bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-Throughput Identification and Validation of In Situ-Expressed Genes of
Lactococcus lactis
Authors: Bachmann Herwig , Kleerebezem Michiel , van Hylckama Vlieg Johan E. T. 
Published: June 2008

ABSTRACT

Understanding the functional response of bacteria to their natural environment is one of the current challenges in microbiology. Over the past decades several techniques have been developed to study gene expression in complex natural habitats. Most of these methods, however, are laborious, and validation of results under in situ conditions is cumbersome. Here we report the improvement of the recombinase-based in vivo expression technology (R-IVET) by the implementation of two additional reporter genes. The first one is an α-galactosidase gene (
melA
), which facilitates the rapid identification of in vivo-induced genes. Second, the bacterial luciferase genes (
luxAB
) are transcriptionally coupled to the resolvase gene, which allows rapid validation and characterization of in vivo-induced genes. The system is implemented and validated in the industrially important lactic acid bacterium
Lactococcus lactis
. We demonstrate the applicability of the advanced R-IVET system by the identification and validation of lactococcal promoter elements that are induced in minimal medium compared to the commonly used rich laboratory medium M17. R-IVET screening led to the identification of 19 promoters that predominantly control expression of genes involved in amino acid and nucleotide metabolism and in transport functions. Furthermore, the luciferase allows high-resolution transcription analysis and enabled the identification of complex medium constituents and specific molecules involved in promoter control. Rapid target validation exemplifies the high-throughput potential of the extended R-IVET system. The system can be applied to other bacterial species, provided that the reporter genes used are functional in the organism of interest.
Exopolysaccharides Produced by IntestinalBifidobacteriumStrains Act as Fermentable Substrates for Human Intestinal Bacteria
Authors: Salazar Nuria , Gueimonde Miguel , Hernández-Barranco Ana María , Ruas-Madiedo Patricia , de los Reyes-Gavilán Clara G. 
Published: June 2008

ABSTRACTEleven exopolysaccharides (EPS) isolated from different human intestinalBifidobacteriumstrains were tested in fecal slurry batch cultures and compared with glucose and the prebiotic inulin for their abilities to act as fermentable substrates for intestinal bacteria. During incubation, the increases in levels of short-chain fatty acids (SCFA) were considerably more pronounced in cultures with EPS, glucose, and inulin than in controls without carbohydrates added, indicating that the substrates assayed were fermented by intestinal bacteria. Shifts in molar proportions of SCFA during incubation with EPS and inulin caused a decrease in the acetic acid-to-propionic acid ratio, a possible indicator of the hypolipidemic effect of prebiotics, with the lowest values for this parameter being obtained for EPS from the speciesBifidobacterium longumand fromBifidobacterium pseudocatenulatumstrain C52. This behavior was contrary to that found with glucose, a carbohydrate not considered to be a prebiotic and for which a clear increase of this ratio was obtained during incubation. Quantitative real-time PCR showed that EPS exerted a moderate bifidogenic effect, which was comparable to that of inulin for some polymers but which was lower than that found for glucose. PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments using universal primers was employed to analyze microbial groups other than bifidobacteria. Changes in banding patterns during incubation with EPS indicated microbial rearrangements ofBacteroidesandEscherichia colirelatives. Moreover, the use of EPS fromB. pseudocatenulatumin fecal cultures from some individuals accounted for the prevalence ofDesulfovibrioandFaecalibacterium prausnitzii, whereas incubation with EPS fromB. longumsupported populations close toAnaerostipes,Prevotella, and/orOscillospira. Thus, EPS synthesized by intestinal bifidobacteria could act as fermentable substrates for microorganisms in the human gut environment, modifying interactions among intestinal populations.
Protein Kinase A Regulates Growth, Sporulation, and Pigment Formation in
Aspergillus fumigatus
Authors: Grosse Christina , Heinekamp Thorsten , Kniemeyer Olaf , Gehrke Alexander , Brakhage Axel A. 
Published: June 2008

ABSTRACT

Aspergillus fumigatus
is an opportunistic human pathogenic fungus causing severe infections in immunocompromised patients. Cyclic AMP (cAMP) signal transduction plays an important role in virulence. A central component of this signaling cascade is protein kinase A (PKA), which regulates cellular processes by phosphorylation of specific target proteins. Here we describe the generation and analysis of
A. fumigatus
mutants expressing the gene encoding the catalytic subunit of PKA,
pkaC1
, under control of an inducible promoter. Strains overexpressing
pkaC1
showed high PKA activity, reduced growth, sporulation deficiency, and formation of a dark pigment in the mycelium. These data indicate that cAMP-PKA signaling is involved in the regulation of important processes, such as growth, asexual reproduction, and biosynthesis of secondary metabolites. Furthermore, elevated PKA activity led to increased expression of the
pksP
gene. The polyketide synthase PksP is an essential enzyme for production of dihydroxynaphthalene-melanin in
A. fumigatus
and contributes to virulence. Our results suggest that increased
pksP
expression is responsible for pigment formation in the mycelium. Comparative proteome analysis of the
pkaC1
-overexpressing strain and the wild-type strain led to the identification of proteins regulated by the cAMP-PKA signal transduction pathway. We showed that elevated PKA activity resulted in activation of stress-associated proteins and of enzymes involved in protein biosynthesis and glucose catabolism. In contrast, proteins which were involved in nucleotide and amino acid biosynthesis were downregulated, as were enzymes involved in catabolism of carbon sources other than glucose.
Borrelia burgdorferi
Sensu Stricto Is Clonal in Patients with Early Lyme Borreliosis
Authors: Hanincová Klára , Liveris Dionysios , Sandigursky Sabina , Wormser Gary P. , Schwartz Ira 
Published: June 2008

ABSTRACT

Lyme borreliosis, the most commonly reported vector-borne disease in North America, is caused by the spirochete
Borrelia burgdorferi
. Given the extensive genetic polymorphism of
B
.
burgdorferi
, elucidation of the population genetic structure of the bacterium in clinical samples may be relevant for understanding disease pathogenesis and may have applicability for the development of diagnostic tests and vaccine preparations. In this investigation, the genetic polymorphism of the 16S-23S rRNA (
rrs
-
rrlA
) intergenic spacer and
ospC
was investigated at the sequence level in 127 clinical isolates obtained from patients with early Lyme borreliosis evaluated in suburban New York City. Sixteen distinct
rrs
-
rrlA
and 16 distinct
ospC
alleles were identified, representing virtually all of the genotypes previously found in questing
Ixodes scapularis
nymphs in this region. In addition, a new
ospC
group was identified in a single patient. The strong linkage observed between the chromosome-located
rrs
-
rrlA
and plasmid-borne
ospC
genes suggests a clonal structure of
B
.
burgdorferi
in these isolates, despite evidence of recombination at
ospC
.
Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine
Authors: Matthies Anastasia , Clavel Thomas , Gütschow Michael , Engst Wolfram , Haller Dirk , Blaut Michael , Braune Annett 
Published: June 2008

ABSTRACT

The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the
Coriobacteriaceae
. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.
Fibrinogen Induces Biofilm Formation by
Streptococcus suis
and Enhances Its Antibiotic Resistance
Authors: Bonifait Laetitia , Grignon Louis , Grenier Daniel 
Published: June 2008

ABSTRACT

In this study, we showed that supplementing the culture medium with fibrinogen induced biofilm formation by
Streptococcus suis
in a dose-dependent manner. Biofilm-grown
S. suis
cells were much more resistant to penicillin G than planktonic cells.
S. suis
bound fibrinogen to its surface, a property that likely contributes to biofilm formation.
Intracellular pH Homeostasis Plays a Role in the Tolerance of
Debaryomyces hansenii
and
Candida zeylanoides
to Acidified Nitrite
Authors: Mortensen Henrik Dam , Jacobsen Tomas , Koch Anette Granly , Arneborg Nils 
Published: June 2008

ABSTRACT

The effects of acidified-nitrite stress on the growth initiation and intracellular pH (pH
i
) of individual cells of
Debaryomyces hansenii
and
Candida zeylanoides
were investigated. Our results show that 200 μg/ml of nitrite caused pronounced growth inhibition and intracellular acidification of
D. hansenii
at an external pH (pH
ex
) value of 4.5 but did not at pH
ex
5.5. These results indicate that nitrous acid as such plays an important role in the antifungal effect of acidified nitrite. Furthermore, both yeast species experienced severe growth inhibition and a pH
i
decrease at pH
ex
4.5, suggesting that at least some of the antifungal effects of acidified nitrite may be due to intracellular acidification. For
C. zeylanoides
, this phenomenon could be explained in part by the uncoupling effect of energy generation from growth.
Debaryomyces hansenii
was more tolerant to acidified nitrite at pH
ex
5.5 than
C. zeylanoides
, as determined by the rate of growth initiation. In combination with the fact that
D. hansenii
was able to maintain pH
i
homeostasis at pH
ex
5.5 but
C. zeylanoides
was not, our results suggest that the ability to maintain pH
i
homeostasis plays a role in the acidified-nitrite tolerance of
D. hansenii
and
C. zeylanoides
. Possible mechanisms underlying the different abilities of the two yeast species to maintain their pH
i
homeostasis during acidified-nitrite stress, comprising the intracellular buffer capacity and the plasma membrane ATPase activity, were investigated, but none of these mechanisms could explain the difference.
Oral Administration of
Lactobacillus
Strains Isolated from Breast Milk as an Alternative for the Treatment of Infectious Mastitis during Lactation
Authors: Jiménez E. , Fernández L. , Maldonado A. , Martín R. , Olivares M. , Xaus J. , Rodríguez J. M. 
Published: June 2008

ABSTRACT

In this study, 20 women with staphylococcal mastitis were randomly divided in two groups. Those in the probiotic group daily ingested 10 log
10
CFU of
Lactobacillus salivarius
CECT5713 and the same quantity of
Lactobacillus gasseri
CECT5714 for 4 weeks, while those in the control one only ingested the excipient. Both lactobacillus strains were originally isolated from breast milk. On day 0, the mean staphylococcal counts in the probiotic and control groups were similar (4.74 and 4.81 log
10
CFU/ml, respectively), but lactobacilli could not be detected. On day 30, the mean staphylococcal count in the probiotic group (2.96 log
10
CFU/ml) was lower than that of the control group (4.79 log
10
CFU/ml).
L. salivarius
CECT5713 and
L. gasseri
CECT5714 were isolated from the milk samples of 6 of the 10 women of the probiotic group. At day 14, no clinical signs of mastitis were observed in the women assigned to the probiotic group, but mastitis persisted throughout the study period in the control group women. In conclusion,
L. salivarius
CECT5713 and
L. gasseri
CECT5714 appear to be an efficient alternative for the treatment of lactational infectious mastitis during lactation.
Molecular Comparison of
Mycobacterium avium
Isolates from Clinical and Environmental Sources
Authors: Hilborn Elizabeth D. , Yakrus Mitchell A. , Covert Terry C. , Harris Stephanie I. , Donnelly Sandra F. , Schmitt Michael T. , Toney Sean , Bailey Stephanie A. , Stelma Gerard N. 
Published: June 2008

ABSTRACT

We collected
Mycobacterium avium
isolates from clinical and drinking-water sources and compared isolates among themselves and to each other using molecular methods. Four clinical isolates were related to water isolates. Groups of indistinguishable clinical isolates were identified. The groups of identical clinical isolates suggest a common source of exposure.
Diversity of Uncultured
Epsilonproteobacteria
from Terrestrial Sulfidic Caves and Springs
Authors: Porter Megan L. , Engel Annette Summers 
Published: June 2008

ABSTRACT

This study expands the phylogenetic diversity of
Epsilonproteobacteria
using the 16S rRNA gene framework. Of the 73 lineages defined by sequence similarities at or greater than 99%, most were found at only one site. In contrast, eight lineages were retrieved from sites spanning geographic distances from 1,000 to >10,000 km.
Aspergillus nidulans
ArfB Plays a Role in Endocytosis and Polarized Growth
Authors: Lee Soo Chan , Schmidtke Sabrina N. , Dangott Lawrence J. , Shaw Brian D. 
Published: June 2008

ABSTRACT

Filamentous fungi undergo polarized growth throughout most of their life cycles. The Spitzenkörper is an apical organelle composed primarily of vesicles that is unique to filamentous fungi and is likely to act as a vesicle supply center for tip growth. Vesicle assembly and trafficking are therefore important for hyphal growth. ADP ribosylation factors (Arfs), a group of small GTPase proteins, play an important role in nucleating vesicle assembly. Little is known about the role of Arfs in filamentous hyphal growth. We found that
Aspergillus nidulans
is predicted to encode six Arf family proteins. Analysis of protein sequence alignments suggests that
A. nidulans
ArfB shares similarity with ARF6 of
Homo sapiens
and Arf3p of
Saccharomyces cerevisiae
. An
arfB
null allele (
arfB
disrupted by a transposon [
arfB
::Tn]) was characterized by extended isotropic growth of germinating conidia followed by cell lysis or multiple, random germ tube emergence, consistent with a failure to establish polarity. The mutant germ tubes and hyphae that do form initially meander abnormally off of the axis of polarity and frequently exhibit dichotomous branching at cell apices, consistent with a defect in polarity maintenance. FM4-64 staining of the
arfB
::Tn strain revealed that another phenotypic characteristic seen for
arfB
::Tn is a reduction and delay in endocytosis. ArfB is myristoylated at its N terminus. Green fluorescent protein-tagged ArfB (ArfB::GFP) localizes to the plasma membrane and endomembranes and mutation (ArfB
G2A
::GFP) of the N-terminal myristoylation motif disperses the protein to the cytoplasm rather than to the membranes. These results demonstrate that ArfB functions in endocytosis to play important roles in polarity establishment during isotropic growth and polarity maintenance during hyphal extension.
Amino Acid Residues in the GIY-YIG Endonuclease II of Phage T4 Affecting Sequence Recognition and Binding as Well as Catalysis
Authors: Lagerbäck Pernilla , Carlson Karin 
Published: June 2008

ABSTRACT
Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate.
The ε Subunit of DNA Polymerase III Is Involved in the Nalidixic Acid-Induced SOS Response in
Escherichia coli
Authors: Pohlhaus Jennifer Reineke , Long David T. , O'Reilly Erin , Kreuzer Kenneth N. 
Published: June 2008

ABSTRACT

Quinolone antibacterial drugs such as nalidixic acid target DNA gyrase in
Escherichia coli
. These inhibitors bind to and stabilize a normally transient covalent protein-DNA intermediate in the gyrase reaction cycle, referred to as the cleavage complex. Stabilization of the cleavage complex is necessary but not sufficient for cell killing—cytotoxicity apparently results from the conversion of cleavage complexes into overt DNA breaks by an as-yet-unknown mechanism(s). Quinolone treatment induces the bacterial SOS response in a RecBC-dependent manner, arguing that cleavage complexes are somehow converted into double-stranded breaks. However, the only proteins known to be required for SOS induction by nalidixic acid are RecA and RecBC. In hopes of identifying additional proteins involved in the cytotoxic response to nalidixic acid, we screened for
E. coli
mutants specifically deficient in SOS induction upon nalidixic acid treatment by using a
dinD
::
lacZ
reporter construct. From a collection of SOS partially constitutive mutants with disruptions of 47 different genes, we found that
dnaQ
insertion mutants are specifically deficient in the SOS response to nalidixic acid.
dnaQ
encodes DNA polymerase III ε subunit, the proofreading subunit of the replicative polymerase. The deficient response to nalidixic acid was rescued by the presence of the wild-type
dnaQ
gene, confirming involvement of the ε subunit. To further characterize the SOS deficiency of
dnaQ
mutants, we analyzed the expression of several additional SOS genes in response to nalidixic acid using real-time PCR. A subset of SOS genes lost their response to nalidixic acid in the
dnaQ
mutant strain, while two tested SOS genes (
recA
and
recN
) continued to exhibit induction. These results argue that the replication complex plays a role in modulating the SOS response to nalidixic acid and that the response is more complex than a simple on/off switch.
fslE
Is Necessary for Siderophore-Mediated Iron Acquisition in
Francisella tularensis
Schu S4
Authors: Ramakrishnan Girija , Meeker Alexis , Dragulev Bojan 
Published: June 2008

ABSTRACT

Strains of
Francisella tularensis
secrete a siderophore in response to iron limitation. Siderophore production is dependent on
fslA
, the first gene in an operon that appears to encode biosynthetic and export functions for the siderophore. Transcription of the operon is induced under conditions of iron limitation. The
fsl
genes lie adjacent to the
fur
homolog on the chromosome, and there is a canonical Fur box sequence in the promoter region of
fslA
. We generated a Δ
fur
mutant of the Schu S4 strain of
F. tularensis tularensis
and determined that siderophore production was now constitutive and no longer regulated by iron levels. Quantitative reverse transcriptase PCR analysis with RNA from Schu S4 and the mutant strain showed that Fur represses transcription of
fslA
under iron-replete conditions. We determined that
fslE
(locus FTT0025 in the Schu S4 genome), located downstream of the siderophore biosynthetic genes, is also under Fur regulation and is transcribed as part of the
fslABCDEF
operon. We generated a defined in-frame deletion of
fslE
and found that the mutant was defective for growth under iron limitation. Using a plate-based growth assay, we found that the mutant was able to secrete a siderophore but was defective in utilization of the siderophore. FslE belongs to a family of proteins that has no known homologs outside of the
Francisella
species, and the
fslE
gene product has been previously localized to the outer membrane of
F. tularensis
strains. Our data suggest that FslE may function as the siderophore receptor in
F. tularensis
.
NagA-Dependent Uptake ofN-Acetyl-Glucosamine andN-Acetyl-Chitin Oligosaccharides across the Outer Membrane ofCaulobacter crescentus
Authors: Eisenbeis Simone , Lohmiller Stefanie , Valdebenito Marianne , Leicht Stefan , Braun Volkmar 
Published: June 2008

ABSTRACTAmong the 67 predicted TonB-dependent outer membrane transporters ofCaulobacter crescentus, NagA was found to be essential for growth onN-acetyl-β-d-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a β-barrel composed of 22 antiparallel β-strands linked by large surface loops and very short periplasmic turns. Mutations intonB1andexbBD, known to be required for maltose transport via MalA inC. crescentus, and in two additional predictedtonBgenes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake.nagAis located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since anagAinsertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc intotonBandexbBDmutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc)3and (GlcNAc)5requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.