bluuebunny/crossref_metadata_2025 huggingface dataset:
SDG714, a Histone H3K9 Methyltransferase, Is Involved in Tos17 DNA Methylation and Transposition in Rice
Authors: Ding Yong , Wang Xia , Su Lei , Zhai JiXian , Cao ShouYun , Zhang DongFen , Liu ChunYan , Bi YuPing , Qian Qian , Cheng ZhuKuan , Chu ChengCai , Cao XiaoFeng
Published: January 2007

Although the role of H3K9 methylation in rice (Oryza sativa) is unclear, in Arabidopsis thaliana the loss of histone H3K9 methylation by mutation of Kryptonite [also known as SU(VAR)3-9 homolog] reduces genome-wide DNA methylation and increases the transcription of transposable elements. Here, we report that rice SDG714 (for SET Domain Group Protein714) encodes a histone H3K9-specific methyltransferase. The C terminus of SDG714 confers enzymatic activity and substrate specificity, whereas the N terminus localizes it in the nucleus. Loss-of-function mutants of SDG714 (SDG714IR transformants) generated by RNA interference display a mostly glabrous phenotype as a result of the lack of macro trichomes in glumes, leaves, and culms compared with control plants. These mutants also show decreased levels of CpG and CNG cytosine methylation as well as H3K9 methylation at the Tos17 locus, a copia-like retrotransposon widely used for the generation of rice mutants. Most interestingly, loss of function of SDG714 can enhance transcription and cause the transposition of Tos17. Together, these results suggest that histone H3K9 methylation mediated by SDG714 is involved in DNA methylation, the transposition of transposable elements, and genome stability in rice.
Stomatal Development and Patterning Are Regulated by Environmentally Responsive Mitogen-Activated Protein Kinases inArabidopsis
Authors: Wang Huachun , Ngwenyama Njabulo , Liu Yidong , Walker John C. , Zhang Shuqun
Published: January 2007

Stomata are specialized epidermal structures that regulate gas (CO2 and O2) and water vapor exchange between plants and their environment. In Arabidopsis thaliana, stomatal development is preceded by asymmetric cell divisions, and stomatal distribution follows the one-cell spacing rule, reflecting the coordination of cell fate specification. Stomatal development and patterning are regulated by both genetic and environmental signals. Here, we report that Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6, two environmentally responsive mitogen-activated protein kinases (MAPKs), and their upstream MAPK kinases, MKK4 and MKK5, are key regulators of stomatal development and patterning. Loss of function of MKK4/MKK5 or MPK3/MPK6 disrupts the coordinated cell fate specification of stomata versus pavement cells, resulting in the formation of clustered stomata. Conversely, activation of MKK4/MKK5-MPK3/MPK6 causes the suppression of asymmetric cell divisions and stomatal cell fate specification, resulting in a lack of stomatal differentiation. We further establish that the MKK4/MKK5-MPK3/MPK6 module is downstream of YODA, a MAPKKK. The establishment of a complete MAPK signaling cascade as a key regulator of stomatal development and patterning advances our understanding of the regulatory mechanisms of intercellular signaling events that coordinate cell fate specification during stomatal development.
Characterization of
Campylobacter jejuni
Biofilms under Defined Growth Conditions
Authors: Reeser Ryan J. , Medler Robert T. , Billington Stephen J. , Jost B. Helen , Joens Lynn A.
Published: January 2007

Campylobacter jejuni
is a major cause of human diarrheal disease in many industrialized countries and is a source of public health and economic burden.
C. jejuni
, present as normal flora in the intestinal tract of commercial broiler chickens and other livestock, is probably the main source of human infections. The presence of
C. jejuni
in biofilms found in animal production watering systems may play a role in the colonization of these animals. We have determined that
C. jejuni
can form biofilms on a variety of abiotic surfaces commonly used in watering systems, such as acrylonitrile butadiene styrene and polyvinyl chloride plastics. Furthermore,
C. jejuni
biofilm formation was inhibited by growth in nutrient-rich media or high osmolarity, and thermophilic and microaerophilic conditions enhanced biofilm formation. Thus, nutritional and environmental conditions affect the formation of
C. jejuni
biofilms. Both flagella and quorum sensing appear to be required for maximal biofilm formation, as
C. jejuni flaAB
and
luxS
mutants were significantly reduced in their ability to form biofilms compared to the wild-type strain.
Inactivation of Influenza A Virus on Copper versus Stainless Steel Surfaces
Authors: Noyce J. O. , Michels H. , Keevil C. W.
Published: January 2007

Influenza A virus particles (2 × 10
6
) were inoculated onto copper or stainless steel and incubated at 22°C at 50 to 60% relative humidity. Infectivity of survivors was determined by utilizing a defined monolayer with fluorescent microscopy analysis. After incubation for 24 h on stainless steel, 500,000 virus particles were still infectious. After incubation for 6 h on copper, only 500 particles were active.
Characterization of the Population of the Sulfur-Oxidizing Symbiont ofCodakia orbicularis(Bivalvia, Lucinidae) by Single-Cell Analyses
Authors: Caro Audrey , Gros Olivier , Got Patrice , De Wit Rutger , Troussellier Marc
Published: January 2007

We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills ofCodakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 μm in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes ofCodakiarevealed that around 80% of the symbionts hosted byCodakiamaintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host.
Monitoring Bacterial Communities in Raw Milk and Cheese by Culture-Dependent and -Independent 16S rRNA Gene-Based Analyses
Authors: Delbès Céline , Ali-Mandjee Leila , Montel Marie-Christine
Published: January 2007

The diversity and dynamics of bacterial populations in Saint-Nectaire, a raw-milk, semihard cheese, were investigated using a dual culture-dependent and direct molecular approach combining single-strand conformation polymorphism (SSCP) fingerprinting and sequencing of 16S rRNA genes. The dominant clones, among 125 16S rRNA genes isolated from milk, belonged to members of the
Firmicutes
(58% of the total clones) affiliated mainly with the orders
Clostridiales
and the
Lactobacillales
, followed by the phyla
Proteobacteria
(21.6%),
Actinobacteria
(16.8%), and
Bacteroidetes
(4%). Sequencing the 16S rRNA genes of 126 milk isolates collected from four culture media revealed the presence of 36 different species showing a wider diversity in the
Gammaproteobacteria
phylum and
Staphylococcus
genus than that found among clones. In cheese, a total of 21 species were obtained from 170 isolates, with dominant species belonging to the
Lactobacillales
and subdominant species affiliated with the
Actinobacteria
,
Bacteroidetes
(
Chryseobacterium
sp.), or
Gammaproteobacteria
(
Stenotrophomonas
sp.). Fingerprinting DNA isolated from milk by SSCP analysis yielded complex patterns, whereas analyzing DNA isolated from cheese resulted in patterns composed of a single peak which corresponded to that of lactic acid bacteria. SSCP fingerprinting of mixtures of all colonies harvested from plate count agar supplemented with crystal violet and vancomycin showed good potential for monitoring the subdominant
Proteobacteria
and
Bacteroidetes
(
Flavobacteria
) organisms in milk and cheese. Likewise, analyzing culturable subcommunities from cheese-ripening bacterial medium permitted assessment of the diversity of halotolerant
Actinobacteria
and
Staphylococcus
organisms. Direct and culture-dependent approaches produced complementary information, thus generating a more accurate view of milk and cheese microbial ecology.
Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable
Mycobacterium paratuberculosis
Bacteria within 48 Hours
Authors: Stanley Emma C. , Mole Richard J. , Smith Rebecca J. , Glenn Sarah M. , Barer Michael R. , McGowan Michael , Rees Catherine E. D.
Published: January 2007

The
FASTPlaque
TB assay is an established diagnostic aid for the rapid detection of
Mycobacterium tuberculosis
from human sputum samples. Using the
FASTPlaque
TB assay reagents, viable
Mycobacterium avium
subsp.
paratuberculosis
cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect
M. avium
subsp.
paratuberculosis
alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify
M. avium
subsp.
paratuberculosis
-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either
M. avium
subsp.
paratuberculosis
or
M. tuberculosis
complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable
M. avium
subsp.
paratuberculosis
in milk samples in just 48 h.
Piezotolerant Small-Colony Variants with Increased Thermotolerance, Antibiotic Susceptibility, and Low Invasiveness in a Clonal
Staphylococcus aureus
Population
Authors: Karatzas Kimon A. G. , Zervos Angelos , Tassou Chrysoula C. , Mallidis Costas G. , Humphrey Tom J.
Published: January 2007

Following a pressure treatment of a clonal
Staphylococcus aureus
culture with 400 MPa for 30 min, piezotolerant variants were isolated. Among 21 randomly selected survivors, 9 were piezotolerant and all formed small colonies on several agar media. The majority of the isolates showed increased thermotolerance, impaired growth, and reduced antibiotic resistance compared to the wild type. However, several nonpiezotolerant isolates also demonstrated impaired growth and the small-colony phenotype. In agglutination tests for the detection of protein A and fibrinogen, the piezotolerant variants showed weaker agglutination reactions than the wild type and the other isolates. All variants also showed defective production of the typical
S. aureus
golden color, a characteristic which has previously been linked with virulence. They were also less able to invade intestinal epithelial cells than the wild type. These
S. aureus
variants showed phenotypic similarities to previously isolated
Listeria monocytogenes
piezotolerant mutants that contained mutations in
ctsR
. Because of these similarities, possible alterations in the
ctsR
hypermutable regions of the
S. aureus
variants were investigated through amplified fragment length polymorphism analysis. No mutations were identified, and subsequently we sequenced the
ctsR
and
hrcA
genes of three representative variants, finding no mutations. This work demonstrates that
S. aureus
probably possesses a strategy resulting in an abundance of multiple-stress-resistant variants within clonal populations. This strategy, however, seems to involve genes and regulatory mechanisms different from those previously reported for
L. monocytogenes
. We are in the process of identifying these mechanisms.
Campylobacter insulaenigrae
Isolates from Northern Elephant Seals (
Mirounga angustirostris
) in California
Authors: Stoddard Robyn A. , Miller William G. , Foley Janet E. , Lawrence Judy , Gulland Frances M. D. , Conrad Patricia A. , Byrne Barbara A.
Published: January 2007

There are only two reports in the literature demonstrating the presence of
Campylobacter
spp. in marine mammals. One report describes the isolation of a new species,
Campylobacter insulaenigrae
sp. nov., from three harbor seals (
Phoca vitulina
) and a harbor porpoise (
Phocoena phocoena
) in Scotland, and the other describes the isolation of
Campylobacter jejuni
,
Campylobacter lari
, and an unknown
Campylobacter
species from northern elephant seals (
Mirounga angustirostris
) in California. In this study, 72 presumptive
C. lari
and unknown
Campylobacter
species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were
C. insulaenigrae
, with one isolate being similar to but distinct from both
Campylobacter upsaliensis
and
Campylobacter helveticus
. Phylogenetic analysis identified two
C. insulaenigrae
clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all
Campylobacter
species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of
Campylobacter
isolates from marine mammals.
Factors Affecting Survival of Bacteriophage on Tomato Leaf Surfaces
Authors: Iriarte F. B. , Balogh B. , Momol M. T. , Smith L. M. , Wilson M. , Jones J. B.
Published: January 2007

The ability of bacteriophage to persist in the phyllosphere for extended periods is limited by many factors, including sunlight irradiation, especially in the UV zone, temperature, desiccation, and exposure to copper bactericides. The effects of these factors on persistence of phage and formulated phage (phage mixed with skim milk) were evaluated. In field studies, copper caused significant phage reduction if applied on the day of phage application but not if applied 4 or 7 days in advance. Sunlight UV was evaluated for detrimental effects on phage survival on tomato foliage in the field. Phage was applied in the early morning, midmorning, early afternoon, and late evening, while UVA plus UVB irradiation and phage populations were monitored. The intensity of UV irradiation positively correlated with phage population decline. The protective formulation reduced the UV effect. In order to demonstrate direct effects of UV, phage suspensions were exposed to UV irradiation and assayed for effectiveness against bacterial spot of tomato. UV significantly reduced phage ability to control bacterial spot. Ambient temperature had a pronounced effect on nonformulated phage but not on formulated phages. The effects of desiccation and fluorescent light illumination on phage were investigated. Desiccation caused a significant but only slight reduction in phage populations after 60 days, whereas fluorescent light eliminated phages within 2 weeks. The protective formulation eliminated the reduction caused by both of these factors. Phage persistence was dramatically affected by UV, while the other factors had less pronounced effects. Formulated phage reduced deleterious effects of the studied environmental factors.
Cloning and Characterization of a Novel β-Transaminase fromMesorhizobiumsp. Strain LUK: a New Biocatalyst for the Synthesis of Enantiomerically Pure β-Amino Acids
Authors: Kim Juhan , Kyung Dohyun , Yun Hyungdon , Cho Byung-Kwan , Seo Joo-Hyun , Cha Minho , Kim Byung-Gee
Published: January 2007

A novel β-transaminase gene was cloned fromMesorhizobiumsp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.5-kb gene fragment was obtained by colony hybridization of a cosmid library. Through Southern blotting and sequence analysis of the selected cosmid clone, the structural gene of the enzyme (1,335 bp) was identified, which encodes a protein of 47,244 Da with a theoretical pI of 6.2. The deduced amino acid sequence of the β-transaminase showed the highest sequence similarity with glutamate-1-semialdehyde aminomutase of transaminase subgroup II. The β-transaminase showed higher activities towardd-β-aminocarboxylic acids such as 3-aminobutyric acid, 3-amino-5-methylhexanoic acid, and 3-amino-3-phenylpropionic acid. The β-transaminase has an unusually broad specificity for amino acceptors such as pyruvate and α-ketoglutarate/oxaloacetate. The enantioselectivity of the enzyme suggested that the recognition mode of β-aminocarboxylic acids in the active site is reversed relative to that of α-amino acids. After comparison of its primary structure with transaminase subgroup II enzymes, it was proposed that R43 interacts with the carboxylate group of the β-aminocarboxylic acids and the carboxylate group on the side chain of dicarboxylic α-keto acids such as α-ketoglutarate and oxaloacetate. R404 is another conserved residue, which interacts with the α-carboxylate group of the α-amino acids and α-keto acids. The β-transaminase was used for the asymmetric synthesis of enantiomerically pure β-aminocarboxylic acids. (3S)-Amino-3-phenylpropionic acid was produced from the ketocarboxylic acid ester substrate by coupled reaction with a lipase using 3-aminobutyric acid as amino donor.
Diagnostic Real-Time PCR Assays for the Detection of Emetic
Bacillus cereus
Strains in Foods and Recent Food-Borne Outbreaks
Authors: Fricker Martina , Messelhäußer Ute , Busch Ulrich , Scherer Siegfried , Ehling-Schulz Monika
Published: January 2007

Cereulide-producing
Bacillus cereus
can cause an emetic type of food-borne disease that mimics the symptoms provoked by
Staphylococcus aureus
. Based on the recently discovered genetic background for cereulide formation, a novel 5′ nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic
B. cereus
in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens
B. cereus
and
S. aureus
. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic
B. cereus
and 14
S. aureus
strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic
B. cereus
by using the real-time PCR assay.
Zooxanthellae Harvested by Ciliates Associated with Brown Band Syndrome of Corals Remain Photosynthetically Competent
Authors: Ulstrup Karin E. , Kühl Michael , Bourne David G.
Published: January 2007

Brown band syndrome is a new coral affliction characterized by a local accumulation of yet-unidentified ciliates migrating as a band along the branches of coral colonies. In the current study, morphologically intact zooxanthellae (=
Symbiodinium
) were observed in great numbers inside the ciliates (>50 dinoflagellates per ciliate). Microscale oxygen measurements and variable chlorophyll
a
fluorescence analysis along with microscopic observations demonstrated that zooxanthellae within the ciliates are photosynthetically competent and do not become compromised during the progression of the brown band zone. Zooxanthellae showed similar trends in light acclimation in a comparison of rapid light curve and steady-state light curve measures of variable chlorophyll
a
fluorescence. Extended light exposure of steady-state light curves resulted in higher quantum yields of photosystem II. The brown band tissue exhibited higher photosynthetically active radiation absorptivity, indicating more efficient light absorption due to a higher density of zooxanthellae in the ciliate-dominated zone. This caused relatively higher gross photosynthesis rates in the zone with zooxanthella-containing ciliates compared to healthy coral tissue. The observation of photosynthetically active intracellular zooxanthellae in the ciliates suggests that the latter can benefit from photosynthates produced by ingested zooxanthellae and from photosynthetic oxygen production that alleviates diffusion limitation of oxic respiration in the densely populated brown band tissue. It remains to be shown whether the zooxanthellae form a stable symbiotic association with the ciliate or are engulfed incidentally during grazing on coral tissue and then maintained as active inside the ciliate for a period before being digested and replaced by new zooxanthellae.
Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry
Authors: Monincová Marta , Prokop Zbyněk , Vévodová Jitka , Nagata Yuji , Damborský Jiří
Published: January 2007

1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from
Sphingobium japonicum
UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (
k
cat
= 0.005 s
−1
) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.
Construction of an
Escherichia coli
K-12 Mutant for Homoethanologenic Fermentation of Glucose or Xylose without Foreign Genes
Authors: Kim Youngnyun , Ingram L. O. , Shanmugam K. T.
Published: January 2007

Conversion of lignocellulosic feedstocks to ethanol requires microorganisms that effectively ferment both hexose and pentose sugars. Towards this goal, recombinant organisms have been developed in which heterologous genes were added to platform organisms such as
Saccharomyces cerevisiae
,
Zymomonas mobilis
, and
Escherichia coli
. Using a novel approach that relies only on native enzymes, we have developed a homoethanologenic alternative,
Escherichia coli
strain SE2378. This mutant ferments glucose or xylose to ethanol with a yield of 82% under anaerobic conditions. An essential mutation in this mutant was mapped within the
pdh
operon (
pdhR aceEF lpd
), which encodes components of the pyruvate dehydrogenase complex. Anaerobic ethanol production by this mutant is apparently the result of a novel pathway that combines the activities of pyruvate dehydrogenase (typically active during aerobic, oxidative metabolism) with the fermentative alcohol dehydrogenase.
Antisense-RNA-Mediated Decreased Synthesis of Small, Acid-Soluble Spore Proteins Leads to Decreased Resistance of
Clostridium perfringens
Spores to Moist Heat and UV Radiation
Authors: Raju Deepa , Setlow Peter , Sarker Mahfuzur R.
Published: January 2007

Previous work has suggested that a group of α/β-type small, acid-soluble spore proteins (SASP) is involved in the resistance of
Clostridium perfringens
spores to moist heat. However, this suggestion is based on the analysis of
C. perfringens
spores lacking only one of the three genes encoding α/β-type SASP in this organism. We have now used antisense RNA to decrease levels of α/β-type SASP in
C. perfringens
spores by ∼90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of α/β-type SASP in the resistance of
C. perfringens
spores.
The Iron-Sulfur Cluster Proteins Isa1 and Isa2 Are Required for the Function but Not for the De Novo Synthesis of the Fe/S Clusters of Biotin Synthase in
Saccharomyces cerevisiae
Authors: Mühlenhoff Ulrich , Gerl Mathias J. , Flauger Birgit , Pirner Heike M. , Balser Sandra , Richhardt Nadine , Lill Roland , Stolz Jürgen
Published: January 2007

The yeast
Saccharomyces cerevisiae
is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both
isa
mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of
isa
mutants to utilize desthiobiotin, since this defect was not cured by overexpression of
BIO2
. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.
Novel Method for Preparing Spheroplasts from Cells with an Internal Cellulosic Cell Wall
Authors: Kwok Alvin C. M. , Mak Carmen C. M. , Wong Francis T. W. , Wong Joseph T. Y.
Published: January 2007

Protoplast and spheroplast preparations allow the transfer of macromolecules into cells and provide the basis for the generation of engineered organisms.Crypthecodinium cohniicells harvested from polyethylene glycol-containing agar plates possessed significantly lower levels of cellulose in their cortical layers, which facilitated the delivery of fluorescence-labeled oligonucleotides into these cells.
In Vivo and In Vitro Anaerobic Mating in
Candida albicans
Authors: Dumitru Raluca , Navarathna Dhammika H. M. L. P. , Semighini Camile P. , Elowsky Christian G. , Dumitru Razvan V. , Dignard Daniel , Whiteway Malcolm , Atkin Audrey L. , Nickerson Kenneth W.
Published: January 2007

Candida albicans
cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37°C, block production of farnesol, and permit in vitro mating at 37°C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for
C. albicans
mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous
hisG
marker from one of the parental strains.
Following Temperature Stress, Export of Heat Shock mRNA Occurs Efficiently in Cells with Mutations in Genes Normally Important for mRNA Export
Authors: Rollenhagen Christiane , Hodge Christine A. , Cole Charles N.
Published: January 2007

Heat shock leads to accumulation of polyadenylated RNA in nuclei of
Saccharomyces cerevisiae
cells, transcriptional induction of heat shock genes, and efficient export of polyadenylated heat shock mRNAs. These studies were conducted to examine the requirements for export of mRNA following heat shock. We used in situ hybridization to detect
SSA4
mRNA (encoding Hsp70) and flow cytometry to measure the amount of Ssa4p-green fluorescent protein (GFP) produced following heat shock. Npl3p and Yra1p are mRNA-binding proteins recruited to nascent mRNAs and are essential for proper mRNA biogenesis and export. Heat shock mRNA was exported efficiently in temperature-sensitive
npl3
,
yra1
, and
npl3 yra1
mutant strains. Nevertheless, Yra1p was recruited to heat shock mRNA, as were Nab2p and Npl3p. Interestingly, Yra1p was not recruited to heat shock mRNA in
yra1
-
1
cells, suggesting that Npl3p is required for recruitment of Yra1p. The THO complex, which functions in transcription elongation and in recruitment of Yra1p, was not required for heat shock mRNA export, although normal mRNA export is impaired in growing cells lacking THO complex proteins. Taken together, these studies indicate that export following heat shock depends upon fewer factors than does mRNA export in growing cells. Furthermore, even though some mRNA-binding proteins are dispensable for efficient export of heat shock mRNA, those that are present in nuclei of heat shocked cells were recruited to heat shock mRNA.
Copine A Is Required for Cytokinesis, Contractile Vacuole Function, and Development inDictyostelium
Authors: Damer Cynthia K. , Bayeva Marina , Kim Pamela S. , Ho Lilian K. , Eberhardt Eric S. , Socec Catherine I. , Lee Jennifer S. , Bruce Emily A. , Goldman-Yassen Adam E. , Naliboff Lauren C.
Published: January 2007

Copines make up a family of soluble, calcium-dependent, membrane binding proteins found in a variety of eukaryotic organisms. In an earlier study, we identified six copine genes in theDictyostelium discoideumgenome and focused our studies oncpnA. Our previous localization studies of green fluorescent protein-tagged CpnA inDictyosteliumsuggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created acpnA−knockout strain, and here we report the initial characterization of the mutant phenotype. ThecpnA−cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions,cpnA−cells appeared to aggregate into mounds and form fingers with normal timing; however, they were delayed or arrested in the finger stage. When placed in water,cpnA−cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for thecpnA−cells were similar to those seen for wild-type cells. These studies indicate that CpnA plays a role in cytokinesis and contractile vacuole function and is required for normal development, specifically in the later stages prior to culmination. We also used real-time reverse transcription-PCR to determine the expression patterns of all six copine genes during development. The six copine genes were expressed in vegetative cells, with each gene exhibiting a distinct pattern of expression throughout development. All of the copine genes exceptcpnFshowed an upregulation of mRNA expression at one or two developmental transitions, suggesting that copines may be important regulators ofDictyosteliumdevelopment.
Cellular Processes and Pathways That Protect
Saccharomyces cerevisiae
Cells against the Plasma Membrane-Perturbing Compound Chitosan
Authors: Zakrzewska Anna , Boorsma Andre , Delneri Daniela , Brul Stanley , Oliver Stephen G. , Klis Frans M.
Published: January 2007

Global fitness analysis makes use of a genomic library of tagged deletion strains. We used this approach to study the effect of chitosan, which causes plasma membrane stress. The data were analyzed using T-profiler, which was based on determining the sensitivities of groups of deletion strains to chitosan, as defined by Gene Ontology (GO) and by genomic synthetic lethality screens, in combination with
t
statistics. The chitosan-hypersensitive groups included a group of deletion strains characterized by a defective HOG (high-osmolarity glycerol) signaling pathway, indicating that the HOG pathway is required for counteracting chitosan-induced stress. Consistent with this, activation of this pathway in wild-type cells by hypertonic conditions offered partial protection against chitosan, whereas hypotonic conditions sensitized the cells to chitosan. Other chitosan-hypersensitive groups were defective in RNA synthesis and processing, actin cytoskeleton organization, protein N-glycosylation, ergosterol synthesis, endocytosis, or cell wall formation, predicting that these cellular functions buffer the cell against the deleterious effect of chitosan. These predictions were supported by showing that tunicamycin, miconazole, and staurosporine (which target protein N-glycosylation, ergosterol synthesis, and the cell wall integrity pathway, respectively) sensitized
Saccharomyces cerevisiae
cells to chitosan. Intriguingly, the GO-defined group of deletion strains belonging to the “cytosolic large ribosomal subunit” was more resistant to chitosan. We propose that global fitness analysis of yeast in combination with T-profiler is a powerful tool to identify specific cellular processes and pathways that are required for survival under stress conditions.
Spliced Leader RNA Gene Transcription in
Trypanosoma brucei
Requires Transcription Factor TFIIH
Authors: Lee Ju Huck , Nguyen Tu N. , Schimanski Bernd , Günzl Arthur
Published: January 2007

Trypanosomatid parasites share a gene expression mode which differs greatly from that of their human and insect hosts. In these unicellular eukaryotes, protein-coding genes are transcribed polycistronically and individual mRNAs are processed from precursors by spliced leader (SL)
trans
splicing and polyadenylation. In
trans
splicing, the SL RNA is consumed through a transfer of its 5′-terminal part to the 5′ end of mRNAs. Since all mRNAs are
trans
spliced, the parasites depend on strong and continuous SL RNA synthesis mediated by RNA polymerase II. As essential factors for SL RNA gene transcription in
Trypanosoma brucei
, the general transcription factor (GTF) IIB and a complex, consisting of the TATA-binding protein-related protein 4, the small nuclear RNA-activating protein complex, and TFIIA, were recently identified. Although
T. brucei
TFIIA and TFIIB are extremely divergent to their counterparts in other eukaryotes, their characterization suggested that trypanosomatids do form a class II transcription preinitiation complex at the SL RNA gene promoter and harbor orthologues of other known GTFs. TFIIH is a GTF which functions in transcription initiation, DNA repair, and cell cycle control. Here, we investigated whether a
T. brucei
TFIIH is important for SL RNA gene transcription and found that silencing the expression of the highly conserved TFIIH subunit XPD in
T. brucei
affected SL RNA gene synthesis in vivo, and depletion of this protein from extract abolished SL RNA gene transcription in vitro. Since we also identified orthologues of the TFIIH subunits XPB, p52/TFB2, and p44/SSL1 copurifying with TbXPD, we concluded that the parasite harbors a TFIIH which is indispensable for SL RNA gene transcription.
Enhanced Post-Stationary-Phase Survival of a Clinical Thymidine-Dependent Small-Colony Variant of
Staphylococcus aureus
Results from Lack of a Functional Tricarboxylic Acid Cycle
Authors: Chatterjee Indranil , Herrmann Mathias , Proctor Richard A. , Peters Georg , Kahl Barbara C.
Published: January 2007

The mechanisms underlying the persistence of the
Staphylococcus aureus
small-colony variant (SCV) are not fully elucidated. In this study, clinical thymidine-dependent SCVs displayed altered expression of
citB
,
clpC
, and
arcA
genes, reduced acetate catabolization, and enhanced survival. These results implicate the importance of changes in tricarboxylic acid cycle and acetic acid metabolism in SCV survival and persistence.
Products of theEscherichia coliAcid Fitness Island Attenuate Metabolite Stress at Extremely Low pH and Mediate a Cell Density-Dependent Acid Resistance
Authors: Mates Aaron K. , Sayed Atef K. , Foster John W.
Published: January 2007

Escherichia colihas an ability, rare among theEnterobacteriaceae, to survive extreme acid stress under various host (e.g., human stomach) and nonhost (e.g., apple cider) conditions. Previous microarray studies have exposed a cluster of 12 genes at 79 centisomes collectively called an acid fitness island (AFI). Four AFI genes,gadA,gadX,gadW, andgadE, were already known to be involved in an acid resistance system that consumes an intracellular proton through the decarboxylation of glutamic acid. However, roles for the other eight AFI gene products were either unknown or subject to conflicting findings. Two new aspects of acid resistance are described that require participation of five of the remaining eight AFI genes. YhiF (a putative regulatory protein), lipoprotein Slp, and the periplasmic chaperone HdeA protectedE. colifrom organic acid metabolites produced during fermentation once the external pH was reduced to pH 2.5. HdeA appears to handle protein damage caused when protonated organic acids diffuse into the cell and dissociate, thereby decreasing internal pH. In contrast, YhiF- and Slp-dependent systems appear to counter the effects of the organic acids themselves, specifically succinate, lactate, and formate, but not acetate. A second phenomenon was defined by two other AFI genes,yhiDandhdeD, encoding putative membrane proteins. These proteins participate in an acid resistance mechanism exhibited only at high cell densities (>108CFU per ml). Density-dependent acid resistance does not require any demonstrable secreted factor and may involve cell contact-dependent activation. These findings further define the complex physiology ofE. coliacid resistance.
Mutational Analysis of the
Escherichia coli
DEAD Box Protein CsdA
Authors: Turner Anne-Marie W. , Love Cheraton F. , Alexander Rebecca W. , Jones Pamela G.
Published: January 2007

The
Escherichia coli
cold shock protein CsdA is a member of the DEAD box family of ATP-dependent RNA helicases, which share a core of nine conserved motifs. The DEAD (Asp-Glu-Ala-Asp) motif for which this family is named has been demonstrated to be essential for ATP hydrolysis. We show here that CsdA exhibits in vitro ATPase and helicase activities in the presence of short RNA duplexes with either 3′ or 5′ extensions at 15°C. In contrast to wild-type CsdA, a DQAD variant of CsdA (Glu-157→Gln) had no detectible helicase or ATPase activity at 15°C in vitro. A plasmid encoding the DQAD variant was also unable to suppress the impaired growth of the
csdA
null mutant at 15°C. Plasmid-encoded CsdAΔ444, which lacks most of the carboxy-terminal extension, enhanced the growth of a
csdA
null mutant at 25°C but not at 15°C; this truncated protein also has limited in vitro activity at 15°C. These results support the physiological function of CsdA as a DEAD box ATP-dependent RNA helicase at low temperature.
Identification of the Initial Steps in
d
-Lysine Catabolism in
Pseudomonas putida
Authors: Revelles Olga , Wittich Rolf-Michael , Ramos Juan L.
Published: January 2007

Pseudomonas putida
uses
l
-lysine as the sole carbon and nitrogen source which preferentially requires its metabolism through two parallel pathways. In one of the pathways δ-aminovalerate is the key metabolite, whereas in the other
l
-lysine is racemized to
d
-lysine, and
l
-pipecolate and α-aminoadipate are the key metabolites. All the genes and enzymes involved in the
d
-lysine pathway, except for those involved in the conversion of
d
-lysine into Δ
1
-piperideine-2-carboxylate, have been identified previously (30). In this study we report that the conversion of
d
-lysine into Δ
1
-piperideine-2-carboxylate can be mediated by a
d
-lysine aminotransferase (PP3590) and a
d
-lysine dehydrogenase (PP3596). From a physiological point of view PP3596 plays a major role in the catabolism of
d
-lysine since its inactivation leads to a marked reduction in the growth rate with
l
- or
d
-lysine as the sole carbon and nitrogen source, whereas inactivation of PP3590 leads only to slowed growth. The gene encoding PP3590, called here
amaC
, forms an operon with
dpkA
, the gene encoding the enzyme involved in conversion of Δ
1
-piperideine-2-carboxylate to
l
-pipecolate in the
d
-lysine catabolic pathway. The gene encoding PP3596, called here
amaD
, is the fifth gene in an operon made up of seven open reading frames (ORFs) encoding PP3592 through PP3597. The
dpkA amaC
operon was transcribed divergently from the operon ORF3592 to ORF3597. Both promoters were mapped by primer extension analysis, which showed that the divergent −35 hexamers of these operon promoters were adjacent to each other. Transcription of both operons was induced in response to
l
- or
d
-lysine in the culture medium.
MalE of Group A
Streptococcus
Participates in the Rapid Transport of Maltotriose and Longer Maltodextrins
Authors: Shelburne Samuel A. , Fang Han , Okorafor Nnaja , Sumby Paul , Sitkiewicz Izabela , Keith David , Patel Payal , Austin Celest , Graviss Edward A. , Musser James M. , Chow Dar-Chone
Published: January 2007

Study of the maltose/maltodextrin binding protein MalE in
Escherichia coli
has resulted in fundamental insights into the molecular mechanisms of microbial transport. Whether gram-positive bacteria employ a similar pathway for maltodextrin transport is unclear. The maltodextrin binding protein MalE has previously been shown to be key to the ability of group A
Streptococcus
(GAS) to colonize the oropharynx, the major site of GAS infection in humans. Here we used a multifaceted approach to elucidate the function and binding characteristics of GAS MalE. We found that GAS MalE is a central part of a highly efficient maltodextrin transport system capable of transporting linear maltodextrins that are up to at least seven glucose molecules long. Of the carbohydrates tested, GAS MalE had the highest affinity for maltotriose, a major breakdown product of starch in the human oropharynx. The thermodynamics and fluorescence changes induced by GAS MalE-maltodextrin binding were essentially opposite those reported for
E. coli
MalE. Moreover, unlike
E. coli
MalE, GAS MalE exhibited no specific binding of maltose or cyclic maltodextrins. Our data show that GAS developed a transport system optimized for linear maltodextrins longer than two glucose molecules that has several key differences from its well-studied
E. coli
counterpart.
Phage 3396 from a
Streptococcus dysgalactiae
subsp.
equisimilis
Pathovar May Have Its Origins in
Streptococcus pyogenes
Authors: Davies Mark R. , McMillan David J. , Van Domselaar Gary H. , Jones Malcolm K. , Sriprakash Kadaba S.
Published: January 2007

Streptococcus dysgalactiae
subsp.
equisimilis
strains (group G streptococcus [GGS]) are largely defined as commensal organisms, which are closely related to the well-defined human pathogen, the group A streptococcus (GAS). While lateral gene transfers are emerging as a common theme in these species, little is known about the mechanisms and role of these transfers and their effect on the population structure of streptococci in nature. It is now becoming evident that bacteriophages are major contributors to the genotypic diversity of GAS and, consequently, are pivotal to the GAS strain structure. Furthermore, bacteriophages are strongly associated with altering the pathogenic potential of GAS. In contrast, little is know about phages from GGS and their role in the population dynamics of GGS. In this study we report the first complete genome sequence of a GGS phage, Φ3396. Exhibiting high homology to the GAS phage Φ315.1, the chimeric nature of Φ3396 is unraveled to reveal evidence of extensive ongoing genetic diversity and dissemination of streptococcal phages in nature. Furthermore, we expand on our recent findings to identify inducible Φ3396 homologues in GAS from a region of endemicity for GAS and GGS infection. Together, these findings provide new insights into not only the population structure of GGS but also the overall population structure of the streptococcal genus and the emergence of pathogenic variants.
Properties of
R
-Citramalyl-Coenzyme A Lyase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of
Chloroflexus aurantiacus
Authors: Friedmann Silke , Alber Birgit E. , Fuchs Georg
Published: January 2007

The autotrophic CO
2
fixation pathway (3-hydroxypropionate cycle) in
Chloroflexus aurantiacus
results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO
2
acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO
2
) to β-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both
S-
and
R-
citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the
S-
and
R-
citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth.
S-
Citramalyl-CoA lyase activity was found to be due to
l
-malyl-CoA lyase/β-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO
2
fixation pathway, forms acetyl-CoA and glyoxylate from
l
-malyl-CoA, and condenses glyoxylate with propionyl-CoA to β-methylmalyl-CoA.
R-
Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg
2
or Mn
2+
ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in
C. aurantiacus
. Genes similar to those involved in CO
2
fixation in
C. aurantiacus
, including an
R-
citramalyl-CoA lyase gene, were found in
Roseiflexus
sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the γ-proteobacterium
Congregibacter litoralis
. This may indicate that part of the reactions may be involved in a different metabolic process.