Mutational Analysis of cj0183 Campylobacter jejuni Promoter

Salamasznska-Guz, Agnieszka; Grodzik, Marta; Klimuszko, D.

doi:10.1007/s00284-013-0420-8

Cited by 3 publications

(3 citation statements)

References 16 publications

(38 reference statements)

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“…Apparently a strongly conserved −10 and −16 regions are not sufficient to activate the ggt transcription without additional transcription factors, as seen by real-time RT-PCR and luciferase assay ( Figures 1 and 4 ). These results suggest that although a conserved consensus sequence for the −35 region of sigma 70 regulated promoters in C. jejuni is lacking the region upstream of the −16 region is essential to activate sigma 70 regulated promoters as has been observed already ( Dugar et al, 2013 ; Salamasznska-Guz et al, 2013 ). Using the previously obtained RacR binding consensus ( van der Stel et al, 2015 ), a potential RacR binding site was found ~80-bp upstream of the transcriptional start site (TSS) ( Dugar et al, 2013 ), besides that, a palindromic sequence was found ~60-bp upstream of the TSS, which could be a potential regulatory element.…”

Section: Discussionsupporting

confidence: 72%

The Campylobacter jejuni RacRS two-component system activates the glutamate synthesis by directly upregulating Î³-glutamyltranspeptidase (GGT)

Stel

Mourik

Łaniewski³

et al. 2015

Front. Microbiol.

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The highly conserved enzyme γ-glutamyltranspeptidase (GGT) plays an important role in metabolism of glutathione and glutamine. Yet, the regulation of ggt transcription in prokaryotes is poorly understood. In the human pathogen Campylobacter jejuni, GGT is important as it contributes to persistent colonization of the gut. Here we show that the GGT activity in C. jejuni is dependent on a functional RacRS (reduced ability to colonize) two-component system. Electrophoretic mobility shift and luciferase reporter assays indicate that the response regulator RacR binds to a promoter region ~80 bp upstream of the ggt transcriptional start site, which contains a recently identified RacR DNA binding consensus sequence. RacR needs to be phosphorylated to activate the transcription of the ggt gene, which is the case under low oxygen conditions in presence of alternative electron acceptors. A functional GGT and RacR are needed to allow C. jejuni to grow optimally on glutamine as sole carbon source under RacR inducing conditions. However, when additional carbon sources are present C. jejuni is capable of utilizing glutamine independently of GGT. RacR is the first prokaryotic transcription factor known to directly up-regulate both the cytoplasmic [glutamine-2-oxoglutarate aminotransferase (GOGAT)] as well as the periplasmic (GGT) production of glutamate.

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Section: Discussionsupporting

confidence: 72%

The Campylobacter jejuni RacRS two-component system activates the glutamate synthesis by directly upregulating Î³-glutamyltranspeptidase (GGT)

Stel

Mourik

Łaniewski³

et al. 2015

Front. Microbiol.

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“…The C. jejuni 81–176 null mutant was complemented by inserting the Mycobacterium smegmatis wild‐type tlyA II into the 121‐bp intergenic region between cj0652 and cj0653c (Javed et al, ; Wösten, Boeve, Koot, van Nuenen, & van der Zeijst, ) under control of the C. jejuni cj0183 gene promoter (Sałamaszyńska‐Guz, Grodzik, & Klimuszko, ). This created strain 81‐176Δ cj0588 :: Myco_tlyA II (Table ).…”

Section: Methodsmentioning

confidence: 99%

Virulence properties of Campylobacter jejuni are enhanced by displaying a mycobacterial TlyA methylation pattern in its rRNA

Sałamaszyńska-Guz

Serafińska

Bącal

et al. 2020

Cellular Microbiology

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Campylobacter jejuni is a bacterial pathogen that is generally acquired as a zoonotic infection from poultry and animals. Adhesion of C. jejuni to human colorectal epithelial cells is weakened after loss of its cj0588 gene. The Cj0588 protein belongs to the type I group of TlyA (TlyA I ) enzymes, which 2 0 -O-methylate nucleotide C1920 in 23S rRNA. Slightly longer TlyA II versions of the methyltransferase are found in actinobacterial species including Mycobacterium tuberculosis, and methylate not only C1920 but also nucleotide C1409 in 16S rRNA. Loss of TlyA function attenuates virulence of both M. tuberculosis and C. jejuni. We show here that the traits impaired in C. jejuni null strains can be rescued by complementation not only with the original cj0588 (tlyA I ) but also with a mycobacterial tlyA II gene. There are, however, significant differences in the recombinant phenotypes. While cj0588 restores motility, biofilm formation, adhesion to and invasion of human epithelial cells and stimulation of IL-8 production in a C. jejuni null strain, several of these properties are further enhanced by the mycobacterial tlyA II gene, in some cases to twice the original wild-type level.These findings strongly suggest that subtle changes in rRNA modification patterns can affect protein synthesis in a manner that has serious consequences for bacterial pathogenicity. K E Y W O R D S bacterial motility, biofilms, capreomycin resistance, epithelial cell invasion, rRNA 2 0 -Omethylation

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“…The C. jejuni 405Δ cj0588 null-mutant was complemented in trans with plasmid-encoded versions of the cj0588 gene. A fragment containing the cj0588 gene was amplified by PCR from C. jejuni 81-176 using primers C2 and C5R (Table S2 ) and ligated into the Xba I-digested shuttle vector p0183 under C. jejuni promoter control to form plasmid pMW0588 (Salamasznska-Guz et al, 2013 ). This plasmid was used to transform E. coli before being moved to C. jejuni 405 forming strain 405Δ cj0588 +pMW0588 (Table S1 ).…”

Section: Methodsmentioning

confidence: 99%

Biofilm Formation and Motility Are Promoted by Cj0588-Directed Methylation of rRNA in Campylobacter jejuni

Sałamaszyńska-Guz

Rose

Lykkebo

et al. 2018

Front. Cell. Infect. Microbiol.

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Numerous bacterial pathogens express an ortholog of the enzyme TlyA, which is an rRNA 2′-O-methyltransferase associated with resistance to cyclic peptide antibiotics such as capreomycin. Several other virulence traits have also been attributed to TlyA, and these appear to be unrelated to its methyltransferase activity. The bacterial pathogen Campylobacter jejuni possesses the TlyA homolog Cj0588, which has been shown to contribute to virulence. Here, we investigate the mechanism of Cj0588 action and demonstrate that it is a type I homolog of TlyA that 2′-O-methylates 23S rRNA nucleotide C1920. This same specific function is retained by Cj0588 both in vitro and also when expressed in Escherichia coli. Deletion of the cj0588 gene in C. jejuni or substitution with alanine of K80, D162, or K188 in the catalytic center of the enzyme cause complete loss of 2′-O-methylation activity. Cofactor interactions remain unchanged and binding affinity to the ribosomal substrate is only slightly reduced, indicating that the inactivated proteins are folded correctly. The substitution mutations thus dissociate the 2′-O-methylation function of Cj0588/TlyA from any other putative roles that the protein might play. C. jejuni strains expressing catalytically inactive versions of Cj0588 have the same phenotype as cj0588-null mutants, and show altered tolerance to capreomycin due to perturbed ribosomal subunit association, reduced motility and impaired ability to form biofilms. These functions are reestablished when methyltransferase activity is restored and we conclude that the contribution of Cj0588 to virulence in C. jejuni is a consequence of the enzyme's ability to methylate its rRNA.

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Mutational Analysis of cj0183 Campylobacter jejuni Promoter

Cited by 3 publications

References 16 publications

The Campylobacter jejuni RacRS two-component system activates the glutamate synthesis by directly upregulating Î³-glutamyltranspeptidase (GGT)

The Campylobacter jejuni RacRS two-component system activates the glutamate synthesis by directly upregulating Î³-glutamyltranspeptidase (GGT)

Virulence properties of Campylobacter jejuni are enhanced by displaying a mycobacterial TlyA methylation pattern in its rRNA

Biofilm Formation and Motility Are Promoted by Cj0588-Directed Methylation of rRNA in Campylobacter jejuni

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