Phage resistance profiling identifies new genes required for biogenesis and modification of the corynebacterial cell envelope
Abstract
Bacteria of the order Corynebacteriales including pathogens such as Mycobacterium tuberculosi and Corynebacterium diphtheriae are characterized by their complex, multi-layered envelope. In addition to a peptidoglycan layer, these organisms possess an additional polysaccharide layer made of arabinogalactan and an outer membrane layer composed predominantly of long-chain fatty acids called mycolic acids. This so-called mycolata envelope structure is both a potent barrier against antibiotic entry into cells and a target of several antibacterial therapeutics. A better understanding of the mechanisms underlying mycolata envelope assembly therefore promises to reveal new ways of disrupting this unique structure for the development of antibiotics and antibiotic potentiators. Because they engage with receptors on the cell surface during infection, bacteriophages have long been used as tools to uncover important aspects of host envelope assembly. However, surprisingly little is known about the interactions between Corynebacteriales phages and their hosts. We therefore made use of the phages Cog and CL31 that infect Corynebacterium glutamicum (Cglu), a model member of the Corynebacteriales, to discover host factors important for phage infection. A high-density transposon library of Cglu was challenged with these phages followed by transposon sequencing to identify resistance loci. The analysis identified an important role for mycomembrane proteins in phage infection as well as components of the arabinogalactan and mycolic acid synthesis pathways. Importantly, the approach also implicated a new gene (cgp_0396) in the process of arabinogalactan modification and identified a conserved new factor (AhfA, Cpg_0475) required for mycolic acid synthesis in Cglu.
Data availability
Sequencing data generated from this study have been deposited in the NCBI Sequence Read Archive under BioProject PRJNA834153. All other data generated or analyzed during this study are provided in the manuscript and supporting files.
-
Phenotypic profiling of a Corynebacterium glutamicum transposon libraryNCBI BioProject PRJNA610521.
Article and author information
Author details
Funding
Howard Hughes Medical Institute
- Thomas G Bernhardt
Life Sciences Research Foundation
- Amelia C McKitterick
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, McKitterick & Bernhardt
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,025
- views
-
- 302
- downloads
-
- 6
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Genetics and Genomics
- Microbiology and Infectious Disease
Polyamines are biologically ubiquitous cations that bind to nucleic acids, ribosomes, and phospholipids and, thereby, modulate numerous processes, including surface motility in Escherichia coli. We characterized the metabolic pathways that contribute to polyamine-dependent control of surface motility in the commonly used strain W3110 and the transcriptome of a mutant lacking a putrescine synthetic pathway that was required for surface motility. Genetic analysis showed that surface motility required type 1 pili, the simultaneous presence of two independent putrescine anabolic pathways, and modulation by putrescine transport and catabolism. An immunological assay for FimA—the major pili subunit, reverse transcription quantitative PCR of fimA, and transmission electron microscopy confirmed that pili synthesis required putrescine. Comparative RNAseq analysis of a wild type and ΔspeB mutant which exhibits impaired pili synthesis showed that the latter had fewer transcripts for pili structural genes and for fimB which codes for the phase variation recombinase that orients the fim operon promoter in the ON phase, although loss of speB did not affect the promoter orientation. Results from the RNAseq analysis also suggested (a) changes in transcripts for several transcription factor genes that affect fim operon expression, (b) compensatory mechanisms for low putrescine which implies a putrescine homeostatic network, and (c) decreased transcripts of genes for oxidative energy metabolism and iron transport which a previous genetic analysis suggests may be sufficient to account for the pili defect in putrescine synthesis mutants. We conclude that pili synthesis requires putrescine and putrescine concentration is controlled by a complex homeostatic network that includes the genes of oxidative energy metabolism.
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.