Genetic Mapping by Duplication Segregation in Salmonella enterica

Genetics ◽  
2001 ◽  
Vol 157 (2) ◽  
pp. 491-502
Author(s):  
Eva M Camacho ◽  
Josep Casadesús
Keyword(s):  
Salmonella Enterica ◽  
Replication Fork ◽  
Limiting Factor ◽  
Multiplicity Of Infection ◽  
Dominant Marker ◽  
Host Chromosome ◽  
Transposon Insertion ◽  
Large Size ◽  
Chromosomal Duplication ◽  
Duplication Segregation

Abstract MudP and MudQ elements were used to induce duplications in Salmonella enterica by formation of a triple crossover between two transduced fragments and the host chromosome. The large size (36 kb) of MudP and MudQ is a favorable trait for duplication formation, probably because homology length is a limiting factor for the central crossover. Additional requirements are a multiplicity of infection of 2 or higher in the infecting phage suspensions (which reflects the need of two transduced fragments) and an exponentially growing recipient (which reflects the need of a chromosome replication fork). We describe a set of 11 strains of S. enterica, each carrying a chromosomal duplication with known endpoints. The collection covers all the Salmonella chromosome except the terminus. For mapping, a dominant marker (e.g., a transposon insertion in or near the locus to be mapped) is transduced into the 11-strain set. Several transductants from each cross are grown nonselectively, and haploid segregants are scored for the presence of the marker. If all the segregants contain the transduced marker, it maps outside the duplication interval. If the marker is found only in a fraction of the segregants, it maps within the duplicated region.

scholarly journals OmpR and LeuO Positively Regulate the Salmonella enterica Serovar Typhi ompS2 Porin Gene

2004 ◽  
Vol 186 (10) ◽  
pp. 2909-2920 ◽  
Author(s):  
Marcos Fernández-Mora ◽  
José Luis Puente ◽  
Edmundo Calva
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Phosphorylation Site ◽  
Regulatory Region ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Upstream Regulatory Region ◽  
Wild Type ◽  
Transposon Insertion

ABSTRACT The Salmonella enterica serovar Typhi ompS2 gene codes for a 362-amino-acid outer membrane protein that contains motifs common to the porin superfamily. It is expressed at very low levels compared to the major OmpC and OmpF porins, as observed for S. enterica serovar Typhi OmpS1, Escherichia coli OmpN, and Klebsiella pneumoniae OmpK37 quiescent porins. A region of 316 bp, between nucleotides −413 and −97 upstream of the transcriptional start point, is involved in negative regulation, as its removal resulted in a 10-fold increase in ompS2 expression in an S. enterica serovar Typhi wild-type strain. This enhancement in expression was not observed in isogenic mutant strains, which had specific deletions of the regulatory ompB (ompR envZ) operon. Furthermore, ompS2 expression was substantially reduced in the presence of the OmpR D55A mutant, altered in the major phosphorylation site. Upon random mutagenesis, a mutant where the transposon had inserted into the upstream regulatory region of the gene coding for the LeuO regulator, showed an increased level of ompS2 expression. Augmented expression of ompS2 was also obtained upon addition of cloned leuO to the wild-type strain, but not in an ompR isogenic derivative, consistent with the notion that the transposon insertion had increased the cellular levels of LeuO and with the observed dependence on OmpR. Moreover, LeuO and OmpR bound in close proximity, but independently, to the 5′ upstream regulatory region. Thus, the OmpR and LeuO regulators positively regulate ompS2.

scholarly journals A whole-genome screen identifies Salmonella enterica serovar Typhi genes involved in fluoroquinolone susceptibility

2020 ◽  
Vol 75 (9) ◽  
pp. 2516-2525
Author(s):  
A Keith Turner ◽  
Sabine E Eckert ◽  
Daniel J Turner ◽  
Muhammud Yasir ◽  
Mark A Webber ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Insertion Site ◽  
Whole Genome ◽  
Genome Screen ◽  
Polysaccharide Biosynthesis ◽  
Salmonella Enterica Serovar Typhi ◽  
Transposon Insertion ◽  
Associated Functions ◽  
The Impact ◽  
Insertion Mutations

Abstract Objectives A whole-genome screen at sub-gene resolution was performed to identify candidate loci that contribute to enhanced or diminished ciprofloxacin susceptibility in Salmonella enterica serovar Typhi. Methods A pool of over 1 million transposon insertion mutants of an S. Typhi Ty2 derivative were grown in a sub-MIC concentration of ciprofloxacin, or without ciprofloxacin. Transposon-directed insertion site sequencing (TraDIS) identified relative differences between the mutants that grew following the ciprofloxacin treatment compared with the untreated mutant pool, thereby indicating which mutations contribute to gain or loss of ciprofloxacin susceptibility. Results Approximately 88% of the S. Typhi strain’s 4895 annotated genes were assayed, and at least 116 were identified as contributing to gain or loss of ciprofloxacin susceptibility. Many of the identified genes are known to influence susceptibility to ciprofloxacin, thereby providing method validation. Genes were identified that were not known previously to be involved in susceptibility, and some of these had no previously known phenotype. Susceptibility to ciprofloxacin was enhanced by insertion mutations in genes coding for efflux, other surface-associated functions, DNA repair and expression regulation, including phoP, barA and marA. Insertion mutations that diminished susceptibility were predominantly in genes coding for surface polysaccharide biosynthesis and regulatory genes, including slyA, emrR, envZ and cpxR. Conclusions A genomics approach has identified novel contributors to gain or loss of ciprofloxacin susceptibility in S. Typhi, expanding our understanding of the impact of fluoroquinolones on bacteria and of mechanisms that may contribute to resistance. The data also demonstrate the power of the TraDIS technology for antibacterial research.

scholarly journals Reduction of Experimental Salmonella and Campylobacter Contamination of Chicken Skin by Application of Lytic Bacteriophages

2003 ◽  
Vol 69 (8) ◽  
pp. 5032-5036 ◽  
Author(s):  
D. Goode ◽  
V. M. Allen ◽  
P. A. Barrow
Keyword(s):  
Campylobacter Jejuni ◽  
Salmonella Enterica ◽  
Unit Area ◽  
Multiplicity Of Infection ◽  
Chicken Skin

ABSTRACT Lytic bacteriophages, applied to chicken skin that had been experimentally contaminated with Salmonella enterica serovar Enteritidis or Campylobacter jejuni at a multiplicity of infection (MOI) of 1, increased in titer and reduced the pathogen numbers by less than 1 log10 unit. Phages applied at a MOI of 100 to 1,000 rapidly reduced the recoverable bacterial numbers by up to 2 log10 units over 48 h. When the level of Salmonella contamination was low (< log10 2 per unit area of skin) and the MOI was 105, no organisms were recovered. By increasing the number of phage particles applied (i.e., MOI of 107), it was also possible to eliminate other Salmonella strains that showed high levels of resistance because of restriction but to which the phages were able to attach.

scholarly journals Salmonella-Induced Filament Formation Is a Dynamic Phenotype Induced by Rapidly Replicating Salmonella enterica Serovar Typhimurium in Epithelial Cells

2005 ◽  
Vol 73 (2) ◽  
pp. 1204-1208 ◽  
Author(s):  
Cheryl L. Birmingham ◽  
Xiuju Jiang ◽  
Maikke B. Ohlson ◽  
Samuel I. Miller ◽  
John H. Brumell
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Host Cells ◽  
Multiplicity Of Infection ◽  
Tubular Structures ◽  
Filament Formation ◽  
Dynamic Nature ◽  
Density Growth ◽  
Serovar Typhimurium ◽  
Bacterial Replication

ABSTRACT Salmonella enterica serovar Typhimurium has the fascinating ability to form tubular structures known as Salmonella-induced filaments (Sifs) in host cells. Here, we show that the prevalence of the Sif phenotype in HeLa cells is affected by host cell density, growth, and the multiplicity of infection. Sif formation was observed in cells that displayed rapid intracellular bacterial replication and was found to be dynamic, being maximal 8 to 10 h postinfection and declining thereafter. The virulence factors SpvB and SseJ were found to negatively modulate Sif formation. Our findings demonstrate the complex and dynamic nature of the Sif phenotype.

scholarly journals Assemblage of medium and large size mammals in an urban Semideciduous Seasonal Forest fragment in Cerrado biome

2013 ◽  
Vol 13 (2) ◽  
pp. 76-80 ◽  
Author(s):  
Paulo Vitor dos Santos Bernardo ◽  
Fabiano Rodrigues de Melo
Keyword(s):  
Urban Area ◽  
Fragment Size ◽  
Domestic Animals ◽  
Present Species ◽  
Limiting Factor ◽  
Sampling Effort ◽  
Forest Fragment ◽  
Wild Mammals ◽  
Large Size ◽  
Semideciduous Seasonal Forest

Nowadays, the processes of deforestation and loss of habitats represent a major threat to many species of mammals. These processes cause changes in natural landscapes by decreasing area, connectivity, and fragment size, and increasing edge effects and number of fragments. Understanding which and how many species persist in disturbed fragments may indicate the species' minimum requirements and might contribute to their conservation. Here we show how the mammalian fauna of medium and large size (higher than 1 kg) are structured in a semideciduous seasonal forest fragment of 36.5 ha in the urban area of Jataí, Goiás. We performed the sampling with 30 sand track plots (1 x 1 m). We analyzed the relative record frequency and built a collector's curve to demonstrate the sampling effort. With a total effort of 600 track plots × days, we recorded twelve species of mammals with our tracks sampling method, from which only the wild mammals were included in the analyzes (11 species). The estimated species richness reached 13 species (SD (Standard Deviation) = ±1, CI (Confidence Interval) = ±2 (11 – 15 species). The species with the highest relative record frequency was Didelphis albiventris and the species with the lowest was Tamandua tetradactyla. The fragment size must be a limiting factor to the richness and to the occurrence of species, as it may not be sufficient to allow the persistence of a population or an individual. Disturbances that originated from houses, like domestic animals and movement of people, also contributed to the removal and extinction of species. To conserve the species in the fragment, we suggest the prevention of entrance of people and of domestic animals. We also recommend increased connectivity of the fragment with the landscape external to the urban area in order to allow the movement of the currently present species.

scholarly journals Identification and Genetic Characterization of PmrA-Regulated Genes and Genes Involved in Polymyxin B Resistance in Salmonella enterica Serovar Typhimurium

2002 ◽  
Vol 70 (12) ◽  
pp. 6770-6778 ◽  
Author(s):  
Rita Tamayo ◽  
Sara S. Ryan ◽  
Andrea J. McCoy ◽  
John S. Gunn
Keyword(s):  
Salmonella Enterica ◽  
Lipid A ◽  
Regulatory System ◽  
Enteric Bacteria ◽  
Polymyxin B ◽  
High Iron ◽  
Transposon Insertion ◽  
Mucosal Surfaces ◽  
Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium encounters antimicrobial peptides (AP) within the phagosomes of professional phagocytes and at intestinal mucosal surfaces. Salmonella serovar Typhimurium utilizes the two-component regulatory system PmrA-PmrB, which is activated in response to the environmental conditions encountered in vivo, to regulate resistance to several AP, including polymyxin B (PM). Random MudJ transposon mutagenesis was used to identify PmrA-PmrB-regulated genes, as well as genetic loci necessary for PM resistance. Three different phenotypic classes of genes were identified: those necessary for PM resistance and regulated by PmrA, those necessary for PM resistance and not regulated by PmrA, and PmrA-regulated genes not required for PM resistance. Loci identified as necessary for PM resistance showed between 6- and 192-fold increased sensitivities to PM, and transposon insertion sites include surA, tolB, and gnd. PmrA-regulated loci identified included dgoA and yibD and demonstrated 500- and 2,500-fold activation by PmrA, respectively. The role of the identified loci in aminoarabinose modification of lipid A was determined by paper chromatography. The gnd mutant demonstrated a loss of aminoarabinose from lipid A, which was suggested to be due to a polar effect on the downstream gene pmrE. The remaining PMs mutants (surA and tolB), as well as the two PmrA-regulated gene (yibD and dgoA) mutants, retained aminoarabinose on lipid A. yibD, dgoA, and gnd (likely affecting pmrE) played no role in PmrA-regulated resistance to high iron concentrations, while surA and tolB mutations grew poorly on high iron media. All PMs mutants identified in this study demonstrated a defect in virulence compared to wild-type Salmonella serovar Typhimurium when administered orally to mice, while the PmrA-regulated gene (yibD and dgoA) mutants showed normal virulence in mice. These data broaden our understanding of in vivo gene regulation, lipopolysaccharide modification, and mechanisms of resistance to AP in enteric bacteria.

The supply of exogenous deoxyribonucleotides accelerates the speed of the replication fork in early S-phase

2001 ◽  
Vol 114 (4) ◽  
pp. 747-750
Author(s):  
J. Malinsky ◽  
K. Koberna ◽  
D. Stanek ◽  
M. Masata ◽  
I. Votruba ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Hela Cells ◽  
Replication Fork ◽  
S Phase ◽  
Limiting Factor ◽  
Replication Forks ◽  
Replication Process ◽  
Average Speed ◽  
Rate Limiting

Earlier studies have established that the average speed of a replication fork is two to three times slower in early S-phase than in late S-phase and that the intracellular 2′-deoxyribonucleoside 5′-triphosphate pools grow during S-phase. In this study, the effect of the exogenous 2′-deoxyribonucleoside 5′-triphosphate (dNTP) supply on the average replication speed in a synchronised population of human HeLa cells was tested. The speed of replication fork movement was measured on extended DNA fibers labelled with 2′-deoxythymidine analogues 5-chloro-2′-deoxyuridine and 5-iodo-2′-deoxyuridine. We show that the introduction of exogenous dNTPs accelerates the replication process at the beginning of DNA synthesis only. In late S-phase, the administration of additional dNTPs has no effect on the speed of replication forks. The availability of 2′-deoxynucleotides seems to be a rate-limiting factor for DNA replication during early S-phase.

scholarly journals Effect of a bacteriophage T5virus on growth of Shiga toxigenic Escherichia coli and Salmonella strains in individual and mixed cultures

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan D. Niu ◽  
Hui Liu ◽  
Roger P. Johnson ◽  
Tim A. McAllister ◽  
Kim Stanford
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Biocontrol Agent ◽  
Sampling Time ◽  
Mixed Cultures ◽  
Broth Culture ◽  
Great Promise ◽  
Multiplicity Of Infection ◽  
Post Harvest ◽  
Zoonotic Pathogens

AbstractA previously isolated a bacteriophage, vB_EcoS_AKFV33 of T5virus, demonstrated great potential in biocontrol of Shiga toxigenic Escherichia coli (STEC) O157. This study further evaluated its potential as a biocontrol agent in broth culture against other important non-O157 serogroups of STEC and Salmonella. AKFV33 was capable of lysing isolates of STEC serogroups O26 (n = 1), O145 (n = 1) and Salmonella enterica serovars (n = 6). In a broth culture microplate system, efficacy of AKFV33 for killing STEC O26:H11, O145:NM and Salmonella was improved (P < 0.05) at a lower multiplicity of infection and sampling time (6–10 h), when STEC O157:H7 was also included in the culture. This phage was able to simultaneously reduce numbers of STEC and Salmonella in mixtures with enhanced activity (P < 0.05) against O157:H7 and O26:H11, offering great promise for control of multiple zoonotic pathogens at both pre and post-harvest.

Utilizing Dark Field Electron Microscopy to Produce Lantern Slides

1974 ◽  
Vol 32 ◽  
pp. 116-117
Author(s):  
J. N. Meador ◽  
C. N. Sun ◽  
H. J. White
Keyword(s):  
Electron Microscope ◽  
Electron Micrographs ◽  
Dark Field ◽  
Limiting Factor ◽  
Clinical Laboratories ◽  
Field Electron ◽  
Time Element ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Dark Field Micrographs

The electron microscope is being utilized more and more in clinical laboratories for pathologic diagnosis. One of the major problems in the utilization of the electron microscope for diagnostic purposes is the time element involved. Recent experimentation with rapid embedding has shown that this long phase of the process can be greatly shortened. In rush cases the making of projection slides can be eliminated by taking dark field electron micrographs which show up as a positive ready for use. The major limiting factor for use of dark field micrographs is resolution. However, for conference purposes electron micrographs are usually taken at 2.500X to 8.000X. At these low magnifications the resolution obtained is quite acceptable.

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