When bacterial cells come in contact, antagonism mediated by the delivery of toxins frequently ensues. The potential for such encounters to have long-term beneficial consequences in recipient cells has not been investigated. Here, we examined the effects of intoxication by DddA, a cytosine deaminase delivered via the type VI secretion system (T6SS) of Burkholderia cenocepacia. Despite its killing potential, we observed that several bacterial species resist DddA and instead accumulate mutations. These mutations can lead to the acquisition of antibiotic resistance, indicating that even in the absence of killing, interbacterial antagonism can have profound consequences on target populations. Investigation of additional toxins from the deaminase superfamily revealed that mutagenic activity is a common feature of these proteins, including a representative we show targets single-stranded DNA and displays a markedly divergent structure. Our findings suggest that a surprising consequence of antagonistic interactions between bacteria could be the promotion of adaptation via the action of directly mutagenic toxins.
Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase-isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase-isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy and tandem mass spectrometry. Our results show that MpeV is the lyase-isomerase that covalently attaches a doubly-linked phycourobilin to two cysteine residues (C50, C61) on the β-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization and stability. These data provide novel insights into β-PE biosynthesis and advance our understanding of the mechanisms guiding lyase-isomerases.
Edited by Chris Whitfield Phycoerythrin (PE) is a green light-absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due to its prosthetic groups, or phycoerythrobilins (PEBs), that are covalently attached to the protein chain by specific bilin lyases. Only two PE lyases have been identified and characterized so far, and the other bilin lyases are unknown. Here, using in silico analyses, markerless deletion, biochemical assays with purified and recombinant proteins, and site-directed mutagenesis, we examined the role of a putative lyase-encoding gene, cpeF, in the cyanobacterium Fremyella diplosiphon. Analyzing the phenotype of the cpeF deletion, we found that cpeF is required for proper PE biogenesis, specifically for ligation of the doubly linked PEB to Cys-48/Cys-59 residues of the CpeB subunit of PE. We also show that in a heterologous host, CpeF can attach PEB to Cys-48/ Cys-59 of CpeB, but only in the presence of the chaperone-like protein CpeZ. Additionally, we report that CpeF likely ligates the A ring of PEB to Cys-48 prior to the attachment of the D ring to Cys-59. We conclude that CpeF is the bilin lyase responsible for attachment of the doubly ligated PEB to Cys-48/Cys-59 of CpeB and together with other specific bilin lyases contributes to the post-translational modification and assembly of PE into mature light-harvesting complexes. This work was supported by National Science Foundation Grants MCB-1029414 and MCB-1818187 (to D. M. K.) and MCB-1244339 and MCB-0843664 (to W. M. S.) and by a University of New Orleans Graduate School performance and accountability fellowship (to C. M. K.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S3 and Tables S1-S4.
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