Characterization of membrane vesicles in Alteromonas macleodii indicates potential roles in their copiotrophic lifestyle

Abstract

Bacterial membrane vesicles (MVs) are abundant in the oceans, but their potential functional roles remain unclear. In this study we characterized MV production and protein content of six strains of Alteromonas macleodii, a cosmopolitan marine bacterium. Alteromonas macleodii strains varied in their MV production rates, with some releasing up to 30 MVs per cell per generation. Microscopy imaging revealed heterogenous MV morphologies, including some MVs aggregated within larger membrane structures. Proteomic characterization revealed that A. macleodii MVs are rich in membrane proteins related to iron and phosphate uptake, as well as proteins with potential functions in biofilm formation. Furthermore, MVs harbored ectoenzymes, such as aminopeptidases and alkaline phosphatases, which comprised up to 20% of the total extracellular enzymatic activity. Our results suggest that A. macleodii MVs may support its growth through generation of extracellular 'hotspots' that facilitate access to essential substrates. This study provides an important basis to decipher the ecological relevance of MVs in heterotrophic marine bacteria.

Keywords: EVs; extracellular enzymes; iron uptake; marine bacteria; membrane transporters; moonlighting proteins.

Figure 1.

Distinct morphological shapes in A. macleodii MVs, visualized using cryo-EM. (A) strain BS11–protrusions of potentially lipopolysaccharide or proteins surrounding the MVs. (B) strain HOT1A3–outer inner-MVs. (C) strain BGP6–micron-large membrane-bound structures. (D) strain BS11–tube-shaped membranous structures. Note the larger scale bars in panels C and D.

Figure 2.

Size characterization of MVs in A. macleodii strains. Each plot represents the NTA size distribution of MVs; the shaded area representing the standard deviation between the technical replicates of the NTA runs.

Figure 3.

Production of MVs by A. macleodii. (A) Total cell (circles) and MV (triangles) abundances in six different strains of A. macleodii. The standard errors between the biological replicates are smaller than the symbols. (B) Calculated production rates of MVs during the early (0–24 h) and late (24–72 h) growth stages of each strain. The color coding of the individual strains is the same for panels A and B.

Figure 4.

Proportions of subcellular localizations among the most abundant proteins in the cellular (‘C’) and MV (‘M’) fractions in each A. macleodii strain. The color code represents the different subcellular origins, predicted by CELLO2GO (A) and psortdb (B).

Figure 5.

Proportion of selected COG categories among the most abundant MV-associated proteins in each A. macleodii strain. Included COG categories are: E—Amino acid transport and metabolism; G—Carbohydrate transport and metabolism; N—Cell motility; M—Cell wall/membrane/envelope biogenesis; H—Coenzyme transport and metabolism; C—Energy production and conversion; W—Extracellular structures; S—Function unknown; R—General function prediction only; P—Inorganic ion transport and metabolism; U—Intracellular trafficking, secretion, and vesicular transport; I—Lipid transport and metabolism; F—Nucleotide transport and metabolism; O—Posttranslational modification, protein turnover, chaperones; L—Replication, recombination and repair; K—Transcription; J—Translation, ribosomal structure and biogenesis.

Figure 6.

Extracellular enzymatic activity (EEA) of different size fractions in culture of A. macleodii type strain ATCC27126. The bulk represents EEA of non-filtered samples (i.e. bacterial cells + ectoenzymes). The color coding of the individual fractions is the same for all plots. AGase—alpha-glucosidase, BGase—beta-glucosidase, APA—alkaline phosphatase, LAPase—L-aminopeptidase.