bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Characterization ofhsp20, Encoding a Small Heat Shock Protein ofBifidobacterium breveUCC2003
Authors: Ventura Marco , Canchaya Carlos , Zhang Ziding , Fitzgerald Gerald F. , van Sinderen Douwe 
Published: May 2007

ABSTRACTSmall heat shock proteins (sHSPs) are members of a diverse family of stress proteins that are important in cells to protect proteins under stressful conditions. Genome analysis ofBifidobacterium breveUCC2003 revealed a single sHSP-encoding gene, which was classified as ahsp20gene by comparative analyses. Genomic surveillance of available genome sequences indicated thathsp20homologs are not widely distributed in bacteria. In members of the genusBifidobacterium, this gene appears to be present in only 7 of the 30 currently described species. Moreover, phylogenetic analysis using all available bacterial and eukaryotic sHSP sequences revealed a close relationship between bifidobacterial HSP20 and the class B sHSPs found in members of the divisionFirmicutes. The results of this comparative analysis and variation in codon usage content suggest thathsp20was acquired by certain bifidobacteria through horizontal gene transfer. Analysis by slot blot, Northern blot, and primer extension experiments showed that transcription ofhsp20is strongly induced in response to severe heat shock regimens and by osmotic shock.
Postinoculation Protozoan Establishment and Association Patterns of Methanogenic Archaea in the Ovine Rumen
Authors: Ohene-Adjei Samuel , Teather Ronald M. , Ivan Michael , Forster Robert J. 
Published: May 2007

ABSTRACT

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa,
Isotricha
and
Dasytricha
spp.,
Entodinium
spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the
Entodinium
, and the type A inoculations clustered primarily with uncultured phylotypes.
Polyplastron multivesiculatum
was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (
Methanosphaera stadtmanae
,
Methanobrevibacter ruminantium
, and
Methanobacterium bryantii
). Furthermore, the
Isotricha
and
Dasytricha
inoculation treatment resulted primarily in archaeal clones affiliated with
Methanobrevibacter smithii
. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.
Luciferase Detection during Stationary Phase in
Lactococcus lactis
Authors: Bachmann Herwig , Santos Filipe , Kleerebezem Michiel , van Hylckama Vlieg Johan E. T. 
Published: May 2007

ABSTRACT

The luminescence signal of
luxAB
-encoded bacterial luciferase strongly depends on the metabolic state of the host cell, which restricts the use of this reporter system to metabolically active bacteria. Here we show that in stationary-phase cells of
Lactococcus lactis
, detection of luciferase is significantly improved by the addition of riboflavin or flavin mononucleotide to whole-cell assay systems.
Characterization of Three Classes of Membrane Proteins Involved in Fungal Azole Resistance by Functional Hyperexpression inSaccharomyces cerevisiae
Authors: Lamping Erwin , Monk Brian C. , Niimi Kyoko , Holmes Ann R. , Tsao Sarah , Tanabe Koichi , Niimi Masakazu , Uehara Yoshimasa , Cannon Richard D. 
Published: May 2007

ABSTRACTThe study of eukaryotic membrane proteins has been hampered by a paucity of systems that achieve consistent high-level functional protein expression. We report the use of a modified membrane protein hyperexpression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, the major facilitator superfamily transporter CaMdr1p, and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotypes of five pathogenic fungi and to express human P glycoprotein (HsAbcb1p). The hyperexpression system consists of a set of plasmids that direct the stable integration of a single copy of the expression cassette at the chromosomalPDR5locus of a modified hostSaccharomyces cerevisiaestrain, ADΔ. Overexpression of heterologous proteins at levels of up to 29% of plasma membrane protein was achieved. Membrane proteins were expressed with or without green fluorescent protein (GFP), monomeric red fluorescent protein, His, FLAG/His, Cys, or His/Cys tags. Most GFP-tagged proteins tested were correctly trafficked within the cell, and His-tagged proteins could be affinity purified. Kinetic analysis of ABC transporters indicated that the apparentKmvalue and theVmaxvalue of ATPase activities were not significantly affected by the addition of His tags. The efflux properties of seven fungal drug pumps were characterized by their substrate specificities and their unique patterns of inhibition by eight xenobiotics that chemosensitizedS. cerevisiaestrains overexpressing ABC drug pumps to fluconazole. The modified hyperexpression system has wide application for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening.
Use of RecombinantEntamoeba histolyticaCysteine Proteinase 1 To Identify a Potent Inhibitor of Amebic Invasion in a Human Colonic Model
Authors: Meléndez-López Samuel G. , Herdman Scott , Hirata Ken , Choi Min-Ho , Choe Youngchool , Craik Charles , Caffrey Conor R. , Hansell Elisabeth , Chávez-Munguía Bibiana , Chen Yen Ting , Roush William R. , McKerrow James , Eckmann Lars , Guo Jianhua , Stanley Samuel L. , Reed Sharon L. 
Published: May 2007

ABSTRACTCysteine proteinases are key virulence factors of the protozoan parasiteEntamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of theE. histolyticagenome project has revealed more than 40 genes encoding cysteine proteinases. We have focused onE. histolyticacysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasiveE. histolyticaand is highly expressed and released. Recombinant EhCP1 was expressed inEscherichia coliand refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.
Genomic and Population Analyses of the Mating Type Loci in
Coccidioides
Species Reveal Evidence for Sexual Reproduction and Gene Acquisition
Authors: Mandel M. Alejandra , Barker Bridget M. , Kroken Scott , Rounsley Steven D. , Orbach Marc J. 
Published: May 2007

ABSTRACT

Coccidioides
species, the fungi responsible for the valley fever disease, are known to reproduce asexually through the production of arthroconidia that are the infectious propagules. The possible role of sexual reproduction in the survival and dispersal of these pathogens is unexplored. To determine the potential for mating of
Coccidioides
, we analyzed genome sequences and identified mating type loci characteristic of heterothallic ascomycetes.
Coccidioides
strains contain either a
MAT1-1
or a
MAT1-2
idiomorph, which is 8.1 or 9 kb in length, respectively, the longest reported for any ascomycete species. These idiomorphs contain four or five genes, respectively, more than are present in the
MAT
loci of most ascomycetes. Along with their cDNA structures, we determined that all genes in the
MAT
loci are transcribed. Two genes frequently found in common sequences flanking
MAT
idiomorphs,
APN2
and
COX13
, are within the
MAT
loci in
Coccidioides
, but the
MAT1-1
and
MAT1-2
copies have diverged dramatically from each other. Data indicate that the acquisition of these genes in the
MAT
loci occurred prior to the separation of
Coccidioides
from
Uncinocarpus reesii
. An analysis of 436
Coccidioides
isolates from patients and the environment indicates that in both
Coccidioides immitis
and
C. posadasii
, there is a 1:1 distribution of
MAT
loci, as would be expected for sexually reproducing species. In addition, an analysis of isolates obtained from 11 soil samples demonstrated that at three sampling sites, strains of both mating types were present, indicating that compatible strains were in close proximity in the environment.
Type IV Fimbrial Biogenesis Is Required for Protease Secretion and Natural Transformation inDichelobacter nodosus
Authors: Han Xiaoyan , Kennan Ruth M. , Parker Dane , Davies John K. , Rood Julian I. 
Published: May 2007

ABSTRACTThe objective of this study was to develop an understanding of the molecular mechanisms by which type IV fimbrial biogenesis, natural transformation, and protease secretion are linked in the ovine foot rot pathogen,Dichelobacter nodosus. We have shown that like theD. nodosusfimbrial subunit FimA, the pilin-like protein PilE and the FimN, FimO, and FimP proteins, which are homologs of PilB, PilC, and PilD fromPseudomonas aeruginosa, are essential for fimbrial biogenesis and natural transformation, indicating that transformation requires an intact type IV fimbrial apparatus. The results also showed that extracellular protease secretion in thefimN,fimO,fimP, andpilEmutants was significantly reduced, which represents the first time that PilB, PilC, and PilE homologs have been shown to be required for the secretion of unrelated extracellular proteins in a type IV fimbriate bacterium. Quantitative real-time PCR analysis of the three extracellular protease genesaprV2,aprV5, andbprVshowed that the effects on protease secretion were not mediated at the transcriptional level. Bioinformatic analysis did not identify a classical type II secretion system, and the putative fimbrial biogenesis genepilQwas the only outer membrane secretin gene identified. Based on these results, it is postulated that inD. nodosus, protease secretion occurs by a type II secretion-related process that directly involves components of the type IV fimbrial biogenesis machinery, which represents the only type II secretion system encoded by the small genome of this highly evolved pathogen.
Genetic Interactions of DNA Repair Pathways in the PathogenNeisseria meningitidis
Authors: Davidsen Tonje , Tuven Hanne K. , Bjørås Magnar , Rødland Einar A. , Tønjum Tone 
Published: May 2007

ABSTRACTThe current increase in the incidence and severity of infectious diseases mandates improved understanding of the basic biology and DNA repair profiles of virulent microbes. In our studies of the major pathogen and model organismNeisseria meningitidis, we constructed a panel of mutants inactivating genes involved in base excision repair, mismatch repair, nucleotide excision repair (NER), translesion synthesis, and recombinational repair pathways. The highest spontaneous mutation frequency among theN. meningitidissingle mutants was found in the MutY-deficient strain as opposed tomutSmutants inEscherichia coli, indicating a role for meningococcal MutY in antibiotic resistance development. Recombinational repair was recognized as a major pathway counteracting methyl methanesulfonate-induced alkylation damage in theN. meningitidis. In contrast to what has been shown in other species, meningococcal NER did not contribute significantly to repair of alkylation-induced DNA damage, and meningococcal recombinational repair may thus be one of the main pathways for removal of abasic (apurinic/apyrimidinic) sites and strand breaks in DNA. Conversely, NER was identified as the main meningococcal defense pathway against UV-induced DNA damage.N. meningitidisRecA single mutants exhibited only a moderate decrease in survival after UV exposure as opposed toE. coli recAstrains, which are extremely UV sensitive, possibly reflecting the lack of a meningococcal SOS response. In conclusion, distinct differences betweenN. meningitidisand established DNA repair characteristics inE. coliand other species were identified.
Overproduction of Penicillin-Binding Protein 2 and Its Inactive Variants Causes Morphological Changes and Lysis inEscherichia coli
Authors: Legaree Blaine A. , Adams Calvin B. , Clarke Anthony J. 
Published: May 2007

ABSTRACTPenicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, includingEscherichia coliandPseudomonas aeruginosa. In the course of earlier studies withP. aeruginosaPBP 2, we observed thatE. coliwas sensitive to the overexpression of its gene,pbpA. In this study, we examinedE. colioverproducing bothP. aeruginosaandE. coliPBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However,E. colioverproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted withE. coliMHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.
Two Genes Encoding Protein Kinases of the HstK Family Are Involved in Synthesis of the Minor Heterocyst-Specific Glycolipid in the Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Shi Lei , Li Jian-Hong , Cheng Yong , Wang Li , Chen Wen-Li , Zhang Cheng-Cai 
Published: May 2007

ABSTRACT

The filamentous cyanobacterium
Anabaena
sp. strain PCC 7120 can fix N
2
under oxic conditions, and the activity of nitrogen fixation occurs exclusively in heterocysts, cells differentiated from vegetative cells in response to a limitation of a combined-nitrogen source in the growth medium. At the late stages of heterocyst differentiation, an envelope layer composed of two glycolipids is formed to limit the entry of oxygen so that the oxygen-sensitive nitrogenase can function. The genome of
Anabaena
sp. strain PCC 7120 possesses a family of 13 genes (the
hstK
family), all encoding proteins with a putative Ser/Thr kinase domain at their N termini and a His-kinase domain at their C termini. In this study, we showed that the double mutant D4.3 strain, in which two members of this gene family,
pkn44
(all1625) and
pkn30
(all3691), were both inactivated, failed to fix N
2
in the presence of oxygen (Fox
−
). In an environment without oxygen, a low level of nitrogenase activity was detectable (Fix
+
). Heterocyst development in the mutant D4.3 was delayed by 24 h and arrested at a relatively early stage without the formation of the glycolipid layer (Hgl
−
). Only the minor species of the two heterocyst-specific glycolipids (HGLs) was missing in the mutant. We propose that DevH, a putative transcription factor, coordinates the synthesis of both HGLs, while Pkn44/Pkn30 and the previously characterized PrpJ may represent two distinct regulatory pathways involved in the synthesis of the minor HGL and the major HGL, respectively.
Complex Interplay between Type 1 Fimbrial Expression and Flagellum-Mediated Motility of Uropathogenic
Escherichia coli
Authors: Lane M. Chelsea , Simms Amy N. , Mobley Harry L. T. 
Published: May 2007

ABSTRACT

Type 1 fimbriae and flagella have been previously shown to contribute to the virulence of uropathogenic
Escherichia coli
(UPEC) within the urinary tract. In this study, the relationship between motility and type 1 fimbrial expression was tested for UPEC strain CFT073 by examining the phenotypic effect of fimbrial expression on motility and the effect that induction of motility has on type 1 fimbrial expression. While constitutive expression of type 1 fimbriae resulted in a significant decrease in motility and flagellin expression (
P
< 0.0001), a loss of type 1 fimbrial expression did not result in increased motility. Additionally, hypermotility and flagellar gene over- and underexpression were not observed to affect the expression of type 1 fimbriae. Hence, it appeared that the relationship between type 1 fimbrial expression and motility is unidirectional, where the overexpression of type 1 fimbriae dramatically affects motility and flagellum expression but not vice versa. Moreover, the constitutive expression of type 1 fimbriae in UPEC cystitis isolate F11 and the laboratory strain
E. coli
K-12 MG1655 also resulted in decreased motility, suggesting that this phenomenon is not specific to CFT073 or UPEC in general. Lastly, by analyzing the repression of motility caused by constitutive type 1 fimbrial expression, it was concluded that the synthesis and presence of type 1 fimbriae at the bacterial surface is only partially responsible for the repression of motility, as evidenced by the partial restoration of motility in the CFT073
fim
L-ON Δ
fimAICDFGH
mutant. Altogether, these data provide further insight into the complex interplay between type 1 fimbrial expression and flagellum-mediated motility.
Identification of a Novel Streptococcal Gene Cassette Mediating SOS Mutagenesis in
Streptococcus uberis
Authors: Varhimo Emilia , Savijoki Kirsi , Jalava Jari , Kuipers Oscar P. , Varmanen Pekka 
Published: May 2007

ABSTRACT

Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the
Streptococcaceae
family. Exposure to UV light was found to increase mutations to antibiotic resistance in
Streptococcus uberis
cultures. The mutational spectra revealed mainly G:C→A:T transitions, and Northern analyses demonstrated increased expression of a Y-family DNA polymerase resembling UmuC under DNA-damaging conditions. In the absence of the Y-family polymerase,
S. uberis
cells were sensitive to UV light and to mitomycin C. Furthermore, the UV-induced mutagenesis was almost completely abolished in cells deficient in the Y-family polymerase. The gene encoding the Y-family polymerase was localized in a four-gene operon including two hypothetical genes and a gene encoding a HdiR homolog. Electrophoretic mobility shift assays demonstrated that
S. uberis
HdiR binds specifically to an inverted repeat sequence in the promoter region of the four-gene operon. Database searches revealed conservation of the gene cassette in several
Streptococcus
species, including at least one genome each of
Streptococcus pyogenes
,
Streptococcus agalactiae
,
Streptococcus mitis
,
Streptococcus sanguinis
, and
Streptococcus thermophilus
strains. In addition, the
umuC
operon was localized in several mobile DNA elements of
Streptococcus
and
Lactococcus
species. We conclude that the
hdiR-umuC
-ORF3-ORF4 operon represents a novel gene cassette capable of mediating SOS mutagenesis among members of the
Streptococcaceae
.
What Is the Benefit to
Escherichia coli
of Having Multiple Toxin-Antitoxin Systems in Its Genome?
Authors: Tsilibaris Virginie , Maenhaut-Michel Geneviève , Mine Natacha , Van Melderen Laurence 
Published: May 2007

ABSTRACT

The
Escherichia coli
K-12 chromosome encodes at least five proteic toxin-antitoxin (TA) systems. The
mazEF
and
relBE
systems have been extensively characterized and were proposed to be general stress response modules. On one hand,
mazEF
was proposed to act as a programmed cell death system that is triggered by a variety of stresses. On the other hand,
relBE
and
mazEF
were proposed to serve as growth modulators that induce a dormancy state during amino acid starvation. These conflicting hypotheses led us to test a possible synergetic effect of the five characterized
E. coli
TA systems on stress response. We compared the behavior of a wild-type strain and its derivative devoid of the five TA systems under various stress conditions. We were unable to detect TA-dependent programmed cell death under any of these conditions, even under conditions previously reported to induce it. Thus, our results rule out the programmed-cell-death hypothesis. Moreover, the presence of the five TA systems advantaged neither recovery from the different stresses nor cell growth under nutrient-limited conditions in competition experiments. This casts a doubt on whether TA systems significantly influence bacterial fitness and competitiveness during non-steady-state growth conditions.