Microbes spontaneously release membrane vesicles (MVs), which play roles in nutrient acquisition and microbial interactions. Iron is indispensable for microbes, but is a difficult nutrient to acquire. However, whether MVs are also responsible for efficient iron uptake and therefore involved in microbial interaction remains to be elucidated. Here, we used a Gram-positive strain, Dietzia sp. DQ12-45-1b, to analyze the function of its MVs in heme-iron recycling and sharing between species. We determined the structure and constituent of MVs and showed that DQ12-45-1b releases MVs originating from the mycomembrane. When comparing proteomes of MVs between iron-limiting and iron-rich conditions, we found that under iron-limiting conditions, heme-binding proteins are enriched. Next, we proved that MVs participate in extracellular heme capture and transport, especially in heme recycling from environmental hemoproteins. Finally, we found that the heme carried in MVs is utilized by multiple species, and we further verified that membrane fusion efficiency and species evolutionary distance determine heme delivery. Together, our findings strongly suggest that MVs act as a newly identified pathway for heme recycling, and represent a public good shared between phylogenetically closely related species.

Summary Membrane vesicles (MVs) released from bacteria act as extracellular vehicles carrying various functional cargoes between cells. MVs with different cargoes play multiple roles in stress adaptation, nutrient acquisition and microbial interactions. However, previous studies have primarily focused on MVs from Gram‐negative bacteria, while the characteristics of cargoes in MVs from Gram‐positive bacteria and their involvement in microbial interactions remain to be elucidated. Here, we used a Gram‐positive strain, Dietzia sp. DQ12‐45‐1b from Corynebacteriales, to analyse the characteristics and functions of MVs. We identified the ‘antioxidant’ canthaxanthin is stored within MVs by LC–MS/MS. In addition, nearly the entire genomic content of strain DQ12‐45‐1b are evenly distributed in MVs, suggesting that MVs from DQ12‐45‐1b might involve in horizontal gene transfer. Finally, the mycobactin‐type siderophores were detected in MVs. The iron‐loaded MVs effectively mediate iron binding and delivery to homologous bacteria from the order Corynebacteriales, but not to more distantly related species from the orders Pseudomonadales, Bacillales and Enterobacterales. These results revealed that the iron‐loaded MVs are shared between homologous species. Together, we report the Gram‐positive bacterium Dietzia sp. DQ12‐45‐1b released MVs that contain canthaxanthin, DNA and siderophores and prove that MVs act as public goods between closely related species.

-Alkanes are ubiquitous in nature and are widely used by microorganisms as carbon sources. Alkane hydroxylation by alkane monooxygenases is a critical step in the aerobic biodegradation of -alkanes, which plays important roles in natural alkane attenuation and is used in industrial and environmental applications. The alkane oxidation operon,, in the alkane-degrading strain sp. strain DQ12-45-1b is negatively autoregulated by the TetR family repressor AlkX via a product positive feedback mechanism. To predict the gene regulation mechanism, we determined the 3.1-Å crystal structure of an AlkX homodimer in a non-DNA-bound state. The structure showed traceable long electron density deep inside a hydrophobic cavity of each monomer along the long axis of the helix bundle, and further gas chromatography-mass spectrometry analysis of AlkX revealed that it contained the-derived long-chain fatty acid molecules as a ligand. Moreover, an unusual structural feature of AlkX is an extra helix, α6', forming a lid-like structure with α6 covering the inducer-binding pocket and occupying the space between the two symmetrical DNA-binding motifs in one dimer, indicating a distinct conformational transition mode in modulating DNA binding. Sequence alignment of AlkX homologs from strains showed that the residues involved in DNA and inducer binding are highly conserved, suggesting that the regulation mechanisms of-alkane hydroxylation are possibly a common characteristic of strains. With -alkanes being ubiquitous in nature, many bacteria from terrestrial and aquatic environments have evolved-alkane oxidation functions. Alkane hydroxylation by alkane monooxygenases is a critical step in the aerobic biodegradation of -alkanes, which plays important roles in natural alkane attenuation and petroleum-contaminating environment bioremediation. The gene regulation of the most common alkane hydroxylase, AlkB, has been studied widely in Gram-negative bacteria but has been less explored in Gram-positive bacteria. Our previous study showed that the TetR family regulator (TFR) AlkX negatively autoregulated the alkane oxidation operon,, in the Gram-positive strain sp. strain DQ12-45-1b. Although TFRs are one of the most common transcriptional regulator families in bacteria, the TFR involved in-alkane metabolism has been reported only recently. In this study, we determined the crystal structure of AlkX, which implies a distinct DNA/ligand binding mode. Our results shed light upon the regulation mechanism of the common alkane degradation process in nature.

Nonhomologous end joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNA Here, we established a proof-of-concept single-homology-arm linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNA by NHEJ could be used to generate nonreplicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach in sp. strain DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA single homolog to the sequence in the chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% of prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, an strain also showed NHEJ activity when the Ku and LigD of sp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for nonmodel microorganisms. Many nonmodel Gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway in sp. DQ12-45-1b was evaluated and was used to successfully knock out 11 genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the single-homology-arm linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse nonmodel prokaryotic organisms.