bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Rapid, Sensitive, and Field-Deployable Razor Ex BioDetection System and Quantitative PCR Assay for Detection of Phymatotrichopsis omnivora Using Multiple Gene Targets
Authors: Arif M. , Fletcher J. , Marek S. M. , Melcher U. , Ochoa-Corona F. M.
Published: January 2013

A validated, multigene-based method using real-time quantitative PCR (qPCR) and the Razor Ex BioDetection system was developed for detection of
Phymatotrichopsis omnivora.
This soilborne fungus causes Phymatotrichopsis root rot of cotton, alfalfa, and other dicot crops in the southwestern United States and northern Mexico, leading to significant crop losses and limiting the range of crops that can be grown in soils where the fungus is established. It is on multiple lists of regulated organisms. Because
P. omnivora
is difficult to isolate, accurate and sensitive culture-independent diagnostic tools are needed to confirm infections by this fungus. Specific PCR primers and probes were designed based on
P. omnivora
nucleotide sequences of the genes encoding rRNA internal transcribed spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II (RPB2). PCR products were cloned and sequenced to confirm their identity. All primer sets allowed early detection of
P. omnivora
in infected but asymptomatic plants. A modified rapid DNA purification method, which facilitates a quick (∼30-min) on-site assay capability for
P. omnivora
detection, was developed. Combined use of three target genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a multigene-based, field-deployable, rapid, and reliable identification method for a fungal plant pathogen and should serve as a model for the development of field-deployable assays of other phytopathogens.
Quantitative Detection of Viable Bifidobacterium bifidum BF-1 Cells in Human Feces by Using Propidium Monoazide and Strain-Specific Primers
Authors: Fujimoto Junji , Watanabe Koichi
Published: January 2013

We developed a PCR-based method to detect and quantify viable
Bifidobacterium bifidum
BF-1 cells in human feces. This method (PMA-qPCR) uses propidium monoazide (PMA) to distinguish viable from dead cells and quantitative PCR using a BF-1-specific primer set designed from the results of randomly amplified polymorphic DNA analysis. During long-term culture (10 days), the number of viable BF-1 cells detected by counting the number of CFU on modified MRS agar, by measuring the ATP contents converted to CFU, and by using PMA-qPCR decreased from about 10
10
to 10
6
cells/ml; in contrast, the total number of (viable and dead) BF-1 cells detected by counting 4′,6-diamidino-2-phenylindolee (DAPI)-stained cells and by using qPCR without PMA and reverse transcription-qPCR remained constant. The number of viable BF-1 cells in fecal samples detected by using PMA-qPCR was highly and significantly correlated with the number of viable BF-1 cells added to the fecal samples, within the range of 10
5.3
to 10
10.3
cells/g feces (wet weight) (
r
> 0.99,
P
< 0.001). After 12 healthy subjects ingested 10
10.3
to 10
11.0
CFU of BF-1 in a fermented milk product daily for 28 days, 10
4.5 ï¿½ 1.5
(mean ï¿½ standard deviation [SD]) BF-1 CFU/g was detected in fecal samples by using strain-specific selective agar; in contrast, 10
6.2 ï¿½ 0.4
viable BF-1 cells/g were detected by using PMA-qPCR, and a total of 10
7.6 ï¿½ 0.7
BF-1 cells/g were detected by using qPCR without PMA. Thus, the number of viable BF-1 cells detected by PMA-qPCR was about 50 times higher (
P
< 0.01) than that detected by the culture-dependent method. We conclude that strain-specific PMA-qPCR can be used to quickly and accurately evaluate viable BF-1 in feces.
Constitutive Expression of the Cytochrome P450 EthABCD Monooxygenase System Enables Degradation of Synthetic Dialkyl Ethers in Aquincola tertiaricarbonis L108
Authors: Schuster Judith , Purswani Jessica , Breuer Uta , Pozo Clementina , Harms Hauke , Mï¿½ller Roland H. , Rohwerder Thore
Published: January 2013

In
Rhodococcus ruber
IFP 2001,
Rhodococcus zopfii
IFP 2005, and
Gordonia
sp. strain IFP 2009, the cytochrome P450 monooxygenase EthABCD catalyzes hydroxylation of methoxy and ethoxy residues in the fuel oxygenates methyl
tert
-butyl ether (MTBE), ethyl
tert
-butyl ether (ETBE), and
tert
-amyl methyl ether (TAME). The expression of the IS
3
-type transposase-flanked
eth
genes is ETBE dependent and controlled by the regulator EthR (C. Malandain et al., FEMS Microbiol. Ecol. 72:289–296, 2010). In contrast, we demonstrated by reverse transcription-quantitative PCR (RT-qPCR) that the betaproteobacterium
Aquincola tertiaricarbonis
L108, which possesses the
ethABCD
genes but lacks
ethR
, constitutively expresses the P450 system at high levels even when growing on nonether substrates, such as glucose. The mutant strain
A. tertiaricarbonis
L10, which is unable to degrade dialkyl ethers, resulted from a transposition event mediated by a rolling-circle IS
91
-type element flanking the
eth
gene cluster in the wild-type strain L108. The constitutive expression of Eth monooxygenase is likely initiated by the housekeeping sigma factor σ
70
, as indicated by the presence in strain L108 of characteristic −10 and −35 binding sites upstream of
ethA
which are lacking in strain IFP 2001. This enables efficient degradation of diethyl ether, diisopropyl ether, MTBE, ETBE, TAME, and
tert
-amyl ethyl ether (TAEE) without any lag phase in strain L108. However, ethers with larger residues,
n
-hexyl methyl ether, tetrahydrofuran, and alkyl aryl ethers, were not attacked by the Eth system at significant rates in resting-cell experiments, indicating that the residue in the ether molecule which is not hydroxylated also contributes to the determination of substrate specificity.
Cytochrome P450 Initiates Degradation ofcis-Dichloroethene by Polaromonas sp. Strain JS666
Authors: Nishino Shirley F. , Shin Kwanghee A. , Gossett James M. , Spain Jim C.
Published: January 2013

Polaromonassp. strain JS666 grows oncis-1,2-dichoroethene (cDCE) as the sole carbon and energy source under aerobic conditions, but the degradation mechanism and the enzymes involved are unknown. In this study, we established the complete pathway forcDCE degradation through heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. Several lines of evidence indicate that a cytochrome P450 monooxygenase catalyzes the initial step ofcDCE degradation. Both the transient accumulation of dichloroacetaldehyde incDCE-degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 support a pathway for degradation ofcDCE through dichloroacetaldehyde. The mechanism minimizes the formation ofcDCE epoxide. The molecular phylogeny of the cytochrome P450 gene and the organization of neighboring genes suggest that thecDCE degradation pathway recently evolved in a progenitor capable of degrading 1,2-dichloroethane either by the recruitment of the cytochrome P450 monooxygenase gene from an alkane catabolic pathway or by selection for variants of the P450 in a preexisting 1,2-dichloroethane catabolic pathway. The results presented here add yet another role to the broad array of productive reactions catalyzed by cytochrome P450 enzymes.
Homeostasis and Catabolism of Choline and Glycine Betaine: Lessons from Pseudomonas aeruginosa
Authors: Wargo Matthew J.
Published: January 2013

Most sequenced bacteria possess mechanisms to import choline and glycine betaine (GB) into the cytoplasm. The primary role of choline in bacteria appears to be as the precursor to GB, and GB is thought to primarily act as a potent osmoprotectant. Choline and GB may play accessory roles in shaping microbial communities, based on their limited availability and ability to enhance survival under stress conditions. Choline and GB enrichment near eukaryotes suggests a role in the chemical relationships between these two kingdoms, and some of these interactions have been experimentally demonstrated. While many bacteria can convert choline to GB for osmoprotection, a variety of soil- and water-dwelling bacteria have catabolic pathways for the multistep conversion of choline, via GB, to glycine and can thereby use choline and GB as sole sources of carbon and nitrogen. In these choline catabolizers, the GB intermediate represents a metabolic decision point to determine whether GB is catabolized or stored as an osmo- and stress protectant. This minireview focuses on this decision point inPseudomonas aeruginosa, which aerobically catabolizes choline and can use GB as an osmoprotectant and a nutrient source.P. aeruginosais an experimentally tractable and ecologically relevant model to study the regulatory pathways controlling choline and GB homeostasis in choline-catabolizing bacteria. The study ofP. aeruginosaassociations with eukaryotes and other bacteria also makes this a powerful model to study the impact of choline and GB, and their associated regulatory and catabolic pathways, on host-microbe and microbe-microbe relationships.
Genome Instability in Lactobacillus rhamnosus GG
Authors: Sybesma Wilbert , Molenaar Douwe , van IJcken Wilfred , Venema Koen , Kort Remco
Published: January 2013

We describe here a comparative genome analysis of three dairy product isolates of
Lactobacillus rhamnosus
GG (LGG) and the ATCC 53103 reference strain to the published genome sequence of
L. rhamnosus
GG. The analysis showed that in two of three isolates, major DNA segments were missing from the genomic islands LGGISL1,2. The deleted DNA segments consist of 34 genes in one isolate and 84 genes in the other and are flanked by identical insertion elements. Among the missing genes are the
spaCBA
genes, which encode pilin subunits involved in adhesion to mucus and persistence of the strains in the human intestinal tract. Subsequent quantitative PCR analyses of six commercial probiotic products confirmed that two more products contain a heterogeneous population of
L. rhamnosus
GG variants, including genotypes with or without
spaC
. These results underline the relevance for quality assurance and control measures targeting genome stability in probiotic strains and justify research assessing the effect of genetic rearrangements in probiotics on the outcome of
in vitro
and
in vivo
efficacy studies.
Influence of Therapeutic Ceftiofur Treatments of Feedlot Cattle on Fecal and Hide Prevalences of Commensal Escherichia coli Resistant to Expanded-Spectrum Cephalosporins, and Molecular Characterization of Resistant Isolates
Authors: Schmidt John W. , Griffin Dee , Kuehn Larry A. , Brichta-Harhay Dayna M.
Published: January 2013

In the United States, the
bla
CMY-2
gene contained within incompatibility type A/C (IncA/C) plasmids is frequently identified in extended-spectrum-cephalosporin-resistant (ESC
r
)
Escherichia coli
strains from both human and cattle sources. Concerns have been raised that therapeutic use of ceftiofur in cattle may increase the prevalence of ESC
r
E. coli
. We report that herd ESC
r
E. coli
fecal and hide prevalences throughout the residency of cattle at a feedlot, including during the period of greatest ceftiofur use at the feedlot, were either not significantly different (
P
≥ 0.05) or significantly less (
P
< 0.05) than the respective prevalences at arrival. Longitudinal sampling of cattle treated with ceftiofur demonstrated that once the transient increase of ESC
r
E. coli
shedding that follows ceftiofur injection abated, ceftiofur-injected cattle were no more likely than untreated members of the same herd to shed ESC
r
E. coli
. Pulsed-field gel electrophoresis (PFGE) genotyping, antibiotic resistance phenotyping, screening for presence of the
bla
CMY-2
gene, and plasmid replicon typing were performed on 312 ESC
r
E. coli
isolates obtained during six sampling periods spanning the 10-month residence of cattle at the feedlot. The identification of only 26 unique PFGE genotypes, 12 of which were isolated during multiple sampling periods, suggests that clonal expansion of feedlot-adapted
bla
CMY-2
E. coli
strains contributed more to the persistence of
bla
CMY-2
than horizontal transfer of IncA/C plasmids between
E. coli
strains at this feedlot. We conclude that therapeutic use of ceftiofur at this cattle feedlot did not significantly increase the herd prevalence of ESC
r
E. coli
.
Identification of Homophenylalanine Biosynthetic Genes from the Cyanobacterium Nostoc punctiforme PCC73102 and Application to Its Microbial Production by Escherichia coli
Authors: Koketsu Kento , Mitsuhashi Satoshi , Tabata Kazuhiko
Published: January 2013

l-Homophenylalanine (
l
-Hph) is a useful chiral building block for synthesis of several drugs, including angiotensin-converting enzyme inhibitors and the novel proteasome inhibitor carfilzomib. While the chemoenzymatic route of synthesis is fully developed, we investigated microbial production of
l
-Hph to explore the possibility of a more efficient and sustainable approach to
l
-Hph production. We hypothesized that
l
-Hph is synthesized from
l
-Phe via a mechanism homologous to 3-methyl-2-oxobutanoic acid conversion to 4-methyl-2-oxopentanoic acid during leucine biosynthesis. Based on bioinformatics analysis, we found three putative homophenylalanine biosynthesis genes,
hphA
(Npun_F2464),
hphB
(Npun_F2457), and
hphCD
(Npun_F2458), in the cyanobacterium
Nostoc punctiforme
PCC73102, located around the gene cluster responsible for anabaenopeptin biosynthesis. We constructed
Escherichia coli
strains harboring
hphABCD
-expressing plasmids and achieved the fermentative production of
l
-Hph from
l
-Phe. To our knowledge, this is the first identification of the genes responsible for homophenylalanine synthesis in any organism. Furthermore, to improve the low conversion efficiency of the initial strain, we optimized the expression of
hphA
,
hphB
, and
hphCD
, which increased the yield to ∼630 mg/liter. The
l
-Hph biosynthesis and
l
-Leu biosynthesis genes from
E. coli
were also compared. This analysis revealed that HphB has comparatively relaxed substrate specificity and can perform the function of LeuB, but HphA and HphCD show tight substrate specificity and cannot complement the LeuA and LeuC/LeuD functions, and vice versa. Finally, the range of substrate tolerance of the
l
-Hph-producing strain was examined, which showed that
m
-fluorophenylalanine,
o
-fluorophenylalanine, and
l
-tyrosine were accepted as substrates and that the corresponding homoamino acids were generated.
Direct Assessment of Metabolite Utilization by Pseudomonas aeruginosa during Growth on Artificial Sputum Medium
Authors: Behrends Volker , Geier Benedikt , Williams Huw D. , Bundy Jacob G.
Published: January 2013

We grew
Pseudomonas aeruginosa
in LB and artificial sputum medium (ASM) (filtered and unfiltered) and quantified metabolite utilization and excretion by nuclear magnetic resonance (NMR) spectroscopy (metabolic footprinting or extracellular metabolomics). Utilization rates were similar between media, but there were differences in excretion—e.g., acetate was produced only in unfiltered ASM.
Stochasticity in Colonial Growth Dynamics of Individual Bacterial Cells
Authors: Koutsoumanis Konstantinos P. , Lianou Alexandra
Published: January 2013

Conventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells of
Salmonella enterica
serotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (μ
max
) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (
N
0
). For bacterial populations with
N
0
of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations.
Photosynthetic Bradyrhizobium sp. Strain ORS285 Is Capable of Forming Nitrogen-Fixing Root Nodules on Soybeans (Glycine max)
Authors: Giraud Eric , Xu Lei , Chaintreuil Clï¿½mence , Gargani Daniel , Gully Djamel , Sadowsky Michael J.
Published: January 2013

The ability of photosynthetic
Bradyrhizobium
strains ORS285 and ORS278 to nodulate soybeans was investigated. While the
nod
gene-deficient ORS278 strain induced bumps only on soybean roots, the
nod
gene-containing ORS285 strain formed nitrogen-fixing nodules. However, symbiotic efficiencies differed drastically depending on both the soybean genotype used and the culture conditions tested.
A Hydrolase from Lactobacillus sakei Moonlights as a Transaminase
Authors: Sinz Quirin , Freiding Simone , Vogel Rudi F. , Schwab Wilfried
Published: January 2013

Enzymatic transamination of amino acids yields α-keto acids and is the initial step for the production of volatile compounds that contribute to the sensory perception of fermented foods such as salami.
Lactobacillus sakei
is one of the lactic acid bacterial strains commonly used in starter cultures. Although the genome sequence of
L. sakei
23K lacks genes encoding typical branched-chain amino acid transaminases, transamination activity and the formation of amino acid-derived volatile metabolites could be demonstrated. A protein purified from
L. sakei
is held responsible for the transamination activity. By heterologous expression of the corresponding gene in
Escherichia coli
, we were able to characterize the transamination side activity of an enzyme annotated as a putative acylphosphatase (AcP). A transamination side activity of hen egg white lysozyme (HEWL) was also discovered. Both enzymes showed substrate specificity toward branched-chain and aromatic amino acids. AcP also accepted l-methionine. Activity was optimal at neutral pH for both enzymes, whereas AcP showed a significantly higher temperature optimum (55ï¿½C) than that of HEWL (37ï¿½C). Kinetic parameters revealed high affinity toward
l
-leucine for AcP (

K
m

= 1.85 mM) and toward
l
-isoleucine for HEWL (

K
m

= 3.79 mM). AcP seems to play a major role in the metabolism of amino acids in
L. sakei
.
High Frequency of Virulence Factor Genes
tdh
,
trh
, and
tlh
in Vibrio parahaemolyticus Strains Isolated from a Pristine Estuary
Authors: Gutierrez West Casandra K. , Klein Savannah L. , Lovell Charles R.
Published: January 2013

Virulence factor genes encoding the thermostable direct hemolysin (
tdh
) and the thermostable direct hemolysin-related hemolysin (
trh
) are strongly correlated with virulence of the emergent human pathogen
Vibrio parahaemolyticus
. The gene encoding the thermolabile hemolysin (
tlh
) is also considered a signature molecular marker for the species. These genes are typically reported in very low percentages (1 to 2%) of nonclinical strains.
V. parahaemolyticus
strains were isolated from various niches within a pristine estuary (North Inlet, SC) and were screened for these genes using both newly designed PCR primers and more commonly used primers. DNA sequences of
tdh
and
trh
were recovered from 48% and 8.3%, respectively, of these North Inlet strains. The recovery of pathogenic
V. parahaemolyticus
strains in such high proportions from an estuarine ecosystem that is virtually free of anthropogenic influences indicates the potential for additional, perhaps environmental roles of the
tdh
and
trh
genes.
Glucose Triggers ATP Secretion from Bacteria in a Growth-Phase-Dependent Manner
Authors: Hironaka Ippei , Iwase Tadayuki , Sugimoto Shinya , Okuda Ken-ichi , Tajima Akiko , Yanaga Katsuhiko , Mizunoe Yoshimitsu
Published: January 2013

ATP modulates immune cell functions, and ATP derived from gut commensal bacteria promotes the differentiation of T helper 17 (Th17) cells in the intestinal lamina propria. We recently reported that
Enterococcus gallinarum
, isolated from mice and humans, secretes ATP. We have since found and characterized several ATP-secreting bacteria. Of the tested enterococci,
Enterococcus mundtii
secreted the greatest amount of ATP (>2 μM/10
8
cells) after overnight culture. Glucose, not amino acids and vitamins, was essential for ATP secretion from
E. mundtii
. Analyses of energy-deprived cells demonstrated that glycolysis is the most important pathway for bacterial ATP secretion. Furthermore, exponential-phase
E. mundtii
and
Enterococcus faecalis
cells secrete ATP more efficiently than stationary-phase cells. Other bacteria, including
Pseudomonas aeruginosa
,
Escherichia coli
, and
Staphylococcus aureus
, also secrete ATP in exponential but not stationary phase. These results suggest that various gut bacteria, including commensals and pathogens, might secrete ATP at any growth phase and modulate immune cell function.
Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols
Authors: Wu Xi , Zhang Chong , Orita Izumi , Imanaka Tadayuki , Fukui Toshiaki , Xing Xin-Hui
Published: January 2013

A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon
Thermococcus kodakarensis
KOD1 (
Tk
ADH). The gene,
tk0845
, which encodes an aldo-keto reductase, was heterologously expressed in
Escherichia coli
. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90ï¿½C and a half-life of 4.5 h at 95ï¿½C. The apparent

K
m

values for the cofactors NAD(P)
+
and NADPH were similar within a range of 66 to 127 μM.
Tk
ADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the
meta
and
para
positions with high enantioselectivity, yielding the corresponding (
R
)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee).
Tk
ADH could also reduce 2,2,2-trifluoroacetophenone to (
R
)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H
2
O–20% 2-propanol and H
2
O–50%
n
-hexane or
n
-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols.
Microfluidic Chip-Based Detection and Intraspecies Strain Discrimination of Salmonella Serovars Derived from Whole Blood of Septic Mice
Authors: Patterson Adriana S. , Heithoff Douglas M. , Ferguson Brian S. , Soh H. Tom , Mahan Michael J. , Plaxco Kevin W.
Published: January 2013

Salmonellais a zoonotic pathogen that poses a considerable public health and economic burden in the United States and worldwide. Resultant human diseases range from enterocolitis to bacteremia to sepsis and are acutely dependent on the particular serovar ofSalmonella entericasubsp.enterica, which comprises over 99% of human-pathogenicS. entericaisolates. Point-of-care methods for detection and strain discrimination ofSalmonellaserovars would thus have considerable benefit to medical, veterinary, and field applications that safeguard public health and reduce industry-associated losses. Here we describe a single, disposable microfluidic chip that supports isothermal amplification and sequence-specific detection and discrimination ofSalmonellaserovars derived from whole blood of septic mice. The integrated microfluidic electrochemical DNA (IMED) chip consists of an amplification chamber that supports loop-mediated isothermal amplification (LAMP), a rapid, single-temperature amplification method as an alternative to PCR that offers advantages in terms of sensitivity, reaction speed, and amplicon yield. The amplification chamber is connected via a microchannel to a detection chamber containing a reagentless, multiplexed (here biplex) sensing array for sequence-specific electrochemical DNA (E-DNA) detection of the LAMP products. Validation of the IMED device was assessed by the detection and discrimination ofS. entericasubsp.entericaserovars Typhimurium and Choleraesuis, the causative agents of enterocolitis and sepsis in humans, respectively. IMED chips conferred rapid (under 2 h) detection and discrimination of these strains at clinically relevant levels (<1,000 CFU/ml) from whole, unprocessed blood collected from septic animals. The IMED-based chip assay shows considerable promise as a rapid, inexpensive, and portable point-of-care diagnostic platform for the detection and strain-specific discrimination of microbial pathogens.
Frequent Occurrence of Extended-Spectrum Beta-Lactamase- and Transferable AmpC Beta-Lactamase-Producing Escherichia coli on Domestic Chicken Meat in Sweden
Authors: Bï¿½rjesson Stefan , Egervï¿½rn Maria , Lindblad Mats , Englund Stina
Published: January 2013

Forty-four percent of Swedish chicken meat fillets were contaminated with extended-spectrum or transferable AmpC beta-lactamase-producing
Escherichia coli
strains. Isolates from Swedish chicken meat and broilers were closely related to isolates from chicken meat imported into Sweden; these results indicate a common source of the contamination.
Distinct and Redundant Roles of the Two MYST Histone Acetyltransferases Esa1 and Sas2 in Cell Growth and Morphogenesis of Candida albicans
Authors: Wang Xiongjun , Chang Peng , Ding Jianping , Chen Jiangye
Published: January 2013

Candida albicans
is associated with humans, as both a harmless commensal organism and a pathogen. Adaption to human body temperature is extremely important for its growth and morphogenesis.
Saccharomyces cerevisiae
Esa1, a member of the MYST family HATs (histone acetyltransferases) and the catalytic subunit of the NuA4 complex, and its homologues in other eukaryotes have been shown to be essential for cell growth. To investigate the functional roles of two MYST family HATs, Esa1 and Sas2 in
C. albicans
, we deleted
ESA1
and
SAS2
in the
C. albicans
genome and performed cell growth analyses. Our results demonstrated that
C. albicans
Esa1 is not essential for general growth but is essential for filamentous growth. The
esa1/esa1
mutant cells exhibited sensitivity to thermal, genotoxic, and oxidative stresses but tolerance to cold, osmotic, and cell wall stresses. In contrast, the
sas2
/
sas2
mutant adapted to growth at higher temperatures and promoted filament formation at lower temperatures, resembling the phenotype of a
C. albicans
strain overexpressing
ESA1
. Cells with deletions of both
ESA1
and
SAS2
were inviable, reflecting the functional redundancy in cell growth.
C. albicans
Esa1 and Sas2 have distinct and synergistic effects on histone acetylation at H4K5, H4K12, and H4K16. Esa1 contributes mainly to acetylation of H4K5 and H4K12, whereas Sas2 contributes to acetylation of H4K16. Our findings suggest that
C. albicans
Esa1 and Sas2 play opposite roles in cell growth and morphogenesis and contribute coordinately to histone acetylation and gene regulation.
Effects of a Defective Endoplasmic Reticulum-Associated Degradation Pathway on the Stress Response, Virulence, and Antifungal Drug Susceptibility of the Mold Pathogen Aspergillus fumigatus
Authors: Krishnan Karthik , Feng Xizhi , Powers-Fletcher Margaret V. , Bick Gregory , Richie Daryl L. , Woollett Laura A. , Askew David S.
Published: January 2013

Proteins that are destined for release outside the eukaryotic cell, insertion into the plasma membrane, or delivery to intracellular organelles are processed and folded in the endoplasmic reticulum (ER). An imbalance between the level of nascent proteins entering the ER and the organelle's ability to manage that load results in the accumulation of unfolded proteins. Terminally unfolded proteins are disposed of by ER-associated degradation (ERAD), a pathway that transports the aberrant proteins across the ER membrane into the cytosol for proteasomal degradation. The ERAD pathway was targeted in the mold pathogen
Aspergillus fumigatus
by deleting the
hrdA
gene, encoding the
A. fumigatus
ortholog of Hrd1, the E3 ubiquitin ligase previously shown to contribute to ERAD in other species. Loss of HrdA was associated with impaired degradation of a folding-defective ERAD substrate, CPY*, as well as activation of the unfolded-protein response (UPR). The Δ
hrdA
mutant showed resistance to voriconazole and reduced thermotolerance but was otherwise unaffected by a variety of environmental stressors. A double-deletion mutant deficient in both HrdA and another component of the same ERAD complex, DerA, was defective in secretion and showed hypersensitivity to ER, thermal, and cell wall stress. However, the Δ
hrdA
Δ
derA
mutant remained virulent in mouse and insect infection models. These data demonstrate that HrdA and DerA support complementary ERAD functions that promote survival under conditions of ER stress but are dispensable for virulence in the host environment.
Reduction of Ribosome Level Triggers Flocculation of Fission Yeast Cells
Authors: Li Rongpeng , Li Xuesong , Sun Lei , Chen Feifei , Liu Zhenxing , Gu Yuyu , Gong Xiaoyan , Liu Zhonghua , Wei Hua , Huang Ying , Yuan Sheng
Published: January 2013

Deletion of ribosomal protein L32 genes resulted in a nonsexual flocculation of fission yeast. Nonsexual flocculation also occurred when two other ribosomal protein genes,
rpl21-2
and
rpl9-2
, were deleted. However, deletion of two nonribosomal protein genes,
mpg
and
fbp
, did not cause flocculation. Overall transcript levels of
rpl32
in
rpl32-1
Δ and
rpl32-2
Δ cells were reduced by 35.9% and 46.9%, respectively, and overall ribosome levels in
rpl32-1
Δ and
rpl32-2
Δ cells dropped 31.1% and 27.8%, respectively, compared to wild-type cells. Interestingly, ribosome protein expression levels and ribosome levels were also reduced greatly in sexually flocculating diploid YHL6381/WT (
h
+
/h
−
) cells compared to a mixture of YHL6381 (
h
+
) and WT (
h
−
) nonflocculating haploid cells. Transcriptome analysis indicated that the reduction of ribosomal levels in sexual flocculating cells was caused by more-extensive suppression of ribosomal biosynthesis gene expression, while the reduction of ribosomal levels caused by deleting ribosomal protein genes in nonsexual flocculating cells was due to an imbalance between ribosomal proteins. We propose that once the reduction of ribosomal levels is below a certain threshold value, flocculation is triggered.
Conserved Gene Regulatory Function of the Carboxy-Terminal Domain of Dictyostelid C-Module-Binding Factor
Authors: Schmith Anika , Groth Marco , Ratka Josephine , Gatz Sara , Spaller Thomas , Siol Oliver , Glöckner Gernot , Winckler Thomas
Published: January 2013

C-module-binding factor A (CbfA) is a jumonji-type transcription regulator that is important for maintaining the expression and mobility of the retrotransposable element TRE5-A in the social amoeba
Dictyostelium discoideum
. CbfA-deficient cells have lost TRE5-A retrotransposition, are impaired in the ability to feed on bacteria, and do not enter multicellular development because of a block in cell aggregation. In this study, we performed Illumina RNA-seq of growing CbfA mutant cells to obtain a list of CbfA-regulated genes. We demonstrate that the carboxy-terminal domain of CbfA alone is sufficient to mediate most CbfA-dependent gene expression. The carboxy-terminal domain of CbfA from the distantly related social amoeba
Polysphondylium pallidum
restored the expression of CbfA-dependent genes in the
D. discoideum
CbfA mutant, indicating a deep conservation in the gene regulatory function of this domain in the dictyostelid clade. The CbfA-like protein CbfB displays ∼25% sequence identity with CbfA in the amino-terminal region, which contains a JmjC domain and two zinc finger regions and is thought to mediate chromatin-remodeling activity. In contrast to CbfA proteins, where the carboxy-terminal domains are strictly conserved in all dictyostelids, CbfB proteins have completely unrelated carboxy-terminal domains. Outside the dictyostelid clade, CbfA-like proteins with the CbfA-archetypical JmjC/zinc finger arrangement and individual carboxy-terminal domains are prominent in filamentous fungi but are not found in yeasts, plants, and metazoans. Our data suggest that two functional regions of the CbfA-like proteins evolved at different rates to allow the occurrence of species-specific adaptation processes during genome evolution.
Two Roles for Aconitase in the Regulation of Tricarboxylic Acid Branch Gene Expression in Bacillus subtilis
Authors: Pechter Kieran B. , Meyer Frederik M. , Serio Alisa W. , Stülke Jörg , Sonenshein Abraham L.
Published: January 2013

Previously, it was shown that an aconitase (
citB
) null mutation results in a vast overaccumulation of citrate in the culture fluid of growing
Bacillus subtilis
cells, a phenotype that causes secondary effects, including the hyperexpression of the
citB
promoter.
B. subtilis
aconitase is a bifunctional protein; to determine if either or both activities of aconitase were responsible for this phenotype, two strains producing different mutant forms of aconitase were constructed, one designed to be enzymatically inactive (C450S [
citB2
]) and the other designed to be defective in RNA binding (R741E [
citB7
]). The
citB2
mutant was a glutamate auxotroph and accumulated citrate, while the
citB7
mutant was a glutamate prototroph. Unexpectedly, the
citB7
strain also accumulated citrate. Both mutant strains exhibited overexpression of the
citB
promoter and accumulated high levels of aconitase protein. These strains and the
citB
null mutant also exhibited increased levels of citrate synthase protein and enzyme activity in cell extracts, and the major citrate synthase (
citZ
) transcript was present at higher-than-normal levels in the
citB
null mutant, due at least in part to a >3-fold increase in the stability of the
citZ
transcript compared to the wild type. Purified
B. subtilis
aconitase bound to the
citZ
5′ leader RNA
in vitro
, but the mutant proteins did not. Together, these data suggest that wild-type aconitase binds to and destabilizes the
citZ
transcript in order to maintain proper cell homeostasis by preventing the overaccumulation of citrate.
Regulation, Integrase-Dependent Excision, and Horizontal Transfer of Genomic Islands in Legionella pneumophila
Authors: Lautner Monika , Schunder Eva , Herrmann Vroni , Heuner Klaus
Published: January 2013

Legionella pneumophila
is a Gram-negative freshwater agent which multiplies in specialized nutrient-rich vacuoles of amoebae. When replicating in human alveolar macrophages,
Legionella
can cause Legionnaires' disease. Recently, we identified a new type of conjugation/type IVA secretion system (T4ASS) in
L. pneumophila
Corby (named
trb
-
tra
). Analogous versions of
trb
-
tra
are localized on the genomic islands Trb-1 and Trb-2. Both can exist as an episomal circular form, and Trb-1 can be transferred horizontally to other
Legionella
strains by conjugation. In our current work, we discovered the importance of a site-specific integrase (Int-1,
lpc2818
) for the excision and conjugation process of Trb-1. Furthermore, we identified the genes
lvrRABC
(
lpc2813
to
lpc2816
) to be involved in the regulation of Trb-1 excision. In addition, we demonstrated for the first time that a
Legionella
genomic island (LGI) of
L. pneumophila
Corby (LpcGI-2) encodes a functional type IV secretion system. The island can be transferred horizontally by conjugation and is integrated site specifically into the genome of the transconjugants. LpcGI-2 generates three different episomal forms. The predominant episomal form, form A, is generated integrase dependently (Lpc1833) and transferred by conjugation in a
pilT
-dependent manner. Therefore, the genomic islands Trb-1 and LpcGI-2 should be classified as integrative and conjugative elements (ICEs). Coculture studies of
L. pneumophila
wild-type and mutant strains revealed that the
int-1
and
lvrRABC
genes (located on Trb-1) as well as
lpc1833
and
pilT
(located on LpcGI-2) do not influence the
in vivo
fitness of
L. pneumophila
in
Acanthamoeba castellanii
.
Streptococcus pneumoniae Folate Biosynthesis Responds to Environmental CO2Levels
Authors: Burghout Peter , Zomer Aldert , van der Gaast-de Jongh Christa E. , Janssen-Megens Eva M. , Françoijs Kees-Jan , Stunnenberg Hendrik G. , Hermans Peter W. M.
Published: January 2013

Although carbon dioxide (CO2) is known to be essential forStreptococcus pneumoniaegrowth, it is poorly understood how this respiratory tract pathogen adapts to the large changes in environmental CO2levels it encounters during transmission, host colonization, and disease. To identify the molecular mechanisms that facilitate pneumococcal growth under CO2-poor conditions, we generated a randomS. pneumoniaeR6marinertransposon mutant library representing mutations in 1,538 different genes and exposed it to CO2-poor ambient air. With Tn-seq, we found mutations in two genes that were involved inS. pneumoniaeadaptation to changes in CO2availability. The genepca, encoding pneumococcal carbonic anhydrase (PCA), was absolutely essential forS. pneumoniaegrowth under CO2-poor conditions. PCA catalyzes the reversible hydration of endogenous CO2to bicarbonate (HCO3−) and was previously demonstrated to facilitate HCO3−-dependent fatty acid biosynthesis. The genefolCthat encodes the dihydrofolate/folylpolyglutamate synthase was required at the initial phase of bacterial growth under CO2-poor culture conditions. FolC compensated for the growth-phase-dependent decrease inS. pneumoniaeintracellular long-chain (n> 3) polyglutamyl folate levels, which was most pronounced under CO2-poor growth conditions. In conclusion,S. pneumoniaeadaptation to changes in CO2availability involves the retention of endogenous CO2and the preservation of intracellular long-chain polyglutamyl folate pools.
The HtrA Protease of Streptococcus pneumoniae Controls Density-Dependent Stimulation of the Bacteriocin
blp
Locus via Disruption of Pheromone Secretion
Authors: Kochan Travis J. , Dawid Suzanne
Published: January 2013

All fully sequenced strains of
Streptococcus pneumoniae
(pneumococcus) contain a version of the
blp
locus which is responsible for the regulation and secretion of a variable repertoire of pneumococcal bacteriocins called pneumocins and their associated immunity proteins. Pneumocins mediate intra- and interspecies competition
in vitro
and have been shown to provide a competitive advantage
in vivo
. Pneumocin production is stimulated by extracellular accumulation of the peptide pheromone, BlpC. Both BlpC and the functional pneumocins are secreted out of the cell via the Blp transporter, BlpAB. The conserved surface-expressed serine protease, HtrA, has been shown to limit activation of the locus and secretion of functional pneumocins. In this work, we demonstrate that
htrA
mutants stimulate the
blp
locus at lower cell density and to a greater extent than strains expressing wild-type HtrA. This effect is not due to direct proteolytic degradation of secreted pheromone by the protease, but instead is a result of HtrA-mediated disruption of peptide processing and secretion. Because pneumocins are secreted through the same transporter as the pheromone, this finding explains why pheromone supplementation cannot completely restore pneumocin inhibition to strains expressing high levels of HtrA despite restoration of
blp
transcriptional activity. HtrA restricts pneumocin production to high cell density by limiting the rate of accumulation of BlpC in the environment. Importantly, HtrA does not interfere with the ability of a strain to sense environmental pheromones, which is necessary for the induction of protective immunity in the face of pneumocin-secreting competitors.
Identification of the Mutation Responsible for the Temperature-Sensitive Lipopolysaccharide O-Antigen Defect in the Pseudomonas aeruginosa Cystic Fibrosis Isolate 2192
Authors: Davis Michael R. , Muszyński Artur , Lollett Ivonne V. , Pritchett Christopher L. , Carlson Russell W. , Goldberg Joanna B.
Published: January 2013

Pseudomonas aeruginosa
in the lungs of cystic fibrosis (CF) patients is characterized by a series of genotypic and phenotypic changes that reflect the transition from acute to chronic infection. These include the overproduction of the exopolysaccharide alginate and the loss of complete lipopolysaccharide (LPS). LPS is a major component of the Gram-negative outer membrane and is composed of lipid A, core oligosaccharide, and O antigen. In this report, we show that the LPS defect of the
P. aeruginosa
chronic infection isolate 2192 is temperature sensitive. When grown at 25°C, 2192 expresses serotype O1 LPS with a moderate chain length and in reduced amounts relative to those of a wild-type serotype O1 laboratory strain (stO1). In contrast, 2192 expresses no LPS O antigen when grown at 37°C. This is the first time that a temperature-sensitive defect in O-antigen production has been reported. Using complementation analyses with a constructed
wbpM
deletion mutant of stO1, we demonstrate that the temperature-sensitive O-antigen production defect in 2192 is due to a mutation in
wbpM
, which encodes a UDP-4,6-GlcNAc dehydratase involved in O-antigen synthesis. The mutation, a deletion of a single amino acid (V636) from the extreme C terminus of WbpM, renders the protein less stable than its wild-type counterpart. This residue of WbpM, which is critical for stability and function, is located outside of the recognized domains of the protein and may provide insight into the structure-function relationship of this enzyme, which is found in all 20 serotypes of
P. aeruginosa
. We also identify a promoter of
wbpM
, map a transcriptional start site of
wbpM
, and show that mucoidy plays a role in the loss of expression of high-molecular-weight LPS in this CF isolate.
Biochemical Properties of MutT2 Proteins from Mycobacterium tuberculosis and M. smegmatis and Their Contrasting Antimutator Roles in Escherichia coli
Authors: Sang Pau Biak , Varshney Umesh
Published: January 2013

Mycobacterium tuberculosis
, the causative agent of tuberculosis, is at increased risk of accumulating damaged guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP because of its residency in the oxidative environment of the host macrophages. By hydrolyzing the oxidized guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role in allowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we purified recombinant
M. tuberculosis
MutT2 (
Mtu
MutT2) and
M. smegmatis
MutT2 (
Msm
MutT2) proteins from
M. tuberculosis
(a slow grower) and
M. smegmatis
(fast growing model mycobacteria), respectively, for their biochemical characterization. Distinct from the
Escherichia coli
MutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (

K
m

and
V
max
) revealed that while
Mtu
MutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP,
Msm
MutT2 hydrolyzes them nearly equally. Also,
Msm
MutT2 is about 14 times more efficient than
Mtu
MutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP. Consistent with these observations,
Msm
MutT2 but not
Mtu
MutT2 rescues
E. coli
for MutT deficiency by decreasing both the mutation frequency and A-to-C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins.
AmrZ Modulates Pseudomonas aeruginosa Biofilm Architecture by Directly Repressing Transcription of thepslOperon
Authors: Jones Christopher J. , Ryder Cynthia R. , Mann Ethan E. , Wozniak Daniel J.
Published: January 2013

Pseudomonas aeruginosastrains recovered from chronic pulmonary infections in cystic fibrosis patients are frequently mucoid. Such strains express elevated levels of alginate but reduced levels of the aggregative polysaccharide Psl; however, the mechanistic basis for this regulation is not completely understood. ElevatedpslAexpression was observed in anamrZnull mutant and in strains expressing a DNA-binding-deficient AmrZ. AmrZ is a transcription factor that positively regulates twitching motility and alginate synthesis, two phenotypes involved inP. aeruginosabiofilm development. AmrZ bound directly to thepslApromoterin vitro, and molecular analyses indicate that AmrZ repressespslexpression by binding to a site overlapping the promoter. Altered expression ofamrZin nonmucoid strains impacted biofilm structure and architecture, as structured microcolonies were observed with low AmrZ production and flat biofilms withamrZoverexpression. These biofilm phenotypes correlated with Psl levels, since we observed elevated Psl production inamrZmutants and lower Psl production inamrZ-overexpressing strains. These observations support the hypothesis that AmrZ is a multifunctional regulator mediating transition ofP. aeruginosabiofilm infections from colonizing to chronic biofilms through repression of thepsloperon while activating thealgDoperon.
GenR, an IclR-Type Regulator, Activates and Represses the Transcription ofgenGenes Involved in 3-Hydroxybenzoate and Gentisate Catabolism in Corynebacterium glutamicum
Authors: Chao Hongjun , Zhou Ning-Yi
Published: January 2013

The genes required for 3-hydroxybenzoate and gentisate catabolism inCorynebacterium glutamicumare closely clustered in three operons. GenR, an IclR-type regulator, can activate the transcription ofgenKHandgenDFMoperons in response to 3-hydroxybenzoate and gentisate, and it can repress its own expression. Footprinting analyses demonstrated that GenR bound to four sites with different affinities. Two GenR-binding sites (DFMn01 and DFMn02) were found to be located between positions −41 and −84 upstream of the −35 and −10 regions of thegenDFMpromoter, which was involved in positive regulation ofgenDFMtranscription. The GenR binding site R-KHn01 (located between positions −47 and −16) overlapped the −35 region of thegenKHpromoter sequence and is involved in positive regulation of its transcription. The binding site R-KHn02, at which GenR binds to its own promoter, was found within a footprint extending from position −44 to −67. It appeared to be involved in negative regulation of the activity of thegenRpromoter. A consensus motif with a 5-bp imperfect palindromic sequence [ATTCC-N7(5)-GGAAT] was identified among all four GenR binding sites and found to be necessary to GenR regulation through site-directed mutagenesis. The results reveal a new regulatory function of the IclR family in the catabolism of aromatic compounds.
Right-sided infective endocarditis presenting with purpuric skin rash and cardiac failure in a patient without fever
Authors: Shetty Ranjan K , Vivek G , Naha Kushal , Bekkam Sreedivya
Published: January 2013

We describe the case of a 70-year-old woman who presented with symptoms of cardiac failure, and was found on clinical examination to have a purpuric rash over her lower extremities. Further evaluation of cardiac failure revealed tricuspid regurgitation with two large tricuspid valve vegetations. Blood cultures yielded a growth ofStreptococcus viridans. She was treated with an intravenous ceftriaxone and gentamicin and made a complete recovery with resolution of the rash.