bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Fluorescence-Based Screening for Wolbachia Endosymbionts in Drosophila Germ Line and Somatic Tissues
Authors: Casper-Lindley Catharina , Kimura Scott , Saxton Daniel S. , Essaw Yonathan , Simpson Isaac , Tan Vinson , Sullivan William
Published: May 2011

Wolbachia
is a globally distributed bacterial endosymbiont present in arthropods and nematodes. The advent of sensitive PCR-based approaches has greatly facilitated the identification of
Wolbachia
-infected individuals and analysis of population infection levels. Here, a complementary visual fluorescence-based
Wolbachia
screening approach is described. Through the use of the fluorescent dye Syto-11,
Wolbachia
can be efficiently detected in various
Drosophila
tissues, including ovaries. Syto-11 also stains
Wolbachia
in other insects. Because
Wolbachia
is inherited through the maternal germ line, bacteria reside in the ovaries of flies in infected populations. An advantage of this staining approach is that it informs about
Wolbachia
titer as well as its tissue and cellular distribution. Using this method, the infection status of insect populations in two central California locations was determined, and variants with unusually low or high
Wolbachia
titers were isolated. In addition, a variant with ovarioles containing both infected and uninfected egg chambers was identified. Syto-11 staining of
Cardinium
- and
Spiroplasma
-infected insects was also analyzed.
Different IS
629
Transposition Frequencies Exhibited by Escherichia coli O157:H7 Strains in the Stepwise Evolutionary Model
Authors: Rump Lydia V. , Fischer Markus , González-Escalona Narjol
Published: May 2011

The insertion sequence IS
629
, which is highly prevalent in
Escherichia coli
O157:H7 genomes, was found to be absent in O157:H− strains, which are on a divergent pathway in the emergence of O157:H7. Although O157:H− is deficient in IS
629
, it permits IS
629
transposition, with an excision frequency higher than that of ancestral O55:H7 strains but significantly lower than that of pathogenic O157:H7 strains.
Cultivation-Independent Detection of Autotrophic Hydrogen-Oxidizing Bacteria by DNA Stable-Isotope Probing
Authors: Pumphrey Graham M. , Ranchou-Peyruse Anthony , Spain Jim C.
Published: May 2011

Knallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of13CO2was H2dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. AhydBhomolog encoding a putative high-affinity NiFe hydrogenase was amplified from13C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H2concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation.
Methylotrophy in a Lake: from Metagenomics to Single-Organism Physiology
Authors: Chistoserdova Ludmila
Published: May 2011

This review provides a brief summary of ongoing studies in Lake Washington (Seattle, WA) directed at an understanding of the content and activities of microbial communities involved in methylotrophy. One of the findings from culture-independent approaches, including functional metagenomics, is the prominent presence of
Methylotenera
species in the site and their inferred activity in C
1
metabolism, highlighting the local environmental importance of this group. Comparative analyses of individual genomes of
Methylophilaceae
from Lake Washington provide insights into their genomic divergence and suggest significant metabolic flexibility.
Diversity, Abundance, and Potential Activity of Nitrifying and Nitrate-Reducing Microbial Assemblages in a Subglacial Ecosystem
Authors: Boyd Eric S. , Lange Rachel K. , Mitchell Andrew C. , Havig Jeff R. , Hamilton Trinity L. , Lafrenière Melissa J. , Shock Everett L. , Peters John W. , Skidmore Mark
Published: May 2011

Subglacial sediments sampled from beneath Robertson Glacier (RG), Alberta, Canada, were shown to harbor diverse assemblages of potential nitrifiers, nitrate reducers, and diazotrophs, as assessed by
amoA
,
narG
, and
nifH
gene biomarker diversity. Although archaeal
amoA
genes were detected, they were less abundant and less diverse than bacterial
amoA
, suggesting that bacteria are the predominant nitrifiers in RG sediments. Maximum nitrification and nitrate reduction rates in microcosms incubated at 4°C were 280 and 18.5 nmol of N per g of dry weight sediment per day, respectively, indicating the potential for these processes to occur
in situ
. Geochemical analyses of subglacial sediment pore waters and bulk subglacial meltwaters revealed low concentrations of inorganic and organic nitrogen compounds. These data, when coupled with a C/N atomic ratio of dissolved organic matter in subglacial pore waters of ∼210, indicate that the sediment communities are N limited. This may reflect the combined biological activities of organic N mineralization, nitrification, and nitrate reduction. Despite evidence of N limitation and the detection of
nifH
, we were unable to detect biological nitrogen fixation activity in subglacial sediments. Collectively, the results presented here suggest a role for nitrification and nitrate reduction in sustaining microbial life in subglacial environments. Considering that ice currently covers 11% of the terrestrial landmass and has covered significantly greater portions of Earth at times in the past, the demonstration of nitrification and nitrate reduction in subglacial environments furthers our understanding of the potential for these environments to contribute to global biogeochemical cycles on glacial-interglacial timescales.
Impact of Internal RNA on Aggregation and Electrokinetics of Viruses: Comparison between MS2 Phage and Corresponding Virus-Like Particles
Authors: Dika C. , Duval J. F. L. , Ly-Chatain H. M. , Merlin C. , Gantzer C.
Published: May 2011

We compare for the first time the electrokinetic and aggregation properties of MS2 phage (pH 2.5 to 7, 1 to 100 mM NaNO
3
electrolyte concentration) with those of the corresponding virus-like particles (VLPs), which lack entirely the inner viral RNA component. In line with our previous work (J. Langlet, F. Gaboriaud, C. Gantzer, and J. F. L. Duval, Biophys. J. 94:3293-3312, 2008), it is found that modifying the content of RNA within the virus leads to very distinct electrohydrodynamic and aggregation profiles for MS2 and MS2 VLPs. Under the given pH and concentration conditions, MS2 VLPs exhibit electrophoretic mobility larger in magnitude than that of MS2, and both have similar isoelectric point (IEP) values (∼4). The electrokinetic results reflect a greater permeability of MS2 VLPs to electroosmotic flow, developed within/around these soft particles during their migration under the action of the applied electrical field. Results also support the presence of some remaining negatively charged component within the VLPs. In addition, MS2 phage systematically forms aggregates at pH values below the IEP, regardless of the magnitude of the solution ionic strength, whereas MS2 VLPs aggregate under the strict condition where the pH is relatively equal to the IEP at sufficiently low salt concentrations (<10 mM). It is argued that the stability of VLPs against aggregation and the differences between electrokinetics of MS2 and corresponding VLPs conform to recently developed formalisms for the stability and electrohydrodynamics of soft multilayered particles. The differences between the surface properties of these two kinds of particles reported here suggest that VLPs may not be appropriate for predicting the behavior of pathogenic viruses in aqueous media.
Persistence of Avian Influenza Viruses in Lake Sediment, Duck Feces, and Duck Meat
Authors: Nazir Jawad , Haumacher Renate , Ike Anthony C. , Marschang Rachel E.
Published: May 2011

The persistence of 3 low-pathogenicity avian influenza viruses (LPAIV) (H4N6, H5N1, and H6N8) and one human influenza virus (H1N1) as well as Newcastle disease virus (NDV) and enteric cytopathogenic bovine orphan (ECBO) virus was investigated in lake sediment, duck feces, and duck meat at 30, 20, 10, and 0°C using a germ carrier technique. Virus-loaded germ carriers were incubated in each substrate, and residual infectivity of the eluted virus was quantified on cell culture after regular intervals for a maximum of 24 weeks. Data were analyzed by a linear regression model to calculate
T
90
values (time required for 90% loss of virus infectivity) and estimated persistence of the viruses. In general, the persistence of all of the viruses was highest in lake sediment, followed by feces, and was the lowest in duck meat at all temperatures. For the avian influenza virus subtypes,
T
90
values in sediment ranged from 5 to 11, 13 to 18, 43 to 54, and 66 to 394 days at 30, 20, 10, and 0°C, respectively, which were 2 to 5 times higher than the
T
90
values of the viruses in the feces and meat. Although the individual viruses vary in tenacity, the survival time of influenza viruses was shorter than that of NDV and ECBO virus in all substrates. The results of this study suggest that lake sediment may act as a long-term source of influenza viruses in the aquatic habitat, while the viruses may remain infectious for extended periods of time in duck feces and meat at low temperatures, allowing persistence of the viruses in the environment over winter.
Detection of Known and Novel Adenoviruses in Cattle Wastes via Broad-Spectrum Primers
Authors: Sibley Samuel D. , Goldberg Tony L. , Pedersen Joel A.
Published: May 2011

The critical assessment of bovine adenoviruses (BAdV) as indicators of environmental fecal contamination requires improved knowledge of their prevalence, shedding dynamics, and genetic diversity. We examined DNA extracted from bovine and other animal waste samples collected in Wisconsin for atadenoviruses and mastadenoviruses using novel, broad-spectrum PCR primer sets. BAdV were detected in 13% of cattle fecal samples, 90% of cattle urine samples, and 100% of cattle manure samples; 44 percent of BAdV-positive samples contained bothAtadenovirusandMastadenovirusDNA. Additionally, BAdV were detected in soil, runoff water from a cattle feedlot, and residential well water. Overall, we detected 8 of 11 prototype BAdV, plus bovine, rabbit, and porcine mastadenoviruses that diverged significantly from previously reported genotypes. The prevalence of BAdV shedding by cattle supports targeting AdV broadly as indicators of the presence of fecal contamination in aqueous environments. Conversely, several factors complicate the use of AdV for fecal source attribution. Animal AdV infecting a given livestock host were not monophyletic, recombination among livestock mastadenoviruses was detected, and the genetic diversity of animal AdV is still underreported. These caveats highlight the need for continuing genetic surveillance for animal AdV and for supporting data when BAdV detection is invoked for fecal source attribution in environmental samples. To our knowledge, this is the first study to report natural BAdV excretion in urine, BAdV detection in groundwater, and recombination in AdV of livestock origin.
Control of Bacterial Persister Cells by Trp/Arg-Containing Antimicrobial Peptides
Authors: Chen Xi , Zhang Mi , Zhou Chunhui , Kallenbach Neville R. , Ren Dacheng
Published: May 2011

Persister cells are dormant phenotypic variants inherent in a bacterial population. They play important roles in chronic infections and present great challenges to therapy due to extremely enhanced tolerance to antibiotics compared to that of normal cells of the same genotype. In this study, we report that cationic membrane-penetrating peptides containing various numbers of arginine and tryptophan repeats are effective in killing persister cells of
Escherichia coli
HM22, a hyper-persister producer. The activities of three linear peptides [(RW)

n

-NH
2
, where
n
is 2, 3, or 4] and a dendrimeric peptide, (RW)
4D
, in killing bacterial persisters were compared. Although the dendrimeric peptide (RW)
4D
requires a lower threshold to kill planktonic persisters, octameric peptide (RW)
4
-NH
2
is the most effective against planktonic persister cells at high concentrations. For example, treatment with 80 μM (RW)
4
-NH
2
for 60 min led to a 99.7% reduction in the number of viable persister cells. The viability of persister cells residing in surface-attached biofilms was also significantly reduced by (RW)
4
-NH
2
and (RW)
4D
. These two peptides were also found to significantly enhance the susceptibility of biofilm cells to ofloxacin. The potency of (RW)
4
-NH
2
was further marked by its ability to disperse and kill preformed biofilms harboring high percentages of persister cells. Interestingly, approximately 70% of the dispersed cells were found to have lost their intrinsic tolerance and become susceptible to ampicillin if not killed directly by this peptide. These results are helpful for better understanding the activities of these peptides and may aid in future development of more effective therapies of chronic infections.
Assembly of Minicellulosomes on the Surface of Bacillus subtilis
Authors: Anderson Timothy D. , Robson Scott A. , Jiang Xiao Wen , Malmirchegini G. Reza , Fierobe Henri-Pierre , Lazazzera Beth A. , Clubb Robert T.
Published: May 2011

To cost-efficiently produce biofuels, new methods are needed to convert lignocellulosic biomass into fermentable sugars. One promising approach is to degrade biomass using cellulosomes, which are surface-displayed multicellulase-containing complexes present in cellulolytic
Clostridium
and
Ruminococcus
species. In this study we created cellulolytic strains of
Bacillus subtilis
that display one or more cellulase enzymes. Proteins containing the appropriate cell wall sorting signal are covalently anchored to the peptidoglycan by coexpressing them with the
Bacillus anthracis
sortase A (SrtA) transpeptidase. This approach was used to covalently attach the Cel8A endoglucanase from
Clostridium thermocellum
to the cell wall. In addition, a Cel8A-dockerin fusion protein was anchored on the surface of
B. subtilis
via noncovalent interactions with a cell wall-attached cohesin module. We also demonstrate that it is possible to assemble multienzyme complexes on the cell surface. A three-enzyme-containing minicellulosome was displayed on the cell surface; it consisted of a cell wall-attached scaffoldin protein noncovalently bound to three cellulase-dockerin fusion proteins that were produced in
Escherichia coli
.
B. subtilis
has a robust genetic system and is currently used in a wide range of industrial processes. Thus, grafting larger, more elaborate minicellulosomes onto the surface of
B. subtilis
may yield cellulolytic bacteria with increased potency that can be used to degrade biomass.
Saccharomyces cerevisiae Genome Shuffling through Recursive Population Mating Leads to Improved Tolerance to Spent Sulfite Liquor
Authors: Pinel Dominic , D'Aoust Frédéric , del Cardayre Stephen B. , Bajwa Paramjit K. , Lee Hung , Martin Vincent J. J.
Published: May 2011

Spent sulfite liquor (SSL) is a waste effluent from sulfite pulping that contains monomeric sugars which can be fermented to ethanol. However, fermentative yeasts used for the fermentation of the sugars in SSL are adversely affected by the inhibitory substances in this complex feedstock. To overcome this limitation, evolutionary engineering of
Saccharomyces cerevisiae
was carried out using genome-shuffling technology based on large-scale population cross mating. Populations of UV-light-induced yeast mutants more tolerant than the wild type to hardwood spent sulfite liquor (HWSSL) were first isolated and then recursively mated and enriched for more-tolerant populations. After five rounds of genome shuffling, three strains were isolated that were able to grow on undiluted HWSSL and to support efficient ethanol production from the sugars therein for prolonged fermentation of HWSSL. Analyses showed that greater HWSSL tolerance is associated with improved viability in the presence of salt, sorbitol, peroxide, and acetic acid. Our results showed that evolutionary engineering through genome shuffling will yield robust yeasts capable of fermenting the sugars present in HWSSL, which is a complex substrate containing multiple sources of inhibitors. These strains may not be obtainable through classical evolutionary engineering and can serve as a model for further understanding of the mechanism behind simultaneous tolerance to multiple inhibitors.
Development of a Multilocus Sequence Typing Tool for High-Resolution Genotyping of Enterocytozoon bieneusi
Authors: Feng Yaoyu , Li Na , Dearen Theresa , Lobo Maria L. , Matos Olga , Cama Vitaliano , Xiao Lihua
Published: May 2011

Thus far, genotyping of
Enterocytozoon bieneusi
has been based solely on DNA sequence analysis of the internal transcribed spacer (ITS) of the rRNA gene. Both host-adapted and zoonotic (human-pathogenic) genotypes of
E. bieneusi
have been identified. In this study, we searched for microsatellite and minisatellite sequences in the whole-genome sequence database of
E. bieneusi
isolate H348. Seven potential targets (MS1 to MS7) were identified. Testing of the seven targets by PCR using two human-pathogenic
E. bieneusi
genotypes (A and Peru10) led to the selection of four targets (MS1, MS3, MS4, and MS7). Further analysis of the four loci with an additional 24 specimens of both host-adapted and zoonotic
E. bieneusi
genotypes indicated that most host-adapted genotypes were not amplified by PCR targeting these loci. In contrast, 10 or 11 of the 13 specimens of the zoonotic genotypes were amplified by PCR at each locus. Altogether, 12, 8, 7, and 11 genotypes of were identified at MS1, MS3, MS4, and MS7, respectively. Phylogenetic analysis of the nucleotide sequences obtained produced a genetic relationship that was similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included most
E. bieneusi
genotypes in humans. Thus, a multilocus sequence typing tool was developed for high-resolution genotyping of
E. bieneusi.
Data obtained in the study should also have implications for understanding the taxonomy of
Enterocytozoon
spp., the public health significance of
E. bieneusi
in animals, and the sources of human
E. bieneusi
infections.
Role Played by Exosporium Glycoproteins in the Surface Properties of Bacillus cereus Spores and in Their Adhesion to Stainless Steel
Authors: Lequette Yannick , Garénaux Estelle , Tauveron Grégoire , Dumez Sylvain , Perchat Stéphane , Slomianny Christian , Lereclus Didier , Guérardel Yann , Faille Christine
Published: May 2011

Bacillus cereus
spores are surrounded by a loose-fitting layer called the exosporium, whose distal part is mainly formed from glycoproteins. The role played by the exosporium glycoproteins of
B. cereus
ATCC 14579 (BclA and ExsH) was investigated by considering hydrophobicity and charge, as well as the properties of spore adhesion to stainless steel. The absence of BclA increased both the isoelectric point (IEP) and hydrophobicity of whole spores while simultaneously reducing the interaction between spores and stainless steel. However, neither the hydrophobicity nor the charge associated with BclA could explain the differences in the adhesion properties. Conversely, ExsH, another exosporium glycoprotein, did not play a significant role in spore surface properties. The monosaccharide analysis of
B. cereus
ATCC 14579 showed different glycosylation patterns on ExsH and BclA. Moreover, two specific glycosyl residues, namely, 2-
O
-methyl-rhamnose (2-Me-Rha) and 2,4-
O
-methyl-rhamnose (2,4-Me-Rha), were attached to BclA, in addition to the glycosyl residues already reported in
B. anthracis
.
Evaluation of Physical Coverings Used To Control Escherichia coli O157:H7 at the Compost Heap Surface
Authors: Shepherd Marion W. , Kim Jinkyung , Jiang Xiuping , Doyle Michael P. , Erickson Marilyn C.
Published: May 2011

Throughout four field trials, compost heaps covered with finished compost maintained temperatures under the physical covering that were ca. 7 to 15.5°C higher, resulting in rapid
Escherichia coli
O157:H7 reduction, than those of the heaps covered with fresh straw or left uncovered. Our results validated recommendations made by the U.S. Environmental Protection Agency for covering fresh compost.
Cholesterol Degradation by Gordonia cholesterolivorans
Authors: Drzyzga O. , Fernández de las Heras L. , Morales V. , Navarro Llorens J. M. , Perera J.
Published: May 2011

This paper reports physiological and genetic data about the type strain
Gordonia cholesterolivorans
, a strain that is able to degrade steroid compounds containing a long carbon side chain such as cholesterol (C
27
), cholestenone (C
27
), ergosterol (C
28
), and stigmasterol (C
29
). The length of the carbon side chain appears to be of great importance for this bacterium, as the strain is unable to grow using steroids with a shorter or nonaliphatic carbon side chain such as cholic acid (C
24
), progesterone (C
21
), testosterone, androsterone, 4-androstene-3,17-dione (all C
19
), and further steroids. This study also demonstrates that the degradation of cholesterol is a quite common feature of the genus
Gordonia
by comparing
Gordonia
cholesterolivorans
with some other species of this genus (e.g.,
G. sihwensis
,
G. hydrophobica
,
G. australis
, and
G. neofelifaecis
). Pyrosequencing of the genome of
G. cholesterolivorans
led to the identification of two conventional cholesterol oxidase genes on an 8-kb and a 12.8-kb genomic fragment with genetic organizations that are quite unique as compared to the genomes of other cholesterol-degrading bacteria sequenced so far. The identified two putative cholesterol oxidases of
G. cholesterolivorans
are both intracellularly acting enzymes of the class I type. Whereas one of these two cholesterol oxidases (ChoOx-1) shows high identity with an oxidoreductase of the opportunistic pathogen
G. bronchialis
and is not transcribed during growth with cholesterol, the other one (ChoOx-2) appears phylogenetically closer to cholesterol oxidases from members of the genus
Rhodococcus
and is transcribed constitutively. By using targeted gene disruption, a
G. cholesterolivorans
ChoOx-2 gene mutant strain that was unable to grow with steroids was obtained.
Role of Hsl7 in Morphology and Pathogenicity and Its Interaction with Other Signaling Components in the Plant Pathogen Ustilago maydis
Authors: Lovely C. Ben , Aulakh Kavita Burman , Perlin Michael H.
Published: May 2011

The phytopathogenic fungus
Ustilago maydis
undergoes a dimorphic transition in response to mating pheromone, host, and environmental cues. On a solid medium deficient in ammonium (SLAD [0.17% yeast nitrogen base without ammonium sulfate or amino acids, 2% dextrose, 50 μM ammonium sulfate]),
U. maydis
produces a filamentous colony morphology, while in liquid SLAD, the cells do not form filaments. The p21-activated protein kinases (PAKs) play a substantial role in regulating the dimorphic transition in fungi. The PAK-like Ste20 homologue Smu1 is required for a normal response to pheromone, via upregulation of pheromone expression, and virulence, and its disruption affects both processes. Our experiments suggest that Smu1 also regulates cell length and the filamentous response on solid SLAD medium. Yeast two-hybrid analysis suggested an Hsl7 homologue as a potential interacting partner of Smu1, and a unique open reading frame for such an arginine methyltransferase was detected in the
U. maydis
genome sequence. Hsl7 regulates cell length and the filamentous response to solid SLAD in a fashion opposite to that of Smu1, but neither overexpression nor disruption of
hsl7
attenuates virulence. Simultaneous disruption of
hsl7
and overexpression of
smu1
lead to a hyperfilamentous response on solid SLAD. Moreover, only this double mutant strain forms filaments in liquid SLAD. The double mutant strain was also significantly reduced in virulence. A similar filamentous response in both solid and liquid SLAD was observed in strains lacking another PAK-like protein kinase involved in cytokinesis and polar growth, Cla4. Our data suggest that Hsl7 may regulate cell cycle progression, while both Smu1 and Cla4 appear to be involved in the filamentous response in
U. maydis
.
Do Red and Green Make Brown?: Perspectives on Plastid Acquisitions within Chromalveolates
Authors: Dorrell Richard G. , Smith Alison G.
Published: May 2011

The chromalveolate “supergroup” is of key interest in contemporary phycology, as it contains the overwhelming majority of extant algal species, including several phyla of key importance to oceanic net primary productivity such as diatoms, kelps, and dinoflagellates. There is also intense current interest in the exploitation of these algae for industrial purposes, such as biodiesel production. However, the evolution of the constituent species, and in particular the origin and radiation of the chloroplast genomes, remains poorly understood. In this review, we discuss current theories of the origins of the extant red alga-derived chloroplast lineages in the chromalveolates and the potential ramifications of the recent discovery of large numbers of green algal genes in chromalveolate genomes. We consider that the best explanation for this is that chromalveolates historically possessed a cryptic green algal endosymbiont that was subsequently replaced by a red algal chloroplast. We consider how changing selective pressures acting on ancient chromalveolate lineages may have selectively favored the serial endosymbioses of green and red algae and whether a complex endosymbiotic history facilitated the rise of chromalveolates to their current position of ecological prominence.
Effects of Fluconazole on the Secretome, the Wall Proteome, and Wall Integrity of the Clinical Fungus Candida albicans
Authors: Sorgo Alice G. , Heilmann Clemens J. , Dekker Henk L. , Bekker Martijn , Brul Stanley , de Koster Chris G. , de Koning Leo J. , Klis Frans M.
Published: May 2011

Fluconazole is a commonly used antifungal drug that inhibits Erg11, a protein responsible for 14α-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14α-methylated sterols, which causes increased membrane fluidity and drug permeability. Surface-grown and planktonic cultures of
Candida albicans
responded similarly to fluconazole at 0.5 mg/liter, showing reduced biomass formation, severely reduced ergosterol levels, and almost complete inhibition of hyphal growth. There was no evidence of cell leakage. Mass spectrometric analysis of the secretome showed that its composition was strongly affected and included 17 fluconazole-specific secretory proteins. Relative quantification of
14
N-labeled query walls relative to a reference standard mixture of
15
N-labeled yeast and hyphal walls in combination with immunological analysis revealed considerable fluconazole-induced changes in the wall proteome as well. They were, however, similar for both surface-grown and planktonic cultures. Two major trends emerged: (i) decreased incorporation of hypha-associated wall proteins (Als3, Hwp1, and Plb5), consistent with inhibition of hyphal growth, and (ii) increased incorporation of putative wall repair-related proteins (Crh11, Pga4, Phr1, Phr2, Pir1, and Sap9). As exposure to the wall-perturbing drug Congo red led to a similar response, these observations suggested that fluconazole affects the wall. In keeping with this, the resistance of fluconazole-treated cells to wall-perturbing compounds decreased. We propose that fluconazole affects the integrity of both the cellular membranes and the fungal wall and discuss its potential consequences for antifungal therapy. We also present candidate proteins from the secretome for clinical marker development.
Activity Control of the ClpC Adaptor McsB in Bacillus subtilis
Authors: Elsholz A. K. W. , Hempel K. , Michalik S. , Gronau K. , Becher D. , Hecker M. , Gerth U.
Published: May 2011

Controlled protein degradation is an important cellular reaction for the fast and efficient adaptation of bacteria to ever-changing environmental conditions. In the low-GC, Gram-positive model organism
Bacillus subtilis
, the AAA+ protein ClpC requires specific adaptor proteins not only for substrate recognition but also for chaperone activity. The McsB adaptor is activated particularly during heat stress, allowing the controlled degradation of the CtsR repressor by the ClpCP protease. Here we report how the McsB adaptor becomes activated by autophosphorylation on specific arginine residues during heat stress. In nonstressed cells McsB activity is inhibited by ClpC as well as YwlE.
Comparative Genomic Analysis of the Hexuronate Metabolism Genes and Their Regulation in Gammaproteobacteria
Authors: Suvorova Inna A. , Tutukina Maria N. , Ravcheev Dmitry A. , Rodionov Dmitry A. , Ozoline Olga N. , Gelfand Mikhail S.
Published: May 2011

The hexuronate metabolism in
Escherichia coli
is regulated by two related transcription factors from the FadR subfamily of the GntR family, UxuR and ExuR. UxuR controls the
d
-glucuronate metabolism, while ExuR represses genes involved in the metabolism of all hexuronates. We use a comparative genomics approach to reconstruct the hexuronate metabolic pathways and transcriptional regulons in gammaproteobacteria. We demonstrate differences in the binding motifs of UxuR and ExuR, identify new candidate members of the UxuR/ExuR regulons, and describe the links between the UxuR/ExuR regulons and the adjacent regulons UidR, KdgR, and YjjM. We provide experimental evidence that two predicted members of the UxuR regulon,
yjjM
and
yjjN
, are the subject of complex regulation by this transcription factor in
E. coli
.
TdaA Regulates Tropodithietic Acid Synthesis by Binding to the
tdaC
Promoter Region
Authors: Geng Haifeng , Belas Robert
Published: May 2011

Silicibacter
sp. TM1040, a member of the marine
Roseobacter
clade, produces the antibiotic and quorum signaling molecule tropodithietic acid (TDA), encoded by
tdaABCDEF
. Here, we showed that an LysR-type transcriptional regulator, TdaA, is a positive regulator of
tdaCDE
gene expression and binds to the
tdaC
promoter region.
Genetic Variation of Adenylation Domains of the Anabaenopeptin Synthesis Operon and Evolution of Substrate Promiscuity
Authors: Christiansen Guntram , Philmus Benjamin , Hemscheidt Thomas , Kurmayer Rainer
Published: May 2011

Anabaenopeptins (AP) are bioactive cyclic hexapeptides synthesized nonribosomally in cyanobacteria. APs are characterized by several conserved motifs, including the ureido bond, N-methylation in position 5, and
d
-Lys in position 2. All other positions of the AP molecule are variable, resulting in numerous structural variants. We have identified a nonribosomal peptide synthetase (NRPS) operon from
Planktothrix agardhii
strain CYA126/8 consisting of five genes (
apnA
to
apnE
) encoding six NRPS modules and have confirmed its role in AP synthesis by the generation of a mutant via insertional inactivation of
apnC
. In order to correlate the genetic diversity among adenylation domains (A domains) with AP structure variation, we sequenced the A domains of all six NRPS modules from seven
Planktothrix
strains differing in the production of AP congeners. It is remarkable that single strains coproduce APs bearing either of the chemically divergent amino acids Arg and Tyr in exocyclic position 1. Since the A domain of the initiation module (the ApnA A
1
domain) has been proposed to activate the amino acid incorporated into exocyclic position 1, we decided to analyze this domain both biochemically and phylogenetically. Only ApnA A
1
enzymes from strains producing AP molecules containing Arg or Tyr in position 1 were found to activate these two chemically divergent amino acids
in vitro
. Phylogenetic analysis of
apn
A domain sequences revealed that strains with a promiscuous ApnA A
1
domain are derived from an ancestor that activates only Arg. Surprisingly, positive selection appears to affect only three codons within the
apnA
A
1
gene, suggesting that this remarkable promiscuity has evolved from point mutations only.
The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola
Authors: Bian Jiang , Shen Hongwu , Tu Youbin , Yu Aiming , Li Chunhao
Published: May 2011

Thiamine pyrophosphate (TPP), a biologically active form of thiamine (vitamin B
1
), is an essential cofactor in all living systems. Microorganisms either synthesize TPP via
de novo
biosynthesis pathways or uptake exogenous thiamine from the environment via specific transporters. The oral spirochete
Treponema denticola
is an important pathogen that is associated with human periodontal diseases. It lacks a
de novo
TPP biosynthesis pathway and needs exogenous TPP for growth, suggesting that it may obtain exogenous TPP via a thiamine transporter. In this study, we identified a gene cluster that encodes a TPP ABC transporter which consists of a TPP-binding protein (TDE0143), a transmembrane permease (TDE0144), and a cytosolic ATPase (TDE0145). Transcriptional and translational analyses showed that the genes encoding these three proteins are cotranscribed and form an operon (

tbpABC
Td

) that is initiated by a σ
70
-like promoter. The expression level of this operon is negatively regulated by exogenous TPP and is mediated by a TPP-sensing riboswitch (Td

thi-

box
). Genetic and biochemical studies revealed that the
TDE0143
deletion mutant (
T. denticola
Δ
tbpA
) had a decreased ability to transport exogenous TPP, and the mutant failed to grow when exogenous TPP was insufficient. These results taken together indicate that the

tbpABC
Td

operon encodes an ABC transporter that is required for the uptake of exogenous TPP and that the expression of this operon is regulated by a TPP-binding riboswitch via a feedback inhibition mechanism.
Genomes of Three Methylotrophs from a Single Niche Reveal the Genetic and Metabolic Divergence of the Methylophilaceae
Authors: Lapidus Alla , Clum Alicia , LaButti Kurt , Kaluzhnaya Marina G. , Lim Sujung , Beck David A. C. , Glavina del Rio Tijana , Nolan Matt , Mavromatis Konstantinos , Huntemann Marcel , Lucas Susan , Lidstrom Mary E. , Ivanova Natalia , Chistoserdova Ludmila
Published: May 2011

The genomes of three representatives of the family
Methylophilaceae
,
Methylotenera mobilis
JLW8,
Methylotenera versatilis
301, and
Methylovorus glucosetrophus
SIP3-4, all isolated from a single study site, Lake Washington in Seattle, WA, were completely sequenced. These were compared to each other and to the previously published genomes of
Methylobacillus flagellatus
KT and an unclassified
Methylophilales
strain, HTCC2181. Comparative analysis revealed that the core genome of
Methylophilaceae
may be as small as approximately 600 genes, while the pangenome may be as large as approximately 6,000 genes. Significant divergence between the genomes in terms of both gene content and gene and protein conservation was uncovered, including the varied presence of certain genes involved in methylotrophy. Overall, our data demonstrate that metabolic potentials can vary significantly between different species of
Methylophilaceae
, including organisms inhabiting the very same environment. These data suggest that genetic divergence among the members of this family may be responsible for their specialized and nonredundant functions in C
1
cycling, which in turn suggests means for their successful coexistence in their specific ecological niches.
Using Single-Nucleotide Polymorphisms To Discriminate Disease-Associated from Carried Genomes of Neisseria meningitidis
Authors: Katz Lee S. , Sharma Nitya V. , Harcourt Brian H. , Thomas Jennifer Dolan , Wang Xin , Mayer Leonard W. , Jordan I. King
Published: May 2011

Neisseria meningitidis
is one of the main agents of bacterial meningitis, causing substantial morbidity and mortality worldwide. However, most of the time
N. meningitidis
is carried as a commensal not associated with invasive disease. The genomic basis of the difference between disease-associated and carried isolates of
N. meningitidis
may provide critical insight into mechanisms of virulence, yet it has remained elusive. Here, we have taken a comparative genomics approach to interrogate the difference between disease-associated and carried isolates of
N. meningitidis
at the level of individual nucleotide variations (i.e., single nucleotide polymorphisms [SNPs]). We aligned complete genome sequences of 8 disease-associated and 4 carried isolates of
N. meningitidis
to search for SNPs that show mutually exclusive patterns of variation between the two groups. We found 63 SNPs that distinguish the 8 disease-associated genomes from the 4 carried genomes of
N. meningitidis
, which is far more than can be expected by chance alone given the level of nucleotide variation among the genomes. The putative list of SNPs that discriminate between disease-associated and carriage genomes may be expected to change with increased sampling or changes in the identities of the isolates being compared. Nevertheless, we show that these discriminating SNPs are more likely to reflect phenotypic differences than shared evolutionary history. Discriminating SNPs were mapped to genes, and the functions of the genes were evaluated for possible connections to virulence mechanisms. A number of overrepresented functional categories related to virulence were uncovered among SNP-associated genes, including genes related to the category “symbiosis, encompassing mutualism through parasitism.”
Lateral Transfer of Genes and Gene Fragments in Staphylococcus Extends beyond Mobile Elements
Authors: Chan Cheong Xin , Beiko Robert G. , Ragan Mark A.
Published: May 2011

The widespread presence of antibiotic resistance and virulence among
Staphylococcus
isolates has been attributed in part to lateral genetic transfer (LGT), but little is known about the broader extent of LGT within this genus. Here we report the first systematic study of the modularity of genetic transfer among 13
Staphylococcus
genomes covering four distinct named species. Using a topology-based phylogenetic approach, we found, among 1,354 sets of homologous genes examined, strong evidence of LGT in 368 (27.1%) gene sets, and weaker evidence in another 259 (19.1%). Within-gene and whole-gene transfer contribute almost equally to the topological discordance of these gene sets against a reference phylogeny. Comparing genetic transfer in single-copy and in multicopy gene sets, we observed a higher frequency of LGT in the latter, and a substantial functional bias in cases of whole-gene transfer (little such bias was observed in cases of fragmentary genetic transfer). We found evidence that lateral transfer, particularly of entire genes, impacts not only functions related to antibiotic, drug, and heavy-metal resistance, as well as membrane transport, but also core informational and metabolic functions not associated with mobile elements. Although patterns of sequence similarity support the cohesion of recognized species, LGT within
S. aureus
appears frequently to disrupt clonal complexes. Our results demonstrate that LGT and gene duplication play important parts in functional innovation in staphylococcal genomes.
Role of High-Fidelity Escherichia coli DNA Polymerase I in Replication Bypass of a Deoxyadenosine DNA-Peptide Cross-Link
Authors: Yamanaka Kinrin , Minko Irina G. , Finkel Steven E. , Goodman Myron F. , Lloyd R. Stephen
Published: May 2011

Reaction of bifunctional electrophiles with DNA in the presence of peptides can result in DNA-peptide cross-links. In particular, the linkage can be formed in the major groove of DNA via the exocyclic amino group of adenine (
N
6
-dA). We previously demonstrated that an A family human polymerase, Pol ν, can efficiently and accurately synthesize DNA past
N
6
-dA-linked peptides. Based on these results, we hypothesized that another member of that family,
Escherichia coli
polymerase I (Pol I), may also be able to bypass these large major groove DNA lesions. To test this, oligodeoxynucleotides containing a site-specific
N
6
-dA dodecylpeptide cross-link were created and utilized for
in vitro
DNA replication assays using
E. coli
DNA polymerases. The results showed that Pol I and Pol II could efficiently and accurately bypass this adduct, while Pol III replicase, Pol IV, and Pol V were strongly inhibited. In addition, cellular studies were conducted using
E. coli
strains that were either wild type or deficient in all three DNA damage-inducible polymerases, i.e., Pol II, Pol IV, and Pol V. When single-stranded DNA vectors containing a site-specific
N
6
-dA dodecylpeptide cross-link were replicated in these strains, the efficiencies of replication were comparable, and in both strains, intracellular bypass of the lesion occurred in an error-free manner. Collectively, these findings demonstrate that despite its constrained active site, Pol I can catalyze DNA synthesis past
N
6
-dA-linked peptide cross-links and is likely to play an essential role in cellular bypass of large major groove DNA lesions.
Functional Organization of MobB, a Small Protein Required for Efficient Conjugal Transfer of Plasmid R1162
Authors: Meyer Richard
Published: May 2011

MobB is a small (molecular weight = 15,097) protein encoded by the broad-host-range plasmid R1162 and is required for its efficient transfer by conjugation. The C-terminal half of the protein contains a membrane domain essential for transfer. This region can be replaced by a putative membrane domain from another, unrelated protein, and thus is likely to function independently from the rest of MobB. The other, functionally active region of MobB, identified by mutagenesis, is at the N-terminal end. One mutation affecting this region inhibits replication, suggesting that this part of the protein is contacting and sequestering the relaxase-linked primase. The overall organization reflects a multimeric and bipolar organization, with molecules of MobB anchored in the membrane at one end and engaging the relaxase at the other. This arrangement could increase the transfer frequency by raising the probability of contact between the relaxase and the membrane-embedded, coupling protein for type IV secretion.
Genome Sequence of Lactococcus garvieae 21881, Isolated in a Case of Human Septicemia
Authors: Aguado-Urda Mónica , López-Campos Guillermo H. , Blanco M. Mar , Fernández-Garayzábal José F. , Cutuli M. Teresa , Aspiroz Carmen , López-Alonso Victoria , Gibello Alicia
Published: May 2011

Lactococcus garvieae
is a Gram-positive bacterium considered an important opportunistic emerging human pathogen and also a well-recognized fish pathogen. Here, we present the draft genome sequence of
Lactococcus garvieae
strain 21881 (2,164,557 bp, with a G+C content of 37.9%), which represents the first report of a genome sequence on
Lactococcus garvieae
.
Functional Characterization of Seven γ-Glutamylpolyamine Synthetase Genes and the
bauRABCD
Locus for Polyamine and β-Alanine Utilization in Pseudomonas aeruginosa PAO1
Authors: Yao Xiangyu , He Weiqing , Lu Chung-Dar
Published: May 2011

Pseudomonas aeruginosa
and many other bacteria can utilize biogenic polyamines, including diaminopropane (DAP), putrescine (Put), cadaverine (Cad), and spermidine (Spd), as carbon and/or nitrogen sources. Transcriptome analysis in response to exogenous Put and Spd led to the identification of a list of genes encoding putative enzymes for the catabolism of polyamines. Among them,
pauA1
to
pauA6
,
pauB1
to
pauB4
,
pauC
, and
pauD1
and
pauD2
(
p
oly
a
mine
u
tilization) encode enzymes homologous to
Escherichia coli
PuuABCD of the γ-glutamylation pathway in converting Put into GABA. A series of unmarked
pauA
mutants was constructed for growth phenotype analysis. The results revealed that it requires specific combinations of
pauA
knockouts to abolish utilization of different polyamines and support the importance of γ-glutamylation for polyamine catabolism in
P. aeruginosa
. Another finding was that the list of Spd-inducible genes overlaps almost completely with that of Put-inducible ones except the
pauA3B2
operon and the
bauABCD
operon (
β
-
a
lanine
u
tilization). Mutation analysis led to the conclusion that
pauA3B2
participate in catabolism of DAP, which is related to the aminopropyl moiety of Spd, and that
bauABCD
are essential for growth on β-alanine derived from DAP (or Spd) catabolism via the γ-glutamylation pathway. Measurements of the
pauA3-lacZ
and
bauA-lacZ
expression indicated that these two promoters were differentially induced by Spd, DAP, and β-alanine but showed no apparent response to Put, Cad, and GABA. Induction of the
pauA3
and
bauA
promoters was abolished in the
bauR
mutant. The recombinant BauR protein was purified to demonstrate its interactions with the
pauA3
and
bauA
regulatory regions
in vitro
. In summary, the present study support that the γ-glutamylation pathway for polyamine utilization is evolutionarily conserved in
E. coli
and
Pseudomonas
spp. and is further expanded in
Pseudomonas
to accommodate a more diverse metabolic capacity in this group of microorganisms.
Quorum Sensing in Chromobacterium violaceum: DNA Recognition and Gene Regulation by the CviR Receptor
Authors: Stauff Devin L. , Bassler Bonnie L.
Published: May 2011

The bacterial pathogen
Chromobacterium violaceum
uses a LuxIR-type quorum-sensing system to detect and respond to changes in cell population density. CviI synthesizes the autoinducer C
10
-homoserine lactone (C
10
-HSL), and CviR is a cytoplasmic DNA binding transcription factor that activates gene expression following binding to C
10
-HSL. A number of behaviors are controlled by quorum sensing in
C. violaceum
. However, few genes have been shown to be directly controlled by CviR, in part because the DNA motif bound by CviR is not well characterized. Here, we define the DNA sequence required for promoter recognition by CviR. Using
in vivo
data generated from a library of point mutations in a CviR-regulated promoter, we find that CviR binds to a palindrome with the ideal sequence CTGNCCNNNNGGNCAG. We constructed a position weight matrix using these
in vivo
data and scanned the
C. violaceum
genome to predict CviR binding sites. We measured direct activation of the identified promoters by CviR and found that CviR controls the expression of the promoter for a chitinase, a type VI secretion-related gene, a transcriptional regulator gene, a guanine deaminase gene, and
cviI
. Indeed, regulation of
cviI
expression by CviR generates a canonical quorum-sensing positive-feedback loop.
Complete Genome Sequence of Melissococcus plutonius ATCC 35311
Authors: Okumura Kayo , Arai Rie , Okura Masatoshi , Kirikae Teruo , Takamatsu Daisuke , Osaki Makoto , Miyoshi-Akiyama Tohru
Published: May 2011

We report the first completely annotated genome sequence of
Melissococcus plutonius
ATCC 35311.
M. plutonius
is a one-genus, one-species bacterium and the etiological agent of European foulbrood of the honeybee. The genome sequence will provide new insights into the molecular mechanisms underlying its pathogenicity.