bluuebunny/crossref_metadata_2025 huggingface dataset:
Insights into the Effect of Soil pH on N
2
O and N
2
Emissions and Denitrifier Community Size and Activity
Authors: Čuhel Jiří , Šimek Miloslav , Laughlin Ronnie J. , Bru David , Chèneby Dominique , Watson Catherine J. , Philippot Laurent
Published: January 2010

The objective of this study was to investigate how changes in soil pH affect the N
2
O and N
2
emissions, denitrification activity, and size of a denitrifier community. We established a field experiment, situated in a grassland area, which consisted of three treatments which were repeatedly amended with a KOH solution (alkaline soil), an H
2
SO
4
solution (acidic soil), or water (natural pH soil) over 10 months. At the site, we determined field N
2
O and N
2
emissions using the
15
N gas flux method and collected soil samples for the measurement of potential denitrification activity and quantification of the size of the denitrifying community by quantitative PCR of the
narG
,
napA
,
nirS
,
nirK
, and
nosZ
denitrification genes. Overall, our results indicate that soil pH is of importance in determining the nature of denitrification end products. Thus, we found that the N
2
O/(N
2
O + N
2
) ratio increased with decreasing pH due to changes in the total denitrification activity, while no changes in N
2
O production were observed. Denitrification activity and N
2
O emissions measured under laboratory conditions were correlated with N fluxes
in situ
and therefore reflected treatment differences in the field. The size of the denitrifying community was uncoupled from
in situ
N fluxes, but potential denitrification was correlated with the count of NirS denitrifiers. Significant relationships were observed between
nirS
,
napA
, and
narG
gene copy numbers and the N
2
O/(N
2
O + N
2
) ratio, which are difficult to explain. However, this highlights the need for further studies combining analysis of denitrifier ecology and quantification of denitrification end products for a comprehensive understanding of the regulation of N fluxes by denitrification.
Prevalence ofBacillus anthracis-Like Organisms and Bacteriophages in the Intestinal Tract of the EarthwormEisenia fetida
Authors: Schuch R. , Pelzek A. J. , Kan S. , Fischetti V. A.
Published: January 2010

Stable infection ofBacillus anthracislaboratory strains with environmental bacteriophages confers survival phenotypes in soil and earthworm intestinal niches (R. Schuch and V. A. Fischetti, PLoS One 4:e6532, 2009). Here, the natural occurrence of two suchB. anthracis-infective bacteriophages, Wip1 and Wip4, was examined in the intestines ofEisenia fetidaearthworms as part of a 6-year longitudinal study at a Pennsylvania forest site. The Wip1 tectivirus was initially dominant before being supplanted by the Wip4 siphovirus, which was then dominant for the next 3 years. In a host range analysis of a wide-ranging group ofBacillusspecies and related organisms, Wip1 and Wip4 were both infective only towardB. anthracisand certainB. cereusstrains. The natural host of Wip4 remained constant for 3 years and was aB. cereusstrain that expressed aB. anthracis-like surface polysaccharide at septal positions on the cell surface. Next, a novel metagenomic approach was used to determine the extent to which suchB. cereus- andB. anthracis-like strains are found in worms from two geographical locations. Three different enrichment strategies were used for metagenomic DNA isolation, based either on the ability ofB. cereussensu lato to form heat-resistant spores, the sensitivity ofB. anthracisto the PlyG lysin, or the selective amplification of environmental phages cocultured withB. anthracis. Findings from this work indicate thatB. cereussensu lato and its phages are common inhabitants of earthworm intestines.
Metabolic Flux Control at the Pyruvate Node in an AnaerobicEscherichia coliStrain with an Active Pyruvate Dehydrogenase
Authors: Wang Qingzhao , Ou Mark S. , Kim Y. , Ingram L. O. , Shanmugam K. T.
Published: January 2010

During anaerobic growth ofEscherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant ofE. coliwith a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In apflmutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvatein vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. Apflmutant with LDH and PDH* activities, as well as apfl ldhdouble mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (YATP) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lowerYATPsuggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering ofE. coliand related bacteria for the production of various biobased products at high rates and yields.
Presence of Novel, Potentially Homoacetogenic Bacteria in the Rumen as Determined by Analysis of Formyltetrahydrofolate Synthetase Sequences from Ruminants
Authors: Henderson Gemma , Naylor Graham E. , Leahy Sinead C. , Janssen Peter H.
Published: January 2010

Homoacetogens produce acetate from H
2
and CO
2
via the Wood-Ljungdahl pathway. Some homoacetogens have been isolated from the rumen, but these organisms are expected to be only part of the full diversity present. To survey the presence of rumen homoacetogens, we analyzed sequences of formyltetrahydrofolate synthetase (FTHFS), a key enzyme of the Wood-Ljungdahl pathway. A total of 275 partial sequences of genes encoding FTHFS were PCR amplified from rumen contents of a cow, two sheep, and a deer. Phylogenetic trees were constructed using these FTHFS gene sequences and the translated amino acid sequences, together with other sequences from public databases and from novel nonhomoacetogenic bacteria isolated from the rumen. Over 90% of the FTHFS sequences fell into 34 clusters defined with good bootstrap support. Few rumen-derived FTHFS sequences clustered with sequences of known homoacetogens. Conserved residues were identified in the deduced FTHFS amino acid sequences from known homoacetogens, and their presence in the other sequences was used to determine a “homoacetogen similarity” (HS) score. A homoacetogen FTHFS profile hidden Markov model (HoF-HMM) was used to assess the homology of rumen and homoacetogen FTHFS sequences. Many clusters had low HS scores and HoF-HMM matches, raising doubts about whether the sequences originated from homoacetogens. In keeping with these findings, FTHFS sequences from nonhomoacetogenic bacterial isolates grouped in these clusters with low scores. However, sequences that formed 10 clusters containing no known isolates but representing 15% of our FTHFS sequences from rumen samples had high HS scores and HoF-HMM matches and so could represent novel homoacetogens.
Molecular Monitoring and Isolation of Previously Uncultured Bacterial Strains from the Sheep Rumen
Authors: Koike S. , Handa Y. , Goto H. , Sakai K. , Miyagawa E. , Matsui H. , Ito S. , Kobayashi Y.
Published: January 2010

To estimate the contribution of uncultured bacterial groups to fiber degradation, we attempted to retrieve both ecological and functional information on uncultured groups in the rumen. Among previously reported uncultured bacteria, fiber-associated groups U2 and U3, belonging to the low-GC Gram-positive bacterial group, were targeted. PCR primers and fluorescence
in situ
hybridization (FISH) probe targeting 16S rRNA genes or rRNA were designed and used to monitor the distribution of targets. The population size of group U2 in the rumen was as high as 1.87%, while that of group U3 was only 0.03%. Strong fluorescence signals were observed from group U2 cells attached to plant fibers in the rumen. These findings indicate the ecological significance of group U2 in the rumen. We succeeded in enriching group U2 using rumen-incubated rice straw as the inoculum followed by incubation in an appropriate medium with an agent inhibitory for Gram-negative bacteria. Consequently, we successfully isolated two strains, designated B76 and R-25, belonging to group U2. Both strains were Gram-positive short rods or cocci that were 0.5 to 0.8 μm in size. Strain B76 possessed xylanase and α-
l
-arabinofuranosidase activity. In particular, the xylanase activity of strain B76 was higher than that of xylanolytic
Butyrivibrio fibrisolvens
H17c grown on cellobiose. Strain R-25 showed an α-
l
-arabinofuranosidase activity higher than that of strain B76. These results suggest that strains B76 and R-25 contribute to hemicellulose degradation in the rumen.
High Abundance of Ammonia-OxidizingArchaeain Coastal Waters, Determined Using a Modified DNA Extraction Method
Authors: Urakawa Hidetoshi , Martens-Habbena Willm , Stahl David A.
Published: January 2010

Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition ofNitrosopumilus maritimuscells to track the recovery of DNA from marineArchaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizingArchaeaat different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers ofArchaeaincreased with depth, often constituting as much as 20% of the total bacterial community.
Inactivation of
Vibrio anguillarum
by Attached and Planktonic
Roseobacter
Cells
Authors: D'Alvise Paul W. , Melchiorsen Jette , Porsby Cisse H. , Nielsen Kristian F. , Gram Lone
Published: January 2010

The purpose of the present study was to investigate the inhibition of
Vibrio
by
Roseobacter
in a combined liquid-surface system. Exposure of
Vibrio anguillarum
to surface-attached roseobacters (10
7
CFU/cm
2
) resulted in significant reduction or complete killing of the pathogen inoculated at 10
2
to 10
4
CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of
V. anguillarum
.
Hitherto Unknown [Fe-Fe]-Hydrogenase Gene Diversity in Anaerobes and Anoxic Enrichments from a Moderately Acidic Fen
Authors: Schmidt Oliver , Drake Harold L. , Horn Marcus A.
Published: January 2010

Newly designed primers for [Fe-Fe]-hydrogenases indicated that (i) fermenters, acetogens, and undefined species in a fen harbor hitherto unknown hydrogenases and (ii)
Clostridium
- and
Thermosinus
-related primary fermenters, as well as secondary fermenters related to sulfate or iron reducers might be responsible for hydrogen production in the fen. Comparative analysis of [Fe-Fe]-hydrogenase and 16S rRNA gene-based phylogenies indicated the presence of homologous multiple hydrogenases per organism and inconsistencies between 16S rRNA gene- and [Fe-Fe]-hydrogenase-based phylogenies, necessitating appropriate qualification of [Fe-Fe]-hydrogenase gene data for diversity analyses.
Requirement of the
galU
Gene for Polysaccharide Production by and Pathogenicity and Growth
In Planta
of
Xanthomonas citri
subsp.
citri
Authors: Guo Yinping , Sagaram Uma Shankar , Kim Jeong-soon , Wang Nian
Published: January 2010

Xanthomonas citri
subsp.
citri
is the causal agent of citrus canker, which has a significant impact on citrus production. In this study, we characterized the
galU
gene of
X. citri
subsp.
citri
. Two
galU
mutants (F6 and D12) were identified in an
X. citri
subsp.
citri
EZ-Tn5  Tnp transposon library. Rescue cloning, sequence analysis, and Southern blot analysis indicated that both of these mutants had a single copy of the EZ-Tn5 transposon inserted in
galU
in the chromosome. Further study showed that
galU
was required for biosynthesis of extracellular polysaccharides (EPS; xanthan gum) and capsular polysaccharide (CPS) and biofilm formation. Mutation of
galU
resulted in a loss of pathogenicity for grapefruit. The loss of pathogenicity of a
galU
mutant resulted from its inability to grow
in planta
rather than from the effect on virulence genes. Quantitative reverse transcription-PCR assays indicated that mutation of
galU
did not impair the expression of key virulence genes, such as
pthA
of
X. citri
subsp.
citri
. Although D12 had a growth rate similar to that of the wild-type strain in nutrient broth, no D12 population became established in the intercellular spaces of citrus leaves. Coinoculation of a
galU
mutant with the wild-type strain did not promote growth of the
galU
mutant
in planta
. Defects in EPS and CPS production, pathogenicity, and growth
in planta
of the
galU
mutant were complemented to the wild-type level using plasmid pCGU2.1 containing an intact
galU
gene. These data indicate that the
galU
gene contributes to
X. citri
subsp.
citri
growth in intercellular spaces and is involved in EPS and CPS synthesis and biofilm formation.
The RFX Protein RfxA Is an Essential Regulator of Growth and Morphogenesis in Penicillium marneffei
Authors: Bugeja Hayley E. , Hynes Michael J. , Andrianopoulos Alex
Published: January 2010

Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. The opportunistic human pathogen
Penicillium marneffei
is a dimorphic fungus, displaying vegetative (proliferative) multicellular hyphal growth at 25°C and unicellular yeast growth at 37°C.
P. marneffei
also undergoes asexual development into differentiated multicellular conidiophores bearing uninucleate spores. These morphogenetic processes require regulated changes in cell polarity establishment, cell cycle dynamics, and nuclear migration. The RFX (regulatory factor X) proteins are a family of transcriptional regulators in eukaryotes. We sought to determine how the sole
P. marneffei
RFX protein, RfxA, contributes to the regulation of morphogenesis. Attempts to generate a haploid
rfxA
deletion strain were unsuccessful, but we did isolate an
rfxA
+
/
rfxA
Δ heterozygous diploid strain. The role of RfxA was assessed using conditional overexpression, RNA interference (RNAi), and the production of dominant interfering alleles. Reduced RfxA function resulted in defective mitoses during growth at 25°C and 37°C. This was also observed for the heterozygous diploid strain during growth at 37°C. In contrast, overexpression of
rfxA
caused growth arrest during conidial germination. The data show that
rfxA
must be precisely regulated for appropriate nuclear division and to maintain genome integrity. Perturbations in
rfxA
expression also caused defects in cellular proliferation and differentiation. The data suggest a role for RfxA in linking cellular division with morphogenesis, particularly during conidiation and yeast growth, where the uninucleate state of these cell types necessitates coupling of nuclear and cellular division tighter than that observed during multinucleate hyphal growth.
The TEA Transcription Factor Tec1 Confers Promoter-Specific Gene Regulation by Ste12-Dependent and -Independent Mechanisms
Authors: Heise Barbara , van der Felden Julia , Kern Sandra , Malcher Mario , Brückner Stefan , Mösch Hans-Ulrich
Published: January 2010

In
Saccharomyces cerevisiae
, the TEA transcription factor Tec1 is known to regulate target genes together with a second transcription factor, Ste12. Tec1-Ste12 complexes can activate transcription through Tec1 binding sites (TCSs), which can be further combined with Ste12 binding sites (PREs) for cooperative DNA binding. However, previous studies have hinted that Tec1 might regulate transcription also without Ste12. Here, we show that
in vivo
, physiological amounts of Tec1 are sufficient to stimulate TCS-mediated gene expression and transcription of the
FLO11
gene in the absence of Ste12.
In vitro
, Tec1 is able to bind TCS elements with high affinity and specificity without Ste12. Furthermore, Tec1 contains a C-terminal transcriptional activation domain that confers Ste12-independent activation of TCS-regulated gene expression. On a genome-wide scale, we identified 302 Tec1 target genes that constitute two distinct classes. A first class of 254 genes is regulated by Tec1 in a Ste12-dependent manner and is enriched for genes that are bound by Tec1 and Ste12
in vivo
. In contrast, a second class of 48 genes can be regulated by Tec1 independently of Ste12 and is enriched for genes that are bound by the stress transcription factors Yap6, Nrg1, Cin5, Skn7, Hsf1, and Msn4. Finally, we find that combinatorial control by Tec1-Ste12 complexes stabilizes Tec1 against degradation. Our study suggests that Tec1 is able to regulate TCS-mediated gene expression by Ste12-dependent and Ste12-independent mechanisms that enable promoter-specific transcriptional control.
Farnesol Induces Hydrogen Peroxide Resistance in Candida albicans Yeast by Inhibiting the Ras-Cyclic AMP Signaling Pathway
Authors: Deveau Aurélie , Piispanen Amy E. , Jackson Angelyca A. , Hogan Deborah A.
Published: January 2010

Farnesol, a
Candida albicans
cell-cell signaling molecule that participates in the control of morphology, has an additional role in protection of the fungus against oxidative stress. In this report, we show that although farnesol induces the accumulation of intracellular reactive oxygen species (ROS), ROS generation is not necessary for the induction of catalase (Cat1)-mediated oxidative-stress resistance. Two antioxidants, α-tocopherol and, to a lesser extent, ascorbic acid effectively reduced intracellular ROS generation by farnesol but did not alter farnesol-induced oxidative-stress resistance. Farnesol inhibits the Ras1-adenylate cyclase (Cyr1) signaling pathway to achieve its effects on morphology under hypha-inducing conditions, and we demonstrate that farnesol induces oxidative-stress resistance by a similar mechanism. Strains lacking either Ras1 or Cyr1 no longer exhibited increased protection against hydrogen peroxide upon preincubation with farnesol. While we also observed the previously reported increase in the phosphorylation level of Hog1, a known regulator of oxidative-stress resistance, in the presence of farnesol, the
hog1
/
hog1
mutant did not differ from wild-type strains in terms of farnesol-induced oxidative-stress resistance. Analysis of Hog1 levels and its phosphorylation states in different mutant backgrounds indicated that mutation of the components of the Ras1-adenylate cyclase pathway was sufficient to cause an increase of Hog1 phosphorylation even in the absence of farnesol or other exogenous sources of oxidative stress. This finding indicates the presence of unknown links between these signaling pathways. Our results suggest that farnesol effects on the Ras-adenylate cyclase cascade are responsible for many of the observed activities of this fungal signaling molecule.
Sterol Regulatory Element Binding Proteins in Fungi: Hypoxic Transcription Factors Linked to Pathogenesis
Authors: Bien Clara M. , Espenshade Peter J.
Published: January 2010

Sterol regulatory element binding proteins (SREBPs) are membrane-bound transcription factors whose proteolytic activation is controlled by the cellular sterol concentration. Mammalian SREBPs are activated in cholesterol-depleted cells and serve to regulate cellular lipid homeostasis. Recent work demonstrates that SREBP is functionally conserved in fungi. While the ability to respond to sterols is conserved, fungal SREBPs are hypoxic transcription factors required for adaptation to a low-oxygen environment. In the fission yeast
Schizosaccharomyces pombe
, oxygen regulates the SREBP homolog Sre1 by independently controlling both its proteolytic activation and its degradation. SREBP is also required for adaptation to hypoxia in the human pathogens
Cryptococcus neoformans
and
Aspergillus fumigatus
. In these organisms, SREBP is required for virulence and resistance to antifungal drugs, making the SREBP pathway a potential target for antifungal therapy.
Bug22p, a Conserved Centrosomal/Ciliary Protein Also Present in Higher Plants, Is Required for an Effective Ciliary Stroke in Paramecium
Authors: Laligné C. , Klotz C. , Garreau de Loubresse N. , Lemullois M. , Hori M. , Laurent F. X. , Papon J. F. , Louis B. , Cohen J. , Koll F.
Published: January 2010

Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in
Paramecium
, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed
per se
. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In
Paramecium
, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.
Complete Genome Sequence of the Plant Pathogen
Erwinia amylovora
Strain ATCC 49946
Authors: Sebaihia M. , Bocsanczy A. M. , Biehl B. S. , Quail M. A. , Perna N. T. , Glasner J. D. , DeClerck G. A. , Cartinhour S. , Schneider D. J. , Bentley S. D. , Parkhill J. , Beer S. V.
Published: January 2010

Erwinia amylovora
causes the economically important disease fire blight that affects rosaceous plants, especially pear and apple. Here we report the complete genome sequence and annotation of strain ATCC 49946. The analysis of the sequence and its comparison with sequenced genomes of closely related enterobacteria revealed signs of pathoadaptation to rosaceous hosts.
Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by
Methylobacterium extorquens
AM1
Authors: Okubo Yoko , Yang Song , Chistoserdova Ludmila , Lidstrom Mary E.
Published: January 2010

Methylobacterium extorquens
AM1 is a facultative methylotroph capable of growth on both single-carbon and multicarbon compounds. Mutants defective in a pathway involved in converting acetyl-coenzyme A (CoA) to glyoxylate (the ethylmalonyl-CoA pathway) are unable to grow on both C
1
and C
2
compounds, showing that both modes of growth have this pathway in common. However, growth on C
2
compounds via the ethylmalonyl-CoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/β-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still grows on C
2
compounds. Since glyoxylate is toxic to this bacterium, it seemed likely that a system is in place to keep it from accumulating. In this study, we have addressed this question and have shown by microarray analysis, mutant analysis, metabolite measurements, and
13
C-labeling experiments that
M. extorquens
AM1 contains an additional malyl-CoA/β-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate metabolism during growth on C
2
compounds. In addition, an alternative pathway appears to be responsible for consuming part of the glyoxylate, converting it to glycine, methylene-H
4
F, and serine. Mutants lacking either pathway have a partial defect for growth on ethylamine, while mutants lacking both pathways are unable to grow appreciably on ethylamine. Our results suggest that the malate synthase reaction is a bottleneck for growth on C
2
compounds by this bacterium, which is partially alleviated by this alternative route for glyoxylate consumption. This strategy of multiple enzymes/pathways for the consumption of a toxic intermediate reflects the metabolic versatility of this facultative methylotroph and is a model for other metabolic networks involving high flux through toxic intermediates.
A Superfamily 3 DNA Helicase Encoded by Plasmid pSSVi from the Hyperthermophilic ArchaeonSulfolobus solfataricusUnwinds DNA as a Higher-Order Oligomer and Interacts with Host Primase
Authors: Guo Xin , Huang Li
Published: January 2010

Replication proteins encoded by nonconjugative plasmids from the hyperthermophilic archaea of the orderSulfolobalesshow great diversity in amino acid sequence. We have biochemically characterized ORF735, a replication protein from pSSVi, an integrative nonconjugative plasmid fromSulfolobus solfataricusP2. We show that ORF735 is a DNA helicase of superfamily 3. It unwound double-stranded DNA (dsDNA) in a 3′-to-5′ direction in the presence of ATP over a wide range of temperatures, from 37°C to 75°C, and possessed DNA-stimulated ATPase activity. ORF735 existed in solution as a salt-stable dimer and was capable of assembling into a salt-sensitive oligomer that was significantly larger than a hexamer in the presence of a divalent cation (Mg2+) and an adenine nucleotide (ATP, dATP, or ADP) or its analog (ATPγS or AMPPNP). Both N-terminal and C-terminal portions of ORF735 (87 and 160 amino acid residues, respectively, in size) were required for protein dimerization but dispensable for the formation of the higher-order oligomer. The protein unwound DNA only as a large oligomer. Yeast two-hybrid and coimmunoprecipitation assays revealed that ORF735 interacted with the noncatalytic subunit of host primase. These findings provide clues to the functional role of ORF735 in pSSVi DNA replication.
Tn502and Tn512AreresSite Hunters That Provide Evidence of Resolvase-Independent Transposition to Random Sites
Authors: Petrovski Steve , Stanisich Vilma A.
Published: January 2010

In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn502and Tn512, which are members of the Tn5053family. These transposons exhibit targeted and oriented insertion in theparregion of plasmid RP1, sincepar-encoded components, namely, the ParA resolvase and its cognateresregion, are essential for such transposition. Tn502and, under some circumstances, Tn512can transpose whenparis absent, providing evidence for an alternative,par-independent pathway of transposition. We show that the alternative pathway proceeds by a two-step replicative process involving random target selection and orientation of insertion, leading to the formation of cointegrates as the predominant product of the first stage of transposition. Cointegrates remain unresolved because the transposon-encoded (TniR) recombination system is relatively inefficient, as is the host-encoded (RecA) system. In the presence of theres-ParA recombination system, TniR-mediated (and RecA-mediated) cointegrate resolution is highly efficient, enabling resolution both of cointegrates involving functional transposons (Tn502and Tn512) and of defective elements (In0 and In2). These findings implicate the target-encoded accessory functions in the second stage of transposition as well as in the first. We also show that thepar-independent pathway enables the formation of deletions in the target molecule.
Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export
Authors: Minamino Tohru , Shimada Masafumi , Okabe Mayuko , Saijo-Hamano Yumiko , Imada Katsumi , Kihara May , Namba Keiichi
Published: January 2010

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA
C
) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive
Salmonella enterica
mutant, the
flhA
(
G368C
) mutant, which has a mutation in the sequence encoding FlhA
C
. The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA
C
consists of at least three subdomains, a flexible linker, FlhA
CN
, and FlhA
CC
, and that FlhA
CN
becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA
C
not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.
Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor
Authors: Bahl Christopher D. , Morisseau Christophe , Bomberger Jennifer M. , Stanton Bruce A. , Hammock Bruce D. , O'Toole George A. , Madden Dean R.
Published: January 2010

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by
Pseudomonas aeruginosa
that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other α/β hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-Å resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and
cis
-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of α/β hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.
Characterization of the CI Repressor Protein Encoded by the Temperate Lactococcal Phage TP901-1
Authors: Pedersen Margit , Ligowska Małgorzata , Hammer Karin
Published: January 2010

The gene regulatory mechanism determining the developmental pathway of the temperate bacteriophage TP901-1 is regulated by two phage-encoded proteins, CI and MOR. Functional domains of the CI repressor were investigated by introducing linkers of 15 bp at various positions in
cI
and by limited proteolysis of purified CI protein. We show that insertions of five amino acids at positions in the N-terminal half of CI resulted in mutant proteins that could no longer repress transcription from the lytic promoter, P
L
. We confirmed that the N-terminal domain of CI contains the DNA binding site, and we showed that this part of the protein is tightly folded, whereas the central part and the C-terminal part of CI seem to contain more flexible structures. Furthermore, insertions at several different positions in the central part of the CI protein reduced the cooperative binding of CI to the operator sites and possibly altered the interaction with MOR.
Role for CysJ Flavin Reductase in Molybdenum Cofactor-Dependent Resistance of
Escherichia coli
to 6-
N
-Hydroxylaminopurine
Authors: Kozmin Stanislav G. , Wang Jian , Schaaper Roel M.
Published: January 2010

We have previously described a novel
Escherichia coli
detoxification system for the removal of toxic and mutagenic N-hydroxylated nucleobases and related compounds that requires the molybdenum cofactor. Two subpathways (
ycbX
and
yiiM
) were identified, each employing a novel molybdo activity capable of inactivating N-hydroxylated compounds by reduction to the corresponding amine. In the present study, we identify the
cysJ
gene product as one additional component of this system. While the CysJ protein has been identified as the NADPH:flavin oxidoreductase component of the CysJI sulfite reductase complex (CysJ
8
I
4
), we show that the role of CysJ in base analog detoxification is unique and independent of CysI and sulfite reductase. We further show that CysJ functions as a specific partner of the YcbX molybdoenzyme. We postulate that the function of CysJ in this pathway is to provide, via its NADPH:flavin reductase activity, the reducing equivalents needed for the detoxification reaction at the YcbX molybdocenter. In support of the proposed interaction of the CysJ and YcbX proteins, we show that an apparent CysJ-YcbX “hybrid” protein from two
Vibrio
species is capable of compensating for a double
cysJ ycbX
defect in
E. coli
.
Lipopolysaccharide as Shield and Receptor for R-Pyocin-Mediated Killing in
Pseudomonas aeruginosa
Authors: Köhler Thilo , Donner Viviane , van Delden Christian
Published: January 2010

Pseudomonas aeruginosa
produces three different types of bacteriocins: the soluble S-pyocins and the bacteriophage-like F- and R-pyocins. R-pyocins kill susceptible bacteria of the same or closely related species with high efficiency. Five different types of R-pyocins (R1- to R5-pyocins) have been described based on their killing spectra and tail fiber protein sequences. We analyzed the distribution of R-pyocin genes in a collection of clinical
P. aeruginosa
isolates. We found similar percentages of isolates not containing R-pyocins (28%) and isolates containing genes encoding R1-pyocins (25%), R2-pyocins (17%), and R5-pyocins (29%). The R-pyocin-deficient isolates were susceptible to R1-, R2-, and R5-pyocins, while most R2- and R5- pyocin producers were resistant. Determination of the O serotypes revealed that the R-pyocin-susceptible isolates belonged to serotypes O1, O3, and O6, while the R-pyocin-resistant isolates were serotype O10, O11, and O12 isolates. We hypothesized that O-serotype-specific lipopolysaccharide (LPS) packaging densities may account for the distinct accessibilities of R-pyocins to their receptors at the cell surface. Using genetically defined LPS mutants, we showed that the
l
-Rha residue and two distinct
d
-Glc residues of the outer core are part of the receptor sites for R1-, R2-, and R5-pyocins, respectively. To illustrate R-pyocin-mediated intraspecies biological warfare, we monitored the population dynamics of two different R-pyocin-producing
P. aeruginosa
clones of sequential respiratory isolates obtained from a colonized patient. The results of this study highlight the potential role of R-pyocins in shaping bacterial populations during host colonization and support use of these molecules as specific and potent bactericidal agents.
Archaeal Eukaryote-Like Serine/Threonine Protein Kinase Interacts with and Phosphorylates a Forkhead-Associated-Domain-Containing Protein
Authors: Wang Bin , Yang Shifan , Zhang Lei , He Zheng-Guo
Published: January 2010

Protein phosphorylation plays an important role in cell signaling. However, in the
Archaea
, little is known about which proteins are phosphorylated and which kinases are involved. In this study, we identified, for the first time, a typical eukaryote-like Ser/Thr protein kinase and its protein partner, a forkhead-associated (FHA)-domain-containing protein, from the archaeon
Sulfolobus tokodaii
strain 7. This protein kinase, ST1565, physically interacted with the FHA-domain-containing protein, ST0829, both
in vivo
and
in vitro
. ST1565 preferred Mn
2+
as a cofactor for autophosphorylation and for substrate phosphorylation; the optimal temperature for this was 45°C, and the optimal pH was 5.5 to 7.5. The critical amino acid residues of the conserved FHA and kinase domain sites were identified by performing a series of mutation assays. Thr329 was part of a major activation site in the kinase, while Thr326 was a negative regulation site. Several mutants with amino acid substitutions in the conserved FHA domain sites of ST0829 did not physically interact with ST1565. A structural model for the FHA domain demonstrated that the mutation sites were located at the edge of the protein and thus were in the domain that potentially interacts with ST1565. This report describes pioneering work on the third domain of life, the
Archaea
, showing that a protein kinase interacts with and phosphorylates an FHA-domain-containing protein. Our data provide critical information on the structural or functional characteristics of archaeal proteins and could help increase our understanding of fundamental signaling mechanisms in all three domains of life.
Histidine Kinase-Mediated Production and Autoassembly of
Porphyromonas gingivalis
Fimbriae
Authors: Nishikawa Kiyoshi , Duncan Margaret J.
Published: January 2010

Porphyromonas gingivalis
, a Gram-negative oral anaerobe, is strongly associated with chronic adult periodontitis, and it utilizes FimA fimbriae to persistently colonize and evade host defenses in the periodontal crevice. The FimA-related gene cluster (the
fim
gene cluster) is positively regulated by the FimS-FimR two-component system. In this study, comparative analyses between fimbriate type strain ATCC 33277 and fimbria-deficient strain W83 revealed differences in their
fimS
loci, which encode FimS histidine kinase. Using a reciprocal gene exchange system, we established that FimS from W83 is malfunctional. Complementation analysis with chimeric
fimS
constructs revealed that W83 FimS has a defective kinase domain due to a truncated conserved G3 box motif that provides an ATP-binding pocket. The introduction of the functional
fimS
from 33277 restored the production, but not polymerization, of endogenous FimA subunits in W83. Further analyses with a
fimA
-exchanged W83 isogenic strain showed that even the fimbria-deficient W83 retains the ability to polymerize FimA from 33277, indicating the assembly of mature FimA by a primary structure-dependent mechanism. It also was shown that the substantial expression of 33277-type FimA fimbriae in the W83 derivative requires the introduction and expression of the functional 33277
fimS
. These findings indicate that FimSR is the unique and universal regulatory system that activates the
fim
gene cluster in a
fimA
genotype-independent manner.
The Pneumococcal Cell Envelope Stress-Sensing System LiaFSR Is Activated by Murein Hydrolases and Lipid II-Interacting Antibiotics
Authors: Eldholm Vegard , Gutt Beatrice , Johnsborg Ola , Brückner Reinhold , Maurer Patrick , Hakenbeck Regine , Mascher Thorsten , Håvarstein Leiv Sigve
Published: January 2010

In theFirmicutes, two-component regulatory systems of the LiaSR type sense and orchestrate the response to various agents that perturb cell envelope functions, in particular lipid II cycle inhibitors. In the current study, we found that the corresponding system inStreptococcus pneumoniaedisplays similar properties but, in addition, responds to cell envelope stress elicited by murein hydrolases. During competence for genetic transformation, pneumococci attack and lyse noncompetent siblings present in the same environment. This phenomenon, termed fratricide, increases the efficiency of horizontal gene transferin vitroand is believed to stimulate gene exchange also under natural conditions. Lysis of noncompetent target cells is mediated by the putative murein hydrolase CbpD, the key effector of the fratricide mechanism, and the autolysins LytA and LytC. To avoid succumbing to their own lysins, competent attacker cells must possess a protective mechanism rendering them immune. The most important component of this mechanism is ComM, an integral membrane protein of unknown function that is expressed only in competent cells. Here, we show that a second layer of self-protection is provided by the pneumococcal LiaFSR system, which senses the damage inflicted to the cell wall by CbpD, LytA, and LytC. Two members of the LiaFSR regulon,spr0810 and PcpC (spr0351), were shown to contribute to the LiaFSR-coordinated protection against fratricide-induced self-lysis.
Responses of
Escherichia coli
Bacteria to Two Opposing Chemoattractant Gradients Depend on the Chemoreceptor Ratio
Authors: Kalinin Yevgeniy , Neumann Silke , Sourjik Victor , Wu Mingming
Published: January 2010

Escherichia coli
chemotaxis has long served as a simple model of environmental signal processing, and bacterial responses to single chemical gradients are relatively well understood. Less is known about the chemotactic behavior of
E. coli
in multiple chemical gradients. In their native environment, cells are often exposed to multiple chemical stimuli. Using a recently developed microfluidic chemotaxis device, we exposed
E. coli
cells to two opposing but equally potent gradients of major attractants, methyl-aspartate and serine. The responses of
E. coli
cells demonstrated that chemotactic decisions depended on the ratio of the respective receptor number of Tar/Tsr. In addition, the ratio of Tar to Tsr was found to vary with cells’ growth conditions, whereby it depended on the culture density but not on the growth duration. These results provide biological insights into the decision-making processes of chemotactic bacteria that are subjected to multiple chemical stimuli and demonstrate the importance of the cellular microenvironment in determining phenotypic behavior.
Analysis of the
dbpBA
Upstream Regulatory Region Controlled by RpoS in
Borrelia burgdorferi
Authors: Ouyang Zhiming , Haq Shayma , Norgard Michael V.
Published: January 2010

Decorin-binding proteins B and A (DbpB and DbpA) are thought to play important roles in
Borrelia burgdorferi
pathogenesis by serving as adhesins for the extracellular matrix. It has been established that the expression of DbpBA is governed by the Rrp2-RpoN-RpoS regulatory pathway. However, the precise mechanism underlying the control of DbpBA expression has been unclear. In particular, it has been unknown whether RpoS influences DbpBA expression directly or indirectly (through an additional regulatory molecule[s]). Here, employing a wild-type
B. burgdorferi
strain and a
dbpBA
-deficient mutant, we analyzed the 5′ genetic elements of the
dbpBA
operon using deletion analysis, coupled with luciferase reporter assays, quantitative reverse transcription PCR, and immunoblot analyses. A minimal promoter, encompassed within 70 bp upstream of the ATG start codon of
dbpBA
, was identified and found to be necessary and sufficient to initiate
dbpBA
transcription. The minimal
dbpBA
promoter was responsive to environmental stimuli such as temperature, pH, and whole blood. Two
in silico
-identified inverted repeat elements were not involved in the response of
dbpBA
expression to
in vitro
stimulation by environmental factors. The expression of
dbpBA
from the minimal promoter was abolished when
rpoS
was inactivated. In addition, the targeted mutagenesis of a C at position −14 within the extended −10 region of
dbpBA
, which has been postulated to be strategic for Eσ
S
binding in
Escherichia coli
, abolished
dbpBA
expression in
B. burgdorferi
. These combined data suggest that the Rrp2-RpoN-RpoS pathway controls
dbpBA
expression by the direct binding of RpoS to an RpoS-dependent promoter. However, given that there remains a distinct difference between the expression of DbpBA and other genes under the direct control of RpoS (e.g., OspC), our findings do not preclude the existence of another layer of gene regulation that may contribute to the modulation of DbpBA expression via an as-yet unknown mechanism.
Flagellated but Not Hyperfimbriated
Salmonella enterica
Serovar Typhimurium Attaches to and Forms Biofilms on Cholesterol-Coated Surfaces
Authors: Crawford Robert W. , Reeve Kristin E. , Gunn John S.
Published: January 2010

The asymptomatic, chronic carrier state of
Salmonella enterica
serovar Typhi occurs in the bile-rich gallbladder and is frequently associated with the presence of cholesterol gallstones. We have previously demonstrated that salmonellae form biofilms on human gallstones and cholesterol-coated surfaces
in vitro
and that bile-induced biofilm formation on cholesterol gallstones promotes gallbladder colonization and maintenance of the carrier state. Random transposon mutants of
S. enterica
serovar Typhimurium were screened for impaired adherence to and biofilm formation on cholesterol-coated Eppendorf tubes but not on glass and plastic surfaces. We identified 49 mutants with this phenotype. The results indicate that genes involved in flagellum biosynthesis and structure primarily mediated attachment to cholesterol. Subsequent analysis suggested that the presence of the flagellar filament enhanced binding and biofilm formation in the presence of bile, while flagellar motility and expression of type 1 fimbriae were unimportant. Purified
Salmonella
flagellar proteins used in a modified enzyme-linked immunosorbent assay (ELISA) showed that FliC was the critical subunit mediating binding to cholesterol. These studies provide a better understanding of early events during biofilm development, specifically how salmonellae bind to cholesterol, and suggest a target for therapies that may alleviate biofilm formation on cholesterol gallstones and the chronic carrier state.