Alterocin, an Antibiofilm Protein Secreted by Pseudoalteromonas sp. Strain 3J6
This article has been corrected.
VIEW CORRECTIONABSTRACT
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.
INTRODUCTION
Biofilms are microbial communities of cells that are attached to a substratum, to an interface, or to each other and are embedded into a matrix of extracellular polymeric substances that they have produced (1). The biofilm lifestyle is considered predominant for many bacteria in the environment and is adopted in the course of numerous host infections. Within biofilms, bacteria are generally more resistant to antibiotics than their planktonic counterparts (2), and antibiotics are therefore insufficient to clear biofilm infections, leading to chronic infections (1, 3). The need for biofilm-specific therapies, in which antibiofilm compounds would be used in conjunction with conventional antibiotics, leads to the search for antibiofilm molecules active against pathogenic bacteria (3, 4). Pseudomonas aeruginosa is a target organism of particular interest not only one because it is one of the leading causes of nosocomial infections (5) but also because the chronic infections it establishes in the lungs of cystic fibrosis (CF) patients constitute their main cause of morbidity and mortality (6, 7). Biofilms are not only detrimental in medicine, and environmentally friendly antibiofilm molecules and/or their producer organisms are of great interest for a variety of other fields, including, for example, the food industry and aquaculture (8).
Antibiofilm agents notably include quorum-sensing (QS) inhibitors of various natures (e.g., inhibitors of the synthesis of signal molecules [autoinducers], autoinducer analogs, enzymes degrading or modifying autoinducers), natural and synthetic antimicrobial peptides, proteins such as matrix-degrading enzymes, surfactants, free fatty acids, and polysaccharides (4, 9, 10). Marine bacteria constitute a nonnegligible source of antibiofilm polysaccharides, biosurfactants, and QS inhibitors (8, 11). Furthermore, the marine bacteria Pseudoalteromonas tunicata D2 and Marinomonas mediterranea MMB-1 secrete the AlpP and LodA (formerly named marinocine) enzymes, respectively, which are responsible for both antimicrobial and antibiofilm activities due to the hydrogen peroxide they generate as a by-product of their amino acid oxidase activities (8, 12, 13). These enzymes led to the discovery of a large family of proteins encoded by alga-associated marine bacteria and by plant-associated terrestrial microorganisms (8, 14). More generally, marine bacteria from the Pseudoalteromonas genus are often found in association with marine eukaryote organisms, and their ability to produce compounds displaying a variety of biological activities has attracted particular attention (15–18). This genus includes two phylogenetically distinct groups: the pigmented group and the nonpigmented group, with the first group displaying a stronger bioactive potential (16, 18).
The nonpigmented Pseudoalteromonas sp. strain 3J6 was isolated from the Morbihan Gulf, Brittany, France (19), and we previously showed that its culture supernatant displayed antibiofilm activities while being devoid of antimicrobial (biocidal or bacteriostatic) activity against planktonic bacteria (20). Seventy-five percent of the tested marine strains were sensitive to this antibiofilm activity (20–22). Sensitive strains belonged to various genera (Gram negative: Paracoccus, Vibrio, Colwellia, Algibacter, Alteromonas, Pseudoalteromonas, and Sulfitobacter; Gram positive: Bacillus and Micrococcus), while most of the nonsensitive Gram-negative strains were other Pseudoalteromonas strains. The spectrum of action also includes the three tested strains belonging to the human pathogen species P. aeruginosa, Salmonella enterica, and Escherichia coli (20). The antibiofilm activity was lost after proteinase K treatment, indicating that it is due to at least one proteinaceous molecule (21). The first step of biofilm formation is the attachment of bacteria onto the substratum. Pseudoalteromonas sp. 3J6 exoproducts did not impair the attachment of Vibrio sp. strain D66 and strains belonging to the genera Algibacter, Alteromonas, Colwellia, Micrococcus, Sulfitobacter, and Vibrio (23), or else they affected the attachment too mildly to explain the subsequent defect in biofilm formation (Paracoccus sp. strain 4M6 and Vibrio sp. strain D01) (20). The only exception was observed with the Vibrio tapetis CECT4600-GFP strain, the attachment of which onto glass was reduced >5-fold by the Pseudoalteromonas sp. 3J6 culture supernatant (22). Except in the latter case, the Pseudoalteromonas sp. 3J6 exoproducts are thus thought to impair biofilm formation in our experimental conditions by adsorbing onto the glass substrate and subsequently acting mainly at a postattachment step (20). Two effects of the Pseudoalteromonas sp. 3J6 culture supernatant were observed on sensitive strains: it reduced the biomass of the resulting biofilms and/or led to biofilms in which cell viability was decreased. These two phenomena could occur either together or independently from each other (20). Its target in sensitive strains thus remains elusive, and we hoped that identifying the protein or peptide responsible for this activity might provide some clues on its mode of action. This antibiofilm protein or peptide was named alterocin (8, 22). Here, we show that the Pseudoalteromonas sp. 3J6 exoproducts are active against clinical CF P. aeruginosa strains, and we report the sequencing of the genome of Pseudoalteromonas sp. 3J6, in which the alt gene encoding alterocin was identified. Homologs of the alt gene were mostly found in Pseudoalteromonas strains, and the evolutionary history of the alt gene in this genus was examined by comparative genomics.
RESULTS
Activity of Pseudoalteromonas sp. 3J6 exoproducts against clinical strains of P. aeruginosa.
The Pseudoalteromonas sp. 3J6 culture supernatant (SN3J6) was previously shown to be active against the laboratory P. aeruginosa PAO1 strain (20). Since large variabilities were reported between biofilms of P. aeruginosa isolates (24, 25), SN3J6 should be tested against other P. aeruginosa strains forming different types of biofilms. Considering the critical P. aeruginosa role in lung infections of CF patients, we used additional strains isolated from CF patients. For this purpose, a group of four such clinical strains was constituted mainly on the basis of differences in the architecture of their biofilms, including the distribution of matrix components (26), but also on the bases of their mucoid phenotype (mucoid strains overproduce the alginate exopolysaccharide and are responsible for chronic infections [6]), virulence level and antibiotic resistance (M. Simon, A. M. Boukerb, E. Pernet, A. Jouault, E. Portier, S. Pineau, J. Vieillard, E. Bouffartigues, C. Poc-Duclairoir, M. G. J. Feuilloley, O. Lesouhaitier, J. Caillon, S. Chevalier, A. Bazire, and A. Dufour, unpublished data). This panel is composed of the clinical strains MUC-N1, MUC-P2, MUC-P4, and MUC-P5 (Table 1), the genomes of which were recently sequenced (27). To test the effect of SN3J6 on these strains, the bacterial attachment step was performed without or in the presence of SN3J6, and biofilms were then grown under a flow of medium. Without SN3J6, the mucoid MUC-N1 strain formed biofilms with mushroom-like structures containing alginate as the main matrix component (Fig. 1A, left). The MUC-N1 biofilms obtained in the presence of SN3J6 were devoid of mushroom-like structures, and their biovolume was 8.6-fold lower (Fig. 1A). Biofilms of the nonmucoid MUC-N2 strain grown without SN3J6 were devoid of mushroom-like structures, and the stained matrix components were not abundant at the biofilm surface, as seen by the large predominance of the green coloration (bacteria) over the blue (β-polysaccharides) and red (eDNA) colorations (Fig. 1B, left). In the presence of SN3J6, the MUC-N2 biofilm growth was prevented since only dispersed cells and small cell clusters were visible, which led to a 27-fold lower biovolume (Fig. 1B). When using the two other nonmucoid clinical strains of the panel, SN3J6 was active against MUC-P4 but did not impair biofilm formation by MUC-P5, which is thus nonsensitive to alterocin (see Fig. S1 in the supplemental material). Altogether, these data show that SN3J6 is active not only against the P. aeruginosa laboratory strain PAO1 but also against three of the four tested CF clinical strains forming different biofilm types, including one mucoid and two nonmucoid strains. Any of the sensitive strains would be suitable as indicators of antibiofilm activity. In our subsequent experiments, the P. aeruginosa MUC-N1 strain was chosen because it forms biofilms with higher biovolumes than the other strains in the absence of SN3J6, therefore yielding more consistent control biofilms.
TABLE 1
| Strain or plasmid | Characteristicsa | Source or reference |
|---|---|---|
| Strains | ||
| Pseudoalteromonas sp. | ||
| 3J6 | Wild-type alterocin producer strain; Amps | 19 |
| 3J3 | Wild-type strain devoid of antibiofilm activity and not sensitive to alterocin; Amps | 19 |
| 3J3(pOriT) | 3J3 strain containing the pOriT-4EM vector; Ampr | This study |
| 3J3(pOriTalt) | 3J3 strain containing the pOriTalt plasmid; Ampr | This study |
| 3J6(pOriTalt) | 3J6 strain containing the pOriTalt plasmid; Ampr | This study |
| P. aeruginosa | ||
| MUC-N1 | Mucoid clinical strain, biofilms with mushroom-like structures, alginate predominant in matrix | 27; Simon et al.b |
| MUC-N2 | Nonmucoid clinical strain, flat biofilms with Psl and eDNA as main matrix components | 27; Simon et al. |
| MUC-P4 | Nonmucoid clinical strain, biofilms with a few 3D structures, eDNA predominant in matrix | 27; Simon et al. |
| MUC-P5 | Nonmucoid clinical strain, thin biofilms with filamentous cells | 27; Simon et al. |
| E. coli | ||
| TOP10 | F– mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG λ– | Invitrogen |
| GEB883 | E. coli K-12 ΔdapA::erm pir RP4-2 ΔrecA gyrA462 zei298::Tn10; the DAP– strain was used for cloning and as a donor strain for conjugation | 55 |
| Plasmids | ||
| pOriT-4Em | E. coli-Pseudoalteromonas shuttle vector; Ampr Emr | 72 |
| pOriTalt | pOriT-4Em carrying a 605-bp DNA fragment including the alt gene and its promoter region; Ampr Emr | This study |
a
Amp, ampicillin; Em, erythromycin; DAP, diaminopimelate.
b
M. Simon et al., unpublished data.
FIG 1
General characteristics of the Pseudoalteromonas sp. 3J6 genome.
We sequenced the genome of Pseudoalteromonas sp. 3J6, which is ∼4.1-Mb long with an average GC content of 39.93% and contains 3,789 coding DNA sequences (CDS) (Table 2). The sequence seemed complete and not contaminated, as estimated using CheckM (28). To determine whether the Pseudoalteromonas sp. 3J6 strain could belong to a novel species, we determined its genomic relatedness with 58 other Pseudoalteromonas strains, the genome sequences of which were available on the NCBI RefSeq complete genome section at the time of analysis (February 2019), by estimating the average nucleotide identity (ANI) between genomes. The ANI between Pseudoalteromonas sp. 3J6 genome and 57 of the 58 other Pseudoalteromonas genomes were lower than 96% (see Table S1), the typical threshold to determine different bacterial species (29, 30). The only ANI above this threshold was obtained when comparing the genomes of Pseudoalteromonas sp. 3J6 and Pseudoalteromonas undina NCIMB 2128T: the ANI of 96.7% (see Table S1) indicates that Pseudoalteromonas sp. 3J6 is most closely related to a P. undina strain but is not sufficient to firmly conclude that strain 3J6 belongs to the undina species, and it could alternatively belong to a new species. P. undina NCIMB 2128T being part of the nonpigmented Pseudoalteromonas group (31), it at least indicates that Pseudoalteromonas sp. 3J6 is a member of this group.
TABLE 2
| Characteristic | Value |
|---|---|
| Size (bp) | 4,099,791 |
| GC content (%) | 39.93 |
| No. of contigs | 12 |
| Total no. of CDS | 3,789 |
| Average CDS length (bp) | 962.9 |
| Protein coding sequences (%) | 88.55 |
| No. of rRNA operon | 1 |
| No. of tRNAs | 79 |
Locus of the putative alterocin gene.
We observed that the Pseudoalteromonas sp. 3J6 genome is devoid of an alpP-like gene, the product of which could have been responsible for antibiofilm activities (8). This is consistent with previous findings, since AlpP and related proteins lead to both antibacterial and antibiofilm activities (8), whereas no antibacterial activity was ascribed to Pseudoalteromonas sp. 3J6 (20). The antibiofilm activity of the latter thus relies on another protein. To identify it, we concentrated the active molecules by loading SN3J6 onto a Sep-Pak Plus C18 cartridge and eluting the adsorbed molecules with stepwise increased concentrations of acetonitrile. The fraction eluted with 50% acetonitrile was the most active and was named the 3J6 extract (E3J6). Upon SDS-PAGE analysis, a single 13-kDa protein was observed in E3J6. Peptide sequences were obtained from this protein as described in Materials and Methods. None of these sequences matched a protein of known function in Swiss-Prot databases, but seven peptide sequences matched one protein encoded by the Pseudoalteromonas sp. 3J6 genome (Fig. 2B) from a gene located on the second largest contig (contig 11: 791,825 bp). This protein was considered the putative alterocin and its gene was named alt. The alt gene lies in the opposite orientation compared to the upstream and downstream genes (Fig. 2A) and therefore cannot be part of an operon. The surrounding genes P3J6_110063, P3J6_110065, and P3J6_110066 (Fig. 2A) are predicted to encode an aspartate-semialdehyde dehydrogenase, which is involved in amino acid biosynthesis (32), a protein of unknown function, and a TonB-dependent receptor/transporter (TBDR), respectively (see Table S2). The latter belongs to a family of outer membrane proteins responsible for the uptake of iron complexes and other substrates which are often rare nutrients (33). There is thus no obvious relationship between the products of the alt-adjacent genes and alterocin production. A larger view of the alt gene vicinity is provided in Fig. S2 and its accompanying text. In this genomic region, the only gene which could be related to biofilm formation encodes a protein with a predicted diguanylate cyclase GGDEF domain (ORF P3J6_110075). This protein would contribute to the synthesis of cyclic diguanylate (c-di-GMP), a near universal signaling molecule involved in the regulation of bacterial biofilm formation (34, 35).
FIG 2
The alt gene and its product.
The alt gene encodes a 139-residue protein predicted by the SignalP 5.0 server (36) to include a 20-residue N-terminal signal peptide (Fig. 2B) with a likelihood of 0.9073. This signal peptide would be cleaved off by signal peptidase I in the course of translocation across the plasma membrane by the general secretory pathway. The resulting alterocin protein would be composed of 119 amino acids (calculated molecular weight, 13,655.99 Da; pI, 4.82), including four cysteines in its C-terminal half (Fig. 2B), which could lead to the formation of disulfide bridges. The DISULFIND tool (37) indeed predicted two disulfide bridges, with the best ranking connectivity pattern linking residue 65 to residue 81 and residue 109 to residue 188 (confidence of prediction: 6 for each bridge on a scale of 0 = low to 9 = high). The alterocin sequence did not display significant similarities with proteins of known functions using the protein Basic Local Alignment Search Tool (BLAST) program and with conserved domains from the Conserved Domain Database (CDD) (38). When searching for alterocin homologs in the Prokaryotic Genome DataBase (PkGDB) from the MicroScope platform (http://mage.genoscope.cns.fr/microscope), we identified 19 proteins (13 different amino acid sequences) encoded by 18 Pseudoalteromonas strains (≥45% amino acid sequence identities) and one Hahella chejuensis strain (44% amino acid sequence identity). The sequence comparison of the Pseudoalteromonas sp. 3J6 alterocin and its 19 homologs showed that the C-terminal two-thirds of the protein (residues 38 to 119) is the most strongly conserved with numerous residues found in 19 or all of the 20 proteins (Fig. 2B). The four cysteine residues are conserved in all alterocin homologs, consistent with the hypothesis that they could play an important structural role. When searching for alterocin homologs in NCBI sequence databases using the protein BLAST program, 48 proteins displayed more than 40% identity with alterocin. These 48 protein sequences included those of all 19 alterocin homologs found in the PkGDB from MicroScope (corresponding to 13 different sequences), plus 35 additional sequences, in which the residues highlighted on Fig. 2B are also highly conserved. Pseudoalteromonas sp. strains encode the large majority of these proteins (44 of 48), and the four remaining strains are marine Gram-negative bacteria belonging to the Hahella (two strains) and Paraglaciecola (two strains) genera (39, 40). Like the Pseudoalteromonas genus, the Paraglaciecola genus belongs to the Alteromonadales order within the Gammaproteobacteria class, whereas the Hahella genus is a member of the Oceanospirillales order (Gammaproteobacteria class) (https://www.ncbi.nlm.nih.gov/Taxonomy). In contrast, proteins similar to the product of the P3J6_110065 gene located just downstream of the alt gene in the Pseudoalteromonas sp. 3J6 genome were only found in eight other Pseudoalteromonas sp. strains with the BLAST program. Therefore, many Pseudoalteromonas genomes include an alt-like gene without any P3J6_110065-like gene, indicating that the P3J6_110065 gene product is not necessary to the production and/or activity of the alterocin protein. We therefore subsequently focused only on the alt gene.
Expression of the alt gene in the Pseudoalteromonas sp. 3J3 host.
To examine whether the alt gene indeed encodes an antibiofilm protein, we first tried to construct an alt deletion mutant of Pseudoalteromonas sp. 3J6. Unfortunately, we were unable to obtain the expected mutant (data not shown). We thus attempted to introduce the alt gene into another strain, Pseudoalteromonas sp. 3J3 (Table 1), which was isolated from the same environment as the 3J6 strain (19). The 3J3 strain was chosen because (i) it belongs to the same genus as the 3J6 strain (19); (ii) we could not amplify by PCR an alt gene from 3J3 total DNA and, consistently, we did not observe an antibiofilm activity in 3J3 culture supernatants; and (iii) the 3J3 strain is not sensitive to SN3J6 (20, 21). Pseudoalteromonas sp. 3J3 was thus a good candidate strain to express the alt gene and produce the encoded protein. The alt gene with its own promoter region was cloned into the pOriT-4Em vector (designated here as pOriT), yielding the pOriTalt plasmid (Table 1). Both plasmids were introduced into Pseudoalteromonas sp. 3J3, and we examined the effect of the culture supernatants of the two resulting strains, SN3J3(pOriT) and SN3J3(pOriTalt), on the P. aeruginosa MUC-N1 biofilm development. In the presence of SN3J3(pOriTalt), the biofilm biovolume and average thickness were about 2- and 3-fold lower, respectively, than in the presence of SN3J3(pOriT) (Fig. 3). These data show that introducing the alt gene from strain 3J6 into strain 3J3 provides an antibiofilm activity to the exoproducts of the latter.
FIG 3
Alterocin detection in Pseudoalteromonas sp. strain 3J3(pOriTalt) culture supernatants.
SDS-PAGE analyses were performed to verify that Pseudoalteromonas sp. 3J3(pOriTalt) secretes alterocin. Since no band was visible when loading the raw culture supernatants of strains 3J3(pOriTalt) and 3J3(pOriT), we extracted and concentrated the antibiofilm molecule from SN3J3(pOriTalt) using a Sep-Pak Plus C18 cartridge, and the fraction eluted with 50% acetonitrile constituting the E3J3(pOriTalt) extract was resuspended in a volume of water 1,000-fold lower than the used SN volume. Similar extracts were made from SN3J3(pOriT) and SN3J6. A single SDS-PAGE band at about 13 kDa was observed in E3J3(pOriTalt) and E3J6, whereas no band was visible in E3J3(pOriT) (Fig. 4A). To ascertain that this band was due to alterocin, we performed Western blot experiments with rabbit antibodies produced upon inoculation of the peptide P1 that had been chemically synthesized based on the alterocin sequence (Fig. 2B). The antibodies detected the 13-kDa protein in E3J6 and E3J3(pOriTalt), whereas they did not detect any protein in the negative-control E3J3(pOriT) (Fig. 4B). This confirms that Pseudoalteromonas sp. 3J3(pOriTalt) is able to express the alt gene and to secrete alterocin, thereby demonstrating that alterocin is responsible for the antibiofilm activity of the recombinant strain against P. aeruginosa MUC-N1.
FIG 4
The alt gene promoter and expression.
The bacterial σ70 promoter recognition program BPROM (41) predicted a single promoter upstream of the alt gene (Fig. 5, P-35 and P-10). We mapped the transcriptional start site of the alt gene site by 5′-RACE (5′-rapid amplification of cDNA ends) PCR performed on RNAs extracted from Pseudoalteromonas sp. 3J6 transformed by pOriTalt in order to increase the alt gene copy number and thus the alt mRNA level. A single PCR product was obtained, and its sequencing revealed three potential +1 bases: the GGC bases located 54 to 56 bp upstream of the translation initiation codon (Fig. 5). This result did not confirm that the BPROM-predicted promoter is functional since it is located too far away from the mapped transcriptional start site (the putative −10 sequence is lying 50 bp upstream of the first potential +1 base). Although the sequence TTAACC-17 bp-TACCAA was not predicted to constitute a promoter by BPROM, it lies at the appropriate position relative to the +1 bases (Fig. 5) and matches loosely the consensus promoter sequence recognized by the primary σ70 factor (TTGACA-16 to 19 bp-TATAAT). This sequence is thus proposed to constitute a weak promoter where the transcription could be initiated by the RNA polymerase containing σ70. It should be noted that the alt promoter region contains several inverted repeats (IRs) located upstream and downstream of the two promoter-like sequences (Fig. 5), and some of these IRs could be involved in transcription initiation regulation by binding repressor or activator proteins.
FIG 5
Since data presented above suggested that the alt gene expression is regulated at the transcriptional level, we assayed the alt mRNA level by reverse transcription-quantitative PCR (RT-qPCR) at different time points during the growth of Pseudoalteromonas sp. 3J6. The alt mRNA levels were, respectively, 5-, 11-, and 2-fold higher at 5, 10, and 24 h compared to the level at 3 h (Fig. 6). Under our culture conditions, the alt mRNA level thus increased during the stationary phase (5 and 10 h) before decreasing almost down to the exponential-phase level at 24 h (Fig. 6). The transcription of the alt gene seems therefore regulated to reach its peak during the first hours of the stationary phase.
FIG 6
Evolutionary history of the alt gene in the Pseudoalteromonas genus.
To determine the acquisition mode of the alt gene in the Pseudoalteromonas genus, we first searched for alterocin homologs in the 4,418 bacterial genomes from the PkGDB on the MicroScope platform. As mentioned above, Hahella chejuensis KCTC 2396 possesses a gene encoding an alterocin homolog (44% amino acid sequence identity), as well as 18 of 58 (31%) Pseudoalteromonas genomes (≥45% amino acid sequence identity). Interestingly, the P. undina NCIMB 2128T strain, which was the most phylogenetically closely related to Pseudoalteromonas sp. 3J6, is devoid of the alt gene. Compared to Pseudoalteromonas sp. 3J6, the synteny of the alt gene environment is not conserved in the H. chejuensis genome. Parts of the synteny of the genes downstream of the alt gene (region P3J6_110066 to P3J6_110076, including genes for the TonB transporter complex and a TBDR) are conserved in all Pseudoalteromonas genomes. This is the case for the genomes of the 10 pigmented Pseudoalteromonas strains belonging to the species luteoviolacea (nine strains) and citrea (one strain), while the alt gene is located in another region of the genome. The three P. ruthenica genomes and the six genomes of the nonpigmented Pseudoalteromonas strains (including Pseudoalteromonas sp. 3J6) display seven versions of the alt gene environment with different parts of the gene synteny conserved (see Fig. S2), which suggests a differential loss of some genes in the region around the alt gene during the evolutionary history of the Pseudoalteromonas genus.
The alterocin proteins from H. chejuensis and the 19 Pseudoalteromonas strains (including 3J6) allowed computation of a phylogenetic tree where H. chejuensis clustered alone and the Pseudoalteromonas strains clustered together, forming four groups (Fig. 7). Two groups are made only of pigmented Pseudoalteromonas strains: one group consists of P. citrea DSM 8771 and the nine P. luteoviolacea strains, while the second one includes the three P. ruthenica strains. The two other groups contain only nonpigmented Pseudoalteromonas strains: one group with P. haloplanktis ATCC 14393 and P. porphyrae UCD-SED14, and the Pseudoalteromonas sp. 3J6 group, which also includes two other Pseudoalteromonas sp. strains, Bsi20495 and S2292, and P. fuliginea KMM216 (Fig. 7).
FIG 7
The evolutionary history of the alt gene in the Pseudoalteromonas genus was further investigated using two phylogenetic approaches. The first was to construct a 16S rRNA gene tree, including the 58 strains with an available genome plus Pseudoalteromonas sp. 3J6 and 34 additional type strains to determine the position of the alt-containing Pseudoalteromonas strains in the genus (see Fig. S3). The second was to compute a tree based on pairwise genome distances calculated from Mash values between the 59 Pseudoalteromonas genomes, including that of 3J6 (see Fig. S4). In both trees, the P. luteoviolacea strains did not cluster with P. citrea, whereas they belong to the same group in the alterocin tree (Fig. 7). This suggests that they acquired the alt gene from two independent horizontal gene transfer events but likely from the same organism. The other pigmented strains, i.e., the three P. ruthenica strains, clustered together in both 16S rRNA gene and genome trees. Whereas nonpigmented Pseudoalteromonas strains were distributed in two groups in the alterocin tree (Fig. 7), the two corresponding groups were not observed in the 16S rRNA gene tree (see Fig. S3): P. haloplanktis ATCC 14393 did not cluster with P. porphyrae UCD-SED14, and Pseudoalteromonas sp. 3J6 did not cluster with the Pseudoalteromonas sp. strains Bsi20495 and S2292 (see Fig. S3). In contrast, the two corresponding clades were found in the genome-based tree (see Fig. S4). Therefore, the genome-based tree is more consistent with the alterocin tree, suggesting that the genome-based tree may give a finer phylogenetic description of the Pseudoalteromonas evolutionary history than a 16S rRNA-based tree, even though fewer Pseudoalteromonas strains are considered in the genome-based tree. In the latter, several Pseudoalteromonas genomes in the nonpigmented clade and the P. citrea clade do not contain the alt gene. From the observations presented above and since the alt gene is located in a rather conserved region of the genome, an ancestor common to the strains of the nonpigmented group may have acquired the alt gene, and nonpigmented strains lacking this gene may have subsequently lost it.
DISCUSSION
We report here the genome sequence of the Pseudoalteromonas sp. 3J6 strain and the identification of the alterocin gene, the product of which is secreted by this marine bacterium. Alterocin, a predicted 119-residue protein after cleavage of a 20-residue signal peptide, is a novel antibiofilm protein since it is devoid of sequence similarity with proteins of known function and does not contain a known functional domain. Its mode of action thus cannot be deciphered from protein sequence comparisons, and future work will be required to unravel it by combining in-depth analysis of biofilm formation in the presence of alterocin, transcriptomic analyses of bacteria submitted to alterocin, the search for alterocin-insensitive mutants, and their subsequent study.
In the case of ribosomally synthesized peptides (known as RiPPs or RPNPs) secreted by bacteria, the structural genes are generally clustered with other genes forming one or several operons and encoding proteins specifically involved in the production of the mature peptides (modification enzymes, proteases cleaving the leader peptide, and dedicated ATP-binding cassette [ABC] transporters), in the protection of the producer bacteria (immunity systems in the case of antimicrobial peptides), and in dedicated systems regulating the transcription of these genes (42, 43). In contrast, the alt gene is not part of an operon. The P3J6_110065 gene lying immediately downstream of the alt gene in the Pseudoalteromonas sp. 3J6 genome encodes a protein of unknown function, but most of the other Pseudoalteromonas strains possessing an alt-like gene are devoid of a P3J6_110065-like gene, indicating that the P3J6_110065 gene product is unrelated, or at least nonessential, to alterocin production and activity. The other genes of the alt locus encode proteins without an obvious relationship to production of an antibiofilm protein or transcriptional regulation. Therefore, the genetic environment did not help us to understand the alterocin production pathway, suggesting that alterocin is not the object of maturation and export by dedicated proteins. Consistently, the 20-residue signal peptide was strongly predicted to be cleaved off by signal peptidase I, meaning that alterocin is very likely translocated across the plasma membrane by the general secretory pathway and not by a specific transporter. The secretion pathway across the outer membrane remains to be identified. Furthermore, introducing only the alt gene in the Pseudoalteromonas sp. 3J3 strain was sufficient to provide to the latter an antibiofilm activity, which showed that no other gene found specifically in Pseudoalteromonas sp. 3J6 (i.e., absent in the 3J3 strain) is required for alterocin production.
We observed that the alt gene expression is regulated, with the mRNA level increasing during the first 6 hours of the stationary phase before decreasing. We obtained experimental evidence of a single transcription start site, which would result from a promoter only weakly matching the consensus sequence of primary σ70 factor-dependent promoters (7 bases identical out of 12). This promoter could thus be a weak promoter recognized by the primary σ70 factor. Alternatively, a secondary sigma factor could be responsible for the transcription initiation at the mapped start site, as in the cases of genes for several pyocins and tailocins, which are bacteriocins produced by P. aeruginosa strains (44). The Pseudoalteromonas sp. 3J6 genome indeed encodes 12 putative secondary sigma factors (see Table S3), each of them recognizing a specific unknown or hypothetical promoter sequence. In both cases, regulation is expected to occur, either via the action of at least an activator protein on a weak σ70-dependent promoter or via the regulation of a secondary sigma factor activity. Several IRs in the alt promoter region could be binding sites for regulatory proteins. Furthermore, a second promoter was predicted. Although its functionality was not experimentally confirmed, it remains possible that it could be active in conditions other than ours. The pattern of the alt mRNA level in the course of growth (higher level during the first hours of the stationary phase) is consistent with a control by a QS system in response to cell density. There are indeed examples of QS-regulated production of peptidic antimicrobial molecules by Gram-negative bacteria (45, 46). Recent analyses of Pseudoalteromonas genomes indicate that they encode homologs of proteins belonging to three sets of cross-linked QS systems: the Aliivibrio fischeri and P. aeruginosa LuxI-LuxR type systems producing and sensing autoinducers AI-1 (N-acyl homoserine lactones); the Vibrio LuxPQ and CqsS sensing the autoinducers AI-2 (produced by LuxS) and CAI-1, respectively, and both feeding the LuxU-LuxO pathway; and the E. coli QseC-QseB and QseE-QseF two-component systems sensing AI-3 and/or the hormones epinephrine and norepinephrine (47, 48). Whereas several LuxI-LuxR type systems have been shown to be functional in Pseudoalteromonas strains (48, 49), the two other types of systems are not identical to those described in other genera since some proteins are missing in the studied Pseudoalteromonas strains (47, 48). It can thus be speculated that a sophisticated QS network integrating signals provided by several autoinducers is likely to exist in Pseudoalteromonas strains, allowing the achievement of elaborate cell physiology (48). No AI-1 or AI-2 autoinducers were detected in culture supernatants of the Pseudoalteromonas sp. 3J6 strain (50, 51). Consistently, the Pseudoalteromonas sp. 3J6 genome is devoid of genes encoding LuxI, LuxR, and LuxS homologs. However, it includes genes encoding homologs of LuxU, LuxO, and LuxQ and of the two-component system QseC-QseB (see Table S4). Future work will be required to define the QS functioning in Pseudoalteromonas sp. 3J6 and to examine if it controls alterocin production.
The alt gene is not part of the core Pseudoalteromonas genome but is nevertheless present in a nonnegligible proportion of strains (about 30%), indicating that it plays an important, albeit nonessential, role in the physiology of bacteria belonging to this genus. The alt gene is not restricted to one of the two Pseudoalteromonas phylogenetic groups: Pseudoalteromonas sp. 3J6 is affiliated with nonpigmented strains, and the alt gene was also found both in other nonpigmented strains and in pigmented ones. We found alt genes in very few non-Pseudoalteromonas bacterial strains. The latter are marine bacteria belonging to the Hahella and Paraglaciecola genera. This is in sharp contrast for example with the alpP-lodA family genes, which encode antibacterial/antibiofilm amino acid oxidases (8, 12, 13) and were found in a large variety of marine bacteria and terrestrial bacteria belonging to eight phyla and having in common the ability to associate with algae or plants (14).
Consistent with the ecological roles proposed for Pseudoalteromonas, such as influencing the biofilm formation in different ecological niches or being defensive agents for marine flora and fauna (16), alterocin could provide a competitive advantage to the producer strains over other bacteria to colonize living (algae) or inert surfaces and develop a biofilm, and/or to invade a preformed biofilm. The Pseudoalteromonas sp. 3J6 strain tagged with the green fluorescent protein-producing plasmid pCJS10 has indeed been shown to form biofilms and to exert its antibiofilm activity in the course of biofilm formation, inhibiting the development of sensitive strains coinoculated with Pseudoalteromonas sp. 3J6(pCJS10) in attempts to grow two-species biofilms (20). It is, however, puzzling that such an advantage would be of benefit only for Pseudoalteromonas strains and a few other strains of marine bacteria and that the alt gene had not been more broadly disseminated. An explanation for host limitation of the alt gene could come from the fact that no immunity gene, which would protect the alterocin producer strain from its own antibiofilm protein, was found next to the alt gene and from our previous observation that most of the strains of marine bacteria which were nonsensitive to alterocin belonged to the Pseudoalteromonas genus (20, 21). This feature of at least some Pseudoalteromonas strains that are not producers of alterocin could be due to the absence or modification(s) of the alterocin target. This would allow the acquisition of an alt gene without a detrimental effect on the ability of the strain to form its own biofilms while providing a competitive advantage. Consistently, we were able to introduce the alt gene into the Pseudoalteromonas sp. 3J3 strain, which was nonsensitive to alterocin and became an alterocin producer. The resulting 3J3(pOriTalt) strain was still able to form biofilms (data not shown). In contrast, the gain of an alt gene by an alterocin-sensitive strain would modify its lifestyle by preventing it from forming biofilms, which would be too detrimental, thereby limiting the alt gene host range. To test this hypothesis, discovering the alterocin target and searching for its presence and/or variability in Pseudoalteromonas strains and other bacteria will be required. Pseudoalteromonas sp. 3J6 itself is nonsensitive to alterocin at concentrations found in its own culture supernatants (21), consistent with its ability to form biofilms (20, 21). However, it would be interesting to examine whether alterocin could accumulate sufficiently within mature biofilms to affect its own producer cells. In this case, alterocin might contribute to the life cycle of producer bacteria by favoring the dispersal of cells from mature biofilms and the colonization of novel environments. Such an ecological role has been documented for the AlpP and LodA enzymes in the marine bacteria P. tunicata and M. mediterranea, respectively: the hydrogen peroxide synthesized by these proteins leads to the death of AlpP/LodA-producer cells inside biofilm microcolonies, which promotes the dispersal of surviving cells (52, 53). Interestingly, the released bacteria displayed a high level of phenotypic variation, which could be advantageous in colonizing environments under changing conditions.
Alterocin homologs produced by different strains are not identical to each other. Amino acid sequence comparisons showed that the C-terminal part (residues 38 to 119) is more strongly conserved than the N-terminal part (residues 1 to 37) (Fig. 2B). This can provide the basis for future studies of the structure-activity relationship: a mutagenesis study could focus on the most conserved residues to examine which of them play key roles in the antibiofilm activity. The presence of four cysteine residues suggests that two disulfide bridges could be formed, which could be crucial for the tridimensional structuration and activity of the protein. Consistently, these four residues are located within the C-terminal part and are all highly conserved. They would thus be among the residues to prioritize for mutagenesis. It would also be interesting to examine if truncated alterocin versions lacking the whole N-terminal part or portions of it could retain some antibiofilm activity.
In conclusion, we identified a novel antibiofilm protein. Our data raise numerous questions, as discussed above, but they also open up the possibility of applications for alterocin and/or its producer strains in antibiofilm strategies in various domains, including the medical one since they are active against clinical strains of the human pathogen P. aeruginosa.
MATERIALS AND METHODS
Strains and culture conditions.
The strains used in this study are listed in Table 1. Pseudoalteromonas sp. strains were grown in Vaatanen nine-salt solution (VNSS) medium at 20°C under agitation (54). The P. aeruginosa and E. coli strains were grown in Luria-Bertani (LB) medium at 37°C with shaking or on LB agar plates (1.5% agar). For conjugation, strains were grown in modified LB medium (peptone, 10 g liter–1; yeast extract, 5 g liter–1; and sea salt, 15 g liter–1) at 20°C. Diaminopimelate (DAP) was added at a final concentration of 0.3 mM for growth of E. coli GEB883 (55). When necessary, ampicillin was used at 100 μg ml–1 for E. coli and Pseudoalteromonas sp. strains.
Biofilms growth and observation.
Biofilms were grown for 24 h in flow cells under dynamic conditions (2.5 ml h−1 of LB medium) at 37°C as previously described (56), bacteria were stained with 5 μM SYTO 9 green (Molecular Probes/Life Technologies, Saint Aubin, France) and observed by confocal laser scanning microscopy with a TCS-SP2 microscope (Leica Microsystems, Heidelberg, Germany) or a LSM 710 microscope (Zeiss, Oberkochen, Germany) using a 63× or a 40× oil immersion objective, respectively. To visualize matrix compounds, β1-3 and β1-4 polysaccharides (including alginate) were stained with 100 μM calcofluor white (Sigma-Aldrich) (57) and eDNA was detected by 1 using μM 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO; Molecular Probes) (59). Dyes in NaCl 0.9% were injected in flow cell chambers, followed by incubation for 15 min. Biofilms were then rinsed with a LB flow (2.5 ml h−1) for 10 to 15 min and observed immediately. Excitation wavelengths for SYTO 9 green, calcofluor white, and DDAO were 485, 400, and 646 nm, respectively. Emission wavelengths were 498, 410 to 480, and 659 nm, respectively. Fluorescence signal of double- or triple-labeled specimens were acquired simultaneously. Images were taken every micrometer throughout the whole biofilm depth. The Leica LCS or Zen 2011 software was used to visualize and process three-dimensional (3D) image data (volume rendering with shadow projection). Biovolumes and thicknesses were determined with the COMSTAT software (http://www.imageanalysis.dk/) (60). Three image stacks from each of three independent experiments were used for each analysis.
DNA extraction, library preparation, and genome sequencing of Pseudoalteromonas sp. 3J6.
After growth in 200 ml of VNSS medium until the stationary phase, the bacteria were harvested by centrifugation (3,000 × g for 15 min), and the genomic DNA was extracted as described previously (61). The DNA was quantified using a Qubit dsDNA HS kit (Invitrogen, Carlsbad, CA). Libraries were prepared using paired-end and mate-pair strategies. Paired-end libraries were prepared using a Nextera XT DNA sample preparation kit (Illumina, San Diego, CA), and mate-pair libraries were prepared using the Nextera Mate Pair sample preparation kit (Illumina). Template size distribution was verified on a Bioanalyzer 2100 (Agilent, Santa Clara). Paired-end and mate-pair libraries were then sequenced using an Illumina MiSeq sequencer kit v2 (300 cycles). The assembly using the reads from the two libraries was done in SPAdes version 3.1.1 (62), resulting in a genome of 4.1 Mb with 17 contigs (>1 kb). Gene function was annotated as in (61). Briefly, all the genes were subjected to the automatic annotation of the MicroScope platform (63). Selected genes, including the alterocin gene as well as the surrounding genes, were annotated manually by searching for homologs (at least 30% amino acid sequence identity and 80% sequence coverage) with characterized proteins on the MicroScope platform.
Preparation of Pseudoalteromonas culture supernatants and alterocin extract.
Culture supernatants were recovered by centrifugation for 10 min at 10,000 × g and 18°C after 24 h of growth of Pseudoalteromonas sp. 3J6, 3J3(pOriT), and 3J3(pOriTalt) strains in VNSS medium without antibiotic at 20°C under agitation. For Pseudoalteromonas sp. 3J3 carrying the pOriT or pOriTalt plasmid, ampicillin was added for the precultures but omitted in the last culture. The stability of pOriT and pOriTalt in ampicillin-free cultures has been verified as follows. After 24 h of liquid growth without ampicillin, the proportion of bacteria with plasmid was determined by plating dilutions of the cultures and comparing the number of colonies on LB plates with ampicillin and on ampicillin-free LB plates. The experiment was carried out three times. Plasmids pOriT and pOriTalt were maintained in 83 and 66%, respectively, of Pseudoalteromonas sp. 3J3 cells. The culture supernatants were filtered using Steritop-GP (0.22 μm pore size; Millipore, Molsheim, France) and conserved either at 4°C for a rapid use or at –80°C for a longer period. Culture supernatant of Pseudoalteromonas sp. 3J6 (SN3J6) lacking antibiofilm activity was obtained by replacing the pancreatic digest of soy with a pancreatic digest of casein (Biokar Diagnostics, Groupe Solabia, Pantin, France) in the VNSS medium.
The alterocin extracts E3J6 and E3J3(pOriTalt) were obtained by eluting SN3J6 and SN3J3(pOriTalt), respectively, from a Sep-Pak Plus C18 cartridge with acetonitrile (10 or 20%, followed by 50%). The 50% acetonitrile fractions were evaporated using a SpeedVac apparatus (Thermo Fisher Scientific, Bremen, Germany) and then resuspended in sterile ultrapure water.
SDS-PAGE and Western blot analyses.
Culture supernatants or concentrated extracts were suspended in 1 volume of sample buffer, boiled, and electrophoresed on a 1D 15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The gels were stained with Coomassie brilliant blue or used for protein blotting. In the latter case, the proteins were electrotransferred from the polyacrylamide gel onto a nitrocellulose membrane. The membrane was then incubated at room temperature for 30 min in blotting buffer (Tris-buffered saline [TBS] plus 5% [wt/vol] nonfat dry milk) and for 1 h with a 1:100 dilution of anti-alterocin rabbit antibodies in blocking buffer. After a brief wash with TBS, the membranes were incubated with a 1:5,000 dilution of alkaline phosphatase-conjugated antibodies against rabbit immunoglobulin. The revelation was performed with bromochloroindolyl phosphate (BCIP; Thermo Fisher Scientific).
Anti-alterocin antibodies were produced by Eurogentec (Seraing, Belgium) using the synthetic peptide P1 CEERGHNQEISGSTIT (Fig. 2B). This peptide was synthesized with purity of >70%, conjugated to the carrier protein keyhole limpet hemocyanin, and then injected into rabbits on days 0, 7, 10, and 18. Serum samples were collected on days 21 and 28. Antibody production was checked in the first serum sample with an enzyme-linked immunosorbent assay. Antibody purification was performed on affinity column from the second serum sample. P1 was fixed on the TOYOPEARL AF-Amino-650M matrix of the column. A 5-ml volume of serum was then loaded onto the column, and a washing step with phosphate-buffered saline (PBS) was carried out. Antibodies attached to the column were then eluted with 100 mM glycine at pH 2.5. The antibodies were stored at −20°C in a solution of 50% PBS–50% glycerol.
Trypsin digestion and mass spectrometry sequencing of alterocin.
After SDS-PAGE and Coomassie blue straining, the band of interest was cut out, and the gel piece was cut into smaller pieces, washed with distilled water, and destained using acetonitrile. The cysteine residues were reduced by adding 100 μl of 10 mM dithiothreitol at 56°C and alkylated by adding 150 μl of 55 mM iodoacetamide at room temperature. The iodoacetamide solution was replaced by 100 μl of 100 mM NH4HCO3, and gel dehydration was achieved with acetonitrile. After evaporation in a SpeedVac apparatus (Thermo Fisher Scientific), proteins were digested overnight at 37°C by a solution containing 0.9 μg of a modified porcine trypsin (Promega, Madison, WI) prepared in 25 mM NH4HCO3. Finally, a double extraction was performed, first with 5% (vol/vol) formic acid solution and then with 100% (vol/vol) acetonitrile. The resulting peptide mixture was dried under vacuum and resuspended in 50 μl of 1% formic acid solution.
The peptides mixtures were analyzed by using online NanoFlow liquid chromatography-tandem mass spectrometry (LC-MS/MS) on an EASY-nLC II system (Proxeon, Odense, Denmark) connected to the LTQ Orbitrap Discovery instrument (Thermo Fisher Scientific). Next, 10-μl portions of the peptide mixtures were concentrated onto a 2-cm preanalytical column (300-μm inner diameter) packed with 5-μm C18 beads (C18PepMap100; Dionex). These were separated in a 15-cm analytical column (75-μm inner diameter) packed with 3-μm C18 beads (AcclaimPepMap100; Dionex) with a 60-min gradient of 5 to 35% acetonitrile in 0.1% formic acid. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap instrument was operated in data-dependent mode to automatically switch between full scan MS and MS/MS acquisition. Instrument control was done using Tune 2.5.5 and Xcalibur 2.1. For the collision-induced dissociation (CID)-MS/MS top5 method, full-scan MS spectra (from m/z 400 to 2,000) were acquired in an Orbitrap analyzer after accumulation to a target value of 5e+6 in the linear ion trap with resolution r = 30,000. The five most intense peptide ions with charge states of ≥2 were sequentially isolated to a target value of 30,000 and fragmented in the linear ion trap by CID with a normalized collision energy of 35% and wideband activation enabled. The ion selection threshold was 500 counts for MS/MS, and the maximum allowed ion accumulation times were 500 ms for full scans in the Orbitrap and 200 ms for CID-MS/MS measurements in the LTQ. An activation q = 0.25 and activation time of 30 ms were used. Standard MS conditions for all experiments were as follows: spray voltage, 1.7 kV; no sheath and auxiliary gas flow; heated capillary temperature, 200°C; and predictive automatic gain control enabled. Peptide sequences were deduced from the resulting fragment ion spectra using the PEAKS software (Peaks Studio 5.3; Bioinformatics Solutions, Inc.). The resulting peptide sequences were submitted to the Peaks Search option to perform protein identification in the Swiss-Prot database.
DNA and protein sequence analyses.
The BPROM program was used to predict σ70-dependent promoters in the DNA sequence lying upstream of the alt gene (41). The SignalP 5.0 server (http://www.cbs.dtu.dk/services/SignalP/) and DISULFIND tool (http://disulfind.dsi.unifi.it/) were used to predict a signal sequence and disulfide bridges, respectively, in the alterocin protein (36, 37). The ProtParam tool on the ExPASy server (https://web.expasy.org/protparam/) was used to analyze the alterocin protein sequence (64). The protein BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (38) were used to search for proteins similar to alterocin and for functional domains within alterocin, respectively.
Plasmid construction and introduction into Pseudoalteromonas sp. 3J3.
The alt gene and its promoter region were PCR amplified as a DNA fragment extending from bases –129 to +476 (relative to the first possible mapped transcription initiation site [Fig. 5]) using Pseudoalteromonas sp. 3J6 total DNA as the template, the Q5 High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA), and the primers Fpalt (5′-TATgtcgacCTAAAACTAGTGAATAAAGTCG-3′; binding to bases −129 to −108) and Rpalt (5′-TATgtcgacTTACTGACACTTTATAGTTGC-3′; binding to bases +456 to +476, respectively) (lowercase letters correspond to SalI restriction sites). The following steps were programmed: 94°C for 3 min; 35 cycles of 94°C for 1 min, 51°C for 1 min, and 72°C for 2 min; and 10 min at 72°C. The amplicon and the pOriT-4Em vector were both digested with SalI, ligated, and transformed into E. coli Top10 electrocompetent cells (Invitrogen). The resulting pOriTalt plasmid (Table 1) was verified by restriction analysis and sequencing. E. coli GEB833 (55) was then transformed with pOriT-4Em and pOriTalt, and these two plasmids were transferred into Pseudoalteromonas sp. 3J3 by conjugation, as described by Wang et al. (65).
Promoter mapping by 5′-RACE PCR.
Total RNAs were isolated from Pseudoalteromonas sp. 3J6(pOriTalt) grown in VNSS medium by using a MasterPure RNA purification kit (Epicentre Biotechnologies, Madison, WI). The 5′ end of alt mRNA was amplified by using a 5′-RACE kit (Invitrogen) according to the manufacturer’s instructions. The primers used for cDNA synthesis, the first PCR, and the second PCR were GSP1 alt (5′-TTACTGACACTTTATAGTTGC-3′), GSP2 alt (5′-CCATAATTGGTTATACCAATC-3′), and nested GSP alt (5′-CCCCACAAGTGATTGTACT-3′), respectively. The final PCR product of 5′-RACE amplifications was sequenced (GATC Biotech, Cologne, Germany).
mRNA assay by RT-qPCR.
RNA extraction, cDNA synthesis, and qPCR were performed as previously described (66). The primers for the alt mRNA were 5′-CTTTCAGCAAACACAATGGCA-3′ and 5′-GCCTTGTCGTCTTCCACAT-3′, and the primers for the 16S rRNA used as endogenous control were 5′-GACTGAGACACGGCCCAGAC-3′ and 5′-GCAATATTCCCCACTGCTGC-3′. PCRs were performed in triplicate, and the standard deviations were <0.15 threshold cycle (CT). Quantifications were obtained by using the comparative CT (2−ΔΔCT) method (67).
Phylogenetic position of Pseudoalteromonas sp. 3J6 based on the 16S rRNA gene.
We determined the phylogenetic position of Pseudoalteromonas sp. 3J6 among 92 other Pseudoalteromonas strains (58 Pseudoalteromonas strains which have a genome available in public databases and 34 taxonomically characterized Pseudoalteromonas type strains) and an Algicola bacteriolytica strain as the outgroup. The 16S rRNA sequences from the selected species were aligned using the L-INS-i algorithm in MAFFT v.7 (68). The resulting multiple sequence alignment was visualized using Bioedit v.7.0.5.3 (69), and nonaligned regions were removed. A total of 1,341 nucleotide positions were used for the phylogeny, which was accomplished as described previously (61).
ANI and genome phylogeny.
Pairwise genome distances were calculated under the MicroScope platform using Mash, a method that estimates the mutation rate between two sequences (70), after uploading onto the MicroScope platform the 58 Pseudoalteromonas genome sequences that were available at the time of analysis (February 2019) in the NCBI RefSeq complete genome section. The interest in using the Mash distance is that it strongly correlates with the ANI, in accordance with the equation Mash distance ≃ 1 − ANI, which allows the determination of genomic relatedness between prokaryote strains without having to perform the pairwise genome alignments or experimental DNA-DNA hybridizations (30). From the computed Mash distances, a tree was constructed using a neighbor-joining algorithm.
Phylogeny of the alterocin gene and synteny comparison.
We searched for homologs of the alterocin protein sequences of Pseudoalteromonas sp. 3J6 in PkGDB, the database of the MicroScope platform, using the BLASTP algorithm (similarity constraints: at least 30% identity with a coverage of the query of at least 80%) and found 18 Pseudoalteromonas strains and the Oceanospirillales Hahella chejuensis KCTC 2396 strain with an alt gene in their genome. A phylogenetic tree based on the 20 alterocin proteins (including that from 3J6) was then calculated with the Hahella chejuensis protein as the outgroup. The alterocin protein sequences were aligned, and nonaligned regions were trimmed as for the 16S rRNA gene phylogeny. Evolutionary distances were computed using Jones-Taylor-Thornton evolutive model by assuming that a proportion of sites in the sequence are invariant while the remaining sites are gamma distributed (71). A total of 128 amino acid positions were used for the phylogeny. In those 19 genomes, we searched for a set of homologous genes including the alt gene with the same local organization (i.e., synteny conservation) as in the genome of Pseudoalteromonas sp. 3J6 by using the MicroScope platform (63), with correspondence relationships between genomes calculated using protein sequence similarity (parameters: BLASTP Bidirectional Best Hit or at least 30% identity on 80% of the shortest sequence).
Statistical analyses.
P values were determined using a Student t test when comparing two groups or analysis of variance (ANOVA) when comparing more than two groups. GraphPad Prism and Origin softwares were used.
Data availability.
The annotated part of Pseudoalteromonas sp. 3J6 genome, including the alt gene, has been deposited in the European Nucleotide Archive (https://www.ebi.ac.uk/ena) under accession number PRJEB37533.
ACKNOWLEDGMENTS
We thank A. Jack (Université Paris-Sud 11, Paris, France) and X. L. Chen (Shandong University, Jinan, China) for the gifts of E. coli GEB883 and pOriT-4Em, respectively, and E. Duchaud (INRAE, Jouy-en-Josas, France) for critically reading the manuscript.
The LABGeM (CEA/Genoscope and CNRS UMR8030) and the France Génomique and French Bioinformatics Institute national infrastructures (funded as part of the Investissement d’Avenir program managed by Agence Nationale pour la Recherche, contracts ANR-10-INBS-09 and ANR-11-INBS-0013) are acknowledged for support within the MicroScope annotation platform. We thank the Metabomer, Genomer, and ABiMS platforms (SBR, Sorbonne Université) for the contribution of their members (F.G., M.P., and E.C., respectively) to this work. A.G., G.M., A.D., and A.B. are grateful to ANR for its support via the investment expenditure program IDEALG (ANR-10-BTBR-04; http://www.idealg.ueb.eu/). The doctoral fellowships of M.S. and A.J. were funded by the Région Bretagne, France, and the Université de Bretagne-Sud, France. A.G. acknowledges support by the Institut Français de Recherche pour l’Exploitation de la Mer (IFREMER). The LBCM is supported by the Région Bretagne and European FEDER.
Footnote
[This article was published on 1 October 2020 with improper formatting for affiliation “c.” This has been corrected in the version of the article posted on 5 October 2020.]
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REFERENCES
1.
Donlan RM, Costerton JW. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193.
2.
Monds RD, O’Toole GA. 2009. The developmental model of microbial biofilms: ten years of a paradigm up for review. Trends Microbiol 17:73–87.
3.
Taylor PK, Yeung ATY, Hancock REW. 2014. Antibiotic resistance in Pseudomonas aeruginosa biofilms: towards the development of novel anti-biofilm therapies. J Biotechnol 191:121–130.
4.
Pletzer D, Hancock REW. 2016. Antibiofilm peptides: potential as broad-spectrum agents. J Bacteriol 198:2572–2578.
5.
Pendleton JN, Gorman SP, Gilmore BF. 2013. Clinical relevance of the ESKAPE pathogens. Expert Rev Anti Infect Ther 11:297–308.
6.
Lyczak JB, Cannon CL, Pier GB. 2002. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 15:194–222.
7.
Moradali MF, Ghods S, Rehm BH. 2017. Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence. Front Cell Infect Microbiol 7:39.
8.
Desriac F, Rodrigues S, Doghri I, Sablé S, Lanneluc I, Fleury Y, Bazire A, Dufour A. 2018. Antimicrobial and antibiofilm molecules produced by marine bacteria, p 791–819. In La Barre S, Bates S (ed), Blue biotechnology: production and use of marine molecules, vol 2. Wiley-VCH Verlag GmbH, Berlin, Germany.
9.
Rendueles O, Kaplan JB, Ghigo JM. 2013. Antibiofilm polysaccharides. Environ Microbiol 15:334–346.
10.
Li XH, Lee JH. 2017. Antibiofilm agents: a new perspective for antimicrobial strategy. J Microbiol 55:753–766.
11.
Zhao J, Li X, Hou X, Quan C, Chen M. 2019. Widespread existence of quorum sensing inhibitors in marine bacteria: potential drugs to combat pathogens with novel strategies. Mar Drugs 17:275.
12.
James SG, Holmström C, Kjelleberg S. 1996. Purification and characterization of a novel antibacterial protein from the marine bacterium D2. Appl Environ Microbiol 62:2783–2788.
13.
Lucas-Elío P, Gómez D, Solano F, Sanchez-Amat A. 2006. The antimicrobial activity of marinocine, synthesized by Marinomonas mediterranea, is due to hydrogen peroxide generated by its lysine oxidase activity. J Bacteriol 188:2493–2501.
14.
Campillo-Brocal JC, Chacón-Verdú MD, Lucas-Elío P, Sánchez-Amat A. 2015. Distribution in microbial genomes of genes similar to lodA and goxA which encode a novel family of quinoproteins with amino acid oxidase activity. BMC Genomics 16:231.
15.
Holmström C, Kjelleberg S. 1999. Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents. FEMS Microbiol Ecol 30:285–293.
16.
Bowman JP. 2007. Bioactive compound synthetic capacity and ecological significance of marine bacterial genus Pseudoalteromonas. Mar Drugs 5:220–241.
17.
Offret C, Desriac F, Le Chevalier P, Mounier J, Jégou C, Fleury Y. 2016. Spotlight on antimicrobial metabolites from the marine bacteria Pseudoalteromonas: chemodiversity and ecological significance. Mar Drugs 14:129.
18.
Paulsen SS, Strube ML, Bech PK, Gram L, Sonnenschein EC. 2019. Marine chitinolytic Pseudoalteromonas represents an untapped reservoir of bioactive potential. mSystems 4:1–12.
19.
Grasland B, Mitalane J, Briandet R, Quemener E, Meylheuc T, Linossier I, Vallee-Rehel K, Haras D. 2003. Bacterial biofilm in seawater: cell surface properties of early-attached marine bacteria. Biofouling 19:307–313.
20.
Dheilly A, Soum-Soutéra E, Klein GL, Bazire A, Compère C, Haras D, Dufour A. 2010. Antibiofilm activity of the marine bacterium Pseudoalteromonas sp. strain 3J6. Appl Environ Microbiol 76:3452–3461.
21.
Klein GL, Soum-Soutéra E, Guede Z, Bazire A, Compère C, Dufour A. 2011. The anti-biofilm activity secreted by a marine Pseudoalteromonas strain. Biofouling 27:931–940.
22.
Rodrigues S, Paillard C, Dufour A, Bazire A. 2015. Antibiofilm activity of the marine bacterium Pseudoalteromonas sp. 3J6 against Vibrio tapetis, the causative agent of brown ring disease. Probiotics Antimicrob Proteins 7:45–51.
23.
Klein G. 2011. Nouvelles molecules naturelles inhibitrices du développement de biofilms de bactéries marines. PhD thesis. Université de Bretagne Occidentale, Brest, France.
24.
Lee B, Haagensen JA, Ciofu O, Andersen JB, Høiby N, Molin S. 2005. Heterogeneity of biofilms formed by nonmucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis. J Clin Microbiol 43:5247–5255.
25.
Deligianni E, Pattison S, Berrar D, Ternan NG, Haylock RW, Moore JE, Elborn SJ, Dooley JS. 2010. Pseudomonas aeruginosa cystic fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability in vitro. BMC Microbiol 10:38.
26.
Mann EE, Wozniak DJ. 2012. Pseudomonas biofilm matrix composition and niche biology. FEMS Microbiol Rev 36:893–916.
27.
Boukerb AM, Simon M, Pernet E, Jouault A, Portier E, Persyn E, Bouffartigues E, Bazire A, Chevalier S, Feuilloley MGJ, Lesouhaitier O, Caillon J, Dufour A. 2020. Draft genome sequences of four Pseudomonas aeruginosa clinical strains with various biofilm phenotypes. Microbiol Resour Announc 9:e01286-19.
28.
Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043–1055.
29.
Konstantinidis KT, Ramette A, Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361:1929–1940.
30.
Kim M, Oh HS, Park SC, Chun J. 2014. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 64:346–351.
31.
Xie BB, Shu YL, Qin QL, Rong JC, Zhang XY, Chen XL, Shi M, He HL, Zhou BC, Zhang YZ. 2012. Genome sequences of type strains of seven species of the marine bacterium Pseudoalteromonas. J Bacteriol 194:2746–2747.
32.
Karsten WE, Viola RE. 1991. Chemical and kinetic mechanisms of aspartate-β-semialdehyde dehydrogenase from Escherichia coli. Biochim Biophys Acta 1077:209–219.
33.
Schauer K, Rodionov DA, de Reuse H. 2008. New substrates for TonB-dependent transport: do we only see the ‘tip of the iceberg’? Trends Biochem Sci 33:330–338.
34.
Tamayo R, Pratt JT, Camilli A. 2007. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annu Rev Microbiol 61:131–148.
35.
Dahlstrom KM, O’Toole GA. 2017. A symphony of cyclases: specificity in diguanylate cyclase signaling. Annu Rev Microbiol 71:179–195.
36.
Almagro Armenteros JJ, Tsirigos KD, Sønderby CK, Petersen TN, Winther O, Brunak S, von Heijne G, Nielsen H. 2019. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol 37:420–423.
37.
Ceroni A, Passerini A, Vullo A, Frasconi P. 2006. DISULFIND: a disulfide bonding state and cysteine connectivity prediction server. Nucleic Acids Res 34:W177–W181.
38.
Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH. 2017. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res 45:D200–D203.
39.
Jeong H, Yim JH, Lee C, Choi SH, Park YK, Yoon SH, Hur CG, Kang HY, Kim D, Lee HH, Park KH, Park SH, Park HS, Lee HK, Oh TK, Kim JF. 2005. Genomic blueprint of Hahella chejuensis, a marine microbe producing an algicidal agent. Nucleic Acids Res 33:7066–7073.
40.
Shivaji S, Reddy GS. 2014. Phylogenetic analyses of the genus Glaciecola: emended description of the genus Glaciecola, transfer of Glaciecola mesophila, G. agarilytica, G. aquimarina, G. arctica, G. chathamensis, G. polaris and G. psychrophila to the genus Paraglaciecola gen. nov. as Paraglaciecola mesophila comb. nov., P. agarilytica comb. nov., P. aquimarina comb. nov., P. arctica comb. nov., P. chathamensis comb. nov., P. polaris comb. nov. and P. psychrophila comb. nov., and description of Paraglaciecola oceanifecundans sp. nov., isolated from the Southern Ocean. Int J Syst Evol Microbiol 64:3264–3275.
41.
Solovyev V, Salamov A. 2011. Automatic annotation of microbial genomes and metagenomic sequences, p 61–78. In Li RW (ed), Metagenomics and its applications in agriculture, biomedicine, and environmental studies. Nova Science Publishers, Hauppauge, NY.
42.
Li Y, Rebuffat S. 2020. The manifold roles of microbial ribosomal peptide-based natural products in physiology and ecology. J Biol Chem 295:34–54.
43.
Beis K, Rebuffat S. 2019. Multifaceted ABC transporters associated with microcin and bacteriocin export. Res Microbiol 170:399–406.
44.
Chevalier S, Bouffartigues E, Bazire A, Tahrioui A, Duchesne R, Tortuel D, Maillot O, Clamens T, Orange N, Feuilloley MGJ, Lesouhaitier O, Dufour A, Cornelis P. 2019. Extracytoplasmic function sigma factors in Pseudomonas aeruginosa. Biochim Biophys Acta Gene Regul Mech 1862:706–721.
45.
Lu SY, Zhao Z, Avillan JJ, Liu J, Douglas R, Call DR. 2017. Autoinducer-2 quorum sensing contributes to regulation of microcin PDI in Escherichia coli. Front Microbiol 8:2570.
46.
Licciardello G, Caruso A, Bella P, Gheleri R, Strano CP, Anzalone A, Trantas EA, Sarris PF, Almeida NF, Catara V. 2018. The LuxR regulators PcoR and RfiA coregulate antimicrobial peptide and alginate production in Pseudomonas corrugata. Front Microbiol 9:521.
47.
Yu Z, Ding Y, Yin J, Yu D, Zhang J, Zhang M, Ding M, Zhong W, Qiu J, Li J. 2018. Dissemination of genetic acquisition/loss provides a variety of quorum sensing regulatory properties in Pseudoalteromonas. Int J Mol Sci 19:3636.
48.
Yu Z, Yu D, Mao Y, Zhang M, Ding M, Zhang J, Wu S, Qiu J, Yin J. 2019. Identification and characterization of a LuxI/R-type quorum sensing system in Pseudoalteromonas. Res Microbiol 170:243–255.
49.
Dang HT, Komatsu S, Masuda H, Enomoto K. 2017. Characterization of LuxI and LuxR protein homologs of N-acylhomoserine lactone-dependent quorum sensing system in Pseudoalteromonas sp. 520P1. Mar Biotechnol (NY) 19:1–10.
50.
Grasland B. 2002. Etude du rôle des interactions énergétiques et des communications intercellulaires dans la formation de biofilms bactériens en milieu marin. PhD thesis. Université de Bretagne-Sud, Lorient, France.
51.
Dheilly A. 2007. Biofilms bactériens marins multi-espèces: mise en évidence d’un effet antagoniste. PhD thesis. Université de Bretagne-Sud, Lorient, France.
52.
Mai-Prochnow A, Webb JS, Ferrari BC, Kjelleberg S. 2006. Ecological advantages of autolysis during the development and dispersal of Pseudoalteromonas tunicata biofilms. Appl Environ Microbiol 72:5414–5420.
53.
Mai-Prochnow A, Lucas-Elio P, Egan S, Thomas T, Webb JS, Sanchez-Amat A, Kjelleberg S. 2008. Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several Gram-negative bacteria. J Bacteriol 190:5493–5501.
54.
Marden P, Tunlid A, Malmcrona-Friberg K, Odham G, Kjelleberg S. 1985. Physiological and morphological changes during short term starvation of marine bacterial isolates. Arch Microbiol 142:326–332.
55.
Nguyen AN, Disconzi E, Charrière GM, Destoumieux-Garzón D, Bouloc P, Le Roux F, Jacq A. 2018. csrB gene duplication drives the evolution of redundant regulatory pathways controlling expression of the major toxic secreted metalloproteases in Vibrio tasmaniensis LGP32. mSphere 3:e00582-18.
56.
Bazire A, Shioya K, Soum-Soutéra E, Bouffartigues E, Ryder C, Guentas-Dombrowsky L, Hémery G, Linossier I, Chevalier S, Wozniak DJ, Lesouhaitier O, Dufour A. 2010. The sigma factor AlgU plays a key role in formation of robust biofilms by nonmucoid Pseudomonas aeruginosa. J Bacteriol 192:3001–3010.
57.
Chen MY, Lee DJ, Tay JH, Show KY. 2007. Staining of extracellular polymeric substances and cells in bioaggregates. Appl Microbiol Biotechnol 75:467–474.
58.
Reference deleted.
59.
Allesen-Holm M, Barken KB, Yang L, Klausen M, Webb JS, Kjelleberg S, Molin S, Givskov M, Tolker-Nielsen T. 2006. A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol Microbiol 59:1114–1128.
60.
Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin S. 2000. Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146:2395–2407.
61.
Gobet A, Barbeyron T, Matard-Mann M, Magdelenat G, Vallenet D, Duchaud E, Michel G. 2018. Evolutionary evidence of algal polysaccharide degradation acquisition by Pseudoalteromonas carrageenovora 9T to adapt to macroalgal niches. Front Microbiol 9:2740.
62.
Nurk S, Bankevich A, Antipov D, Gurevich A, Korobeynikov A, Lapidus A, Prjibelsky A, Pyshkin A, Sirotkin A, Sirotkin Y, Stepanauskas R, McLean J, Lasken R, Clingenpeel SR, Woyke T, Tesler G, Alekseyev MA, Pevzner PA. 2013. Assembling genomes and mini-metagenomes from highly chimeric reads, p 158–170. In Deng M, Jiang R, Sun F, Zhang X (ed), Research in computational molecular biology. RECOMB 2013. Lecture notes in computer science, vol 7821. Springer, Berlin, Germany.
63.
Vallenet D, Calteau A, Dubois M, Amours P, Bazin A, Beuvin M, Burlot L, Bussell X, Fouteau S, Gautreau G, Lajus A, Langlois J, Planel R, Roche D, Rollin J, Rouy Z, Sabatet V, Médigue C. 2020. MicroScope: an integrated platform for the annotation and exploration of microbial gene functions through genomic, pangenomic and metabolic comparative analysis. Nucleic Acids Res 48:D579–D589.
64.
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. 2005. Protein identification and analysis tools on the ExPASy server, p 571–607. In Walker JM (ed), Proteomics protocols handbook. Humana Press, New York, NY.
65.
Wang P, Yu Z, Li B, Cai X, Zeng Z, Chen X, Wang X. 2015. Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas. Microb Cell Fact 14:11.
66.
Guyard-Nicodème M, Bazire A, Hémery G, Meylheuc T, Mollé D, Orange N, Fito-Boncompte L, Feuilloley M, Haras D, Dufour A, Chevalier S. 2008. Outer membrane modifications of Pseudomonas fluorescens MF37 in response to hyperosmolarity. J Proteome Res 7:1218–1225.
67.
Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408.
68.
Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780.
69.
Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser (Oxf) 41:95–98.
70.
Ondov BD, Treangen TJ, Melsted P, Mallonee AB, Bergman NH, Koren S, Phillippy AM. 2016. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol 17:132.
71.
Jones DT, Taylor WR, Thornton JM. 1992. The rapid generation of mutation data matrices. Comput Appl Biosci 8:275–282.
72.
Yu ZC, Zhao DL, Ran LY, Mi ZH, Wu ZY, Pang X, Zhang XY, Su HN, Shi M, Song XY, Xie BB, Qin QL, Zhou BC, Chen XL, Zhang YZ. 2014. Development of a genetic system for the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913. Microb Cell Fact 13:13.
Information & Contributors
Information
Published In
Applied and Environmental Microbiology
Volume 86 • Number 20 • 1 October 2020
eLocator: e00893-20
Editor: Andrew J. McBain, University of Manchester
PubMed: 32769182
Copyright
© 2020 American Society for Microbiology. All Rights Reserved.
History
Received: 14 April 2020
Accepted: 27 July 2020
Published online: 1 October 2020
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Editor
Andrew J. McBain
Editor
University of Manchester
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2020.
Alterocin, an Antibiofilm Protein Secreted by Pseudoalteromonas sp. Strain 3J6. Appl Environ Microbiol
86:e00893-20.
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