Phosphate limitation induces the intergeneric inhibition of Pseudomonas aeruginosa by Serratia marcescens isolated from paper machines

Abstract

Phosphate is an essential nutrient for heterotrophic bacteria, affecting bacterioplankton in aquatic ecosystems and bacteria in biofilms. However, the influence of phosphate limitation on bacterial competition and biofilm development in multispecies populations has received limited attention in existing studies. To address this issue, we isolated 13 adhesive bacteria from paper machine aggregates. Intergeneric inhibition of Pseudomonas aeruginosa WW5 by Serratia marcescens WW4 was identified under phosphate-limited conditions, but not in Luria-Bertani medium or M9 minimal medium. The viable numbers of the pure S. marcescens WW4 culture decreased over 3 days in the phosphate-limited medium; however, the mortality of S. marcescens WW4 was significantly reduced when it was co-cultured with P. aeruginosa WW5, which appeared to sustain the S. marcescens WW4 biofilm. In contrast, viable P. aeruginosa WW5 cells immediately declined in the phosphate-limited co-culture. To identify the genetic/inhibitory element(s) involved in this process, we inserted a mini-Tn5 mutant of S. marcescens WW4 that lacked inhibitory effect. The results showed that an endonuclease bacteriocin was involved in this intergeneric inhibition by S. marcescens WW4 under phosphate limitation. In conclusion, this study highlights the importance of nutrient limitation in bacterial interactions and provides a strong candidate gene for future functional characterisation.

Fig. 1

The viability of Serratia marcescens WW4 and Pseudomonas aeruginosa WW5 in pure or co-cultures. The viability of S. marcescens WW4 (a) or P. aeruginosa WW5 (b) pure culture in LB, M9 or BM medium was counted at 0, 3, 6, 12, 24, 48 and 72 h. The individual viability of co-cultured S. marcescens WW4 and P. aeruginosa WW5 in BM medium is shown in (c). To confirm the effect of phosphate, the viability of P. aeruginosa WW5 in co-culture is shown in (d), when cultured in phosphate-supplied BM medium (BM+1/2P or BM+P) or phosphate-reduced M9 medium (M9 P-reduced), compared with BM or M9 medium. All data show the means of viable cell numbers (CFU mL⁻¹), and the bars represent the standard error.

Fig. 2

The biofilm of Serratia marcescens WW4 and P. aeruginosa WW5 in chambers, and the adhesion fraction of S. marcescens WW4 in pure or co-cultures. The hill-like biofilm structure of P. aeruginosa WW5 (a) and the filamentous biofilm structure of S. marcescens WW4 (b) were observed in BM medium in the biofilm-forming chambers. The pictures were obtained from 1-day DAPI-stained cultures, and the bars represent 25 μm. (c) The biofilm of S. marcescens WW4 mixing in the absence (×) or presence of equal (▲) or tenfold (▪) P. aeruginosa WW5 was incubated in phosphate-limited chambers. The adhesion fraction of S. marcescens WW4 was photographed at 1, 3 and 6 days using a fluorescence microscope, and the images were analysed using ImageJ software. All data show the means and standard errors of the adhesion proportion (%) of S. marcescens WW4 biofilm.

Fig. 3

The transposon insertion mutant of Serratia marcescens WW4. The diagram (a) shows the inserted transposon-mediated locus on the chromosome of the mini Tn-5 mutant (SM::mTn-5 80) that was associated with phosphate-limited inhibition. The inserted site (▵) was located upstream of a bacteriocin (CDS SMWW4_v1c20360), which was similar to an S-type pyocin with endonuclease activity. The genomic sequences were sequenced by Illumina sequencing and annotated by blastp in GenBank. The viability of Pseudomonas aeruginosa WW5 co-cultured with S. marcescens WW4 (SM WW4) or SM::mTn-5 80 in BM medium is presented in (b). The data show the means of viable P. aeruginosa WW5 cell numbers (CFU mL⁻¹). The bars indicate the standard error.