bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering of Tellurite-Resistant Genetic Tools for Single-Copy Chromosomal Analysis of
Burkholderia
spp. and Characterization of the
Burkholderia thailandensis betBA
Operon
Authors: Kang Yun , Norris Michael H. , Barrett Ashley R. , Wilcox Bruce A. , Hoang Tung T. 
Published: April 2009

ABSTRACT

There are few appropriate single-copy genetic tools for most
Burkholderia
species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents
Burkholderia pseudomallei
and
Burkholderia mallei
, necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Tel
r
) and
Escherichia coli
aspartate-semialdehyde dehydrogenase (

asd
Ec

), were developed to facilitate genetic manipulation in
Burkholderia
species. These systems include one mariner transposon, a mini-Tn
7
-derived site-specific transposon, and six
FRT
reporter fusion vectors based on the
lacZ
,
gfp
, and
luxCDABE
reporter genes. Initially, we showed that the random mariner transposon pBT20-Δ
bla
-Tel
r
-
FRT
efficiently transposed within
Burkholderia cenocepacia
,
Burkholderia thailandensis
,
B. pseudomallei
, and
B. mallei
. We then utilized the mini-Tn
7
-Tel
r
-based transposon vector (mini-Tn7-Tel
r
-
betBA
) and a transposase-containing helper plasmid (pTNS3-

asd
Ec

) to complement the
B. thailandensis
Δ
betBA
mutation. Next, one of the
FRT
-
lacZ
fusion vectors (pFRT1-
lacZ
-Tel
r
) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp-
oriT
-

asd
Ec

) to construct the
B. thailandensis
Δ
betBA
::
FRT
-
lacZ
-Tel
r
reporter fusion strain. The
betBA
operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing β-galactosidase assays on the
B. thailandensis
Δ
betBA
::
FRT
-
lacZ
-Tel
r
fusion strain. Finally, we engineered
B. thailandensis
Δ
betBA
::
FRT
-
gfp
-Tel
r
and Δ
betBA
::
FRT
-
lux
-Tel
r
fusion strains by utilizing fusion vectors pFRT1-
gfp
-Tel
r
and pFRT1-
lux
-Tel
r
, respectively. The induction of the
betBA
operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.
Tracking Functional Guilds: “Dehalococcoides” spp. in European River Basins Contaminated with Hexachlorobenzene
Authors: Taş Neslihan , van Eekert Miriam H. A. , Schraa Gosse , Zhou Jizhong , de Vos Willem M. , Smidt Hauke 
Published: April 2009

ABSTRACTHexachlorobenzene (HCB) has been widely used in chemical manufacturing processes and as a pesticide. Due to its resistance to biological degradation, HCB has mainly accumulated in freshwater bodies and agricultural soils. “Dehalococcoides” spp., anaerobic dechlorinating bacteria that are capable of degrading HCB, were previously isolated from river sediments. Yet there is limited knowledge about the abundance, diversity, and activity of this genus in the environment. This study focused on the molecular analysis of the composition and abundance of activeDehalococcoidesspp. in HCB-contaminated European river basins. 16S rRNA-based real-time quantitative PCR and denaturing gradient gel electrophoresis in combination with multivariate statistics were applied. Moreover, a functional gene array was used to determine reductive dehalogenase (rdh) gene diversity. Spatial and temporal fluctuations were observed not only in the abundance ofDehalococcoidesspp. but also in the composition of the populations andrdhgene diversity. Multivariate statistics revealed thatDehalococcoidessp. abundance is primarily affected by spatial differences, whereas species composition is under the influence of several environmental parameters, such as seasonal changes, total organic carbon and/or nitrogen content, and HCB contamination. This study provides new insight into the natural occurrence and dynamics of activeDehalococcoidesspp. in HCB-contaminated river basins.
Increased Antibiotic Resistance ofEscherichia coliin Mature Biofilms
Authors: Ito Akinobu , Taniuchi Asami , May Thithiwat , Kawata Koji , Okabe Satoshi 
Published: April 2009

ABSTRACTBiofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility ofEscherichia colicells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 μg/ml), kanamycin (25 μg/ml), and ofloxacin (10 μg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced byrpoSexpression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction ofrpoS-mediated stress responses.
Overexpression of a Mutant Form of EhRabA, a Unique Rab GTPase of
Entamoeba histolytica
, Alters Endoplasmic Reticulum Morphology and Localization of the Gal/GalNAc Adherence Lectin
Authors: Welter B. H. , Temesvari L. A. 
Published: April 2009

ABSTRACT

Entamoeba histolytica
is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence.
E. histolytica
possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/
N
-acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in
E. histolytica
.
Transducin Beta-Like Gene
FTL1
Is Essential for Pathogenesis in
Fusarium graminearum
Authors: Ding Shengli , Mehrabi Rahim , Koten Cornelia , Kang Zhensheng , Wei Yangdou , Seong Kyeyong , Kistler H. Corby , Xu Jin-Rong 
Published: April 2009

ABSTRACT

Fusarium head blight caused by
Fusarium graminearum
is an important disease of wheat and barley. In a previous study, we identified several mutants with reduced virulence by insertional mutagenesis. A transducin beta-like gene named
FTL1
was disrupted in one of these nonpathogenic mutants.
FTL1
is homologous to
Saccharomyces cerevisiae SIF2
, which is a component of the Set3 complex involved in late stages of ascospore formation. The Δ
ftl1
mutant was significantly reduced in conidiation and failed to cause typical disease symptoms. It failed to colonize the vascular tissues of rachis or cause necrosis on the rachis of inoculated wheat heads. The Δ
ftl1
mutant also was defective in spreading from infected anthers to ovaries and more sensitive than the wild type to plant defensins MsDef1 and osmotin. However, the activation of two mitogen-activated protein kinases, Mgv1 and Gpmk1, production of deoxynivalenol, and expression of genes known to be important for plant infection in
F. graminearum
were not affected, indicating that the defect of the Δ
ftl1
mutant in plant infection is unrelated to known virulence factors in this pathogen and may involve novel mechanisms. The Δ
ftl1
deletion mutant was significantly reduced in histone deacetylation, and many members of the yeast Set3 complex are conserved in
F. graminearum
.
FTL1
appears to be a component of this well-conserved protein complex that plays a critical role in the penetration and colonization of wheat tissues.
Flavin Mononucleotide-Based Fluorescent Protein as an Oxygen-Independent Reporter in
Candida albicans
and
Saccharomyces cerevisiae
Authors: Tielker D. , Eichhof I. , Jaeger K.-E. , Ernst J. F. 
Published: April 2009

ABSTRACT

Hypoxia is encountered frequently by pathogenic and apathogenic fungi. A codon-adapted gene encoding flavin mononucleotide-based fluorescent protein (Ca
FbFP
) was expressed in
Candida albicans
and
Saccharomyces cerevisiae
. Both species produced CaFbFP and fluoresced even during hypoxia, suggesting that oxygen-independent CaFbFP is a useful, novel tool for monitoring hypoxic gene expression in fungi.
Development of amariner-Based Transposon and Identification ofListeria monocytogenesDeterminants, Including the Peptidyl-Prolyl Isomerase PrsA2, That Contribute to Its Hemolytic Phenotype
Authors: Zemansky Jason , Kline Benjamin C. , Woodward Joshua J. , Leber Jess H. , Marquis Hélène , Portnoy Daniel A. 
Published: April 2009

ABSTRACTListeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogenListeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed aHimar1 marinertransposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar toyjbH), lmo1268 (clpX), lmo1401 (similar toymdB), lmo1575 (similar toytqI), lmo1695 (mprF), lmo1821 (similar toprpC), lmo2219 (prsA2), and lmo2460 (similar tocggR). Some of these genes are involved in previously unexplored areas of research withL. monocytogenes: the genesyjbHandclpXregulate the disulfide stress response inBacillus subtilis, and theprpCphosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate thatprsA2, an extracytoplasmic peptidyl-prolylcis/transisomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown thatprsA2expression is linked to PrfA, the master virulence transcription factor inL. monocytogenespathogenesis, we demonstrate thatprsA2is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role.
α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in
Pseudomonas fluorescens
Authors: Mailloux Ryan J. , Singh Ranji , Brewer Guy , Auger Christopher , Lemire Joseph , Appanna Vasu D. 
Published: April 2009

ABSTRACT

α-Ketoglutarate (KG) is a crucial metabolite in all living organisms, as it participates in a variety of biochemical processes. We have previously shown that this keto acid is an antioxidant and plays a key role in the detoxification of reactive oxygen species (ROS). In an effort to further confirm this intriguing phenomenon,
Pseudomonas fluorescens
was exposed to menadione-containing media, with various amino acids as the sources of nitrogen. Here, we demonstrate that KG dehydrogenase (KGDH) and NAD-dependent glutamate dehydrogenase (GDH) work in tandem to modulate KG homeostasis. While KGDH was sharply decreased in cells challenged with menadione, GDH was markedly increased in cultures containing arginine (Arg), glutamate (Glu), and proline (Pro). When ammonium (NH
4
) was utilized as the nitrogen source, both KGDH and GDH levels were diminished. These enzymatic profiles were reversed when control cells were incubated in menadione media.
13
C nuclear magnetic resonance and high-performance liquid chromatography studies revealed how KG was utilized to eliminate ROS with the concomitant formation of succinate. The accumulation of KG in the menadione-treated cells was dependent on the redox status of the lipoic acid residue in KGDH. Indeed, the treatment of cellular extracts from the menadione-exposed cells with dithiothreitol, a reducing agent, partially restored the activity of KGDH. Taken together, these data reveal that KG is pivotal to the antioxidative defense strategy of
P. fluorescens
and also point to the ROS-sensing role for KGDH.
The
Escherichia coli
Mismatch Repair Protein MutL Recruits the Vsr and MutH Endonucleases in Response to DNA Damage
Authors: Polosina Yaroslava Y. , Mui Justin , Pitsikas Photini , Cupples Claire G. 
Published: April 2009

ABSTRACT

The activities of the Vsr and MutH endonucleases of
Escherichia coli
are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the
mutY
gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.
The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N
α
Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of
Haloferax volcanii
Authors: Humbard Matthew A. , Zhou Guangyin , Maupin-Furlow Julie A. 
Published: April 2009

ABSTRACT

Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N
α
acetylation of the initiator methionine of the α1 protein of 20S core particles (CPs) of
Haloferax volcanii
proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of α1 in
H. volcanii
cells. To further examine this, α1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP “gate” formed by the α1 N terminus were examined for their N
α
acetylation. Both the “gate” deletion and Q2A substitution completely altered the N
α
-acetylation pattern of α1, with the deletion rendering α1 unavailable for N
α
acetylation and the Q2A modification apparently enhancing cleavage of α1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two α1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although α1 proteins with Q2D, Q2P, and Q2T substitutions were N
α
acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of α1 as rings in N
α
-acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these α1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that N
α
acetylation of α1 is important in CP assembly and activity, high levels of α1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP “gate” impairs the ability of cells to overcome salt stress.
A Metabolic Operon in Extraintestinal PathogenicEscherichia coliPromotes Fitness under Stressful Conditions and Invasion of Eukaryotic Cells
Authors: Rouquet Géraldine , Porcheron Gaëlle , Barra Claire , Répérant Maryline , Chanteloup Nathalie K. , Schouler Catherine , Gilot Philippe 
Published: April 2009

ABSTRACTWe identified a carbohydrate metabolic operon (frz) that is highly associated with extraintestinal pathogenicEscherichia coli(ExPEC) strains. Thefrzoperon codes for three subunits of aphosphoenolpyruvate:carbohydrate phosphotransferasesystem (PTS) transporter of the fructose subfamily, for a transcriptional activator of PTSs of the MgA family, for two type II ketose-1,6-bisphosphate aldolases, for a sugar-specific kinase (repressor, open reading frame, kinase family [ROK]), and for a protein of the cupin superfamily. We proved that thefrzoperon promotes bacterial fitness under stressful conditions, such as oxygen restriction, late stationary phase of growth, or growth in serum or in the intestinal tract. Furthermore, we showed thatfrzis involved in adherence to and internalization in human type II pneumocytes, human enterocytes, and chicken liver cells by favoring the ON orientation of thefimoperon promoter and thus acting on the expression of type 1 fimbriae, which are the major ExPEC adhesins. Both the PTS activator and the metabolic enzymes encoded by thefrzoperon are involved in these phenotypes.
Citrate Utilization byCorynebacterium glutamicumIs Controlled by the CitAB Two-Component System through Positive Regulation of the Citrate Transport GenescitHandtctCBA
Authors: Brocker Melanie , Schaffer Steffen , Mack Christina , Bott Michael 
Published: April 2009

ABSTRACTIn this work, the molecular basis of aerobic citrate utilization by the gram-positive bacteriumCorynebacterium glutamicumwas studied. Genome analysis revealed the presence of two putative citrate transport systems. The permease encoded bycitHbelongs to the citrate-Mg2+:H+/citrate-Ca2+:H+symporter family, whereas the permease encoded by thetctCBAoperon is a member of the tripartite tricarboxylate transporter family. The expression ofcitHortctCBAinEscherichia colienabled this species to utilize citrate aerobically, indicating that both CitH and TctABC are functional citrate transporters. Growth tests with the recombinantE. colistrains indicated that CitH is active with Ca2+or Sr2+but not with Mg2+and that TctABC is active with Ca2+or Mg2+but not with Sr2+. We could subsequently show that, with 50 mM citrate as the sole carbon and energy source, theC. glutamicumwild type grew best when the minimal medium was supplemented with CaCl2but that MgCl2and SrCl2also supported growth. Each of the two transporters alone was sufficient for growth on citrate. The expression ofcitHandtctCBAwas activated by citrate in the growth medium, independent of the presence or absence of glucose. This activation was dependent on the two-component signal transduction system CitAB, composed of the sensor kinase CitA and the response regulator CitB. CitAB belongs to the CitAB/DcuSR family of two-component systems, whose members control the expression of genes that are involved in the transport and catabolism of tricarboxylates or dicarboxylates.C. glutamicumCitAB is the first member of this family studied inActinobacteria.
Genomic Sequencing Reveals Regulatory Mutations and Recombinational Events in the Widely Used MC4100 Lineage ofEscherichia coliK-12
Authors: Ferenci Thomas , Zhou Zhemin , Betteridge Thu , Ren Yan , Liu Yu , Feng Lu , Reeves Peter R. , Wang Lei 
Published: April 2009

ABSTRACTThe genome of anEscherichia coliMC4100 strain with a λ placMu50 fusion revealed numerous regulatory differences from MG1655, including one that arose during laboratory storage. The 194 mutational differences between MC4100(MuLac) and other K-12 sequences were mostly allocated to specific lineages, indicating the considerable mutational divergence between K-12 strains.
Conserved Network of Proteins Essential for Bacterial Viability
Authors: Handford Jennifer I. , Ize Bérengère , Buchanan Grant , Butland Gareth P. , Greenblatt Jack , Emili Andrew , Palmer Tracy 
Published: April 2009

ABSTRACTTheyjeE,yeaZ, andygjDgenes are highly conserved in the genomes of eubacteria, andygjDorthologs are also found throughout theArchaeaand eukaryotes. In this study, we have constructed conditional expression strains for each of these genes in the model organismEscherichia coliK12. We show that each gene is essential for the viability ofE. coliunder laboratory growth conditions. Growth of the conditional strains under nonpermissive conditions results in dramatic changes in cell ultrastructure. Deliberate repression of the expression ofyeaZresults in cells with highly condensed nucleoids, while repression ofyjeEandygjDexpression results in at least a proportion of very enlarged cells with an unusual peripheral distribution of DNA. Each of the three conditional expression strains can be complemented by multicopy clones harboring therstAgene, which encodes a two-component-system response regulator, strongly suggesting that these proteins are involved in the same essential cellular pathway. The results of bacterial two-hybrid experiments show that YeaZ can interact with both YjeE and YgjD but that YgjD is the preferred interaction partner. The results of in vitro experiments indicate that YeaZ mediates the proteolysis of YgjD, suggesting that YeaZ and YjeE act as regulators to control the activity of this protein. Our results are consistent with these proteins forming a link between DNA metabolism and cell division.