bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Transcriptional Response of
Bifidobacterium longum
to Human Milk, Formula Milk, and Galactooligosaccharide
Authors: González Rina , Klaassens Eline S. , Malinen Erja , de Vos Willem M. , Vaughan Elaine E.
Published: June 2008

In order to gain insight into the effects of human breast milk on the development of the intestinal bifidobacteria and associated health effects, the transcriptome of
Bifidobacterium longum
LMG 13197 grown in breast milk and formula milk containing galactooligosaccharides (GOS) and long-chain fructooligosaccharides was compared to that obtained in a semisynthetic medium with glucose. Total RNA was isolated from exponentially growing cells and hybridized to a clone library-based microarray. Inserts of clones with significant hybridization signals were sequenced and identified. The
B. longum
transcriptomes obtained during growth on human and formula milk were more similar to each other than to that obtained from growth in semisynthetic medium with glucose. Remarkably, there were only a few genes implicated in carbohydrate metabolism that were similarly upregulated during growth in both human and formula milk although oligosaccharides were added to the formula. Common highly upregulated genes notably included putative genes for cell surface type 2 glycoprotein-binding fimbriae that are implicated in attachment and colonization in the intestine. Genes involved in carbohydrate metabolism formed the dominant group specifically upregulated in breast milk and included putative genes for
N
-acetylglucosamine degradation and for metabolism of mucin and human milk oligosaccharides via the galactose/lacto-
N
-biose gene cluster. This supports the notion that the bifidogenic effect of human milk is to a great extent based on its oligosaccharides. The transcriptional effect of semisynthetic medium containing GOS, which, like human milk, contains a large amount of lactose and galactose, on the
B. longum
transcriptome was also studied and revealed substantial similarity with carbohydrate-utilization genes upregulated during growth in human milk. This knowledge provides leads to optimizing formula milk to better simulate the observed bifidogenic effects of human breast milk.
Streptococcus thermophilus
Core Genome: Comparative Genome Hybridization Study of 47 Strains
Authors: Rasmussen Thomas Bovbjerg , Danielsen Morten , Valina Ondrej , Garrigues Christel , Johansen Eric , Pedersen Martin Bastian
Published: June 2008

A DNA microarray platform based on 2,200 genes from publicly available sequences was designed for
Streptococcus thermophilus
. We determined how single-nucleotide polymorphisms in the 65- to 75-mer oligonucleotide probe sequences affect the hybridization signals. The microarrays were then used for comparative genome hybridization (CGH) of 47 dairy
S. thermophilus
strains. An analysis of the exopolysaccharide genes in each strain confirmed previous findings that this class of genes is indeed highly variable. A phylogenetic tree based on the CGH data showed similar distances for most strains, indicating frequent recombination or gene transfer within
S. thermophilus
. By comparing genome sizes estimated from the microarrays and pulsed-field gel electrophoresis, the amount of unknown DNA in each strain was estimated. A core genome comprised of 1,271 genes detected in all 47 strains was identified. Likewise, a set of noncore genes detected in only some strains was identified. The concept of an industrial core genome is proposed. This is comprised of the genes in the core genome plus genes that are necessary in an applied industrial context.
Impact of
luxS
and Suppressor Mutations on the Gastrointestinal Transit of
Lactobacillus rhamnosus
GG
Authors: Lebeer Sarah , Claes Ingmar J. J. , Verhoeven Tine L. A. , Shen Chong , Lambrichts Ivo , Ceuppens Jan L. , Vanderleyden Jos , De Keersmaecker Sigrid C. J.
Published: June 2008

It is generally believed that probiotic bacteria need to survive gastrointestinal transit to exert a health-promoting effect. In this study, a genuine
luxS
mutant and a
luxS
mutant containing unknown suppressor mutations of the probiotic strain
Lactobacillus rhamnosus
GG were compared to the wild type for survival and persistence in the murine gastrointestinal tract. The LuxS enzyme, catalyzing the production of the autoinducer-2 signaling molecule, also forms an integral part of the activated methyl cycle and the metabolism of methionine and cysteine. The genuine
luxS
mutant CMPG5412 showed drastically reduced persistence in mice, which was related to less survival in simulated gastric juice, indicating that LuxS metabolism is crucial for the gastric stress resistance of
L. rhamnosus
GG. The suppressor mutations in the other
luxS
mutant, CMPG5413, appear to compensate for the metabolic defects of the
luxS
mutation and to restore the resistance to gastric juice but cause a defect in adherence, biofilm formation, and exopolysaccharide production. The shorter residence time of this suppressor mutant in the murine gastrointestinal tract indicates a role for biofilm formation and exopolysaccharides in the persistence capacity of
L. rhamnosus
GG.
Plasmid pCS1966, a New Selection/Counterselection Tool for Lactic Acid Bacterium Strain Construction Based on the
oroP
Gene, Encoding an Orotate Transporter from
Lactococcus lactis
Authors: Solem Christian , Defoor Els , Jensen Peter Ruhdal , Martinussen Jan
Published: June 2008

In this paper we describe the new selection/counterselection vector pCS1966, which is suitable for both sequence-specific integration based on homologous recombination and integration in a bacteriophage attachment site. This plasmid harbors
oroP
, which encodes a dedicated orotate transporter, and can replicate only in
Escherichia coli
. Selection for integration is performed primarily by resistance to erythromycin; alternatively, the ability to utilize orotate as a pyrimidine source in a pyrimidine auxotrophic mutant could be utilized. Besides allowing the cell to utilize orotate, the transporter renders the cell sensitive to 5-fluoroorotate. This sensitivity is used to select for loss of the plasmid. When expressed from its own promoter,
oroP
was toxic to
E. coli
, whereas in
Lactococcus lactis
the level of expression of
oroP
from a chromosomal copy was too low to confer 5-fluoroorotate sensitivity. In order to obtain a plasmid that confers 5-fluoroorotate sensitivity when it is integrated into the chromosome of
L. lactis
and at the same time can be stably maintained in
E. coli
, the expression of the
oroP
gene was controlled from a synthetic promoter conferring these traits. To demonstrate its use, a number of
L. lactis
strains expressing triosephosphate isomerase (
tpiA
) at different levels were constructed.
Improvement of
Lactobacillus plantarum
Aerobic Growth as Directed by Comprehensive Transcriptome Analysis
Authors: Stevens Marc J. A. , Wiersma Anne , de Vos Willem M. , Kuipers Oscar P. , Smid Eddy J. , Molenaar Douwe , Kleerebezem Michiel
Published: June 2008

An aerobic
Lactobacillus plantarum
culture displayed growth stagnation during early growth. Transcriptome analysis revealed that resumption of growth after stagnation correlated with activation of CO
2
-producing pathways, suggesting that a limiting CO
2
concentration induced the stagnation. Analogously, increasing the CO
2
gas partial pressure during aerobic fermentation prevented the temporal growth stagnation.
Functional Analysis of Four Bile Salt Hydrolase and Penicillin Acylase Family Members in
Lactobacillus plantarum
WCFS1
Authors: Lambert Jolanda M. , Bongers Roger S. , de Vos Willem M. , Kleerebezem Michiel
Published: June 2008

Bile salts play an important role in the digestion of lipids in vertebrates and are synthesized and conjugated to either glycine or taurine in the liver. Following secretion of bile salts into the small intestine, intestinal microbes are capable of deconjugating the glycine or taurine from the bile salts, using an enzyme called bile salt hydrolase (Bsh). Intestinal lactobacilli are regarded as major contributors to bile salt hydrolysis in vivo. Since the bile salt-hydrolyzing strain
Lactobacillus plantarum
WCFS1 was predicted to carry four
bsh
genes (
bsh1, bsh2, bsh3
, and
bsh4
), the functionality of these
bsh
genes was explored using
Lactococcus lactis
heterologous overexpression and multiple
bsh
deletion strains. Thus, Bsh1 was shown to be responsible for the majority of Bsh activity in
L. plantarum
WCFS1. In addition,
bsh1
of
L. plantarum
WCFS1 was shown to be involved in conferring tolerance to specific bile salts (i.e., glycocholic acid). Northern blot analysis established that
bsh1, bsh2, bsh3
, and
bsh4
are all expressed in
L. plantarum
WCFS1 during the exponential growth phase. Following biodiversity analysis,
bsh1
appeared to be the only
bsh
homologue that was variable among
L. plantarum
strains; furthermore, the presence of
bsh1
correlated with the presence of Bsh activity, suggesting that Bsh1 is commonly responsible for Bsh activity in
L. plantarum
strains. The fact that
bsh2, bsh3
, and
bsh4
genes appeared to be conserved among
L. plantarum
strains suggests an important role of these genes in the physiology and lifestyle of the species
L. plantarum
. Analysis of these additional
bsh
-like genes in
L. plantarum
WCFS1 suggests that they might encode penicillin acylase rather than Bsh activity, indicating their implication in the conversion of substrates other than bile acids in the natural habitat.
Evaluation of Reuterin Production in Urogenital Probiotic
Lactobacillus reuteri
RC-14
Authors: Cadieux Peter , Wind Anette , Sommer Philip , Schaefer Laura , Crowley Kate , Britton Robert A. , Reid Gregor
Published: June 2008

Classified as a distinct species in 1980,
Lactobacillus reuteri
strains have been used in probiotic formulations for intestinal and urogenital applications. In the former, the primary mechanism of action of
L. reuteri
SD2112 (ATCC 55730) has been purported to be its ability to produce the antibiotic 3-hydroxypropionaldehyde (3-HPA), also known as reuterin. In the vagina, it has been postulated that probiotic
Lactobacillus reuteri
RC-14 does not require reuterin production but mediates a restoration of the normal microbiota via hydrogen peroxide, biosurfactant, lactic acid production, and immune modulation. The aim of the present study was to determine whether strain RC-14 produced reuterin. Using PCR and DNA dot blot analyses, numerous
Lactobacillus
species, including RC-14, were screened for the presence of the gene encoding the large subunit of glycerol dehydratase (
gldC
), the enzyme responsible for reuterin production. In addition, lactobacilli were grown in glycerol-based media and both high-performance liquid chromatography and a colorimetric assay were used to test for the presence of reuterin.
L. reuteri
RC-14 was determined to be negative for
gldC
sequences, as well as for the production of reuterin when cultured in the presence of glycerol. These findings support that the probiotic effects of
L. reuteri
RC-14, repeatedly demonstrated during numerous studies of the intestine and vagina, are independent of reuterin production.
Detection of a Lysozyme Inhibitor in
Proteus mirabilis
by a New Reverse Zymogram Method
Authors: Callewaert Lien , Vanderkelen Lise , Deckers Daphne , Aertsen Abram , Robben Johan , Michiels Chris W.
Published: June 2008

A reverse zymogram method for the detection of bacterial lysozyme inhibitors was developed. This method was validated by using a periplasmic protein extract of
Escherichia coli
containing a known inhibitor and subsequently led to the detection of a new proteinaceous hen egg white lysozyme inhibitor in
Proteus mirabilis
.
Expression of Green Fluorescent Protein Fused to Magnetosome Proteins in Microaerophilic Magnetotactic Bacteria
Authors: Lang Claus , Schüler Dirk
Published: June 2008

The magnetosomes of magnetotactic bacteria are prokaryotic organelles consisting of a magnetite crystal bounded by a phospholipid bilayer that contains a distinct set of proteins with various functions. Because of their unique magnetic and crystalline properties, magnetosome particles are potentially useful as magnetic nanoparticles in a number of applications, which in many cases requires the coupling of functional moieties to the magnetosome membrane. In this work, we studied the use of green fluorescent protein (GFP) as a reporter for the magnetosomal localization and expression of fusion proteins in the microaerophilic
Magnetospirillum gryphiswaldense
by flow cytometry, fluorescence microscopy, and biochemical analysis. Although optimum conditions for high fluorescence and magnetite synthesis were mutually exclusive, we established oxygen-limited growth conditions, which supported growth, magnetite biomineralization, and GFP fluorophore formation at reasonable rates. Under these optimized conditions, we studied the subcellular localization and expression of the GFP-tagged magnetosome proteins MamC, MamF, and MamG by fluorescence microscopy and immunoblotting. While all fusions specifically localized at the magnetosome membrane, MamC-GFP displayed the strongest expression and fluorescence. MamC-GFP-tagged magnetosomes purified from cells displayed strong fluorescence, which was sensitive to detergents but stable under a wide range of temperature and salt concentrations. In summary, our data demonstrate the use of GFP as a reporter for protein localization under magnetite-forming conditions and the utility of MamC as an anchor for magnetosome-specific display of heterologous gene fusions.
Molecular Characterization of the Diversity and Distribution of a Thermal Spring Microbial Community by Using rRNA and Metabolic Genes
Authors: Hall Justine R. , Mitchell Kendra R. , Jackson-Weaver Olan , Kooser Ara S. , Cron Brandi R. , Crossey Laura J. , Takacs-Vesbach Cristina D.
Published: June 2008

The diversity and distribution of a bacterial community from Coffee Pots Hot Spring, a thermal spring in Yellowstone National Park with a temperature range of 39.3 to 74.1°C and pH range of 5.75 to 6.91, were investigated by sequencing cloned PCR products and quantitative PCR (qPCR) of 16S rRNA and metabolic genes. The spring was inhabited by threeAquificaegenera—Thermocrinis, Hydrogenobaculum, andSulfurihydrogenibium—and members of theAlpha-,Beta-, andGammaproteobacteria, Firmicutes, Acidobacteria, Deinococcus-Thermus, and candidate division OP5. The in situ chemical affinities were calculated for 41 potential metabolic reactions using measured environmental parameters and a range of hydrogen and oxygen concentrations. Reactions that use oxygen, ferric iron, sulfur, and nitrate as electron acceptors were predicted to be the most energetically favorable, while reactions using sulfate were expected to be less favorable. Samples were screened for genes used in ammonia oxidation (amoA, bacterial gene only), the reductive tricarboxylic acid (rTCA) cycle (aclB), the Calvin cycle (cbbM), sulfate reduction (dsrAB), nitrogen fixation (nifH), nitrite reduction (nirK), and sulfide oxidation (soxEF1) by PCR. Genes for carbon fixation by the rTCA cycle and nitrogen fixation were detected. AllaclBsequences were phylogenetically related and spatially correlated toSulfurihydrogenibium16S rRNA gene sequences using qPCR (R2= 0.99). This result supports the recent finding of citrate cleavage by enzymes other than ATP citrate lyase in the rTCA cycle of theAquificaceaefamily. We briefly consider potential biochemical mechanisms that may allowSulfurihydrogenibiumandThermocrinisto codominate some hydrothermal environments.
Multiplex Fast Real-Time PCR for Quantitative Detection and Identification of
cos
- and
pac
-Type
Streptococcus thermophilus
Bacteriophages
Authors: del Rio Beatriz , Martín María Cruz , Martínez Noelia , Magadán Alfonso H. , Alvarez Miguel A.
Published: June 2008

The fermentation of milk by
Streptococcus thermophilus
is a widespread industrial process that is susceptible to bacteriophage attack. In this work, a preventive fast real-time PCR method for the detection, quantification, and identification of types of
S. thermophilus
phages in 30 min is described.
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

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

Eleven 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

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

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

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

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

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

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

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

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

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

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

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

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

Among 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.
Cadmium Toxicity in Glutathione Mutants ofEscherichia coli
Authors: Helbig Kerstin , Grosse Cornelia , Nies Dietrich H.
Published: June 2008

The higher affinity of Cd2+for sulfur compounds than for nitrogen and oxygen led to the theoretical consideration that cadmium toxicity should result mainly from the binding of Cd2+to sulfide, thiol groups, and sulfur-rich complex compounds rather than from Cd2+replacement of transition-metal cations from nitrogen- or oxygen-rich biological compounds. This hypothesis was tested by usingEscherichia colifor a global transcriptome analysis of cells synthesizing glutathione (GSH; wild type), γ-glutamylcysteine (ΔgshBmutant), or neither of the two cellular thiols (ΔgshAmutant). The resulting data, some of which were validated by quantitative reverse transcription-PCR, were sorted using the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology system, which groups genes hierarchically with respect to the cellular functions of their respective products. The main difference among the three strains concerned tryptophan biosynthesis, which was up-regulated in wild-type cells upon cadmium shock and strongly up-regulated in ΔgshAcells but repressed in ΔgshBcells containing γ-glutamylcysteine instead of GSH. Overall, however, all threeE. colistrains responded to cadmium shock similarly, with the up-regulation of genes involved in protein, disulfide bond, and oxidative damage repair; cysteine and iron-sulfur cluster biosynthesis; the production of proteins containing sensitive iron-sulfur clusters; the storage of iron; and the detoxification of Cd2+by efflux. General energy conservation pathways and iron uptake were down-regulated. These findings indicated that the toxic action of Cd2+indeed results from the binding of the metal cation to sulfur, lending support to the hypothesis tested.
Global Regulation by (p)ppGpp and CodY inStreptococcus mutans
Authors: Lemos José A. , Nascimento Marcelle M. , Lin Vanessa K. , Abranches Jacqueline , Burne Robert A.
Published: June 2008

The RelA, RelP, and RelQ enzymes are responsible for the production of the alarmone (p)ppGpp inStreptococcus mutans. A strain lacking all three synthetases (ΔrelAPQ) does not grow in minimal medium lacking the branched-chain amino acids (BCAA) leucine or valine but grows well if isoleucine is also omitted. Here, we investigated whether there was a correlation between growth in the absence of leucine and valine with (p)ppGpp pools and the activation of CodY. By using a combination of single, double, and triple mutants lacking the (p)ppGpp synthetase enzymes, we demonstrated that the ability to grow in the absence of leucine or valine required basal levels of (p)ppGpp production by RelP and RelQ. The introduction of acodYmutation into the ΔrelAPQstrain fully restored growth in medium lacking leucine or valine, revealing that the growth-defective phenotype of ΔrelAPQwas directly linked to CodY. Lowering GTP levels through the addition of decoyinine did not alleviate CodY repression or affect the expression of genes involved in BCAA biosynthesis, suggesting thatS. mutansCodY is not activated by GTP. The results of phenotypic studies revealed that thecodYmutant had a reduced capacity to form biofilms and that its growth was more sensitive to low pH, showing a role for CodY in two key virulence properties ofS. mutans. Microarray results revealed the extent of the CodY regulon. Notably, the identification of putative CodY-binding boxes upstream of genes that were downregulated in thecodYmutant indicates that CodY may also function as a transcriptional activator inS. mutans.
Virulence Gene Expression Is Independent of ResDE-Regulated Respiration Control inBacillus anthracis
Authors: Wilson Adam C. , Hoch James A. , Perego Marta
Published: June 2008

The ResDE two-component system regulates the synthesis of several components of the aerobic and anaerobic respiratory pathways in bacilli. The ResD response regulator transcription factor has been implicated in the regulation of virulence factors in a number of gram-positive species, includingBacillus anthracis. The precise deletions ofresDandresEinB. anthracisthat retained the classical respiratory phenotypes did not affect the expression of the gene for the protective antigen of the anthrax toxin,pagA, or that of the toxin regulator,atxA. The results indicate that the loss of ResDE-controlled respiratory capacity does not affect the synthesis of anthrax toxin.
Relative Quantitative Comparisons of the Extracellular Protein Profiles ofStaphylococcus aureusUAMS-1 and ItssarA,agr, andsarA agrRegulatory Mutants Using One-Dimensional Polyacrylamide Gel Electrophoresis and Nanocapillary Liquid Chromatography Coupled with Tandem Mass Spectrometry
Authors: Jones Richard C. , Deck Joanna , Edmondson Ricky D. , Hart Mark E.
Published: June 2008

One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography coupled with mass spectrometry was used to analyze proteins isolated fromStaphylococcus aureusUAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more abundant early on in growth, while proteins known to be secreted into the surrounding milieu were more abundant late in growth. In addition, proteins from spent media and cell lysates of strain UAMS-1 and its isogenicsarA,agr, andsarA agrregulatory mutant strains during exponential growth were identified, and their relative abundances were compared. Extracellular proteins known to be regulated by the global regulatorssarAandagrdisplayed protein levels in accordance with what is known regarding the effects of these regulators. For example, cysteine protease (SspB), endopeptidase (SspA), staphopain (ScpA), and aureolysin (Aur) were higher in abundance in thesarAandsarA agrmutants than in strain UAMS-1. The immunoglobulin G (IgG)-binding protein (Sbi), immunodominant staphylococcal antigen A (IsaA), IgG-binding protein A (Spa), and the heme-iron-binding protein (IsdA) were most abundant in theagrmutant background. Proteins whose abundance was decreased in thesarAmutant included fibrinogen-binding protein (Fib [Efb]), IsaA, lipase 1 and 2, and two proteins identified as putative leukocidin F and S subunits of the two-component leukotoxin family. Collectively, this approach identified 1,263 proteins (matches of two peptides or more) and provided a convenient and reliable way of identifying proteins and comparing their relative abundances.
Burkholderia pseudomallei,B. thailandensis, andB. ambifariaProduce 4-Hydroxy-2-Alkylquinoline Analogues with a Methyl Group at the 3 Position That Is Required for Quorum-Sensing Regulation
Authors: Vial Ludovic , Lépine François , Milot Sylvain , Groleau Marie-Christine , Dekimpe Valérie , Woods Donald E. , Déziel Eric
Published: June 2008

4-Hydroxy-2-alkylquinolines (HAQs), especially 3,4-dihydroxy-2-heptylquinoline (Pseudomonasquinolone signal) and its precursor, 4-hydroxy-2-heptylquinoline, are attracting much attention, mainly because of their role as signaling molecules inPseudomonas aeruginosa. ThepqsABCDEoperon is centrally involved in their biosynthesis. The presence of a homologous operon inBurkholderia pseudomalleiandB. thailandensiswas recently reported. Thus, we have investigated the abilities of 11Burkholderiaspecies to produce HAQ-like molecules by liquid chromatography/mass spectrometry. We have identified 29 different HAQ derivatives produced by the only threeBurkholderiaspecies where apqsABCDEhomologue was found among available sequencedBurkholderiaspecies genomes, includingB. ambifaria, a member of theBurkholderia cepaciacomplex. In contrast with those ofP. aeruginosa,BurkholderiaHAQs typically bear a methyl group, hence their designation as 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs). We identified three families of HMAQs with a saturated or unsaturated alkyl chain at the 2′ position, in contrast with the 1′ position ofP. aeruginosa, including one with anN-oxide group. Furthermore, the operon in these species contains two more genes downstream of thepqsEhomologue, resulting in thehmqABCDEFGoperon. While the inactivation ofhmqAinhibits the production of HMAQs, the methylation of the quinoline ring requires a putative methyltransferase encoded byhmqG. Interestingly,hmqAorhmqGmutations increase the production of acyl homoserine lactones and, consequently, phenotypes under the control of quorum sensing inB. ambifaria: antifungal activity, siderophore production, and proteolytic activity. These results indicate that only HAQs bearing a methyl group (HMAQs) are involved in quorum-sensing regulation.