bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of an
mlrA
Gene-Directed TaqMan PCR Assay for Quantitative Assessment of Microcystin-Degrading Bacteria within Water Treatment Plant Sand Filter Biofilms
Authors: Hoefel Daniel , Adriansen Caroline M. M. , Bouyssou Magali A. C. , Saint Christopher P. , Newcombe Gayle , Ho Lionel
Published: June 2009

We report for the first time a quantitative
mlrA
gene-directed TaqMan PCR assay for the rapid detection of microcystin-degrading bacteria. This was applied, in combination with 16S ribosomal DNA-directed quantitative PCR and denaturing gradient gel electrophoresis, to study virgin sand filter column biofilm development and to correlate
mlr
A
gene abundance with microcystin removal efficiency.
Photoheterotrophic Microbes in the Arctic Ocean in Summer and Winter
Authors: Cottrell Matthew T. , Kirchman David L.
Published: June 2009

Photoheterotrophic microbes, which are capable of utilizing dissolved organic materials and harvesting light energy, include coccoid cyanobacteria (SynechococcusandProchlorococcus), aerobic anoxygenic phototrophic (AAP) bacteria, and proteorhodopsin (PR)-containing bacteria. Our knowledge of photoheterotrophic microbes is largely incomplete, especially for high-latitude waters such as the Arctic Ocean, where photoheterotrophs may have special ecological relationships and distinct biogeochemical impacts due to extremes in day length and seasonal ice cover. These microbes were examined by epifluorescence microscopy, flow cytometry, and quantitative PCR (QPCR) assays for PR and a gene diagnostic of AAP bacteria (pufM). The abundance of AAP bacteria and PR-containing bacteria decreased from summer to winter, in parallel with a threefold decrease in the total prokaryotic community. In contrast, the abundance ofSynechococcusorganisms did not decrease in winter, suggesting that their growth was supported by organic substrates. Results from QPCR assays revealed no substantial shifts in the community structure of AAP bacteria and PR-containing bacteria. However, Arctic PR genes were different from those found at lower latitudes, and surprisingly, they were not similar to those in Antarctic coastal waters. Photoheterotrophic microbes appear to compete successfully with strict heterotrophs during winter darkness below the ice, but AAP bacteria and PR-containing bacteria do not behave as superior competitors during the summer.
Worldwide Prevalence of Class 2 Integrases outside the Clinical Setting Is Associated with Human Impact
Authors: Rodríguez-Minguela Carlos M. , Apajalahti Juha H. A. , Chai Benli , Cole James R. , Tiedje James M.
Published: June 2009

An
intI
-targeted PCR assay was optimized to evaluate the frequency of partial class 2-like integrases relative to putative, environmental IntI elements in clone libraries generated from 17 samples that included various terrestrial, marine, and deep-sea habitats with different exposures to human influence. We identified 169 unique IntI phylotypes (≤98% amino acid identity) relative to themselves and with respect to those previously described. Among these, six variants showed an undescribed, extended, IntI-specific additional domain. A connection between human influence and the dominance of IntI-2-like variants was also observed. IntI phylotypes 80 to 99% identical to class 2 integrases comprised ∼70 to 100% (
n
= 65 to 87) of the IntI elements detected in samples with a high input of fecal waste, whereas IntI2-like sequences were undetected in undisturbed settings and poorly represented (1 to 10%;
n
= 40 to 79) in environments with moderate or no recent fecal or anthropogenic impact. Eleven partial IntI2-like sequences lacking the signature ochre 179 codon were found among samples of biosolids and agricultural soil supplemented with swine manure, indicating a wider distribution of potentially functional IntI2 variants than previously reported. To evaluate IntI2 distribution patterns beyond the usual hosts, namely, the
Enterobacteriaceae
, we coupled PCR assays targeted at
intI
and 16S rRNA loci to G+C fractionation of total DNA extracted from manured cropland. IntI2-like sequences and 16S rRNA phylotypes related to
Firmicutes
(
Clostridium
and
Bacillus
) and
Bacteroidetes
(
Chitinophaga
and
Sphingobacterium
) dominated a low-G+C fraction (∼40 to 45%), suggesting that these groups could be important IntI2 hosts in manured soil. Moreover, G+G fractionation uncovered an additional set of 36 novel IntI phylotypes (≤98% amino acid identity) undetected in bulk DNA and revealed the prevalence of potentially functional IntI2 variants in the low-G+C fraction.
Identification of an Arsenic Resistance and Arsenic-Sensing System in
Campylobacter jejuni
Authors: Wang Liping , Jeon Byeonghwa , Sahin Orhan , Zhang Qijing
Published: June 2009

Arsenic is commonly present in the natural environment and is also used as a feed additive for animal production. Poultry is a major reservoir for
Campylobacter jejuni
, a major food-borne human pathogen causing gastroenteritis. It has been shown that
Campylobacter
isolates from poultry are highly resistant to arsenic compounds, but the molecular mechanisms responsible for the resistance have not been determined, and it is unclear if the acquired arsenic resistance affects the susceptibility of
Campylobacter
spp. to other antimicrobials. In this study, we identified a four-gene operon that contributes to arsenic resistance in
Campylobacter
. This operon encodes a putative membrane permease (ArsP), a transcriptional repressor (ArsR), an arsenate reductase (ArsC), and an efflux protein (Acr3). PCR analysis of various clinical
C. jejuni
isolates indicated a significant association of this operon with elevated resistance to arsenite and arsenate. Gene-specific mutagenesis confirmed the role of the
ars
operon in conferring arsenic resistance. It was further shown that this operon is subject to regulation by ArsR, which directly binds to the
ars
promoter and inhibits the transcription of the operon. Arsenite inhibits the binding of ArsR to the
ars
promoter DNA and induces the expression of the
ars
genes. Mutation of the
ars
genes did not affect the susceptibility of
C. jejuni
to commonly used antibiotics. These results identify the
ars
operon as an important mechanism for arsenic resistance and sensing in
Campylobacter
.
Biochemical Characterization of Haloalkane Dehalogenases DrbA and DmbC, Representatives of a Novel Subfamily
Authors: Jesenská Andrea , Monincová Marta , Koudeláková Táňa , Hasan Khomaini , Chaloupková Radka , Prokop Zbyněk , Geerlof Arie , Damborský Jiří
Published: June 2009

This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes
drbA
from
Rhodopirellula baltica
SH1 and
dmbC
from
Mycobacterium bovis
5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.
Cellular Responses of the Late Blight Pathogen
Phytophthora infestans
to Cyclic Lipopeptide Surfactants and Their Dependence on G Proteins
Authors: van de Mortel Judith E. , Tran Ha , Govers Francine , Raaijmakers Jos M.
Published: June 2009

Oomycete pathogens cause major yield losses for many crop plants, and their control depends heavily on agrochemicals. Cyclic lipopeptides (CLPs) were recently discovered as a new class of natural compounds with strong activities against oomycetes. The CLP massetolide A (Mass A), produced by
Pseudomonas fluorescens
, has zoosporicidal activity, induces systemic resistance, and reduces late blight in tomato. To gain further insight into the modes of action of CLPs, the effects of Mass A on pore formation, mycelial growth, sporangium formation, and zoospore behavior were investigated, as was the involvement of G proteins in the sensitivity of
Phytophthora infestans
to Mass A. The results showed that Mass A induced the formation of transmembrane pores with an estimated size of between 1.2 and 1.8 nm. Dose-response experiments revealed that zoospores were the most sensitive to Mass A, followed by mycelium and cysts. Mass A significantly reduced sporangium formation and caused increased branching and swelling of hyphae. At relatively low concentrations, Mass A induced encystment of zoospores. It had no effect on the chemotactic response of zoospores but did adversely affect zoospore autoaggregation. A loss-of-function transformant of
P. infestans
lacking the G-protein α subunit was more sensitive to Mass A, whereas a gain-of-function transformant required a higher Mass A concentration to interfere with zoospore aggregation. Results indicate that Mass A disturbs various developmental stages in the life cycle of
P. infestans
and suggest that the cellular responses of
P. infestans
to this CLP are, in part, dependent on G-protein signaling.
Involvement of Cell Surface Structures in Size-Independent Grazing Resistance of Freshwater
Actinobacteria
Authors: Tarao Mitsunori , Jezbera Jan , Hahn Martin W.
Published: June 2009

We compared the influences of grazing by the bacterivorous nanoflagellate
Poterioochromonas
sp. strain DS on ultramicrobacterial
Actinobacteria
affiliated with the Luna-2 cluster and ultramicrobacterial
Betaproteobacteria
of the species
Polynucleobacter cosmopolitanus
. These bacteria were almost identical in size (<0.1 μm
3
) and shape. Predation on a
Polynucleobacter
strain resulted in a reduction of >86% relative to the initial bacterial cell numbers within 20 days, while in comparable predation experiments with nine actinobacterial strains, no significant decrease of cell numbers by predation was observed over the period of ≥39 days. The differences in predation mortality between the actinobacterial strains and the
Polynucleobacter
strain clearly demonstrated size-independent grazing resistance for the investigated
Actinobacteria
. Importantly, this size-independent grazing resistance is shared by all nine investigated Luna-2 strains and thus represents a group-specific trait. We investigated if an S-layer, previously observed in an ultrastructure study, was responsible for the grazing resistance of these strains. Experiments aiming for removal of the S-layer or modification of cell surface proteins of one of the grazing-resistant strains by treatment with lithium chloride, EDTA, or formaldehyde resulted in 4.2- to 5.2-fold higher grazing rates in comparison to the levels for untreated cells. These results indicate the protective role of a proteinaceous cell surface structure in the size-independent grazing resistance of the actinobacterial Luna-2 strains, which can be regarded as a group-specific trait.
Coastal Strains of Marine
Synechococcus
Species Exhibit Increased Tolerance to Copper Shock and a Distinctive Transcriptional Response Relative to Those of Open-Ocean Strains
Authors: Stuart Rhona K. , Dupont Chris L. , Johnson D. Aaron , Paulsen Ian T. , Palenik Brian
Published: June 2009

Copper appears to be influencing the distribution and abundance of phytoplankton in marine environments, and cyanobacteria are thought to be the most sensitive of the phytoplankton groups to copper toxicity. By using growth assays of phylogenetically divergent clades, we found that coastal strains of marine
Synechococcus
species were more tolerant to copper shock than open-ocean strains. The global transcriptional response to two levels of copper shock were determined for both a coastal strain and an open-ocean strain of marine
Synechococcus
species using whole-genome expression microarrays. Both strains showed an osmoregulatory-like response, perhaps as a result of increasing membrane permeability. This could have implications for marine carbon cycling if copper shock leads to dissolved organic carbon leakage in
Synechococcus
species. The two strains additionally showed a common reduction in levels of photosynthesis-related gene transcripts. Contrastingly, the open-ocean strain showed a general stress response, whereas the coastal strain exhibited a more specifically oxidative or heavy-metal acclimation response that may be conferring tolerance. In addition, the coastal strain activated more regulatory elements and transporters, many of which are not conserved in other marine
Synechococcus
strains and may have been acquired by horizontal gene transfer. Thus, tolerance to copper shock in some marine
Synechococcus
strains may in part be a result of a generally increased ability to sense and respond in a more stress-specific manner.
Pyrosequencing-Based Assessment of Soil pH as a Predictor of Soil Bacterial Community Structure at the Continental Scale
Authors: Lauber Christian L. , Hamady Micah , Knight Rob , Fierer Noah
Published: June 2009

Soils harbor enormously diverse bacterial populations, and soil bacterial communities can vary greatly in composition across space. However, our understanding of the specific changes in soil bacterial community structure that occur across larger spatial scales is limited because most previous work has focused on either surveying a relatively small number of soils in detail or analyzing a larger number of soils with techniques that provide little detail about the phylogenetic structure of the bacterial communities. Here we used a bar-coded pyrosequencing technique to characterize bacterial communities in 88 soils from across North and South America, obtaining an average of 1,501 sequences per soil. We found that overall bacterial community composition, as measured by pairwise UniFrac distances, was significantly correlated with differences in soil pH (
r
= 0.79), largely driven by changes in the relative abundances of
Acidobacteria
,
Actinobacteria
, and
Bacteroidetes
across the range of soil pHs. In addition, soil pH explains a significant portion of the variability associated with observed changes in the phylogenetic structure within each dominant lineage. The overall phylogenetic diversity of the bacterial communities was also correlated with soil pH (

R
2

= 0.50), with peak diversity in soils with near-neutral pHs. Together, these results suggest that the structure of soil bacterial communities is predictable, to some degree, across larger spatial scales, and the effect of soil pH on bacterial community composition is evident at even relatively coarse levels of taxonomic resolution.
Characterization of Airborne Microbial Communities at a High-Elevation Site and Their Potential To Act as Atmospheric Ice Nuclei
Authors: Bowers Robert M. , Lauber Christian L. , Wiedinmyer Christine , Hamady Micah , Hallar Anna G. , Fall Ray , Knight Rob , Fierer Noah
Published: June 2009

Bacteria and fungi are ubiquitous in the atmosphere. The diversity and abundance of airborne microbes may be strongly influenced by atmospheric conditions or even influence atmospheric conditions themselves by acting as ice nucleators. However, few comprehensive studies have described the diversity and dynamics of airborne bacteria and fungi based on culture-independent techniques. We document atmospheric microbial abundance, community composition, and ice nucleation at a high-elevation site in northwestern Colorado. We used a standard small-subunit rRNA gene Sanger sequencing approach for total microbial community analysis and a bacteria-specific 16S rRNA bar-coded pyrosequencing approach (4,864 sequences total). During the 2-week collection period, total microbial abundances were relatively constant, ranging from 9.6 × 10
5
to 6.6 × 10
6
cells m
−3
of air, and the diversity and composition of the airborne microbial communities were also relatively static. Bacteria and fungi were nearly equivalent, and members of the proteobacterial groups
Burkholderiales
and
Moraxellaceae
(particularly the genus
Psychrobacter
) were dominant. These taxa were not always the most abundant in freshly fallen snow samples collected at this site. Although there was minimal variability in microbial abundances and composition within the atmosphere, the number of biological ice nuclei increased significantly during periods of high relative humidity. However, these changes in ice nuclei numbers were not associated with changes in the relative abundances of the most commonly studied ice-nucleating bacteria.
Homo-
d
-Lactic Acid Fermentation from Arabinose by Redirection of the Phosphoketolase Pathway to the Pentose Phosphate Pathway in
l
-Lactate Dehydrogenase Gene-Deficient
Lactobacillus plantarum
Authors: Okano Kenji , Yoshida Shogo , Tanaka Tsutomu , Ogino Chiaki , Fukuda Hideki , Kondo Akihiko
Published: June 2009

Optically pure
d
-lactic acid fermentation from arabinose was achieved by using the
Lactobacillus plantarum
NCIMB 8826 strain whose
l
-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of
d
-lactic acid was produced from 50 g/liter of arabinose.
Structure-Based Rational Design of a Phosphotriesterase
Authors: Jackson Colin J. , Weir Kahli , Herlt Anthony , Khurana Jeevan , Sutherland Tara D. , Horne Irene , Easton Christopher , Russell Robyn J. , Scott Colin , Oakeshott John G.
Published: June 2009

In silico substrate docking of both stereoisomers of the pesticide chlorfenvinphos (CVP) in the phosphotriesterase from
Agrobacterium radiobacter
identified two residues (F131 and W132) that prevent productive substrate binding and cause stereospecificity. A variant (W131H/F132A) was designed that exhibited ca. 480-fold and 8-fold increases in the rate of
Z
-CVP and
E
-CVP hydrolysis, respectively, eliminating stereospecificity.
Molecular Biological Detection and Quantification of Novel
Fibrobacter
Populations in Freshwater Lakes
Authors: McDonald James E. , de Menezes Alexandre B. , Allison Heather E. , McCarthy Alan J.
Published: June 2009

PCR and quantitative PCR (qPCR) primers targeting the 16S rRNA gene were used to detect and quantify members of the genus
Fibrobacter
in lake water, sediment and colonized cotton taken from two freshwater lakes. Phylogenetic analysis identified two groups of sequences; those clustered with
Fibrobacter succinogenes
, the type species, and a defined cluster of clones loosely associated with several
Fibrobacter
sequences observed previously in clone libraries from freshwater environments. 16S rRNA gene sequences recovered in the same way from soil samples and ovine feces in the surrounding land were all
F. succinogenes
and did not include any from this group of the “freshwater”
Fibrobacteres
. In all cases, nested PCR was required to detect
Fibrobacter
16S rRNA genes, and qPCR analysis of reverse transcribed bacterial community RNA confirmed their very low relative abundance on colonized cotton baits in the water column (at 0, 3, 7, 11, and 13 m) and on the sediment surface (<0.02% of total bacterial rRNA). However, in Esthwaite Water sediment itself, the relative abundance of fibrobacters was 2 orders of magnitude higher (ca. 1% of total bacterial rRNA). The presence of fibrobacters, including the cellulolytic rumen species
F. succinogenes
, on colonized cellulose samples and in lake sediment suggests that these organisms may contribute to the primary degradation of plant and algal biomass in freshwater lake ecosystems.
Aerobic Vanillate Degradation and C
1
Compound Metabolism in
Bradyrhizobium japonicum
Authors: Sudtachat Nirinya , Ito Naofumi , Itakura Manabu , Masuda Sachiko , Eda Shima , Mitsui Hisayuki , Kawaharada Yasuyuki , Minamisawa Kiwamu
Published: June 2009

Bradyrhizobium japonicum
, a symbiotic nitrogen-fixing soil bacterium, has multiple gene copies for aromatic degradation on the genome and is able to use low concentrations of vanillate, a methoxylated lignin monomer, as an energy source. A transcriptome analysis indicated that one set of
vanA1B
,
pcaG1H1
, and genes for C
1
compound catabolism was upregulated in
B. japonicum
USDA110 cells grown in vanillate (N. Ito, M. Itakura, S. Eda, K. Saeki, H. Oomori, T. Yokoyama, T. Kaneko, S. Tabata, T. Ohwada, S. Tajima, T. Uchiumi, E. Masai, M. Tsuda, H. Mitsui, and K. Minamisawa, Microbes Environ.
21:
240-250, 2006). To examine the functions of these genes in vanillate degradation, we tested cell growth and substrate consumption in
vanA1B
,
pcaG1H1
, and
mxaF
mutants of USDA110. The
vanA1B
and
pcaG1H1
mutants were unable to grow in minimal media containing 1 mM vanillate and protocatechuate, respectively, although wild-type USDA110 was able to grow in both media, indicating that the upregulated copies of
vanA1B
and
pcaG1H1
are exclusively responsible for vanillate degradation. Mutating
mxaF
eliminated expression of
gfa
and
flhA
, which contribute to glutathione-dependent C
1
metabolism. The
mxaF
mutant had markedly lower cell growth in medium containing vanillate than the wild-type strain. In the presence of protocatechuate, there was no difference in cell growth between the
mxaF
mutant and the wild-type strain. These results suggest that the C
1
pathway genes are required for efficient vanillate catabolism. In addition, wild-type USDA110 oxidized methanol, whereas the
mxaF
mutant did not, suggesting that the metabolic capability of the C
1
pathway in
B. japonicum
extends to methanol oxidation. The
mxaF
mutant showed normal nodulation and N
2
fixation phenotypes with soybeans, which was not similar to symbiotic phenotypes of methylotrophic rhizobia.
Biochemical Properties of Two Cinnamoyl Esterases Purified from a
Lactobacillus johnsonii
Strain Isolated from Stool Samples of Diabetes-Resistant Rats
Authors: Lai Kin Kwan , Lorca Graciela L. , Gonzalez Claudio F.
Published: June 2009

Cinnamic acids (i.e., ferulic and caffeic acids) that are esterified to the vegetable cell walls should be enzymatically released to be absorbed in a mammal's intestines. A low dosage of ferulic acid in rodent diets stimulates insulin production and alleviates symptoms caused by diabetes (M. Sri Balasubashini, R. Rukkumani, and V. P. Menon, Acta Diabetol. 40:118-122, 2003). Several lactic acid bacteria are able to display ferulic acid esterase (FAE) activity, suggesting that their probiotic activity could be, in part, mediated by the slow release of ferulic acid. In the present work, we describe the isolation of one strain identified as being
Lactobacillus johnsonii
that displayed strong FAE activity in stool samples from diabetes-resistant biobreeding rats. These animals are genetically susceptible to becoming diabetic but do not develop the disease. By using genomic analysis coupled to protein purification and catalytic screening, we were able to purify two proteins with FAE activity. The enzymes displayed 42% sequence identity and a broad range of substrate preferences. High affinities and catalytic efficiencies toward aromatic compounds such as ethyl ferulate (

K
m

= 20 to 60 μM) and chlorogenic acid (

K
m

= 10 to 50 μM) were observed. The strain isolated herein as well as the enzymes studied could be potentially useful for the formulation of probiotics to ameliorate diabetes symptoms.
Quantification of Key Genes Steering the Microbial Nitrogen Cycle in the Rhizosphere of Sorghum Cultivars in Tropical Agroecosystems
Authors: Hai Brigitte , Diallo Ndeye Hélène , Sall Saidou , Haesler Felix , Schauss Kristina , Bonzi Moussa , Assigbetse Komi , Chotte Jean-Luc , Munch Jean Charles , Schloter Michael
Published: June 2009

The effect of agricultural management practices on geochemical cycles in moderate ecosystems is by far better understood than in semiarid regions, where fertilizer availability and climatic conditions are less favorable. We studied the impact of different fertilizer regimens in an agricultural long-term observatory in Burkina Faso at three different plant development stages (early leaf development, flowering, and senescence) of sorghum cultivars. Using real-time PCR, we investigated functional microbial communities involved in key processes of the nitrogen cycle (nitrogen fixation, ammonia oxidation, and denitrification) in the rhizosphere. The results indicate that fertilizer treatments and plant development stages combined with environmental factors affected the abundance of the targeted functional genes in the rhizosphere. While nitrogen-fixing populations dominated the investigated communities when organic fertilizers (manure and straw) were applied, their numbers were comparatively reduced in urea-treated plots. In contrast, ammonia-oxidizing bacteria (AOB) increased not only in absolute numbers but also in relation to the other bacterial groups investigated in the urea-amended plots. Ammonia-oxidizing archaea exhibited higher numbers compared to AOB independent of fertilizer application. Similarly, denitrifiers were also more abundant in the urea-treated plots. Our data imply as well that, more than in moderate regions, water availability might shape microbial communities in the rhizosphere, since low gene abundance data were obtained for all tested genes at the flowering stage, when water availability was very limited.
Differential Activation of Peritoneal Cells by Subcutaneous Treatment of Rats with Cryptococcal Antigens
Authors: Baronetti José L. , Chiapello Laura S. , Garro Ana P. , Masih Diana T.
Published: June 2009

Previous studies in our laboratory have shown that the subcutaneous pretreatment of rats with heat-killed cells (HKC) ofCryptococcus neoformansemulsified in complete Freund adjuvant (CFA) promotes protective immunity against an intraperitoneal challenge withC. neoformans. In contrast, subcutaneous treatment with the capsular polysaccharide (PSC) emulsified in CFA exacerbates the cryptococcal infection. The purpose of this study was to analyze the mechanisms involved in these phenomena. Adherent peritoneal cells from rats treated with HKC-CFA showed upregulated ED2, CD80, and CD86 expression; an increase in the level of production of anticryptococcal metabolites; and the enhanced production of interleukin-12 (IL-12) in comparison with the findings for cells from rats treated with CFA-phosphate-buffered saline (PBS). Adherent peritoneal cells from rats treated with PSC-CFA, however, also presented upregulated ED2, CD80, and CD86 expression compared to the level of expression for peritoneal cells from controls, but these cells showed an increase in arginase activity and decreased levels of production of IL-12 and tumor necrosis factor (TNF) compared with the activity and levels of production by peritoneal cells from CFA-PBS-treated rats. In addition, treatment with HKC-CFA resulted in a rise in the phagocytic and anticryptococcal activities of adherent peritoneal cells compared to those for control rats. However, adherent peritoneal cells from rats treated with PSC-CFA presented a reduction in anticryptococcal activity in comparison with that for cells from animals treated with CFA-PBS. These results show the differential activation between adherent peritoneal cells from HKC-CFA- and PSC-CFA-treated rats, with this differential activation at the primary site of infection possibly being responsible, at least in part, for the phenomena of protection and exacerbation observed in our model.
Characterization of Two Putative Protein Translocation Components in the Apicoplast ofPlasmodium falciparum
Authors: Kalanon Ming , Tonkin Christopher J. , McFadden Geoffrey I.
Published: June 2009

Protein trafficking to the stroma of the apicoplast ofPlasmodium falciparumrequires translocation across several membranes. To further elucidate the mechanisms responsible, we investigated two proteins:P. falciparumTic22 (PfTic22), a putative component of the translocon of the inner chloroplast membrane; and PfsDer1-1, one of two homologues of theP. falciparumsymbiont-derived Der1 (sDer1) protein, a putative component of an endoplasmic reticulum-associated degradation (ERAD) complex in the periplastid membrane. We constructed parasites expressing hemagglutinin (HA)-tagged PfTic22 and PfsDer1-1 under the control of their endogenous promoters using the 3′ replacement strategy. We show that both PfTic22-HA and PfsDer1-1-HA are expressed predominantly during the trophozoite stage of the asexual replication cycle, which corresponds to the most dynamic stages of apicoplast activity. Although both proteins localize to the periphery of the apicoplast, PfTic22-HA is a membrane-associated protein while PfsDer1-1-HA is an integral membrane protein. Phylogenetic analysis indicates that PfsDer1-1 is one of two Der1 paralogues predicted to localize to the apicoplast inP. falciparumand that it has orthologues in diatom algae, supporting the chromalveolate hypothesis. These observations are consistent with putative roles for PfTic22 and PfsDer1-1 in protein translocation into the apicoplast ofP. falciparum.
Phosphatidylinositol-4,5-Bisphosphate and Phospholipase D-Generated Phosphatidic Acid Specify SNARE-Mediated Vesicle Fusion for Prospore Membrane Formation
Authors: Mendonsa Rima , Engebrecht JoAnne
Published: June 2009

The soluble
N
-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of proteins is required for eukaryotic intracellular membrane fusions. Vesicle fusion for formation of the prospore membrane (PSM), a membrane compartment that forms de novo during yeast sporulation, requires SNARE function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P
2
], and the activity of the phospholipase D (PLD) Spo14p, which generates phosphatidic acid (PA). The SNARE syntaxin Sso1p is essential for PSM production while the functionally redundant homolog in vegetative growth, Sso2p, is not. We demonstrate that Sso1p and Sso2p bind similarly in vitro to PA or phosphoinositide-containing liposomes and that the conserved SNARE (H3) domain largely mediates PA-binding. Both green fluorescent protein-Sso fusion proteins localize to the developing PSM in wild-type cells and to the spindle pole body in
spo14
Δ cells induced to sporulate. However, the autoregulatory region of Sso1p binds PI(4,5)P
2
-containing liposomes in vitro with a greater ability than the equivalent region of Sso2p. Overexpression of the phosphatidylinositol-4-phosphate 5-kinase
MSS4
in
sso1
Δ cells induced to sporulate stimulates PSM production; PLD activity is not increased under these conditions, indicating that PI(4,5)P
2
has roles in addition to stimulating PLD in PSM formation. These data suggest that PLD-generated PA and PI(4,5)P
2
collaborate at multiple levels to promote SNARE-mediated fusion for PSM formation.
An Unusual ERAD-Like Complex Is Targeted to the Apicoplast ofPlasmodium falciparum
Authors: Spork Simone , Hiss Jan A. , Mandel Katharina , Sommer Maik , Kooij Taco W. A. , Chu Trang , Schneider Gisbert , Maier Uwe G. , Przyborski Jude M.
Published: June 2009

Many apicomplexan parasites, includingPlasmodium falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event. The exact molecular mechanisms directing the transport of nuclear-encoded proteins to the apicoplast ofP. falciparumare not well understood. Recently, in silico analyses revealed a second copy of proteins homologous to components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) system in organisms with secondary plastids, including the malaria parasiteP. falciparum. These proteins are predicted to be endowed with an apicoplast targeting signal and are suggested to play a role in the transport of nuclear-encoded proteins to the apicoplast. Here, we have studied components of this ERAD-derived putative preprotein translocon complex in malaria parasites. Using transfection technology coupled with fluorescence imaging techniques we can demonstrate that the N terminus of several ERAD-derived components targets green fluorescent protein to the apicoplast. Furthermore, we confirm that full-length PfsDer1-1 and PfsUba1 (homologues of yeast ERAD components) localize to the apicoplast, where PfsDer1-1 tightly associates with membranes. Conversely, PfhDer1-1 (a host-specific copy of the Der1-1 protein) localizes to the ER. Our data suggest that ERAD components have been “rewired” to provide a conduit for protein transport to the apicoplast. Our results are discussed in relation to the nature of the apicoplast protein transport machinery.
The Extent of Migration of the Holliday Junction Is a Crucial Factor for Gene Conversion in
Rhizobium etli
Authors: Castellanos Mildred , Romero David
Published: June 2009

Gene conversion, defined as the nonreciprocal transfer of DNA, is one result of homologous recombination. Three steps in recombination could give rise to gene conversion: (i) DNA synthesis for repair of the degraded segment, (ii) Holliday junction migration, leading to heteroduplex formation, and (iii) repair of mismatches in the heteroduplex. There are at least three proteins (RuvAB, RecG, and RadA) that participate in the second step. Their roles have been studied for homologous recombination, but evidence of their relative role in gene conversion is lacking. In this work, we showed the effect on gene conversion of mutations in
ruvB
,
recG
, and
radA
in
Rhizobium etli
, either alone or in combination, using a cointegration strategy previously developed in our laboratory. The results indicate that the RuvAB system is highly efficient for gene conversion, since its absence provokes smaller gene conversion segments than those in the wild type as well as a shift in the preferred position of conversion tracts. The RecG system possesses a dual role for gene conversion. Inactivation of
recG
leads to longer gene conversion tracts than those in the wild type, indicating that its activity may hinder heteroduplex extension. However, under circumstances where it is the only migration activity present (as in the
ruvB radA
double mutant), conversion segments can still be seen, indicating that RecG can also promote gene conversion. RadA is the least efficient system in
R. etli
but is still needed for the production of detectable gene conversion tracts.
The StkP/PhpP Signaling Couple inStreptococcus pneumoniae: Cellular Organization and Physiological Characterization
Authors: Osaki Makoto , Arcondéguy Tania , Bastide Amandine , Touriol Christian , Prats Hervé , Trombe Marie-Claude
Published: June 2009

InStreptococcus pneumoniae,stkPandphpP, encoding the eukaryotic-type serine-threonine kinase and PP2C phosphatase, respectively, form an operon. PhpP has the features of a so-called “soluble” protein, whereas StkP protein is membrane associated. Here we provide the first genetic and physiological evidence that PhpP and StkP, with antagonist enzymatic activities, constitute a signaling couple. The StkP-PhpP couple signals competence upstream of the competence-specific histidine kinase ComD, receptor for the oligopeptide pheromone “competence stimulating peptide.” We show that PhpP activity is essential in astkP+genetic background, suggesting tight control of StkP activity by PhpP. Proteins PhpP and StkP colocalized to the cell membrane subcellular fraction and likely belong to the same complex, as revealed by coimmunoprecipitation in cellular extracts. Specific coimmunoprecipitation of the N-kinase domain of StkP and PhpP recombinant proteins by PhpP-specific antibodies demonstrates direct interaction between these proteins. Consistently, flow cytometry analysis allowed the determination of the cytoplasmic localization of PhpP and of the N-terminal kinase domain of StkP, in contrast to the periplasmic localization of the StkP C-terminal PASTA (penicillin-binding proteinandserine-threonine kinaseassociated) domain. A signaling route involving interplay between serine, threonine, and histidine phosphorylation is thus described for the first time in this human pathogen.
Multiple Replication Origins ofHalobacteriumsp. Strain NRC-1: Properties of the Conservedorc7-DependentoriC1
Authors: Coker James A. , DasSarma Priya , Capes Melinda , Wallace Tammitia , McGarrity Karen , Gessler Rachael , Liu Jingfang , Xiang Hua , Tatusov Roman , Berquist Brian R. , DasSarma Shiladitya
Published: June 2009

The eukaryote-like DNA replication system of the model haloarchaeonHalobacteriumNRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2andorc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence,oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal toorc7(oriC1) andorc10(oriC2), and the largest peaks on the extrachromosomal elements were nearorc9(oriP1) in both pNRC100 and -200 and nearorc4(oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomalorcgenes showed that replication initiation atoriC1requiresorc7but notorc6andorc8. The initiation sites atoriC1were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. TheoriC1region, Orc binding sites, andorc7gene orthologs were conserved in all sequenced haloarchaea. Serial deletion oforcgenes resulted in the construction of a minimal strain containing not onlyorc2andorc10but alsoorc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.
An Alternative Succinate (2-Oxoglutarate) Transport System in
Rhizobium tropici
Is Induced in Nodules of
Phaseolus vulgaris
Authors: Batista Silvia , Patriarca Eduardo J. , Tatè Rosarita , Martínez-Drets Gloria , Gill Paul R.
Published: June 2009

The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C
4
-dicarboxylates. A previously described
dctA
mutant of
Rhizobium tropici
CIAT899, strain GA1 (
dctA
), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial
dctA
mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn
5
mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a
dctABD
locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of
Ralstonia solanacearum
and
Agrobacterium tumefaciens
. This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated
kgtP
in
R. tropici
. The
dctBD
-like genes in this cluster were found to be required for
kgtP
expression and were designated
kgtSR
. Analysis of a
kgtP
::
lacZ
transcriptional fusion indicated that a
kgtSR
-dependent promoter of
kgtP
was specifically induced by 2-oxoglutarate. The expression of
kgtPp
was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of
Phaseolus vulgaris
. Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by
R. tropici
on roots of
P. vulgaris
.
Structural Basis for the Localization of the Chemotaxis Phosphatase CheZ by CheA
S
Authors: Hao Shufeng , Hamel Damon , Zhou Hongjun , Dahlquist Frederick W.
Published: June 2009

CheA-short interacts with CheZ to localize CheZ to cell poles. The fifth helical region (residues 112 to 133) from the phosphotransfer domain of CheA interacts with CheZ and becomes ordered and helical, although it lacks a stable fold in the CheA fragment comprising residues 98 to 150 alone. One CheA molecule binds to one CheZ dimer.
The
Staphylococcus aureus
LytSR Two-Component Regulatory System Affects Biofilm Formation
Authors: Sharma-Kuinkel Batu K. , Mann Ethan E. , Ahn Jong-Sam , Kuechenmeister Lisa J. , Dunman Paul M. , Bayles Kenneth W.
Published: June 2009

Studies of the
Staphylococcus aureus
LytSR two-component regulatory system have led to the identification of the
cid
and
lrg
operons, which affect murein hydrolase activity, stationary-phase survival, antibiotic tolerance, and biofilm formation. The
cid
gene products enhance murein hydrolase activity and antibiotic tolerance whereas the
lrg
gene products inhibit these processes in a manner believed to be analogous to bacteriophage-encoded holins and antiholins, respectively. Importantly, these operons have been shown to play significant roles in biofilm development by controlling the release of genomic DNA, which then becomes an important structural component of the biofilm matrix. To determine the role of LytSR in biofilm development, a
lytS
knockout mutant was generated from a clinical
S. aureus
isolate (UAMS-1) and the effects on gene expression and biofilm formation were examined. As observed in laboratory isolates, LytSR was found to be required for
lrgAB
expression. Furthermore, the
lytS
mutant formed a more adherent biofilm than the wild-type and complemented strains. Consistent with previous findings, the increased adherence of the mutant was attributed to the increased prevalence of matrix-associated eDNA. Transcription profiling studies indicated that the
lrgAB
operon is the primary target of LytSR-mediated regulation but that this regulatory system also impacts expression of a wide variety of genes involved in basic metabolism. Overall, the results of these studies demonstrate that the LytSR two-component regulatory system plays an important role in
S. aureus
biofilm development, likely as a result of its direct influence on
lrgAB
expression.
Functional and Expression Analyses of the
cop
Operon, Required for Copper Resistance in
Agrobacterium tumefaciens
Authors: Nawapan Sirikan , Charoenlap Nisanart , Charoenwuttitam Anchalee , Saenkham Panatda , Mongkolsuk Skorn , Vattanaviboon Paiboon
Published: June 2009

The copper resistance determinant
copARZ
, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium
Agrobacterium tumefaciens
. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a
copR
-dependent fashion. Analysis of the
copARZ
promoter revealed a putative CopR binding box located within the spacer of the −35 and −10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the
copARZ
operon or
copZ
reduces the level of resistance to copper but not to other metal ions. Also, the
copARZ
operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional
copZ
does not affect the ability of copper ions to induce the
copARZ
promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the
copARZ
operon as a component of the copper resistance machinery in
A. tumefaciens.
Feedback Regulation of Lac Repressor Expression inEscherichia coli
Authors: Oehler Stefan
Published: June 2009

Negative feedback regulation, mediated through repressor binding siteO3, which overlaps thelacIgene, could explain the robustness of the weak expression of Lac repressor. Significant autorepression of Lac repressor has never been ruled out. In the work presented here, the degree of autoregulation of Lac repressor was determined. It is negligible.
Replication Initiator DnaA ofEscherichia coliChanges Its Assembly Form on the Replication Origin during the Cell Cycle
Authors: Nozaki Shingo , Niki Hironori , Ogawa Tohru
Published: June 2009

DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in livingEscherichia colicells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of theoriCregions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from theoriCregion. However, involvement ofdatAin the initiation control was suggested from the observation that, inΔdatAcells, DnaA-EYFP maximally colocalized with theoriCregion earlier in the cell cycle than it did in wild-type cells andoriCconcentration was increased.
Sxy Induces a CRP-S Regulon in
Escherichia coli
Authors: Sinha Sunita , Cameron Andrew D. S. , Redfield Rosemary J.
Published: June 2009

Escherichia coli
is not considered naturally competent, yet it has homologues of the genes that most competent bacteria use for DNA uptake and processing. In
Haemophilus influenzae
and
Vibrio cholerae
, these genes are regulated by the Sxy and cyclic AMP receptor (CRP) proteins. We used microarrays to find out whether similar regulation occurs in
E. coli
. Expression of
sxy
strongly induced 63 transcriptional units, 34 of which required CRP for transcriptional activation and had promoter sites resembling the Sxy- and CRP-dependent CRP-S motif previously characterized in
H. influenzae
. As previously reported,
sxy
expression also induced the sigma-H regulon. Flagellar operons were downregulated by
sxy
expression, although motility remained unaffected. The CRP-S regulon included all of
E. coli
's known competence gene homologues, so we investigated Sxy's effect on competence-associated phenotypes. A
sxy
knockout reduced both “natural” plasmid transformation and competitive fitness in long-term culture. In addition, expression of plasmid-borne
sxy
led to production of type IV pilin, the main subunit of the DNA uptake machinery of most bacteria. Although
H. influenzae
Sxy only weakly activated the
E. coli
Sxy regulon, induction was dramatically improved when it was coexpressed with its cognate CRP, suggesting that intimate interactions between Sxy and CRP are required for transcriptional activation at CRP-S sites.