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Microcin C is a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate. Microcin C is a potent inhibitor of bacterial cell growth. Based on the in vivo kinetics of inhibition of macromolecular synthesis, Microcin C targets translation, through a mechanism that remained undefined. Here, we show that Microcin C is a subject of specific degradation inside the sensitive cell. The product of degradation, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, strongly inhibits translation by blocking the function of aspartyl-tRNA synthetase.Microcins are a class of small (Ͻ10 kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (1). Whereas some microcins are active as unmodified peptides (2), others are produced as polypeptide precursors that are heavily modified by dedicated maturation enzymes (3). Interest is attached to such post-translationally modified microcins due to their highly unusual structures and the fact that they target important cellular processes that are attractive targets for antibacterial drug development.Genes responsible for microcin production are usually plasmidborne. Plasmids encoding microcin structural and maturation genes also encode determinants of immunity specific to the microcin produced. Based on cross-immunity, post-translationally modified microcins can be subdivided into the B, C, and J types. Microcin B (MccB) 4 is a 43-residue peptide with 8 thiazole and oxazole rings that are synthesized by the McbBCD maturation enzyme complex from multiple serine and cysteine residues present in the MccB precursor (4). MccB is a potent inhibitor of DNA gyrase; it traps the enzyme at the stage of DNA strand passage (5). Microcin J, a 21-amino acid peptide, contains an unusual lactam bond between its N-terminal glycine and the ␦-carboxyl group of an internal glutamate; it assumes a highly unusual threaded-lasso structure (6 -8). MccJ inhibits bacterial RNA polymerase by occluding a narrow channel that is used to traffic transcription substrates, NTPs, to the catalytic center of the enzyme (9, 10).The structure of the subject of this study, Microcin C (McC) is shown in Fig. 1A. McC is a heptapeptide containing a modified adenosine monophosphate covalently attached to its C terminus through an N-acylphosphoramidate linkage (11, 12). The phosphoramidate group of the nucleotide part of McC is additionally modified by a propylamine group. Additionally, in mature McC, the peptide moiety, which is encoded by the mccA gene, is modified and the C-terminal asparagine residue specified by mccA is converted to an aspartate (18, 19), through an unknown mechanism. In vivo, McC appears to target translation (12). Guijarro et al. (12) also reported that large concentrations of McC, as well as of synthetic peptide of the same sequence but without the nucleotide modification, mildly inhibit translation in vitro. They therefore concluded that the peptide part of McC is responsible for transl...

Aim: To examine the biocontrol activity of broad-range antagonists Serratia plymuthica IC1270, Pseudomonas fluorescens Q8r1-96 and P. fluorescens B-4117 against tumourigenic strains of Agrobacterium tumefaciens and A. vitis. Methods and Results: Under greenhouse conditions, the antagonists, applied via root soak prior to injecting Agrobacterium strains into the wounded stems, significantly suppressed tumour development on tomato seedlings. A derivative of P. fluorescens Q8r1-96 tagged with a gfp reporter, as well as P. fluorescens B-4117 and S. plymuthica IC1270 marked with rifampicin resistance, stably persisted in tomato tissues for at least 1 month. Mutants of P. fluorescens Q8r1-96 and S. plymuthica IC1270 deficient in 2,4-diacetylphloroglucinol or pyrrolnitrin production, respectively, also proficiently suppressed the tumour development, indicating that these antibiotics are not responsible for the observed biocontrol effect on crown gall disease. The volatile organic compounds (VOCs) produced by the tested P. fluorescens and S. plymuthica strains inhibited the growth of A. tumefaciens and A. vitis strains in vitro. Solid-phase microextraction-gas chromatography-mass spectrometry analysis revealed dimethyl disulfide (DMDS) as the major headspace volatile produced by S. plymuthica IC1270; it strongly suppressed Agrobacterium growth in vitro and was emitted by tomato plants treated with S. plymuthica IC1270. 1-Undecene was the main volatile emitted by the examined P. fluorescens strains, with other volatiles, including DMDS, being detected in only relatively low quantities. Conclusions: S. plymuthica IC1270, P. fluorescens B-4117 and P. fluorescens Q8r1-96 can be used as novel biocontrol agents of pathogenic Agrobacterium. VOCs, and specifically DMDS, might be involved in the suppression of oncogenicity in tomato plants. However, the role of specific volatiles in the biocontrol activity remains to be elucidated. Significance and Impact of the Study: The advantage of applying these antagonists lies in their multiple activities against a number of plant pathogens, including Agrobacterium.

In previous research, volatile organic compounds (VOCs) emitted by various bacteria into the chemosphere were suggested to play a significant role in the antagonistic interactions between microorganisms occupying the same ecological niche and between bacteria and target eukaryotes. Moreover, a number of volatiles released by bacteria were reported to suppress quorum-sensing cell-to-cell communication in bacteria, and to stimulate plant growth. Here, volatiles produced by Pseudomonas and Serratia strains isolated mainly from the soil or rhizosphere exhibited bacteriostatic action on phytopathogenic Agrobacterium tumefaciens and fungi and demonstrated a killing effect on cyanobacteria, flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans). VOCs emitted by the rhizospheric Pseudomonas chlororaphis strain 449 and by Serratia proteamaculans strain 94 isolated from spoiled meat were identified using gas chromatography-mass spectrometry analysis, and the effects of the main headspace compounds—ketones (2-nonanone, 2-heptanone, 2-undecanone) and dimethyl disulfide—were inhibitory toward the tested microorganisms, nematodes, and flies. The data confirmed the role of bacterial volatiles as important compounds involved in interactions between organisms under natural ecological conditions.

We show that volatile organic compounds (VOCs) produced by rhizospheric strains Pseudomonas fluorescens B-4117 and Serratia plymuthica IC1270 may act as inhibitors of the cell-cell communication quorum-sensing (QS) network mediated by N-acyl homoserine lactone (AHL) signal molecules produced by various bacteria, including strains of Agrobacterium, Chromobacterium, Pectobacterium and Pseudomonas. This quorum-quenching effect was observed when AHL-producing bacteria were treated with VOCs emitted by strains B-4117 and IC1270 or with dimethyl disulfide (DMDS), the major volatile produced by strain IC1270. LC-MS/MS analysis revealed that treatment of strains Pseudomonas chlororaphis 449, Pseudomonas aeruginosa PAO1 or Ps. fluorescens 2-79 with VOCs emitted by strain IC1270 or DMDS drastically decreases the amount of AHLs produced by these bacteria. Volatile organic compounds produced by Ps. chlororaphis 449 were able to suppress its own QS-induction activity, suggesting a negative interaction between VOCs and AHL molecules in the same strain. Quantitative RT-PCR analysis showed that treatment of Ps. chlororaphis 449 with VOCs emitted by cells of IC1270, B-4117 or 449 itself, or with DMDS, leads to significant suppression of transcription of AHL synthase genes phzI and csaI. Thus, along with AHLs, bacterial volatiles might be considered another type of signal molecule involved in microbial communication in the rhizosphere.

In the natural environment, bacteria predominantly exist in matrix-enclosed multicellular communities associated with various surfaces, referred to as biofilms. Bacteria in biofilms are extremely resistant to antibacterial agents thus causing serious problems for antimicrobial therapy. In this study, we showed that different plant phenolic compounds, at concentrations that did not or weakly suppressed bacterial growth, increased the capacity of Pseudomonas aeruginosa PAO1 to form biofilms. Biofilm formation of P. aeruginosa PAO1 was enhanced 3- to 7-fold under the action of vanillin and epicatechin, and 2- to 2.5-fold in the presence of 4-hydroxybenzoic, gallic, cinnamic, sinapic, ferulic, and chlorogenic acids. At higher concentrations, these compounds displayed an inhibiting effect. Similar experiments carried out for comparison with Agrobacterium tumefaciens C58 showed the same pattern. Vanillin, 4-hydroxybenzoic, and gallic acids at concentrations within the range of 40 to 400 μg/mL increased the production of N-3-oxo-dodecanoyl-homoserine lactone in P. aeruginosa PAO1 which suggests a possible relationship between stimulation of biofilm formation and Las Quorum Sensing system of this bacterium. Using biosensors to detect N-acyl-homoserine lactones (AHL), we demonstrated that the plant phenolics studied did not mimic AHLs.

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