Construction of targeted single copy lac fusions using λ Red and FLP-mediated site-specific recombination in bacteria

Ellermeier, Craig D.; Janakiraman, Anuradha; Slauch, James M.

doi:10.1016/s0378-1119(02)00551-6

Cited by 280 publications

(336 citation statements)

References 30 publications

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“…Gene disruptions or lacZ reporter fusions to promoters were carried out as described previously (Datsenko & Wanner, 2000;Ellermeier et al, 2002) in strain LB5010 (Bullas & Ryu, 1983). All constructions were transferred to the wild-type strain 14028s by P22 transduction (Davis et al, 1980).…”

Section: Methodsmentioning

confidence: 99%

Dissecting the Salmonella response to copper

et al. 2007

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Section: Methodsmentioning

confidence: 99%

Dissecting the Salmonella response to copper

et al. 2007

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“…Bacterial strains, listed in Table 1, were isogenic derivatives of S. enterica serovar Typhimurium strain 14028, E. coli K-12 strain DH5a or K-12 strain MC4100. Deletions of genes and concomitant insertion of an antibiotic resistance cassette were performed using l Red-mediated recombination (Datsenko & Wanner, 2000;Yu et al, 2000), as described previously (Ellermeier et al, 2002), and were verified by PCR analysis. The end points of each deletion are indicated.…”

Section: Methodsmentioning

confidence: 99%

“…In some cases, antibiotic resistance cassettes were removed using plasmid pCP20 encoding FLP recombinase (Datsenko & Wanner, 2000). The wca-lacZ, dsbL-lacZ and assT-lacZ transcriptional fusions were created using FLP/FRTmediated site-specific recombination, as described previously (Ellermeier et al, 2002). Note that these fusion constructs create null mutations in these genes.…”

Section: Methodsmentioning

confidence: 99%

DsbL and DsbI contribute to periplasmic disulfide bond formation in Salmonella enterica serovar Typhimurium

2009

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Disulfide bond formation in periplasmic proteins is catalysed by the DsbA/DsbB system in most Gram-negative bacteria. Salmonella enterica serovar Typhimurium also encodes a paralogous pair of proteins to DsbA and DsbB, DsbL and DsbI, respectively, downstream of a periplasmic arylsulfate sulfotransferase (ASST). We show that DsbL and DsbI function as a redox pair contributing to periplasmic disulfide bond formation and, as such, affect transcription of the Salmonella pathogenicity island 1 (SPI1) type three secretion system genes and activation of the RcsCDB system, as well as ASST activity. In contrast to DsbA/DsbB, however, the DsbL/DsbI system cannot catalyse the disulfide bond formation required for flagellar assembly. Phylogenic analysis suggests that the assT dsbL dsbI genes are ancestral in the Enterobacteriaceae, but have been lost in many lineages. Deletion of assT confers no virulence defect during acute Salmonella infection of mice.

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“…PCR primers (60 nt) were synthesized with 40 nt of homology to the target gene at the 5¢-end of each primer and 20 nt at the 3¢-end aligning with pDK3/4 plasmid as a source of the antibiotic-resistant cassettes (Table 1) (Datsenko & Wanner, 2000). The FRT scar was used to fuse the lacZY reporter as described (Ellermeier et al, 2002) to construct transcriptional fusions with the SPI-9 genes. The S. Typhi DrpoS, S. Typhi DrpoS/pBRPOS and S. Typhi DrpoS/pBBR5 mutants were previously reported Jofre et al, 2014).…”

Section: Methodsmentioning

confidence: 99%

“…Thus, we constructed the S. Typhi STH2370 DSTY2875 :: lacZY mutant by replacing an internal segment of the STY2875 ORFs by a lac reporter (lacZY) as previously described (Ellermeier et al, 2002). This strain was cultured to logarithmic phase (OD 600 =0.5) under different conditions previously associated with different stages of the infection process, including the presence of glucose, micro-aerophilic conditions, high osmolarity and changes in pH (Bajaj et al, 1996;Ellermeier & Slauch, 2007;Eriksson et al, 2003;Hansen-Wester & Hensel, 2001;Rhen & Dorman, 2005;Rychlik & Barrow, 2005).…”

Section: Spi-9 Orfs Of S Typhi Sth2370 Are Induced Under Low Ph and mentioning

confidence: 99%

SPI-9 of Salmonella enterica serovar Typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture

et al. 2016

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The genomic island 9 (SPI-9) from Salmonella enterica serovar Typhi (S. Typhi) carries three ORFs (STY2876, STY2877, STY2878) presenting 98 % identity with a type 1 secretory apparatus (T1SS), and a single ORF (STY2875) similar to a large RTX-like protein exhibiting repeated Ig domains. BapA, the Salmonella enterica serovar Enteritidis orthologous to S. Typhi STY2875, has been associated with biofilm formation, and is described as a virulence factor in mice. Preliminary in silico analyses revealed that S. Typhi STY2875 ORF has a 600 bp deletion compared with S. Enteritidis bapA, suggesting that S. Typhi STY2875 might be non-functional. At present, SPI-9 has not been studied in S. Typhi. We found that the genes constituting SPI-9 are arranged in an operon whose promoter was up-regulated in high osmolarity and low pH in a RpoS-dependent manner. All the proteins encoded by S. Typhi SPI-9 were located at the membrane fraction, consistent with their putative role as T1SS. Furthermore, SPI-9 contributed to adherence of S. Typhi to epithelial cells when bacteria were grown under high osmolarity or low pH. Under the test conditions, S. Typhi SPI-9 did not participate in biofilm formation. SPI-9 is functional in S. Typhi and encodes an adhesin induced under conditions normally found in the intestine, such as high osmolarity. Hence, this is an example of a locus that might be designated a pseudogene by computational approaches but not by direct biological assays.

show abstract

Construction of targeted single copy lac fusions using λ Red and FLP-mediated site-specific recombination in bacteria

Cited by 280 publications

References 30 publications

Dissecting the Salmonella response to copper

Dissecting the Salmonella response to copper

DsbL and DsbI contribute to periplasmic disulfide bond formation in Salmonella enterica serovar Typhimurium

SPI-9 of Salmonella enterica serovar Typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture

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