bluuebunny/crossref_metadata_2025 huggingface dataset:
Dehalogenation Activities and Distribution of Reductive Dehalogenase Homologous Genes in Marine Subsurface Sediments
Authors: Futagami Taiki , Morono Yuki , Terada Takeshi , Kaksonen Anna H. , Inagaki Fumio
Published: September 2009

Halogenated organic compounds serve as terminal electron acceptors for anaerobic respiration in a diverse range of microorganisms. Here, we report on the widespread distribution and diversity of reductive dehalogenase homologous (
rdhA
) genes in marine subsurface sediments. A total of 32 putative
rdhA
phylotypes were detected in sediments from the southeast Pacific off Peru, the eastern equatorial Pacific, the Juan de Fuca Ridge flank off Oregon, and the northwest Pacific off Japan, collected at a maximum depth of 358 m below the seafloor. In addition, significant dehalogenation activity involving 2,4,6-tribromophenol and trichloroethene was observed in sediment slurry from the Nankai Trough Forearc Basin. These results suggest that dehalorespiration is an important energy-yielding pathway in the subseafloor microbial ecosystem.
Comparison and Utilization of Repetitive-Element PCR Techniques for Typing
Lactobacillus
Isolates from the Chicken Gastrointestinal Tract
Authors: Stephenson David P. , Moore Robert J. , Allison Gwen E.
Published: September 2009

Three repetitive-element PCR techniques were evaluated for the ability to type strains of
Lactobacillus
species commonly identified in the chicken gastrointestinal tract. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) produced species- and strain-specific profiles for
Lactobacillus crispatus
,
Lactobacillus gallinarum
,
Lactobacillus johnsonii
, and
Lactobacillus reuteri
isolates. The technique typed strains within these species equally as well as pulsed-field gel electrophoresis. DNA concentration and quality did not affect the ERIC-PCR profiles, indicating that this method, unlike other high-resolution methods, can be adapted to high-throughput analysis of isolates. Subsequently, ERIC-PCR was used to type
Lactobacillus
species diversity of a large collection of isolates derived from chickens grown under commercial and necrotic enteritis disease induction conditions. This study has illustrated, for the first time, that there is great strain diversity within each
Lactobacillus
species present and has revealed that chickens raised under commercial conditions harbor greater species and strain diversity than chickens raised under necrotic enteritis disease induction conditions.
Rapid Identification of
Vibrio parahaemolyticus
by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
Authors: Hazen Tracy H. , Martinez Robert J. , Chen Yanfeng , Lafon Patricia C. , Garrett Nancy M. , Parsons Michele B. , Bopp Cheryl A. , Sullards M. Cameron , Sobecky Patricia A.
Published: September 2009

Vibrio parahaemolyticus
is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of
V. parahaemolyticus
-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of
V. parahaemolyticus
from other
Vibrio
spp. We identified 30 peaks that were present only in the spectra of the
V. parahaemolyticus
strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of
V. parahaemolyticus
. We detected variation in the MALDI-TOF spectra of
V. parahaemolyticus
strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the
V. parahaemolyticus
strains examined were distinct from those of the other
Vibrio
species examined including the closely related
V. alginolyticus
,
V. harveyi
, and
V. campbellii
. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of
V. parahaemolyticus
.
Microhabitats within Venomous Cone Snails Contain Diverse Actinobacteria
Authors: Peraud Olivier , Biggs Jason S. , Hughen Ronald W. , Light Alan R. , Concepcion Gisela P. , Olivera Baldomero M. , Schmidt Eric W.
Published: September 2009

Actinomycetes can be symbionts in diverse organisms, including both plants and animals. Some actinomycetes benefit their host by producing small molecule secondary metabolites; the resulting symbioses are often developmentally complex. Actinomycetes associated with three cone snails were studied. Cone snails are venomous tropical marine gastropods which have been extensively examined because of their production of peptide-based neurological toxins, but no microbiological studies have been reported on these organisms. A microhabitat approach was used in which dissected tissue from each snail was treated as an individual sample in order to explore bacteria in the tissues separately. Our results revealed a diverse, novel, and highly culturable cone snail-associated actinomycete community, with some isolates showing promising bioactivity in a neurological assay. This suggests that cone snails may represent an underexplored reservoir of novel actinomycetes of potential interest for drug discovery.
Diminished Exoproteome of
Frankia
spp. in Culture and Symbiosis
Authors: Mastronunzio J. E. , Huang Y. , Benson D. R.
Published: September 2009

Frankia
species are the most geographically widespread gram-positive plant symbionts, carrying out N
2
fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three
Frankia
genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from
Frankia
, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of
Frankia
sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in
Casuarina cunninghamiana
and
Casuarina glauca
root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from
Frankia
strains in field-collected root nodules of
Alnus incana
and
Elaeagnus angustifolia
, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the
Frankia
proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.
Combination of Chromogenic Differential Medium and
estA
-Specific PCR for Isolation and Detection of Phytopathogenic
Xanthomonas
spp
Authors: Lee Yung-An , Sung Ai-Ning , Liu Tzu-Fen , Lee Yung-Shan
Published: September 2009

A xanthomonad differential medium (designated Xan-D medium) was developed, on which streaks and colonies of xanthomonads, including 13 species of the genus
Xanthomonas
, turned wet-shining yellow-green and were surrounded with a smaller milky zone and a bigger clear zone in 3 to 4 days. The characteristics could easily be differentiated from those of yellow nonxanthomonads and other bacteria. The mechanism of color change and formation of a milky zone on the medium are mainly due to the Tween 80 hydrolytic capacity of xanthomonads. The gene,
estA
, responsible for Tween 80 hydrolysis was cloned and expressed in
Escherichia coli
, which acquired a capacity to hydrolyze Tween 80 and could turn green and form a milky zone on the Xan-D medium. The nucleotide sequence of
estA
is highly conserved in the xanthomonads, and the sequence was used to design a specific PCR primer set. The PCR amplification using the primer set amplified a 777-bp specific DNA fragment for all xanthomonad strains tested. The Xan-D medium was used to isolate and differentiate
Xanthomonas campestris
pv. campestris from naturally infected cabbages with black rot symptoms for a rapid diagnosis. All isolated
X. campestris
pv. campestris strains developed characteristic colonies and were positive in the PCR with the
estA
primer set. The Xan-D medium was further amended with antibiotics and successfully used for the detection of viable
X. campestris
pv. campestris cells from plant seeds. Although some yellow nonxanthomonads and other saprophytic bacteria from plant seeds could still grow on the medium, they did not interfere with the color development of
X. campestris
pv. campestris. However,
Stenotrophomonas maltophilia
, which is closely related to xanthomonads, existing in a seed lot could also develop yellow-green color but had different colony morphology and was negative in the PCR with the
estA
primer set. Accordingly, the combination of the Xan-D medium with the
estA
-specific PCR is a useful and reliable method for the isolation and detection of viable xanthomonad cells from plant materials.
Edwardsiella ictaluri
Encodes an Acid-Activated Urease That Is Required for Intracellular Replication in Channel Catfish (
Ictalurus punctatus
) Macrophages
Authors: Booth Natha J. , Beekman Judith B. , Thune Ronald L.
Published: September 2009

Genomic analysis indicated that
Edwardsiella ictaluri
encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type
E. ictaluri
and a
ureG
::
kan
urease mutant strain indicated that
E. ictaluri
is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that
E. ictaluri
is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type
E. ictaluri
in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of wild-type
E. ictaluri
was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome.
Composition and Activity of an Autotrophic Fe(II)-Oxidizing, Nitrate-Reducing Enrichment Culture
Authors: Blöthe Marco , Roden Eric E.
Published: September 2009

16S rRNA gene libraries from the lithoautotrophic Fe(II)-oxidizing, nitrate-reducing enrichment culture described by Straub et al. (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl. Environ. Microbiol. 62:1458-1460, 1996) were dominated by a phylotype related (95% 16S rRNA gene homology) to the autotrophic Fe(II) oxidizer
Sideroxydans lithotrophicus
. The libraries also contained phylotypes related to known heterotrophic nitrate reducers
Comamonas badia
,
Parvibaculum lavamentivorans
, and
Rhodanobacter thiooxidans
. The three heterotrophs were isolated and found to be capable of only partial (12 to 24%) Fe(II) oxidation, suggesting that the
Sideroxydans
species has primary responsibility for Fe(II) oxidation in the enrichment culture.
l
-Ribose Production from
l
-Arabinose by Using Purified
l
-Arabinose Isomerase and Mannose-6-Phosphate Isomerase from
Geobacillus thermodenitrificans
Authors: Yeom Soo-Jin , Kim Nam-Hee , Park Chang-Su , Oh Deok-Kun
Published: September 2009

Two enzymes,
l
-arabinose isomerase and mannose-6-phosphate isomerase, from
Geobacillus thermodenitrificans
produced 118 g/liter
l
-ribose from 500 g/liter
l
-arabinose at pH 7.0, 70°C, and 1 mM Co
2+
for 3 h, with a conversion yield of 23.6% and a volumetric productivity of 39.3 g liter
−1
h
−1
.
Reconstruction of Signaling Networks Regulating Fungal Morphogenesis by Transcriptomics
Authors: Meyer Vera , Arentshorst Mark , Flitter Simon J. , Nitsche Benjamin M. , Kwon Min Jin , Reynaga-Peña Cristina G. , Bartnicki-Garcia Salomon , van den Hondel Cees A. M. J. J. , Ram Arthur F. J.
Published: September 2009

Coordinated control of hyphal elongation and branching is essential for sustaining mycelial growth of filamentous fungi. In order to study the molecular machinery ensuring polarity control in the industrial fungusAspergillus niger, we took advantage of the temperature-sensitive (ts) apical-branchingramosa-1mutant. We show here that this strain serves as an excellent model system to study critical steps of polar growth control during mycelial development and report for the first time a transcriptomic fingerprint of apical branching for a filamentous fungus. This fingerprint indicates that several signal transduction pathways, including TORC2, phospholipid, calcium, and cell wall integrity signaling, concertedly act to control apical branching. We furthermore identified the genetic locus affected in theramosa-1mutant by complementation of thetsphenotype. Sequence analyses demonstrated that a single amino acid exchange in the RmsA protein is responsible for induced apical branching of theramosa-1mutant. Deletion experiments showed that the correspondingrmsAgene is essential for the growth ofA. niger, and complementation analyses withSaccharomyces cerevisiaeevidenced that RmsA serves as a functional equivalent of the TORC2 component Avo1p. TORC2 signaling is required for actin polarization and cell wall integrity inS. cerevisiae. Congruently, our microscopic investigations showed that polarized actin organization and chitin deposition are disturbed in theramosa-1mutant. The integration of the transcriptomic, genetic, and phenotypic data obtained in this study allowed us to reconstruct a model for cellular events involved in apical branching.
A Contiguous Compartment Functions as Endoplasmic Reticulum and Endosome/Lysosome in
Giardia lamblia
Authors: Abodeely Marla , DuBois Kelly N. , Hehl Adrian , Stefanic Sasa , Sajid Mohammed , deSouza Wanderley , Attias Marcia , Engel Juan C. , Hsieh Ivy , Fetter Richard D. , McKerrow James H.
Published: September 2009

The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan,
Giardia lamblia
, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The
Giardia
endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes.
Giardia
orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support
Giardia
's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.
Horizontal Chromosome Transfer, a Mechanism for the Evolution and Differentiation of a Plant-Pathogenic Fungus
Authors: Akagi Yasunori , Akamatsu Hajime , Otani Hiroshi , Kodama Motoichiro
Published: September 2009

The tomato pathotype of
Alternaria alternata
produces host-specific AAL toxin and causes Alternaria stem canker on tomato. A polyketide synthetase (PKS) gene,
ALT1
, which is involved in AAL toxin biosynthesis, resides on a 1.0-Mb conditionally dispensable chromosome (CDC) found only in the pathogenic and AAL toxin-producing strains. Genomic sequences of
ALT1
and another PKS gene, both of which reside on the CDC in the tomato pathotype strains, were compared to those of tomato pathotype strains collected worldwide. This revealed that the sequences of both CDC genes were identical among five
A. alternata
tomato pathotype strains having different geographical origins. On the other hand, the sequences of other genes located on chromosomes other than the CDC are not identical in each strain, indicating that the origin of the CDC might be different from that of other chromosomes in the tomato pathotype. Telomere fingerprinting and restriction fragment length polymorphism analyses of the
A. alternata
strains also indicated that the CDCs in the tomato pathotype strains were identical, although the genetic backgrounds of the strains differed. A hybrid strain between two different pathotypes was shown to harbor the CDCs derived from both parental strains with an expanded range of pathogenicity, indicating that CDCs can be transmitted from one strain to another and stably maintained in the new genome. We propose a hypothesis whereby the ability to produce AAL toxin and to infect a plant could potentially be distributed among
A. alternata
strains by horizontal transfer of an entire pathogenicity chromosome. This could provide a possible mechanism by which new pathogens arise in nature.
Overexpression of MitochondrialLeishmania majorAscorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage
Authors: Dolai Subhankar , Yadav Rajesh K. , Pal Swati , Adak Subrata
Published: September 2009

Ascorbate peroxidase fromLeishmania major(LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress-induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl contents in cellular homogenates indicates that overexpression of LmAPX protectsLeishmaniacells against depletion of GSH and oxidative damage of proteins by H2O2or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca2+attained by the LmAPX-overexpressing cells was always below that attained in control cells. Flow cytometry assay data and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H2O2-induced mitochondrial membrane depolarization as well as ATP decrease. Western blot data suggest that overexpression of LmAPX shields against H2O2- or CPT-induced cytochromecand endonuclease G release from mitochondria and subsequently their accumulation in the cytoplasm. Caspase activity assay by flow cytometry shows a lower level of caspase-like protease activity in LmAPX-overexpressing cells under apoptotic stimuli. The data on phosphatidylserine exposed on the cell surface and DNA fragmentation results show that overexpression of LmAPX renders theLeishmaniacells more resistant to apoptosis provoked by H2O2or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria ofL. majorprevents cells from the deleterious effects of oxidative stress, that is, mitochondrial dysfunction and cellular death.
Distinct Subsets of Sit4 Holophosphatases Are Required for Inhibition ofSaccharomyces cerevisiaeGrowth by Rapamycin and Zymocin
Authors: Jablonowski Daniel , Täubert Jens-Eike , Bär Christian , Stark Michael J. R. , Schaffrath Raffael
Published: September 2009

Protein phosphatase Sit4 is required for growth inhibition ofSaccharomyces cerevisiaeby the antifungals rapamycin and zymocin. Here, we show that the rapamycin effector Tap42, which interacts with Sit4, is dispensable for zymocin action. Although Tap42 binding-deficientsit4mutants are resistant to zymocin, these mutations also block interaction between Sit4 and the Sit4-associating proteins Sap185 and Sap190, previously shown to mediate zymocin toxicity. Among the four differentSAPgenes, we found thatSAP190deletions specifically induce rapamycin resistance but that this phenotype is reversed in the additional absence ofSAP155. Similarly, the rapamycin resistance of anrrd1Δ mutant lacking the Sit4 interactor Rrd1 specifically requires the Sit4/Sap190 complex. Thus, Sit4/Sap190 and Sit4/Sap155 holophosphatases apparently play opposing roles following rapamycin treatment, although rapamycin inhibition is operational in the absence of all Sap family members or Sit4. We further identified a Sit4-interacting region on Sap185 insap190Δ cells that mediates Sit4/Sap185 complex formation and is essential for dephosphorylation of Elp1, a subunit of the Elongator complex. This suggests that Sit4/Sap185 and Sit4/Sap190 holophosphatases promote Elongator functions, a notion supported by data showing that their inactivation eliminates Elongator-dependent processes, including tRNA suppression bySUP4and tRNA cleavage by zymocin.
Conservation of the Sterol Regulatory Element-Binding Protein Pathway and Its Pathobiological Importance in
Cryptococcus neoformans
Authors: Chang Yun C. , Ingavale Susham S. , Bien Clara , Espenshade Peter , Kwon-Chung Kyung J.
Published: September 2009

The mammalian sterol regulatory element-binding protein (SREBP) homolog, Sre1, is important for adaptation and growth of
Cryptococcus neoformans
in the mouse brain, where oxygen concentration and nutritional conditions are suboptimal for fungal growth. The extent of conservation of the SREBP pathway in
C. neoformans
or in any other fungi, however, has not been investigated. We generated mutants susceptible to low oxygen and identified six genes that play a role in the SREBP pathway. Three of these genes (
SFB2
,
KAP123
, and
GSK3
) are not known to be involved in the SREBP pathway in other fungi. Furthermore, we show that
C. neoformans
contains an additional gene,
DAM1
, which functions in the SREBP pathway but is yet to be described. Mutants associated with the steps prior to formation of the nuclear Sre1 form dramatically reduced accumulation of the nuclear form under low-oxygen conditions. Concurrently, two mutant strains,
scp1Δ
and
stp1Δ
, and the previously isolated
sre1Δ
strain showed reduction in ergosterol levels, hypersensitivity to several chemical agents, including azole antifungals, CoCl
2
, and compounds producing reactive oxygen or nitrogen species, and most importantly, reduced virulence in mice. Mutants affecting genes involved in later steps of the Sre1 pathway, such as those required for import and phosphorylation of proteins in the nucleus, showed less compelling phenotypes. These findings suggest that the SREBP pathway is highly conserved in
C. neoformans
and it serves as an important link between sterol biosynthesis, oxygen sensing, CoCl
2
sensitivity, and virulence in
C. neoformans
.
Depletion of the Signal Recognition Particle Receptor Inactivates Ribosomes in
Escherichia coli
Authors: Bürk Jonas , Weiche Benjamin , Wenk Meike , Boy Diana , Nestel Sigrun , Heimrich Bernd , Koch Hans-Georg
Published: September 2009

The signal recognition particle (SRP)-dependent cotranslational targeting of proteins to the cytoplasmic membrane in bacteria or the endoplasmic reticulum membrane in eukaryotes is an essential process in most living organisms. Eukaryotic cells have been shown to respond to an impairment of the SRP pathway by (i) repressing ribosome biogenesis, resulting in decreased protein synthesis, and (ii) by increasing the expression of protein quality control mechanisms, such as chaperones and proteases. In the current study, we have analyzed how bacteria like
Escherichia coli
respond to a gradual depletion of FtsY, the bacterial SRP receptor. Our analyses using cell-free transcription/translation systems showed that FtsY depletion inhibits the translation of both SRP-dependent and SRP-independent proteins. This synthesis defect is the result of a multifaceted response that includes the upregulation of the ribosome-inactivating protein ribosome modulation factor (RMF). Although the consequences of these responses in
E. coli
are very similar to some of the effects also observed in eukaryotic cells, one striking difference is that
E. coli
obviously does not reduce the rate of protein synthesis by downregulating ribosome biogenesis. Instead, the upregulation of RMF leads to a direct and reversible inhibition of translation.
Functional Characterization of Flagellin Glycosylation in
Campylobacter jejuni
81-176
Authors: Ewing Cheryl P. , Andreishcheva Ekaterina , Guerry Patricia
Published: September 2009

The major flagellin of
Campylobacter jejuni
strain 81-176, FlaA, has been shown to be glycosylated at 19 serine or threonine sites, and this glycosylation is required for flagellar filament formation. Some enzymatic components of the glycosylation machinery of
C. jejuni
81-176 are localized to the poles of the cell in an FlhF-independent manner. Flagellin glycosylation could be detected in flagellar mutants at multiple levels of the regulatory hierarchy, indicating that glycosylation occurs independently of the flagellar regulon. Mutants were constructed in which each of the 19 serine or threonines that are glycosylated in FlaA was converted to an alanine. Eleven of the 19 mutants displayed no observable phenotype, but the remaining 8 mutants had two distinct phenotypes. Five mutants (mutations S417A, S436A, S440A, S457A, and T481A) were fully motile but defective in autoagglutination (AAG). Three other mutants (mutations S425A, S454A, and S460A) were reduced in motility and synthesized truncated flagellar filaments. The data implicate certain glycans in mediating filament-filament interactions resulting in AAG and other glycans appear to be critical for structural subunit-subunit interactions within the filament.
Repeat-Associated Plasticity in the
Helicobacter pylori
RD Gene Family
Authors: Shak Joshua R. , Dick Jonathan J. , Meinersmann Richard J. , Perez-Perez Guillermo I. , Blaser Martin J.
Published: September 2009

The bacterium
Helicobacter pylori
is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two
H. pylori
strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the
H. pylori
RD gene family. Each gene of this family is composed of a conserved 3′ region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5′ region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42
H. pylori
strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject's stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by
H. pylori
-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding
H. pylori
persistence in its natural host.
Envelope Proteins of the CsmB/CsmF and CsmC/CsmD Motif Families Influence the Size, Shape, and Composition of Chlorosomes in
Chlorobaculum tepidum
Authors: Li Hui , Bryant Donald A.
Published: September 2009

The chlorosome envelope of
Chlorobaculum tepidum
contains 10 proteins that belong to four structural motif families. A previous mutational study (N.-U. Frigaard, H. Li, K. J. Milks, and D. A. Bryant, J. Bacteriol. 186:646-653, 2004) suggested that some of these proteins might have redundant functions. Six multilocus mutants were constructed to test the effects of eliminating the proteins of the CsmC/CsmD and CsmB/CsmF motif families, and the resulting strains were characterized physiologically and biochemically. Mutants lacking all proteins of either motif family still assembled functional chlorosomes, and as measured by growth rates of the mutant strains, light harvesting was affected only at the lowest light intensities tested (9 and 32 μmol photons m
−2
s
−1
). The size, composition, and biogenesis of the mutant chlorosomes differed from those of wild-type chlorosomes. Mutants lacking proteins of the CsmC/CsmD motif family produced smaller chlorosomes than did the wild type, and the

Q
y

absorbance maximum for the bacteriochlorophyll
c
aggregates in these chlorosomes was strongly blueshifted. Conversely, the chlorosomes of mutants lacking proteins of the CsmB/CsmF motif family were larger than wild-type chlorosomes, and the

Q
y

absorption for their bacteriochlorophyll
c
aggregates was redshifted. When CsmH was eliminated in addition to other proteins of either motif family, chlorosomes had smaller diameters. These data show that the chlorosome envelope proteins of the CsmB/CsmF and CsmC/CsmD families play important roles in determining chlorosome size as well as the assembly and supramolecular organization of the bacteriochlorophyll
c
aggregates within the chlorosome.
Identification of Direct Transcriptional Target Genes of ExoS/ChvI Two-Component Signaling in
Sinorhizobium meliloti
Authors: Chen Esther J. , Fisher Robert F. , Perovich Virginia M. , Sabio Erich A. , Long Sharon R.
Published: September 2009

The
Sinorhizobium meliloti
ExoS/ChvI two-component signaling pathway is required for the development of a nitrogen-fixing symbiosis between
S. meliloti
and its plant hosts. ExoS/ChvI also has important roles in regulating succinoglycan production, biofilm formation, motility, nutrient utilization, and the viability of free-living bacteria. Previous microarray experiments with an
exoS96
::Tn
5
mutant indicated that ExoS/ChvI influences the expression of a few hundred genes, complicating the investigation of which downstream genes respond directly or indirectly to ExoS/ChvI regulation. To focus our study of ExoS/ChvI transcriptional target genes, we performed transcriptional profiling with
chvI
gain-of-function and reduced-function strains. The
chvI
gain-of-function strain that we used contains a dominant gain-of-function
chvI
allele in addition to wild-type
chvI
. We identified genes that, relative to their expression level in the wild type, are both upregulated in the
chvI
gain-of-function strain and downregulated in the reduced-function strain or vice versa. Guided by this focused set of genes, we performed gel mobility shift assays and demonstrated that ChvI directly binds the intergenic regions upstream of
ropB1
, SMb21440, and SMc01580. Furthermore, DNase I footprint analysis of the region upstream of SMc01580 identified a specific DNA sequence bound by ChvI and allowed the discovery of a possible motif for ChvI binding. Our results provide insight into the mechanism of how ExoS/ChvI regulates its downstream targets and lay a foundation for studying this conserved pathway with critical roles in free-living and symbiotic bacteria.
Evidence that HumanChlamydia pneumoniaeWas Zoonotically Acquired
Authors: Myers G. S. A. , Mathews S. A. , Eppinger M. , Mitchell C. , O'Brien K. K. , White O. R. , Benahmed F. , Brunham R. C. , Read T. D. , Ravel J. , Bavoil P. M. , Timms P.
Published: September 2009

Zoonotic infections are a growing threat to global health.Chlamydia pneumoniaeis a major human pathogen that is widespread in human populations, causing acute respiratory disease, and has been associated with chronic disease.C. pneumoniaewas first identified solely in human populations; however, its host range now includes other mammals, marsupials, amphibians, and reptiles. Australian koalas (Phascolarctos cinereus) are widely infected with two species ofChlamydia,C. pecorumandC. pneumoniae. Transmission ofC. pneumoniaebetween animals and humans has not been reported; however, two other chlamydial species,C. psittaciandC. abortus, are known zoonotic pathogens. We have sequenced the 1,241,024-bp chromosome and a 7.5-kb cryptic chlamydial plasmid of the koala strain ofC. pneumoniae(LPCoLN) using the whole-genome shotgun method. Comparative genomic analysis, including pseudogene and single-nucleotide polymorphism (SNP) distribution, and phylogenetic analysis of conserved genes and SNPs against the human isolates ofC. pneumoniaeshow that the LPCoLN isolate is basal to human isolates. Thus, we propose based on compelling genomic and phylogenetic evidence that humans were originally infected zoonotically by an animal isolate(s) ofC. pneumoniaewhich adapted to humans primarily through the processes of gene decay and plasmid loss, to the point where the animal reservoir is no longer required for transmission.
The Terminal A Domain of the Fibrillar Accumulation-Associated Protein (Aap) of
Staphylococcus epidermidis
Mediates Adhesion to Human Corneocytes
Authors: Macintosh Robin L. , Brittan Jane L. , Bhattacharya Ritwika , Jenkinson Howard F. , Derrick Jeremy , Upton Mathew , Handley Pauline S.
Published: September 2009

The opportunistic pathogen
Staphylococcus epidermidis
colonizes indwelling medical devices by biofilm formation but is primarily a skin resident. In many
S. epidermidis
strains biofilm formation is mediated by a cell wall-anchored protein, the accumulation-associated protein (Aap). Here, we investigate the role of Aap in skin adhesion. Aap is an LPXTG protein with a domain architecture including a terminal A domain and a B-repeat region.
S. epidermidis
NCTC 11047 expresses Aap as localized, lateral tufts of fibrils on one subpopulation of cells (Fib
+
), whereas a second subpopulation does not express these fibrils of Aap (Fib
−
). Flow cytometry showed that 72% of NCTC 11047 cells expressed Aap and that 28% of cells did not. Aap is involved in the adhesion of Fib
+
cells to squamous epithelial cells from the hand (corneocytes), as the recombinant A-domain protein partially blocked binding to corneocytes. To confirm the role of the Aap A domain in corneocyte attachment, Aap was expressed on the surface of
Lactococcus lactis
MG1363 as sparsely distributed, peritrichous fibrils. The expression of Aap increased corneocyte adhesion 20-fold compared to
L. lactis
carrying Aap without an A domain.
S. epidermidis
isolates from catheters, artificial joints, skin, and the nose also used the A domain of Aap to adhere to corneocytes, emphasizing the role of Aap in skin adhesion. In addition,
L. lactis
expressing Aap with different numbers of B repeats revealed a positive correlation between the number of B repeats and adhesion to corneocytes, suggesting an additional function for the B region in enhancing A-domain-dependent attachment to skin. Therefore, in addition to its established role in biofilm formation, Aap can also promote adhesion to corneocytes and is likely to be an important adhesin in
S. epidermidis
skin colonization.
Genetic and Biochemical Analysis of the Interaction ofBacillus subtilisCodY with Branched-Chain Amino Acids
Authors: Villapakkam Anuradha C. , Handke Luke D. , Belitsky Boris R. , Levdikov Vladimir M. , Wilkinson Anthony J. , Sonenshein Abraham L.
Published: September 2009

Bacillus subtilisCodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.
Motility and Flagellar Glycosylation in
Clostridium difficile
Authors: Twine Susan M. , Reid Christopher W. , Aubry Annie , McMullin David R. , Fulton Kelly M. , Austin John , Logan Susan M.
Published: September 2009

In this study, intact flagellin proteins were purified from strains of
Clostridium difficile
and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of
C. difficile
genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In
C. difficile
strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein.
Laboratory Strains ofBacillus subtilisDo Not Exhibit Swarming Motility
Authors: Patrick Joyce E. , Kearns Daniel B.
Published: September 2009

We redemonstrate that SwrA is essential for swarming motility inBacillus subtilis, and we reassert that laboratory strains ofB. subtilisdo not swarm. Additionally, we find that a number of other genes, previously reported to be required for swarming in laboratory strains, are dispensable for robust swarming motility in an undomesticated strain. We attribute discrepancies in the literature to a lack of reproducible standard experimental conditions, selection for spontaneous swarming suppressors, inadvertent genetic linkage to swarming mutations, and auxotrophy.
Identification of Functional LsrB-Like Autoinducer-2 Receptors
Authors: Pereira Catarina S. , de Regt Anna K. , Brito Patrícia H. , Miller Stephen T. , Xavier Karina B.
Published: September 2009

Although a variety of bacterial species have been reported to use the interspecies communication signal autoinducer-2 (AI-2) to regulate multiple behaviors, the molecular mechanisms of AI-2 recognition and signal transduction remain poorly understood. To date, two types of AI-2 receptors have been identified: LuxP, present inVibriospp., and LsrB, first identified inSalmonella entericaserovar Typhimurium. InS.Typhimurium, LsrB is the ligand binding protein of a transport system that enables the internalization of AI-2. Here, using both sequence analysis and structure prediction, we establish a set of criteria for identifying functional AI-2 receptors. We test our predictions experimentally, assaying key species for their abilities to import AI-2 in vivo, and test their LsrB orthologs for AI-2 binding in vitro. Using these experimental approaches, we were able to identify AI-2 receptors in organisms belonging to phylogenetically distinct families such as theEnterobacteriaceae,Rhizobiaceae, andBacillaceae. Phylogenetic analysis of LsrB orthologs indicates that this pattern could result from one single origin of the functional LsrB gene in a gammaproteobacterium, suggesting possible posterior independent events of lateral gene transfer to theAlphaproteobacteriaandFirmicutes. Finally, we used mutagenesis to show that two AI-2-interacting residues are essential for the AI-2 binding ability. These two residues are conserved in the binding sites of all the functional AI-2 binding proteins but not in the non-AI-2-binding orthologs. Together, these results strongly support our ability to identify functional LsrB-type AI-2 receptors, an important step in investigations of this interspecies signal.
Archaeal Intrinsic Transcription Termination In Vivo
Authors: Santangelo Thomas J. , Cubonová L'ubomíra , Skinner Katherine M. , Reeve John N.
Published: September 2009

Thermococcus kodakarensis
(formerly
Thermococcus kodakaraensis
) strains have been constructed with synthetic and natural DNA sequences, predicted to function as archaeal transcription terminators, identically positioned between a constitutive promoter and a β-glycosidase-encoding reporter gene (TK1761). Expression of the reporter gene was almost fully inhibited by the upstream presence of 5′-TTTTTTTT (T
8
) and was reduced >70% by archaeal intergenic sequences that contained oligo(T) sequences. An archaeal intergenic sequence (t

mcrA

) that conforms to the bacterial intrinsic terminator motif reduced TK1761 expression ∼90%, but this required only the oligo(T) trail sequence and not the inverted-repeat and loop region. Template DNAs were amplified from each
T. kodakarensis
strain, and transcription in vitro by
T. kodakarensis
RNA polymerase was terminated by sequences that reduced TK1761 expression in vivo. Termination occurred at additional sites on these linear templates, including at a 5′-AAAAAAAA (A
8
) sequence that did not reduce TK1761 expression in vivo. When these sequences were transcribed on supercoiled plasmid templates, termination occurred almost exclusively at oligo(T) sequences. The results provide the first in vivo experimental evidence for intrinsic termination of archaeal transcription and confirm that archaeal transcription termination is stimulated by oligo(T) sequences and is different from the RNA hairpin-dependent mechanism established for intrinsic bacterial termination.
Global Transcriptional Response to Spermine, a Component of the Intramacrophage Environment, Reveals Regulation of
Francisella
Gene Expression through Insertion Sequence Elements
Authors: Carlson Paul E. , Horzempa Joseph , O'Dee Dawn M. , Robinson Cory M. , Neophytou Panayiotis , Labrinidis Alexandros , Nau Gerard J.
Published: September 2009

Tularemia is caused by the category A biodefense agent
Francisella tularensis
. This bacterium is associated with diverse environments and a plethora of arthropod and mammalian hosts. How
F. tularensis
adapts to these different conditions, particularly the eukaryotic intracellular environment in which it replicates, is poorly understood. Here, we demonstrate that the polyamines spermine and spermidine are environmental signals that alter bacterial stimulation of host cells. Genomewide analysis showed that
F. tularensis
LVS undergoes considerable changes in gene expression in response to spermine. Unexpectedly, analysis of gene expression showed that multiple members of two classes of
Francisella
insertion sequence (IS) elements, IS
Ftu1
and IS
Ftu2
, and the genes adjacent to these elements were induced by spermine. Spermine was sufficient to activate transcription of these IS elements and of nearby genes in broth culture and in macrophages. Importantly, the virulent strain of
F. tularensis
, Schu S4, exhibited similar phenotypes of cytokine induction and gene regulation in response to spermine. Distinctions in gene expression changes between Schu S4 and LVS at one orthologous locus, however, correlated with differences in IS element location. Our results indicate that spermine and spermidine are novel triggers to alert
F. tularensis
of its eukaryotic host environment. The results reported here also identify an unexpected mechanism of gene regulation controlled by a spermine-responsive promoter contained within IS elements. Different arrangements of these mobile genetic elements among
Francisella
strains may contribute to virulence by conveying new expression patterns for genes from different strains.
YjhS (NanS) Is Required for
Escherichia coli
To Grow on 9-O-Acetylated
N
-Acetylneuraminic Acid
Authors: Steenbergen Susan M. , Jirik Jamie L. , Vimr Eric R.
Published: September 2009

The
nanATEK
-
yhcH
,
yjhATS
, and
yjhBC
operons in
Escherichia coli
are coregulated by environmental
N
-acetylneuraminic acid, the most prevalent sialic acid in nature. Here we show that YjhS (NanS) is a probable 9-O-acetyl
N
-acetylneuraminic acid esterase required for
E. coli
to grow on this alternative sialic acid, which is commonly found in mammalian host mucosal sites.
Mutualistic Biofilm Communities Develop with
Porphyromonas gingivalis
and Initial, Early, and Late Colonizers of Enamel
Authors: Periasamy Saravanan , Kolenbrander Paul E.
Published: September 2009

Porphyromonas gingivalis
is present in dental plaque as early as 4 h after tooth cleaning, but it is also associated with periodontal disease, a late-developing event in the microbial successions that characterize daily plaque development. We report here that
P. gingivalis
ATCC 33277 is remarkable in its ability to interact with a variety of initial, early, middle, and late colonizers growing solely on saliva. Integration of
P. gingivalis
into multispecies communities was investigated by using two in vitro biofilm models. In flow cells, bacterial growth was quantified using fluorescently conjugated antibodies against each species, and static biofilm growth on saliva-submerged polystyrene pegs was analyzed by quantitative real-time PCR using species-specific primers.
P. gingivalis
could not grow as a single species or together with initial colonizer
Streptococcus oralis
but showed mutualistic growth when paired with two other initial colonizers,
Streptococcus gordonii
and
Actinomyces oris
, as well as with
Veillonella
sp. (early colonizer),
Fusobacterium nucleatum
(middle colonizer), and
Aggregatibacter actinomycetemcomitans
(late colonizer). In three-species flow cells,
P. gingivalis
grew with
Veillonella
sp. and
A. actinomycetemcomitans
but not with
S. oralis
and
A. actinomycetemcomitans
. Also, it grew with
Veillonella
sp. and
F. nucleatum
but not with
S. oralis
and
F. nucleatum
, indicating that
P. gingivalis
and
S. oralis
are not compatible. However,
P. gingivalis
grew in combination with
S. gordonii
and
S. oralis
, demonstrating its ability to overcome the incompatibility when cultured with a second initially colonizing species. Collectively, these data help explain the observed presence of
P. gingivalis
at all stages of dental plaque development.