Molecular Microbiology

doi: 10.1111/mmi.70065pmid: N/A

Nhan, Dinh Quan; Michalska, Karolina; Garza‐Sánchez, Fernando; Bartelli, Nicholas L.; Willett, Julia L. E.; Stols, Lucy; Eschenfeldt, William H.; Goulding, Celia W.; Joachimiak, Andrzej; Hayes, Christopher S.

doi: 10.1111/mmi.70053pmid: 41559884

Many Gram‐negative bacterial species use contact‐dependent growth inhibition (CDI) systems to deliver toxic proteins into neighboring competitors. CDI+ strains deploy CdiA effector proteins, which translocate their C‐terminal toxin (CT) domains into target bacteria through a receptor‐mediated delivery pathway. To protect against auto‐intoxication, CDI+ bacteria also produce CdiI immunity proteins that neutralize CT toxin activity. Here, we present the crystal structure of the CT·CdiIO32:H37 complex from Escherichia coli O32:H37. CTO32:H37 adopts the same fold as the tRNase domain of colicin D, and the nucleases share similar catalytic centers. However, unlike colicin D, which cleaves the anticodon loops of tRNAArg isoacceptors, CTO32:H37 exhibits nonspecific RNase activity. Notably, we find that endogenous elongation factor Tu (EF‐Tu) co‐purifies with the over‐produced CT·CdiIO32:H37 complex. Although EF‐Tu does not bind stably to CTO32:H37 in the absence of CdiIO32:H37, the translation factor is required for toxic RNase activity in vitro. AlphaFold 3 modeling and site‐directed mutagenesis indicate that CTO32:H37 interacts with the N‐terminal GTPase domain of EF‐Tu. EF‐Tu appears to stabilize residue Trp52 within the hydrophobic core of the toxin, which in turn supports the RNase active site through an unusual hydrogen‐bonding interaction with the catalytic His67 residue. Thus, EF‐Tu is hijacked as an essential co‐factor to organize the toxin's catalytic center.

Faber, Anne Amalie Winther; Nielsen, Michelle; Madhavan, Vishnu Narayanan; Hansen, Lykke Haastrup; Kirkpatrick, Clare L.

doi: 10.1111/mmi.70055pmid: 41665052

Two‐component systems, comprising a sensor kinase and a response regulator, are very important for bacteria to respond to stress in their environment. In alpha‐proteobacteria, two such systems (ChvIG and NtrYX) help bacteria to respond to cell envelope and acid stress, respectively. In contrast to the majority of two‐component systems, which do not cross talk with one another, ChvIG and NtrYX in Caulobacter are tightly functionally linked and regulate many of the same genes. Moreover, the antagonistic balance between phosphorylated ChvI and non‐phosphorylated NtrX is essential for growth in defined medium. We show that the NtrX‐ChvI balance is also critical for swimming motility in defined medium (M2G) stress conditions, and that loss of motility in M2G in ΔchvI or ΔchvG mutants is rescued by dominant gain‐of‐function mutations in ntrYX. These mutations promote a regulatory switch of NtrX away from the genes that are regulated by NtrX in its non‐phosphorylated form, at least one of which is jointly bound by ChvI and NtrX at the same binding site. The data support a model where these two systems co‐operate tightly as a stress response mediator, almost to the extent that they could be considered a four‐component system, and that ChvI is required for NtrX to regulate some of its target genes.

Goyal, Nisha; Mudgal, Sudhanshu; Saginela, Rahul; Dagur, Hanuman Singh; Eerappa, Rajakumara; Sinha, Krishna Murari

doi: 10.1111/mmi.70057pmid: 41676876

Mycobacterium possesses unique DNA repair and recombination pathways that are absent in E. coli. The pathways are linked to mycobacterial virulence and survival within the host. Cyclic di‐AMP, a secondary messenger present in Mycobacterium regulates DNA recombination, genome stability and SOS response. radA and disA exist in a conserved operon in several bacteria including Mycobacterium suggesting a role of RadA in cyclic di‐AMP mediated DNA damage repair pathways. We show here that RadA is indeed involved in cyclic di‐AMP mediated DSB repair pathways in Mycobacterium and has non‐canonical function in DSB repair as well. Deletion of radA expectedly decreases HR efficiency but interestingly also results in illegitimate recombination outcome GC*. The illegitimate recombination GC* leads to increased stress induced mutagenesis giving rise to drug resistant mutants. We have also found that RadA influences NHEJ repair and in its absence the cells adopt alternative route of DNA repair. Our work shows active participation of RadA in several DNA repair and recombination pathways which could be targeted for potential therapeutics against Mycobacterium.

Zou, Qi; Cronan, John E.

doi: 10.1111/mmi.70054pmid: 41697937

Yamaguchi, Honoka; Ago, Risa; Tahara, Yuhei O.; Inoue, Mari; Niki, Hironori; Miyata, Makoto; Shiomi, Daisuke

doi: 10.1111/mmi.70058pmid: 41721466

Peptidoglycan synthesis and degradation are both essential for bacterial growth, and damaged peptidoglycan must be continuously repaired. In Escherichia coli, peptidoglycan required for cell elongation is synthesized by the Rod complex. Although RodZ is a non‐essential component of this complex, its dysfunction leads to aberrant peptidoglycan synthesis, resulting in defects in cell shape and impaired growth. We previously isolated several suppressor mutants that restore growth in cells with impaired RodZ function (RMR cells). Most suppressor mutations mapped to components in the Rod complex other than RodZ. However, one suppressor mutation was identified in sanA, a gene not previously associated with the Rod complex. This mutation, sanAM27R, represents a loss‐of‐function allele. Here, we show that SanA is associated with PBP1B, a non‐essential yet physiologically important peptidoglycan synthase. Loss of SanA function partially restored the growth of RMR cells, accompanied by enhanced peptidoglycan synthesis and alleviation of structural defects in the cell wall. These findings indicate that SanA contributes to the regulation of peptidoglycan synthesis and cell wall integrity, potentially through functional interplay with PBP1B‐dependent pathways.