Distinct domains of Escherichia coli IgaA connect envelope stress sensing and down-regulation of the Rcs phosphorelay across subcellular compartments

Hussein, Nahla A.; Cho, Seung-Hyun; Laloux, Géraldine; Siam, Rania; Collet, Jean-François

doi:10.1371/journal.pgen.1007398

Cited by 41 publications

(80 citation statements)

References 40 publications

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“…The periplasmic domain of IgaA (see Fig 4B) has previously been found to interact with RcsF [8]. Here, we find that deletion of the periplasmic domain (IgaA Δ384-649 ) fully abrogates the interaction of RcsD and IgaA; T411A modestly restores this interaction, consistent with the mutant leading to increased interaction within the cytoplasmic regions (Fig 4B and S4B Fig).…”

Section: Resultssupporting

confidence: 86%

“…For this reason, the poorly understood IgaA mechanism of action is of key interest. Multiple studies have focused on the interaction of RcsF with IgaA when cell wall stress is detected [3–7], but the downstream action of IgaA is less well understood [8]. In this work we define RcsD as the direct binding partner of IgaA and define the regions in RcsD that are critical for interaction with IgaA.…”

Section: Introductionmentioning

confidence: 99%

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Negative regulation of the Rcs phosphorelay via IgaA contact with the RcsD phosphotransfer protein

Wall

Majdalani

Gottesman

2020

Preprint

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13Two-component systems and phosphorelays play central roles in the ability of bacteria 14 to rapidly respond to changing environments. In E. coli and related enterobacteria, the 15 complex Rcs phosphorelay is a critical player in changing bacterial behavior in response to 16 antimicrobial peptides, beta-lactam antibiotics, and other challenges to the cell surface. The 17Rcs system is unusual in that IgaA, an inner membrane protein, is essential due to its negative 18 regulation of the RcsC/RcsD/RcsB phosphorelay. While it has previously been shown that IgaA 19 transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the 20 phosphorelay was unknown. Here we use in vivo interaction assays and genetic dissection of 21 the critical proteins to demonstrate that IgaA interacts with the phosphorelay protein RcsD, and 22 Author Summary 35The Rcs phosphorelay plays a central role in allowing enterobacteria to sense and respond to 36 antibiotics, host-produced antimicrobials, and interactions with surfaces. A unique negative regulator, 37IgaA, keeps signaling from this pathway under control when it is not needed, but how it controls the 38 phosphorelay has been unclear. We define a set of critical interactions between IgaA and the 39 phosphotransfer protein RcsD. A periplasmic contact between IgaA and RcsD provides a necessary 40 inhibition of Rcs signaling, modulated further by regulated interactions in the cytoplasmic domains of 41 each protein. This multipartite interaction provides a sensitive regulatory switch. 42IgaA negatively regulates the Rcs Phosphorelay via contact with RcsD 4 43 101 cause the loss of ability to de-phosphorylate RcsB that has acquired phosphate from other 102 sources [14][15][16]. In our assay conditions, the rcsC deletion strain produces a signal that is 3-4 103 fold above WT. 104 The rcsD deletion shown, rcsD541, increased PrprA-mCherry expression in a manner 105 similar to the rcsC deletion. rcsD is encoded upstream of rcsB, with the major promoters for 106 rcsB inside the rcsD coding region [17]. This affects the way rcsD deletion alleles can be 107 constructed. In addition, in both Salmonella and E. coli, some transcripts from the rcsD 108 IgaA negatively regulates the Rcs Phosphorelay via contact with RcsD 7 promoter may continue through to rcsB, though apparently at a much lower level [17, 18]. 109 Four different rcsD alleles were examined, each designed to leave the rcsB promoters intact 110 (S1D Fig). These include rcsD carrying an H842A mutation in the phosphotransfer domain 111 active site, as well as rcsD containing stop codons after codon 841 (rcsD841*). The overall 112 amount of PrprA-mCherry expression seems to differ modestly between rcsD541 and rcsD543, 113 even though the deleted regions in both alleles share the same boundaries. rcsD541 also gave 114 higher PrprA-mCherry signal than the point mutants (S1D Fig). When checked by western blot 115 with a polyclonal RcsD antibody, it is evident that rcsD541, rcsD543 and rcsD841* are all dev...

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Section: Resultssupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Negative regulation of the Rcs phosphorelay via IgaA contact with the RcsD phosphotransfer protein

Wall

Majdalani

Gottesman

2020

Preprint

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“…These results indicate that the igaA disruption mutant uncovered in this work activates colanic acid biosynthesis, leads to a mucoidy phenotype, and may be interfering with phage infection by blocking phage receptor accessibility. The IgaA residues 18-164 we mutate in this work overlap with the N-terminal cytoplasmic domain of IgaA that inhibits Rcs signaling in the absence of stress [159]. Recent studies have highlighted that the activation of the Rcs pathway and capsular biosynthesis are essential to survive in diverse environmental contexts, membrane damage and stress-inducing conditions, including those by antibiotic treatment [101,160].…”

Section: Overexpression Of Colanic Acid Biosynthesis Pathway Reduces mentioning

confidence: 99%

“…S1: Map of igaA mutants: igaA mutants and their fitness scores from E. coli K-12 RB-TnSeq screen. Schematic of rcs phosphorelay with the predicted topology of IgaA [159] is shown at the top. RB-TnSeq mutants in IgaA are mapped to the predicted topology of IgaA (top) and also mapped on to igaA nucleotide sequence, with mutant position and fitness scores.…”

Section: Supplementary Notesmentioning

confidence: 99%

High-throughput mapping of the phage resistance landscape inE. coli

Mutalik¹,

Adler²,

Rishi³

et al. 2020

Preprint

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Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and highthroughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phagemediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.3 Introduction:

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“…Interestingly, we previously determined that sending RcsF to the surface is part of a cellular strategy that enables RcsF to detect damage in the cell envelope. Under stress conditions, newly synthesized RcsF molecules fail to interact with BamA 10 : they are not exported to the surface and remain exposed to the periplasm, which allows them to trigger the Rcs signaling cascade by reaching the downstream Rcs partner in the inner membrane 16 . Thus, surface exposure is intimately linked to the function of RcsF.…”

mentioning

confidence: 99%

Structural insight into the formation of lipoprotein-β-barrel complexes by the β-barrel assembly machinery

Létoquart

Rodríguez-Alonso

et al. 2019

Preprint

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20 The b-barrel assembly machinery (BAM) inserts outer membrane b-barrel proteins 21 (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM 22 also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling 23 RcsF-OMP complexes. Although BAM has been the focus of intense research due to its 24 essential activity in generating and maintaining the outer membrane, how it functions 25 assembly of E. coli's diverse set of OMPs, only BamA and BamD are essential and conserved 51 throughout Gram-negative bacteria 1,2 . Despite important structural and functional insights 52 during 15 years of intense scrutiny, crucial questions remain unsolved regarding BAM's 53 mechanism. In particular, the functional importance of BamA cycling between the outward-54 open and inward-open conformations remains unclear, as are the respective contributions of 55 the various BAM components to OMP assembly 9 . 56 57

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Distinct domains of Escherichia coli IgaA connect envelope stress sensing and down-regulation of the Rcs phosphorelay across subcellular compartments

Cited by 41 publications

References 40 publications

Negative regulation of the Rcs phosphorelay via IgaA contact with the RcsD phosphotransfer protein

Negative regulation of the Rcs phosphorelay via IgaA contact with the RcsD phosphotransfer protein

High-throughput mapping of the phage resistance landscape inE. coli

Structural insight into the formation of lipoprotein-β-barrel complexes by the β-barrel assembly machinery

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