bluuebunny/crossref_metadata_2025_split huggingface dataset:
Diverse AvrPtoB Homologs from SeveralPseudomonas syringaePathovars Elicit Pto-Dependent Resistance and Have Similar Virulence Activities
Authors: Lin Nai-Chun , Abramovitch Robert B. , Kim Young Jin , Martin Gregory B. 
Published: January 2006

ABSTRACTAvrPtoB is a type III effector protein fromPseudomonas syringaepv. tomato that physically interacts with the tomato Pto kinase and, depending on the host genotype, either elicits or suppresses programmed cell death associated with plant immunity. We reported previously thatavrPtoB-related sequences are present in diverse gram-negative phytopathogenic bacteria. Here we describe characterization ofavrPtoBhomologs fromP. syringaepv. tomato T1, PT23, and JL1065,P. syringaepv. syringae B728a, andP. syringaepv. maculicola ES4326. TheavrPtoBhomolog fromP. syringaepv. maculicola,hopPmaL, was identified previously. The four new genes identified in this study are designatedavrPtoBT1,avrPtoBPT23,avrPtoBJL1065, andavrPtoBB728a. The AvrPtoB homologs exhibit 52 to 66% amino acid identity with AvrPtoB. Transcripts of each of theavrPtoBhomologs were detected in thePseudomonasstrains from which they were isolated. Proteins encoded by the homologs were detected in all strains exceptP. syringaepv. tomato T1, suggesting that T1 suppresses accumulation of AvrPtoBT1. All of the homologs interacted with the Pto kinase in a yeast two-hybrid system and elicited aPto-dependent defense response when they were delivered into leaf cells by DC3000ΔavrPtoΔavrPtoB, aP. syringaepv. tomato strain with a deletion of bothavrPtoandavrPtoB. Like AvrPtoB, all of the homologs enhanced the ability of DC3000ΔavrPtoΔavrPtoBto form lesions on leaves of two susceptible tomato lines. With the exception of HopPmaL which lacks the C-terminal domain, all AvrPtoB homologs suppressed programmed cell death elicited by the AvrPto-Pto interaction in anAgrobacterium-mediated transient assay. Thus, despite their divergent sequences, AvrPtoB homologs from diverseP. syringaepathovars have conserved avirulence and virulence activities similar to AvrPtoB activity.
Improvement of α-Amylase Production by Modulation of Ribosomal Component Protein S12 in
Bacillus subtilis
168
Authors: Kurosawa Kazuhiko , Hosaka Takeshi , Tamehiro Norimasa , Inaoka Takashi , Ochi Kozo 
Published: January 2006

ABSTRACT

The capacity of ribosomal modification to improve antibiotic production by
Streptomyces
spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (
str
) also enhances α-amylase (and protease) production by a strain of
Bacillus subtilis
as estimated by measuring the enzyme activity. The
str
mutations are point mutations within
rpsL
, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various
rpsL
mutations K56R (which corresponds to position 42 in
E. coli
) was particularly effective at enhancing α-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg
2+
concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in α-amylase production seen in the affected strain.
Recurring Seasonal Dynamics of Microbial Communities in Stream Habitats
Authors: Hullar Meredith A. J. , Kaplan Louis A. , Stahl David A. 
Published: January 2006

ABSTRACT

Recurring seasonal patterns of microbial distribution and abundance in three third-order temperate streams within the southeast Pennsylvania Piedmont were observed over 4 years. Populations associated with streambed sediments and rocks (epilithon) were identified using terminal restriction length polymorphism (tRFLP) and sequencing of 16S rRNA genes selectively amplified with primers for the bacterial domain. Analyses of the relative magnitudes of tRFLP peak areas by using nonmetric multidimensional scaling resolved clear seasonal trends in epilithic and sediment populations. Oscillations between two dominant groups of epilithic genotypes, explaining 86% of the seasonal variation in the data set, were correlated with temperature and dissolved organic carbon. Sequences affiliated with epilithic phototrophs (cyanobacteria and diatom chloroplasts), a
Rhodoferax
sp., and a
Bacillus
species clustered in the summer, whereas sequences most closely related to “
Betaproteobacteria
” (putative
Burkholderia
sp.), and a putative cyanobacterium clustered in the fall/spring. The sediment genotypes also clustered into two groups, and these explained 85% of seasonal variation but correlated only with temperature. A summer tRFLP pattern was characterized by prevalence of “
Betaproteobacteria
,” “
Gammaproteobacteria
,” and a
Bacillus
sp., whereas the winter/spring pattern was characterized by phylotypes most closely related to “
Firmicutes
,” “
Gammaproteobacteria
,” and “
Nitrospirae
.” A close association between these headwater streams and their watersheds was suggested by the recovery of sequences related to microbial populations provisionally attributed to not only freshwaters but also terrestrial habitats.
Prochlorococcus
Ecotype Abundances in the North Atlantic Ocean As Revealed by an Improved Quantitative PCR Method
Authors: Zinser Erik R. , Coe Allison , Johnson Zackary I. , Martiny Adam C. , Fuller Nicholas J. , Scanlan David J. , Chisholm Sallie W. 
Published: January 2006

ABSTRACT

The cyanobacterium
Prochlorococcus
numerically dominates the photosynthetic community in the tropical and subtropical regions of the world's oceans. Six evolutionary lineages of
Prochlorococcus
have been described, and their distinctive physiologies and genomes indicate that these lineages are “ecotypes” and should have different oceanic distributions. Two methods recently developed to quantify these ecotypes in the field, probe hybridization and quantitative PCR (QPCR), have shown that this is indeed the case. To facilitate a global investigation of these ecotypes, we modified our QPCR protocol to significantly increase its speed, sensitivity, and accessibility and validated the method in the western and eastern North Atlantic Ocean. We showed that all six ecotypes had distinct distributions that varied with depth and location, and, with the exception of the deeper waters at the western North Atlantic site, the total
Prochlorococcus
counts determined by QPCR matched the total counts measured by flow cytometry. Clone library analyses of the deeper western North Atlantic waters revealed ecotypes that are not represented in the culture collections with which the QPCR primers were designed, explaining this discrepancy. Finally, similar patterns of relative ecotype abundance were obtained in QPCR and probe hybridization analyses of the same field samples, which could allow comparisons between studies.
Making All Parts of the 16S rRNA of
Escherichia coli
Accessible In Situ to Single DNA Oligonucleotides
Authors: Yilmaz L. Şafak , Ökten Hatice E. , Noguera Daniel R. 
Published: January 2006

ABSTRACT

rRNA accessibility is a major sensitivity issue limiting the design of working probes for fluorescence in situ hybridization (FISH). Previous studies empirically highlighted the accessibility of target sites on rRNA maps by grouping probes into six classes according to their brightness levels. In this study, a recently proposed mechanistic model of FISH, based on the thermodynamics of secondary nucleic acid interactions, was used to evaluate the accessibility of the 16S rRNA of
Escherichia coli
to fluorescein-labeled oligonucleotides when thermodynamic and kinetic barriers were eliminated. To cover the entire 16S rRNA, 109 probes were designed with an average thermodynamic affinity (Δ
G
o
overall
) of −13.5 kcal/mol. Fluorescence intensity was measured by flow cytometry, and a brightness threshold between classes 3 and 4 was used as the requirement for proof of accessibility. While 46% of the probes were above this threshold with conventional 3-h hybridizations, extending the incubation period to 96 h dramatically increased the fraction of bright probes to 86%. Insufficient thermodynamic affinity and/or fluorophore quenching was demonstrated to cause the low fluorescence intensity of the remaining 14% of the probes. In the end, it was proven that every nucleotide in the 16S rRNA of
E. coli
could be targeted with a bright probe and, therefore, that there were no truly inaccessible target regions in the 16S rRNA. Based on our findings and mechanistic modeling, a rational design strategy involving Δ
G
o
overall
, hybridization kinetics, and fluorophore quenching is recommended for the development of bright probes.
Production of Human Papillomavirus Type 16 L1 Virus-Like Particles by RecombinantLactobacillus caseiCells
Authors: Aires Karina Araujo , Cianciarullo Aurora Marques , Carneiro Sylvia Mendes , Villa Luisa Lina , Boccardo Enrique , Pérez-Martinez Gaspar , Perez-Arellano Isabel , Oliveira Maria Leonor Sarno , Ho Paulo Lee 
Published: January 2006

ABSTRACTInfections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on theLactobacillus caseilactose operon promoter (plac) for expression of the HPV-16 L1 protein inL. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis ofL. caseiexpressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on theL. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized withL. caseiexpressing L1 reacted withSpodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs byLactobacillusopens the possibility for development of new live mucosal prophylactic vaccines.
Multicopy Integration and Expression of Heterologous Genes in
Methylobacterium extorquens
ATCC 55366
Authors: Choi Young J. , Bourque Denis , Morel Lyne , Groleau Denis , Míguez Carlos B. 
Published: January 2006

ABSTRACT

High-level expression of chromosomally integrated genes in
Methylobacterium extorquens
ATCC 55366 was achieved under the control of the strong
M. extorquens
AM1 methanol dehydrogenase promoter (P

mxaF

) using the mini-Tn
7
transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for
M. extorquens
was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (
gfp
) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn
7
integration locus in the chromosome of
M. extorquens
, known as the Tn
7
attachment site (
att
Tn
7
site), was identified. This single
att
Tn
7
site was identified in an intergenic region between
glmS
, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and
dhaT
, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the
att
Tn
7
site is a noncoding region of the chromosome make the mini-Tn
7
transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (
bgl
[β-galactosidase],
est
[esterase], and
gfp
[green fluorescent protein]) in
M. extorquens
.
Competitive Fitness of
Listeria monocytogenes
Serotype 1/2a and 4b Strains in Mixed Cultures with and without Food in the U.S. Food and Drug Administration Enrichment Protocol
Authors: Gorski Lisa , Flaherty Denise , Mandrell Robert E. 
Published: January 2006

ABSTRACT

Thirteen different serotypes of the food-borne pathogen
Listeria monocytogenes
have been described. Serotype 4b strains are most often associated with illness, and serotype 1/2a strains are most often isolated from foods and processing plants. Different abilities to respond to stresses have been described for serotype 4b and 1/2a strains. One of the common enrichment protocols used to test foods for the presence
L. monocytogenes
is described in the U.S. Food and Drug Administration (FDA)
Bacterial Analytical Manual
(BAM). We compared three strains of
L. monocytogenes
serotype 4b and five strains of serotype 1/2a in direct competition with each other in two-strain mixed cultures by using the FDA BAM enrichment protocol, which includes both enrichment broth and selective agar, with and without added food to mimic the conditions that occur during attempts to isolate
Listeria
species from contaminated foods. Using a colony immunoblot procedure and analyzing over 112,000 colonies, we observed differences in strain fitness, but these differences were not attributable to serotype or genetic lineage.
Direct and Indirect Effects of Protist Predation on Population Size Structure of a Bacterial Strain with High Phenotypic Plasticity
Authors: Corno Gianluca , Jürgens Klaus 
Published: January 2006

ABSTRACTWe studied the impact of grazing and substrate supply on the size structure of a freshwater bacterial strain (Flectobacillussp.) which showed pronounced morphological plasticity. The cell length varied from 2 to >40 μm and encompassed rods, curved cells, and long filaments. Without grazers and with a sufficient substrate supply, bacteria grew mainly in the form of medium-sized rods (4 to 7 μm), with a smaller proportion (<10%) of filamentous forms. Grazing experiments with the bacterivorous flagellateOchromonassp. showed that freely suspended cells of <7 μm were highly vulnerable to grazers, whereas filamentous cells were resistant to grazing and became enriched during predation. A comparison of long-term growth in carbon-limited chemostats with and without grazers revealed that strikingly different bacterial populations developed: treatments with flagellates were composed of >80% filamentous cells. These attained a biomass comparable to that of populations in chemostats without grazers, which were composed of medium-sized rods and c-shaped cells. Carbon starvation resulted in a fast decrease in cell length and a shift towards small rods, which were highly vulnerable to grazing. Dialysis bag experiments in combination with continuous cultivation revealed that filament formation was significantly enhanced even without direct contact of bacteria with bacterivores and was thus probably stimulated by grazer excretory products.
Modulation of Rat Cecal Microbiota by Administration of Raffinose and Encapsulated
Bifidobacterium breve
Authors: Dinoto Achmad , Suksomcheep Akarat , Ishizuka Satoshi , Kimura Hanae , Hanada Satoshi , Kamagata Yoichi , Asano Kozo , Tomita Fusao , Yokota Atsushi 
Published: January 2006

ABSTRACT

To investigate the effects of administration of raffinose and encapsulated
Bifidobacterium breve
JCM 1192
T
cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated
B. breve
(group CB), and basal diet supplemented with both raffinose and encapsulated
B. breve
(group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP).
B. breve
cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis.
B. breve
was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no
B. breve
signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of
Bifidobacterium animalis
, a
Bifidobacterium
indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.
Abundant and Diverse Fungal Microbiota in the Murine Intestine
Authors: Scupham Alexandra J , Presley Laura L. , Wei Bo , Bent Elizabeth , Griffith Natasha , McPherson Michael , Zhu Feilin , Oluwadara Oluwadayo , Rao Nagesh , Braun Jonathan , Borneman James 
Published: January 2006

ABSTRACTEnteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla:Ascomycota,Basidiomycota,Chytridiomycota, andZygomycota. The largest assemblages of fungal rRNA sequences were related to the generaAcremonium,Monilinia,Fusarium,Cryptococcus/Filobasidium,Scleroderma,Catenomyces,Spizellomyces,Neocallimastix,Powellomyces,Entophlyctis,Mortierella, andSmittiumand the orderMucorales. The majority of bacterial rRNA gene clones were affiliated with the taxaBacteroidetes,Firmicutes,Acinetobacter, andLactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.
New
rfp
- and pES213-Derived Tools for Analyzing Symbiotic
Vibrio fischeri
Reveal Patterns of Infection and
lux
Expression In Situ
Authors: Dunn Anne K. , Millikan Deborah S. , Adin Dawn M. , Bose Jeffrey L. , Stabb Eric V. 
Published: January 2006

ABSTRACT

Genetically altered or tagged
Vibrio fischeri
strains can be observed in association with their mutualistic host
Euprymna scolopes
, providing powerful experimental approaches for studying this symbiosis. Two limitations to such in situ analyses are the lack of suitably stable plasmids and the need for a fluorescent tag that can be used in tandem with green fluorescent protein (GFP). Vectors previously used in
V. fischeri
contain the p15A replication origin; however, we found that this replicon is not stable during growth in the host and is retained by fewer than 20% of symbionts within a day after infection. In contrast, derivatives of
V. fischeri
plasmid pES213 were retained by ∼99% of symbionts even 3 days after infection. We therefore constructed pES213-derived shuttle vectors with a variety of selectable and visual markers. To include a visual tag that can be used in conjunction with GFP, we compared seven variants of the DsRed2 red fluorescent protein (RFP): mRFP1, tdimer2(12), DsRed.T3, DsRed.T4, DsRed.M1, DsRed.T3_S4T, and DsRed.T3(DNT). The last variant was brightest, displaying >20-fold more fluorescence than DsRed2 in
V. fischeri
. RFP expression did not detectably affect the fitness of
V. fischeri
, and cells were readily visualized in combination with GFP-expressing cells in mixed infections. Interestingly, even when inocula were dense enough that most
E. scolopes
hatchlings were infected by two strains, there was little mixing of the strains in the light organ crypts. We also used constitutive RFP in combination with the
luxICDABEG
promoter driving expression of GFP to visualize the spatial and temporal induction of this bioluminescence operon during symbiotic infection. Our results demonstrate the utility of pES213-based vectors and RFP for in situ experimental approaches in studies of the
V. fischeri
-
E. scolopes
symbiosis.
The
Thermotoga maritima
Phenotype Is Impacted by Syntrophic Interaction with
Methanococcus jannaschii
in Hyperthermophilic Coculture
Authors: Johnson M. R. , Conners S. B. , Montero C. I. , Chou C. J. , Shockley K. R. , Kelly R. M. 
Published: January 2006

ABSTRACT

Significant growth phase-dependent differences were noted in the transcriptome of the hyperthermophilic bacterium
Thermotoga maritima
when it was cocultured with the hyperthermophilic archaeon
Methanococcus jannaschii
. For the mid-log-to-early-stationary-phase transition of a
T. maritima
monoculture, 24 genes (1.3% of the genome) were differentially expressed twofold or more. In contrast, methanogenic coculture gave rise to 292 genes differentially expressed in
T. maritima
at this level (15.5% of the genome) for the same growth phase transition. Interspecies H
2
transfer resulted in three- to fivefold-higher
T. maritima
cell densities than in the monoculture, with concomitant formation of exopolysaccharide (EPS)-based cell aggregates. Differential expression of specific sigma factors and genes related to the ppGpp-dependent stringent response suggests involvement in the transition into stationary phase and aggregate formation. Cell aggregation was growth phase dependent, such that it was most prominent during mid-log phase and decayed as cells entered stationary phase. The reduction in cell aggregation was coincidental with down-regulation of genes encoding EPS-forming glycosyltranferases and up-regulation of genes encoding β-specific glycosyl hydrolases; the latter were presumably involved in hydrolysis of β-linked EPS to release cells from aggregates. Detachment of aggregates may facilitate colonization of new locations in natural environments where
T. maritima
coexists with other organisms. Taken together, these results demonstrate that syntrophic interactions can impact the transcriptome of heterotrophs in methanogenic coculture, and this factor should be considered in examining the microbial ecology in anaerobic environments.