H‐NS‐like proteins in <i>Pseudomonas aeruginosa</i> coordinately silence intragenic transcription

Lippa, Andrew M.; Gebhardt, Michael; Dove, Simon L.

doi:10.1111/mmi.14656

Cited by 18 publications

(20 citation statements)

References 97 publications

(149 reference statements)

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“…With ATG 1503602 as putative start codon, olg2 spans 1728 nt and overlaps with two annotated genes. A region of 1536 nt is shared with PA1383, a hypothetical gene predicted to code for an N-terminal Type I signal sequence for cytoplasmic export 55 , which has been shown to be regulated by the transcriptional repressor NrdR 56 , and by both the repressors mvaT and mvaU 57 . Additionally, olg2 overlaps in frame +2 by 34 nt with the galE gene (PA1384), which encodes for a UDP-glucose 4-epimerase.…”

Section: Resultsmentioning

confidence: 99%

Shadow ORFs illuminated: long overlapping genes inPseudomonas aeruginosaare translated and under purifying selection

Kreitmeier

Ardern

Abele

et al. 2021

Preprint

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The existence of overlapping genes (OLGs) with significant coding overlaps revolutionises our understanding of genomic complexity. We report two exceptionally long (957 nt and 1536 nt), evolutionarily novel, translated antisense open reading frames (ORFs) embedded within annotated genes in the medically important Gram-negative bacterium Pseudomonas aeruginosa. Both OLG pairs show sequence features consistent with being genes and transcriptional signals in RNA sequencing data. Translation of both OLGs was confirmed by ribosome profiling and mass spectrometry. Quantitative proteomics of samples taken during different phases of growth revealed regulation of protein abundances, implying biological functionality. Both OLGs are taxonomically highly restricted, and likely arose by overprinting within the genus. Evidence for purifying selection further supports functionality. The OLGs reported here are the longest yet proposed in prokaryotes and are among the best attested in terms of translation and evolutionary constraint. These results highlight a potentially large unexplored dimension of prokaryotic genomes.

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Section: Resultsmentioning

confidence: 99%

Shadow ORFs illuminated: long overlapping genes inPseudomonas aeruginosaare translated and under purifying selection

Kreitmeier

Ardern

Abele

et al. 2021

Preprint

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“…Although this study identified the MvaU binding site as an AT-rich region within the phz2 promoter, subsequent foot-printing analysis failed to identify the specific MvaU binding sequence. Similarly, very few MvaU binding sites have been identified in other Pseudomonas strains [ 10 , 30 ]. These results are probably due to the unique characteristics of MvaU.…”

Section: Discussionmentioning

confidence: 99%

“…MvaU is a small transcriptional regulator without the typical helix-turn-helix (HTH) DNA binding domain. MvaU typically acts as a gene silencer and regulates the expression of more than 300 genes [ 10 , 11 ]. Its binding to DNA requires specific co-factors or unique in vivo conditions that warrant further investigation.…”

Section: Discussionmentioning

confidence: 99%

“…MvaT and MvaU have been identified in Pseudomonas as paralogs of H-NS that share structural and functional similarities to H-NS, despite only sharing 20% sequence similarity with H-NS. MvaT and MvaU are thought to be structurally similar to H-NS, wherein a DNA binding domain is located at the C-terminus of MvaT, and MvaU is comparatively more conserved relative to H-NS, while the multimerization domain located at the N-terminus is more divergent [ [7] , [8] , [9] , [10] ]. Chromatin immunoprecipitation analysis coupled with DNA microarray analysis have shown that MvaU influences the expression of many genes in P. aeruginosa , including genes previously identified that may be important for virulence [ [11] , [12] , [13] ].…”

Section: Introductionmentioning

confidence: 99%

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H-NS family protein MvaU downregulates phenazine-1-carboxylic acid (PCA) biosynthesis via binding to an AT-rich region within the promoter of the phz2 gene cluster in the rhizobacterium Pseudomonas strain PA1201

Fang

Cui

Zhou

et al. 2021

Synthetic and Systems Biotechnology

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Histone-like nucleoid-structuring (H-NS) proteins are key regulators in gene expression silencing and in nucleoid compaction. The H-NS family member proteins MvaU in Pseudomonas aeruginosa are thought to bind the same AT-rich regions of chromosomes and function to coordinate the control of a common set of genes. Here, we explored the molecular mechanism by which MvaU controls PCA biosynthesis in P. aeruginosa PA1201. We present evidence suggesting that MvaU is self-regulated. Deletion of mvaU significantly increased PCA production, and PCA production sharply decreased when mvaU was over-expressed. MvaU transcriptionally repressed phz2 cluster expression and consequently reduced PCA biosynthesis. β-galactosidase assays confirmed that base pairing near the −35 box is required when MvaU regulates PCA production in PA1201. Electrophoretic mobility shift assays (EMSA) and additional point mutation analysis demonstrated that MvaU directly bound to an AT-rich motif within the promoter of the phz2 cluster. Chromatin immunoprecipitation (ChIP) analysis also indicated that MvaU directly bound to the P5 region of the phz2 cluster promoter. MvaU repression of PCA biosynthesis was independent of QscR and OxyR in PA1201 and neither PCA or H 2 O 2 were the environmental signals that induced mvaU expression. These findings detail a new MvaU-dependent regulatory pathway of PCA biosynthesis in PA1201 and provide a foundation to increase PCA fermentation titer by genetic engineering.

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“…The Pseudomonas phage LUZ24 encodes an "early protein," gp4, that binds the H-NS family protein MvaT (36). MvaT is thought to play an important role in the genome organization of P. aeruginosa and in the regulation of gene transcription (15,37). The fold architecture of the MvaT monomer consists of an a-helical N-terminal oligomerization domain (NTD: residues 1-64) tethered to a C-terminal DNA binding domain (DBD) (residues 79-124) by a flexible linker (residues 65-78) ( Figure S1b) (18).…”

Section: Introductionmentioning

confidence: 99%

Novel anti-repression mechanism of H-NS proteins by a phage “early protein”

Bdira

Liang

Volkov

et al. 2021

Preprint

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Bacterial xenogeneic silencers, the H-NS family proteins play central roles in genome organization and in the regulation of foreign genes acquired from phages or through horizontal gene transfer. It is thought that the repression of these genes is directly dependent on their DNA binding modes. H-NS family proteins bind along DNA, forming lateral protein filaments, in which the H-NS protomers adopt a "half-open" conformational state. Under specific environmental conditions and without dissociating from DNA, these proteins adopt an "open" conformational state which can bridge two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the gp4 protein, which binds the H-NS family protein MvaT of Pseudomonas aeruginosa. Binding of gp4 was suggested to inhibit the silencing of phage LUZ24 DNA by MvaT. However, the mechanism by which gp4 modulates MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations by NMR spectroscopy revealed that gp4 acts as an "electrostatic zipper" between the N-terminal and DNA binding domains of MvaT protomers and stabilizes a structure resembling their "half-open" conformation. Based on these observations, we propose that binding of gp4 promotes a half-open bridging-incompetent state of MvaT, resulting in relief of MvaT-mediated gene silencing and adverse effects on P. aeruginosa homeostasis and growth. Conformational switching of H-NS proteins driven by phage "early proteins" provides a novel anti-repression mechanism. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

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H‐NS‐like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription

Cited by 18 publications

References 97 publications

Shadow ORFs illuminated: long overlapping genes inPseudomonas aeruginosaare translated and under purifying selection

Shadow ORFs illuminated: long overlapping genes inPseudomonas aeruginosaare translated and under purifying selection

H-NS family protein MvaU downregulates phenazine-1-carboxylic acid (PCA) biosynthesis via binding to an AT-rich region within the promoter of the phz2 gene cluster in the rhizobacterium Pseudomonas strain PA1201

Novel anti-repression mechanism of H-NS proteins by a phage “early protein”

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