scholarly journals Genetic Tools for Select-Agent-Compliant Manipulation of Burkholderia pseudomallei

2007 ◽  
Vol 74 (4) ◽  
pp. 1064-1075 ◽  
Author(s):  
Kyoung-Hee Choi ◽  
Takehiko Mima ◽  
Yveth Casart ◽  
Drew Rholl ◽  
Ayush Kumar ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Natural Transformation ◽  
The United States ◽  
Broad Host Range ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Site Specific ◽  
Genetic Manipulations ◽  
Type Strains ◽  
Selection Markers

ABSTRACT Because of Burkholderia pseudomallei's classification as a select agent in the United States, genetic manipulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new tools were developed. A temperature-sensitive pRO1600 broad-host-range replicon was isolated and used to construct curable plasmids where the Flp and Cre recombinase genes are expressed from the rhamnose-regulated Escherichia coli PBAD promoter and kanamycin (nptI) and zeocin (ble) selection markers from the constitutive Burkholderia thailandensis ribosomal P S12 or synthetic bacterial P EM7 promoter. Flp and Cre site-specific recombination systems allow in vivo excision and recycling of nptII and ble selection markers contained on FRT or loxP cassettes. Finally, expression of Tn7 site-specific transposase from the constitutive P1 integron promoter allowed development of an efficient site-specific chromosomal integration system for B. pseudomallei. In conjunction with a natural transformation method, the utility of these new tools was demonstrated by isolating an unmarked Δ(amrRAB-oprA) efflux pump mutant. Exploiting natural transformation, chromosomal DNA fragments carrying this mutation marked with zeocin resistance were transferred between the genomes of two different B. pseudomallei strains. Lastly, the deletion mutation was complemented by a chromosomally integrated mini-Tn7 element carrying the amrAB-oprA operon. The new tools allow routine select-agent-compliant genetic manipulations of B. pseudomallei and other Burkholderia species.

STUDIES ON THE PARASITISM AND VARIATION OF ALTERNARIA RAPHANI

1950 ◽  
Vol 28c (3) ◽  
pp. 288-317 ◽  
Author(s):  
R. G. Atkinson
Keyword(s):  
Soil Moisture ◽  
Natural Infection ◽  
The United States ◽  
Nutritional Requirements ◽  
Diseased Plant ◽  
Cultural Variation ◽  
Wild Type ◽  
Rate Of Growth ◽  
Type Strains ◽  
Radish Seed

The natural infection of radish seed with A. Raphani may result in a lack of germination, a pre- or postemergence blight, a distinctive lesioning of cotyledons and hypocotyls, the presence of scablike lesions on table radish, and in the spotting and blighting of leaves, stalks, and siliques. The fungus was isolated from the internal tissues of all parts of dormant radish seed. Although the pathogen has been reported only on radish in Canada and the United States, the present investigation shows that Canadian isolates are capable of causing a severe leaf blight of stocks and wallflowers. Under field conditions at St. Catharines, Ont., most rapid progress of the disease occurred at temperatures within the optimum range for the fungus, i.e., 22° to 26 °C. Experimental evidence suggests that A. Raphani does not establish an overwintering inoculum in the soil by means of diseased plant debris. Increased soil moisture was associated with increased seedling disease. At a high soil moisture content, infection was lowest at 18 °C.; at medium soil moisture, it was lowest at 18 °C. and also at 23 °C., the next highest experimental temperature.Monosporous isolations showed the presence of numerous wild type strains of A. Raphani which were closely related culturally. Five of these studied intensively differed widely in virulence and sporulation, but had similar growth rates and nutritional requirements for maximum growth. Although most isolates of A. Raphani produced only a few spores in ordinary agar cultures, abundant sporulation was obtained by wounding plate cultures and removing the lids of the culture plates. In agar culture, the wild types readily produced mostly appressed variant strains also showing close cultural relations. These variants exhibited wide differences in pathogenicity, rate of growth, and nutritional requirements, but all showed practically complete loss of sporulation either in normal or wounded cultures. The effects of cultural variation of wild type strains on cultural habit, pathogenicity, rate of growth, sporulating capacity, and nutritional requirements were random and unrelated. These data, as well as the spontaneous origin and irreversibility of the variants, favored the view that they arose in culture by mutation in the naturally occurring strains.A. Raphani was shown to be capable of surviving at least 18 months in dry soil cultures with no loss of cultural habit, virulence, or sporulation.Appreciable increase in emergence and decrease in seedling infection was obtained by seed treatments with some of the common fungicidal dusts.

scholarly journals Pathogenic Neisseriae Can Use Hemoglobin, Transferrin, and Lactoferrin Independently of the tonBLocus

2000 ◽  
Vol 182 (19) ◽  
pp. 5586-5591 ◽  
Author(s):  
Pragnya Jasvantrai Desai ◽  
Eric Garges ◽  
Caroline Attardo Genco
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Deletion Mutant ◽  
Iron Transport ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Gram Negative ◽  
Type Strains

ABSTRACT Redundant TonB systems which function in iron transport from TonB-dependent ligands have recently been identified in several gram-negative bacteria. We demonstrate here that in addition to the previously described tonB locus, an alternative system exists for the utilization of iron from hemoglobin, transferrin, or lactoferrin in Neisseria meningitidis andNeisseria gonorrhoeae. Following incubation on media containing hemoglobin, N. meningitidis IR3436 (tonB exbB exbD deletion mutant) and N. gonorrhoeae PD3401 (tonB insertional mutant) give rise to colonies which can grow with hemoglobin. Transfer of Hb+ variants (PD3437 or PD3402) to media containing hemoglobin, transferrin, and/or lactoferrin as sole iron sources resulted in growth comparable to that observed for the wild-type strains. Transformation of N. meningitidis IR3436 or N. gonorrhoeae PD3401 with chromosomal DNA from the Hb+ variants yielded transformants capable of growth with hemoglobin. When we inactivated the TonB-dependent outer membrane hemoglobin receptors (HmbR or HpuB) in the NeisseriaHb+ variants, these strains could not grow with hemoglobin; however, growth was observed with transferrin and/or lactoferrin. These results demonstrate that accumulation of iron from hemoglobin, transferrin, and lactoferrin in the pathogenic neisseriae can occur via a system that is independent of the previously describedtonB locus.

scholarly journals Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli

1982 ◽  
Vol 152 (3) ◽  
pp. 1241-1247
Author(s):  
H Berger ◽  
J Hacker ◽  
A Juarez ◽  
C Hughes ◽  
W Goebel
Keyword(s):  
Escherichia Coli ◽  
High Frequency ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Genetic Determinants ◽  
E Coli ◽  
Gene Banks ◽  
In The Wild ◽  
K 12 ◽  
Type Strains

We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes O4, O6, O18, and O75. The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further subcloned either as SalI fragments in pACYC184 or as BamHI-SalI fragments in a recombinant plasmid (pANN202) containing cistron C (hlyC) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure. These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.

scholarly journals Minicells as a Damage Disposal Mechanism inEscherichia coli

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Camilla U. Rang ◽  
Audrey Proenca ◽  
Christen Buetz ◽  
Chao Shi ◽  
Lin Chao
Keyword(s):  
Wild Type ◽  
Chromosomal Dna ◽  
Cell Level ◽  
Content Type ◽  
E Coli ◽  
Survival And Growth ◽  
First Time ◽  
Antibiotic Level ◽  
Type Strains ◽  
Sister Cells

ABSTRACTMany bacteria produce small, spherical minicells that lack chromosomal DNA and therefore are unable to proliferate. Although minicells have been used extensively by researchers as a molecular tool, nothing is known about why bacteria produce them. Here, we show that minicells helpEscherichia colicells to rid themselves of damaged proteins induced by antibiotic stress. By comparing the survival and growth rates of wild-type strains with theE. coliΔminCmutant, which produces excess minicells, we found that the mutant was more resistant to streptomycin. To determine the effects of producing minicells at the single-cell level, we also tracked the growth ofΔminClineages by microscopy. We were able to show that the mutant increased the production of minicells in response to a higher level of the antibiotic. When we compared two sister cells, in which one produced minicells and the other did not, the daughters of the former had a shorter doubling time at this higher antibiotic level. Additionally, we found that minicells were more likely produced at the mother’s old pole, which is known to accumulate more aggregates. More importantly, by using a fluorescent IbpA chaperone to tag damage aggregates, we found that polar aggregates were contained by and ejected with the minicells produced by the mother bacterium. These results demonstrate for the first time the benefit to bacteria for producing minicells.IMPORTANCEBacteria have the ability to produce minicells, or small spherical versions of themselves that lack chromosomal DNA and are unable to replicate. A minicell can constitute as much as 20% of the cell’s volume. Although molecular biology and biotechnology have used minicells as laboratory tools for several decades, it is still puzzling that bacteria should produce such costly but potentially nonfunctional structures. Here, we show that bacteria gain a benefit by producing minicells and using them as a mechanism to eliminate damaged or oxidated proteins. The elimination allows the bacteria to tolerate higher levels of stress, such as increasing levels of streptomycin. If this mechanism extends from streptomycin to other antibiotics, minicell production could be an overlooked pathway that bacteria are using to resist antimicrobials.

In vitro and in vivo activities of AM-1155, a new fluoroquinolone, against Chlamydia spp.

1997 ◽  
Vol 41 (6) ◽  
pp. 1331-1334 ◽  
Author(s):  
N Miyashita ◽  
Y Niki ◽  
T Kishimoto ◽  
M Nakajima ◽  
T Matsushima
Keyword(s):  
United States ◽  
Body Weight ◽  
Survival Rate ◽  
Chlamydia Pneumoniae ◽  
The United States ◽  
Chlamydia Psittaci ◽  
Wild Type ◽  
Type Strains

The in vitro and in vivo activities of AM-1155, a new quinolone, against Chlamydia spp. were investigated. The MIC of AM-1155 for 10 standard strains of different Chlamydia spp. and 25 wild-type strains of Chlamydia pneumoniae isolated in Japan, which were morphologically different from clinical isolates from the United States, ranged from 0.063 to 0.125 microg/ml. Its activity was almost the same as those of sparfloxacin and tosufloxacin and was 4 and 16 times superior to those of levofloxacin and ciprofloxacin, respectively, but lower than those of clarithromycin and minocycline (range for each, 0.016 to 0.031 microg/ml). The minimal chlamydiacidal concentration of AM-1155 ranged from 0.063 to 0.125 microg/ml, while those of clarithromycin and minocycline ranged from 0.016 to 0.031 microg/ml and 0.016 to 0.063 microg/ml, respectively. The therapeutic effect of a 7-day course of AM-1155 at doses of 5 and 10 mg/kg of body weight administered orally twice daily to mice with experimental Chlamydia psittaci pneumonia was excellent, with a 100% survival rate at 21 days after infection. The efficacy was equal to those of clarithromycin and minocycline and higher than those of ciprofloxacin and ofloxacin.

scholarly journals Oxidative ornithine metabolism supports non-inflammatory C. difficile colonization

2022 ◽  
Author(s):  
Kali M. Pruss ◽  
Fatima Enam ◽  
Eric Battaglioli ◽  
Mary DeFeo ◽  
Oscar R. Diaz ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
The United States ◽  
Wild Type ◽  
Targeted Metabolomics ◽  
Disease Symptoms ◽  
Increased Risk ◽  
Infection And Inflammation ◽  
Gnotobiotic Mice ◽  
Single Operon ◽  
Type Strains

AbstractThe enteric pathogen Clostridioides difficile (Cd) is responsible for a toxin-mediated infection that causes more than 200,000 recorded hospitalizations and 13,000 deaths in the United States every year1. However, Cd can colonize the gut in the absence of disease symptoms. Prevalence of asymptomatic colonization by toxigenic Cd in healthy populations is high; asymptomatic carriers are at increased risk of infection compared to noncolonized individuals and may be a reservoir for transmission of Cd infection2,3. Elucidating the molecular mechanisms by which Cd persists in the absence of disease is necessary for understanding pathogenesis and developing refined therapeutic strategies. Here, we show with gut microbiome metatranscriptomic analysis that mice recalcitrant to Cd infection and inflammation exhibit increased community-wide expression of arginine and ornithine metabolic pathways. To query Cd metabolism specifically, we leverage RNA sequencing in gnotobiotic mice infected with two wild-type strains (630 and R20291) and isogenic toxin-deficient mutants of these strains to differentiate inflammation-dependent versus -independent transcriptional states. A single operon encoding oxidative ornithine degradation is consistently upregulated across non-toxigenic Cd strains. Combining untargeted and targeted metabolomics with bacterial and host genetics, we demonstrate that both diet- and host-derived sources of ornithine provide a competitive advantage to Cd, suggesting a mechanism for Cd persistence within a non-inflammatory, healthy gut.

scholarly journals Development of a System for Genetic Manipulation ofBartonella bacilliformis

1999 ◽  
Vol 65 (8) ◽  
pp. 3441-3448 ◽  
Author(s):  
James M. Battisti ◽  
Michael F. Minnick
Keyword(s):  
Molecular Biology ◽  
Genetic Manipulation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Restriction Enzymes ◽  
Kanamycin Resistance ◽  
Broad Host Range ◽  
Wild Type ◽  
Site Specific ◽  
Suicide Vector

ABSTRACT Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of allBartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymesClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kanr clones of B. bacilliformis. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 andfla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded byfla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG). These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.

scholarly journals The Conjugal Intermediate of Plasmid RSF1010 Inhibits Agrobacterium tumefaciens Virulence and VirB-Dependent Export of VirE2

1998 ◽  
Vol 180 (15) ◽  
pp. 3933-3939 ◽  
Author(s):  
Lisa E. Stahl ◽  
Amy Jacobs ◽  
Andrew N. Binns
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Cell ◽  
Dna Binding Protein ◽  
Tumor Formation ◽  
Broad Host Range ◽  
Wild Type ◽  
Single Stranded Dna ◽  
Crown Gall Disease ◽  
Broad Host Range Plasmid ◽  
Type Strains

ABSTRACT Agrobacterium tumefaciens causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from A. tumefaciens to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 betweenAgrobacterium strains or from Agrobacterium to plants also requires the virB-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of Agrobacterium inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with virE mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.

scholarly journals Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

1989 ◽  
Vol 9 (4) ◽  
pp. 1507-1512 ◽  
Author(s):  
H Zhu ◽  
H Conrad-Webb ◽  
X S Liao ◽  
P S Perlman ◽  
R A Butow
Keyword(s):  
Genetic Analysis ◽  
Rna Processing ◽  
Fold Increase ◽  
Functional Expression ◽  
Rrna Gene ◽  
Yeast Mitochondria ◽  
Wild Type ◽  
Site Specific ◽  
Var1 Gene ◽  
A Site

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

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