Understanding the molecular mechanisms of bacterial antibiotic resistance will help prepare against further emergence of multi-drug resistant strains. MacQ is an enzyme responsible for the multi-drug resistance of Acidovorax sp. strain MR-S7. MacQ has acylase activity against both N-acylhomoserine lactones (AHLs), a class of signalling compounds involved in quorum sensing, and β-lactam antibiotics. Thus, MacQ is crucial as a quencher of quorum sensing as well as in conferring antibiotic resistance in Acidovorax. Here, we report the X-ray structures of MacQ in ligand-free and reaction product complexes. MacQ forms a 170-kDa capsule-shaped molecule via face-to-face interaction with two heterodimers consisting of an α-chain and a β-chain, generated by the self-cleaving activity of a precursor polypeptide. The electron density of the spacer polypeptide in the hollow of the molecule revealed the close orientation of the peptide-bond atoms of Val20SP-Gly21SP to the active-site, implying a role of the residues in substrate binding. In mutational analyses, uncleaved MacQ retained degradation activity against both AHLs and penicillin G. These results provide novel insights into the mechanism of self-cleaving maturation and enzymatic function of N-terminal nucleophile hydrolases.
Pseudomonas putida KT2440 encodes three homologs of the LitR/CarH family (designated PplR1–PplR3), which is an adenosyl B12-dependent light-sensitive MerR family transcriptional regulator. Transcriptome analysis revealed the existence of a number of photo-inducible genes including pplR1, phrB (encoding DNA photolyase), ufaM (furan-containing fatty acid synthase), folE (GTP cyclohydrolase I), cryB (cryptochrome-like protein), and multiple genes without annotated/known function. Transcriptional analysis by quantitative RT-PCR with knockout mutants of pplR1–pplR3 showed that the triple knockout completely abolished the light-inducible transcription in P. putida, which indicates the occurrence of ternary regulation of PplR proteins. DNase I footprint assay showed that PplR1 protein specifically binds to the promoter regions of light-inducible genes, suggesting a consensus PplR1-binding direct repeat, 5′-T(G/A)TACAn12TGTA(C/T)A-3′. The disruption of B12 biosynthesis cluster did not affect the light-inducible transcription; however, disruption of ppSB1-LOV and ppSB2-LOV, encoding blue light photoreceptors adjacently located to pplR3 and pplR2, respectively, led to the complete loss of light-inducible transcription. Overall, the results suggest that the three PplRs and two PpSB-LOVs cooperatively regulate the light-inducible gene expression. The wide distribution of the pplR/ppSB-LOV cognate pair homologs in Pseudomonas spp. and related bacteria suggests that the response and adaptation to light is similarly regulated in the group of non-phototrophic bacteria. Importance LitR/CarH family is a new group of photosensor homologous to MerR-type transcriptional regulators. Proteins of this family are distributed to various non-phototrophic bacteria and grouped into at least five (I-V) classes. Pseudomonas putida retaining three class II LitR proteins exhibited genome-wide response to light. All three paralogs were functional and mediated photo-dependent activation of promoters directing the transcription of light-induced genes or operons. Two LOV (light-oxygen-voltage)-domain proteins adjacently encoded to two litR genes were also essential for the photo-dependent transcriptional control. Despite the difference in light-sensing mechanism, the DNA binding consensus of class II LitR [T(G/A)TA(C/T)A] was the same as that of class I. This is the first study showing the actual involvement of class II LitR in light-induced transcription.
PmlR2, a class II LitR/CarH family transcriptional regulator, and PmSB-LOV, a “short” LOV-type blue light photoreceptor, are adjacently encoded in Pseudomonas mendocina NBRC 14162. An effector protein for the “short” LOV-type photoreceptor in Pseudomonas has not yet been identified. Here, we show that PmlR2 is an effector protein of PmSB-LOV. Transcriptional analyses revealed that the expression of genes located near pmlR2 and its homolog gene, pmlR1, was induced in response to illumination. In vitro DNA–protein binding analyses showed that recombinant PmlR2 directly binds to the promoter region of light-inducible genes. Furthermore PmSB-LOV exhibited a typical LOV-type light-induced spectral change. Gel-filtration chromatography demonstrated that the illuminated PmSB-LOV was directly associated with PmlR2, whereas non-illuminated proteins did not interact. The inhibition of PmlR2 function following PmSB-LOV binding was verified by surface plasmon resonance: the DNA-binding ability of PmlR2 was specifically inhibited in the presence of blue light-illuminated-PmSB-LOV. An In vitro transcription assay showed a dose-dependent reduction in PmlR2 repressor activity in the presence of illuminated PmSB-LOV. Overall, evidence suggests that the DNA-binding activity of PmlR2 is inhibited by its direct association with blue light-activated PmSB-LOV, enabling transcription of light-inducible promoters by RNA polymerase.
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