Alcaligenes ammonioxydans HO-1 antagonizes Bacillus velezensis via hydroxylamine-triggered population response

Abstract

Antagonism is a common behavior seen between microbes in nature. Alcaligenes ammonioxydans HO-1 converts ammonia to nitrogen under aerobic conditions, which leads to the accumulation of extracellular hydroxylamine (HA), providing pronounced growth advantages against many bacterial genera, including Bacillus velezensis V4. In contrast, a mutant variant of A. ammonioxydans, strain 2-29, that cannot produce HA fails to antagonize other bacteria. In this article, we demonstrate that cell-free supernatants derived from the antagonistic HO-1 strain were sufficient to reproduce the antagonistic behavior and the efficiency of this inhibition correlated strongly with the HA content of the supernatant. Furthermore, reintroducing the capacity to produce HA to the 2-29 strain or supplementing bacterial co-cultures with HA restored antagonistic behavior. The HA-mediated antagonism was dose-dependent and affected by the temperature, but not by pH. HA caused a decline in biomass, cell aggregation, and hydrolysis of the cell wall in exponentially growing B. velezensis bulk cultures. Analysis of differential gene expression identified a series of genes modulating multicellular behavior in B. velezensis. Genes involved in motility, chemotaxis, sporulation, polypeptide synthesis, and non-ribosomal peptide synthesis were all significantly downregulated in the presence of HA, whereas autolysis-related genes showed upregulation. Taken together, these findings indicate that HA affects the population response of coexisting strains and also suggest that A. ammonioxydans HO-1 antagonize other bacteria by producing extracellular HA that, in turn, acts as a signaling molecule.

Keywords: Alcaligenes ammonioxydans HO-1; Bacillus velezensis V4; antagonism; autolysis; hydroxylamine; population response.

Figure 1

Contact-independent interbacterial antagonism of A. ammonioxydans HO-1 toward B. velezensis V4. (A) Comparison of antagonistic behavior between A. ammonioxydans HO-1 and 2-29, the latter failing to inhibit B. velezensis V4. (B) A. ammonioxydans HO-1 showed more competitive advantage when co-cultured with B. velezensis V4 with the initial OD₆₀₀ ratios of 1:2, 1:1, and 2:1, respectively. Red arrow indicates B. velezensis V4 colonies, and blue arrow indicates A. ammonioxydans HO-1 or 2-29 colonies. (C) HA production and inhibition effect of HCFS/UCFS (CFS of A. ammonioxydans HO-1/2-29) at different time points in culture. Error bar represents standard deviations of three replicates. (D) The inhibitory capacity of the complementation strain of A. ammonioxydans 2-29 (pBB-dnfR) and UCFS containing specified HA concentrations show inhibition capacity toward B. velezensis V4.

Figure 2

Characteristic of hydroxylamine inhibition toward B. velezensis V4. (A) Linear dose-dependency of HA-mediated inhibition. (B) The effect of temperature on HA-mediated inhibition. Pairwise comparisons between treatments are shown using a letter-based representation. Different letters indicate distinct significant differences. (C) The influence of pH on HA inhibition. Error bars represent standard deviation based on three replicates. Pairwise comparisons between treatments are shown using a letter-based representation. Different letters indicate distinct significant differences. *** means there is a significant difference between respective control and treatment.

Figure 3

The effect of HO-1 co-culture or treatment with HA on the colony morphology and survival kinetics of B. velezensis V4. (A) Changing appearance of B. velezensis V4 colonies during co-culture with A. ammonioxydans HO-1. Red arrows inside yellow circles indicate typical B. velezensis V4 colonies before significant A. ammonioxydans HO-1 growth at 24 h (on the left) and to the same colonies after the growth of A. ammonioxydans HO-1 caused autolysis at 72 h (right). (B) Typical B. velezensis V4 colonies before the treatment with HA (left) and the same plaques following autolysis after HA treatment (right). (C) Survival kinetics of B. velezensis V4 (high initial population density, OD₆₀₀ = 3.0) in V4CFS (as control), HCFS, UCFS, UCFS-2.0 mM HA, 0.9% NaCl-2.0 mM HA, V4CFS-2.0 mM HA. (D) Survival kinetics of B. velezensis V4 (low initial population density, OD₆₀₀ = 0.3) in V4CFS (as control), HCFS, UCFS, UCFS-2.0 mM HA, 0.9% NaCl-2.0 mM HA, V4CFS-2.0 mM HA. (E) Survival kinetics of B. velezensis V4 in V4CFS containing 0.2, 0.5, 1.0, 2.0, and 5.0 mM HA. Error bars represent standard deviations based on three replicates.

Figure 4

Morphology of B. velezensis V4 under different culture conditions. Cell morphology of B. velezensis V4 in V4CFS (A, 2,000× and 5,000×), V4CFS-2.0 mM HA (B, 700× and 10,000×), HCFS (C, 700× and 5,000×), UCFS (D, 2,000× and 5,000×) at 180 min. Green arrows indicate normal morphology of cells with flagellum, purple arrows indicate cell aggregation, blue arrows indicate cell lysis and cellular debris, and yellow arrows indicate biofilm analogs.

Figure 5

Changes in the abundance of genes modulating multicellular behavior in B. velezensis V4 upon exposure to HA. (A) Expression of motility and chemotaxis-related genes in B. velezensis V4 upon exposure to hydroxylamine. (B) Expression of polyketide synthetase (PKS) and non-ribosomal peptide synthetase (NRPS) genes in B. velezensis V4 upon exposure to hydroxylamine. (C) Expression of autolysis-related genes in B. velezensis V4 upon exposure to hydroxylamine. (D) Expression of sporulation-related genes in B. velezensis V4 upon exposure to hydroxylamine. RNA-seq data were normalized to TPM. Error bars indicate standard error based on three replicates. p-values were calculated using a negative binomial distribution-based test, and false discovery rate (p-adjust) was calculated using the Benjamini/Hochberg method. At each time point, ***represents a significant change in transcript abundance (FDR < 0.05).