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An inhibitor/anti-inhibitor system controls the activity of lytic transglycosylase MltF in Pseudomonas aeruginosa

ABSTRACT: Most bacterial cell envelopes contain a cell wall layer made of peptidoglycan. The synthesis of new peptidoglycan is critical for cell growth, division and morphogenesis, and is also coordinated with peptidoglycan hydrolysis to accommodate the new material. However, the enzymes that cleave peptidoglycan must be carefully controlled to avoid autolysis. In recent years, some control mechanisms have begun to emerge, although there are many more questions than answers for how most cell wall hydrolases are regulated. Here, we report a novel cell wall hydrolase control mechanism in Pseudomonas aeruginosa, which we discovered during our characterization of a mutant sensitive to the overproduction of a secretin protein. The mutation affected an uncharacterized Sel1-like repeat protein encoded by the PA3978 locus. In addition to the secretin-sensitivity phenotype, PA3978 disruption also increased resistance to a b-lactam antibiotic used in the clinic. Affinity purification followed by mass spectrometry on the C-terminal flag tagged PA3978 protein revealed that PA3978 binds to the lytic transglycosylase MltF. In vitro and In vivo analysis revealed that the binding occurs on the catalytic domain of the lytic transglycosylase MltF and inhibits its activity. We also discovered another interaction partner of PA3978 encoded by the PA5502 locus. The phenotypes of a deltaPA5502 mutant suggested that PA5502 interferes with the inhibitory function of PA3978 towards MltF, and we confirmed that activity for PA5502 in vitro. Therefore, PA3978 and PA5502 form an inhibitor/anti-inhibitor system that controls MltF activity. We propose to name these proteins IltA (inhibitor of lytic transglycosylase) and LiiA (lytic transglycosylase inhibitor, inhibitor). The mass spectrometry raw files and search results for the affinity purification of PA3978 are deposited here.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Pseudomonas Aeruginosa (ncbitaxon:287)

SUBMITTER: Beatrix Ueberheide

PROVIDER: MSV000093002 | MassIVE |

SECONDARY ACCESSION(S): PXD045827

REPOSITORIES: MassIVE

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Project description:Chromatin-organizing factors, like CTCF and cohesins, have been implicated in the control of complex viral regulatory programs. We investigated the role of CTCF and cohesin in the control of the latent to lytic switch for Kaposi's Sarcoma-Associated Herpesvirus (KSHV). We found that cohesin subunits, but not CTCF, were required for the repression of KSHV immediate early gene transcription. Depletion of cohesin subunits Rad21, SMC1, or SMC3 resulted in lytic cycle gene transcription and viral DNA replication. In contrast, depletion of CTCF failed to induce lytic transcription or DNA replication. ChiP-Seq analysis revealed that cohesins and CTCF bound to several sites within the immediate early control regions for ORF50 and more distal 5' sites that also regulate the divergently transcribed ORF45-46-47 gene cluster. Rad21 depletion led to a robust increase in ORF45 and ORF47 transcripts, with similar kinetics to that observed with chemical induction by sodium butyrate. During latency, the chromatin between the ORF45 and ORF50 transcription start sites was enriched in histone H3K4me3 with elevated H3K9ac at the ORF45 promoter and elevated H3K27me3 at the ORF50 promoter. A paused form of RNA pol II was loosely associated with the ORF45 promoter region during latency, but was converted to an active elongating form upon reactivation induced by Rad21 depletion. Butyrate-induced transcription of ORF45 and ORF47 was resistant to cyclohexamide, suggesting that these genes have immediate early features similar to ORF50. Butyrate-treatment caused the rapid dissociation of cohesins and loss of CTCF binding at the immediate early gene locus, suggesting that cohesins may be a direct target of butyrate-mediated lytic induction. Our findings implicate cohesins as a major repressor of KSHV lytic gene activation, and function coordinately with CTCF to regulate the switch between latent and lytic gene activity. Study of chromatin-organizing factors, like CTCF and cohesins.

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An inhibitor/anti-inhibitor system controls the activity of lytic transglycosylase MltF in Pseudomonas aeruginosa

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