Alterocin, an Antibiofilm Protein Secreted by Pseudoalteromonas sp. Strain 3J6

Abstract

We sought to identify and study the antibiofilm protein secreted by the marine bacterium Pseudoalteromonas sp. strain 3J6. The latter is active against marine and terrestrial bacteria, including Pseudomonas aeruginosa clinical strains forming different biofilm types. Several amino acid sequences were obtained from the partially purified antibiofilm protein, named alterocin. The Pseudoalteromonas sp. 3J6 genome was sequenced, and a candidate alt gene was identified by comparing the genome-encoded proteins to the sequences from purified alterocin. Expressing the alt gene in another nonactive Pseudoalteromonas sp. strain, 3J3, demonstrated that it is responsible for the antibiofilm activity. Alterocin is a 139-residue protein that includes a predicted 20-residue signal sequence, which would be cleaved off upon export by the general secretion system. No sequence homology was found between alterocin and proteins of known functions. The alt gene is not part of an operon and adjacent genes do not seem related to alterocin production, immunity, or regulation, suggesting that these functions are not fulfilled by devoted proteins. During growth in liquid medium, the alt mRNA level peaked during the stationary phase. A single promoter was experimentally identified, and several inverted repeats could be binding sites for regulators. alt genes were found in about 30% of the Pseudoalteromonas genomes and in only a few instances of other marine bacteria of the Hahella and Paraglaciecola genera. Comparative genomics yielded the hypothesis that alt gene losses occurred within the Pseudoalteromonas genus. Overall, alterocin is a novel kind of antibiofilm protein of ecological and biotechnological interest.IMPORTANCE Biofilms are microbial communities that develop on solid surfaces or interfaces and are detrimental in a number of fields, including for example food industry, aquaculture, and medicine. In the latter, antibiotics are insufficient to clear biofilm infections, leading to chronic infections such as in the case of infection by Pseudomonas aeruginosa of the lungs of cystic fibrosis patients. Antibiofilm molecules are thus urgently needed to be used in conjunction with conventional antibiotics, as well as in other fields of application, especially if they are environmentally friendly molecules. Here, we describe alterocin, a novel antibiofilm protein secreted by a marine bacterium belonging to the Pseudoalteromonas genus, and its gene. Alterocin homologs were found in about 30% of Pseudoalteromonas strains, indicating that this new family of antibiofilm proteins likely plays an important albeit nonessential function in the biology of these bacteria. This study opens up the possibility of a variety of applications.

Keywords: Pseudoalteromonas; Pseudomonas aeruginosa; antibiofilm protein; biofilm.

FIG 1

Antibiofilm activity of Pseudoalteromonas sp. 3J6 exoproducts (SN_3J6) on P. aeruginosa clinical strains. P. aeruginosa MUC-N1 (A) and MUC-N2 (B) biofilms obtained without SN_3J6 (control or C) or in the presence of SN_3J6 (+ SN_3J6) during the attachment step. Biofilms were then grown for 24 h under a flow of LB medium, bacteria were stained with SYTO 9 green, β-polysaccharides (including alginate) were visualized with calcofluor white (blue), and eDNA was stained with DDAO (red). Top views of the biofilms are displayed. Scale bars, 50 and 30 μm in the left and middle panels, respectively. The biovolumes were determined by analyzing at least three image stacks from each of three independent biofilms (at least nine stacks in total) using COMSTAT software. ***, P < 0.001 (Student t test).

FIG 2

alt gene locus and alterocin protein. (A) The alt gene encoding alterocin and the surrounding genes are represented in red and gray, respectively, with the arrowhead indicating the transcription orientation. The gene lengths include the stop codons. (B) Sequence of the alterocin protein. The vertical arrow indicates a predicted cleavage site that would allow the release of the protein (numbered +1 to +119) from its signal peptide (–20 to –1). The four cysteine residues are underlined. Residues on a colored background are highly conserved: they are found in all 19 alterocin homologs from the Prokaryotic Genome Database (gray) or in all but one of these 19 homologs (yellow). The sequences underlined in blue correspond to the seven amino acid sequences which were determined from the partially purified alterocin extracted from an SDS-PAGE gel. The sequence underlined in red corresponds to the peptide P1, which was synthesized to generate anti-alterocin antibodies.

FIG 3

Antibiofilm activity of Pseudoalteromonas sp. strain 3J3(pOriTalt) exoproducts compared to Pseudoalteromonas sp. strain 3J3(pOriT) exoproducts. After an attachment step in the presence of culture supernatant of Pseudoalteromonas sp. 3J3(pOriT) (– alt) or of Pseudoalteromonas sp. 3J3(pOriTalt) (+ alt), P. aeruginosa MUC-N1 biofilms were grown for 24 h, stained with SYTO 9 green, and observed by confocal laser scanning microscopy. (A) 3D views of the biofilms. Scale bars, 10 μm. (B) Biofilm biovolumes and average thicknesses determined by analyzing at least three image stacks from each of three independent biofilms (at least nine stacks in total) using COMSTAT software. **, P < 0.01; ***, P < 0.001 (Student t test).

FIG 4

Alterocin detection in culture supernatants of Pseudoalteromonas sp. 3J3 carrying the alt gene and Pseudoalteromonas sp. 3J6. (A) Alterocin was extracted from culture supernatants of Pseudoalteromonas sp. 3J3(pOriT) (strain without alt gene, lane noted “− alt”), Pseudoalteromonas sp. 3J3(pOriTalt) (strain carrying the alt gene, lane noted “+ alt”), and the wild-type alterocin producer Pseudoalteromonas sp. 3J6, and the extracts were analyzed by SDS-PAGE. M, molecular weight markers. (B) Western blot analyses of the same extracts as in panel A, using anti-alterocin antibodies.

FIG 5

Mapping of the alt promoter. The transcriptional start site (+1) was identified by 5′-RACE PCR, as shown by the relevant part of the chromatogram displaying the PCR product sequence. There are three potential +1 bases (underlined GGC bases with vertical lines reaching an arrow indicating the transcription direction), and the sequence is numbered +1 from the first of the two G’s. The sequences noted −35 and −10 are proposed to constitute the σ⁷⁰-dependent promoter yielding the mRNA starting at the +1 bases. The sequences noted P-35 and P-10 were predicted by BPROM to constitute a promoter. The horizontal arrows indicate inverted repeat sequences. RBS, ribosome binding site. M, first codon of the alt gene.

FIG 6

Expression of the alt gene in the course of Pseudoalteromonas sp. 3J6 growth. (A) Growth curve of Pseudoalteromonas sp. 3J6 representative of four different experiments. (B) Relative alt mRNA levels quantified by RT-qPCR from total RNA extracted at 5, 10, and 24 h of growth, compared to the level at 3 h. *, P < 0.05 (ANOVA for four different experiments).

FIG 7

Phylogenetic tree of the Gammaproteobacteria based on the alterocin protein sequences. Only bootstrap values above 50% are shown. The bootstrap value between P. luteoviolacea strains HI1 and CPMOR-1 is 87 (not shown due to a lack of space). The gene label from the MicroScope annotation corresponding to each putative alterocin is indicated in parentheses. The eight Pseudoalteromonas strains that show a similar gene synteny around the alt gene in their genome to that of Pseudoalteromonas sp. 3J6 are in boldface (see Fig. S2). Nonpigmented Pseudoalteromonas strains (16, 18) are in orange boxes. Bar, 0.2 substitution per amino acid position.