Chemotaxis may assist marine heterotrophic bacterial diazotrophs to find microzones suitable for N2 fixation in the pelagic ocean

Abstract

Heterotrophic bacterial diazotrophs (HBDs) are ubiquitous in the pelagic ocean, where they have been predicted to carry out the anaerobic process of nitrogen fixation within low-oxygen microenvironments associated with marine pelagic particles. However, the mechanisms enabling particle colonization by HBDs are unknown. We hypothesized that HBDs use chemotaxis to locate and colonize suitable microenvironments, and showed that a cultivated marine HBD is chemotactic toward amino acids and phytoplankton-derived DOM. Using an in situ chemotaxis assay, we also discovered that diverse HBDs at a coastal site are motile and chemotactic toward DOM from various phytoplankton taxa and, indeed, that the proportion of diazotrophs was up to seven times higher among the motile fraction of the bacterial community compared to the bulk seawater community. Finally, three of four HBD isolates and 16 of 17 HBD metagenome assembled genomes, recovered from major ocean basins and locations along the Australian coast, each encoded >85% of proteins affiliated with the bacterial chemotaxis pathway. These results document the widespread capacity for chemotaxis in diverse and globally relevant marine HBDs. We suggest that HBDs could use chemotaxis to seek out and colonize low-oxygen microenvironments suitable for nitrogen fixation, such as those formed on marine particles. Chemotaxis in HBDs could therefore affect marine nitrogen and carbon biogeochemistry by facilitating nitrogen fixation within otherwise oxic waters, while also altering particle degradation and the efficiency of the biological pump.

Fig. 1. Chemotaxis by Pseudomonas stutzeri.

Chemotaxis index (Ic) of P. stutzeri exposed to A 1 mM concentrations of amino acids and NH₄⁺ and B 1 mg ml⁻¹ cell extracts (pellet (P)) and exudates (supernatant (SN)) of cultured Chlamydomonas reinhardtii (green algae) and Thalassiosira pseudonana (diatom). Treatments significantly different from control (phosphate-buffered saline, PBS) and positive control (10% ZoBell broth) are indicated (*p < 0.05, **p < 0.01, ***p < 0.001; n = 4; ANOVA (one-sided)). Error bars represent standard deviation.

Fig. 2. Diazotrophs in a natural bacterioplankton community are motile and capable of performing chemotaxis.

A Chemotaxis index calculated for each of the treatments. B The relative abundance of metagenomic reads mapping to nifH as a fraction of the total reads in each sample. Bulk (Bulk seawater), FSW (Filtered seawater); significance levels for differences between Bulk and FSW and treatments are indicated (*p < 0.05, **p < 0.01, ***p < 0.001; n = 4; ANOVA (one-sided)). Error bars represent standard deviation. A is a reproduction of data published in [42].

Fig. 3. Diversity and composition of motile and chemotactic diazotrophs.

Analysis of metagenomic reads assigned as nifH obtained from the In situ chemotaxis assay. A Boxplot showing the range and median of the effective number of species calculated for each independent sample replicate. Significance levels reported in A is the difference between FSW and Bulk seawater and phytoplankton treatments (**p < 0.01; ***p < 0.001; n = 4; ANOVA (one-sided)). B Principal coordinate analysis (PCO) of Bray-Curtis similarity. Bulk (Bulk seawater), FSW (Filtered seawater); dotted line represents 60% similarity. C The relative abundance of classified metagenomic reads assigned as nifH combined for the four biological replicates. Blues: Cluster I, Green: Cluster I Cyanobacteria, Yellow, reds, and black: Cluster III, and Gray: Others. B, C are based on averaged relative abundances of four biological replicates.

Fig. 4. Chemotaxis is a prevailing phenotype in HBDs.

The presence (blue squares) or absence (gray squares) of genes assigned to the KEGG pathway for chemotaxis (ko02030) in four marine heterotrophic bacterial diazotroph (HBD) isolates, metagenome assembled genomes (MAGs) of 17 putative HBDs and two cyanobacterial diazotrophs from Tara oceans and AMI, and reference genomes of Trichodesmium erythraeum, UCYN-A, Candidatus Pelagibacter ubique (SAR11), and Azotobacter vinelandii. The genomes and MAGs are clustered based on the proteins present. See Supplementary Information 2 for detailed information of the MAGs, isolates, and reference genomes. Levels of completeness of genomes and MAGs are indicated above the chart.