
,
Beta-
, and
Gammaherpesvirinae
subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in
trans
or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.
Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1
Authors: Bailey Sarah G. , Verrall Elizabeth , Schelcher Celine , Rhie Alex , Doherty Aidan J. , Sinclair Alison J.
Published: August 2009

Epstein-Barr virus (EBV; human herpesvirus 4) poses major clinical problems worldwide. Following primary infection, EBV enters a form of long-lived latency in B lymphocytes, expressing few viral genes, and it persists for the lifetime of the host with sporadic bursts of viral replication. The switch between latency and replication is governed by the action of a multifunctional viral protein Zta (also called BZLF1, ZEBRA, and Z). Using a global proteomic approach, we identified a host DNA damage repair protein that specifically interacts with Zta: 53BP1. 53BP1 is intimately connected with the ATM signal transduction pathway, which is activated during EBV replication. The interaction of 53BP1 with Zta requires the C-terminal ends of both proteins. A series of Zta mutants that show a wild-type ability to perform basic functions of Zta, such as dimer formation, interaction with DNA, and the transactivation of viral genes, were shown to have lost the ability to induce the viral lytic cycle. Each of these mutants also is compromised in the C-terminal region for interaction with 53BP1. In addition, the knockdown of 53BP1 expression reduced viral replication, suggesting that the association between Zta and 53BP1 is involved in the viral replication cycle.
Systemic and Mucosal T-Lymphocyte Activation Induced by Recombinant Adenovirus Vaccines in Rhesus Monkeys
Authors: Sun Yue , Bailer Robert T. , Rao Srinivas S. , Mascola John R. , Nabel Gary J. , Koup Richard A. , Letvin Norman L.
Published: August 2009

The administration of vectors designed to elicited cell-mediated immune responses may have other consequences that are clinically significant. To explore this possibility, we evaluated T-cell activation during the first 2 months after recombinant adenovirus serotype 5 (rAd5) prime or boost immunizations in rhesus monkeys. We also evaluated the kinetics of T-lymphocyte activation in both the systemic and the mucosal compartments after rAd5 administration in monkeys with preexisting immunity to Ad5. The rAd5 immunization induced lower-frequency Gag epitope-specific CD8
+
T cells in the colonic mucosa than in the peripheral blood. There was evidence of an expansion of the simian immunodeficiency virus Gag-specific CD8
+
T-cell responses, but not the Ad5 hexon-specific T-cell responses, following a homologous rAd5 boost. A striking but transient T-lymphocyte activation in both the systemic and the mucosal compartments of rhesus monkeys was observed after rAd5 immunization. These findings indicate that the administration of a vaccine vector such as Ad5 can induce a global activation of T cells.