Genome Analysis, Metabolic Potential, and Predatory Capabilities of Herpetosiphon llansteffanense sp. nov

Abstract

Herpetosiphon spp. are ubiquitous, chemoheterotrophic, filamentous gliding bacteria with the ability to prey on other microbes through a "wolf pack" mechanism. The genus currently comprises four known species (H. aurantiacus, H. geysericola, H. giganteus, and H. gulosus), which produce antimicrobial secondary metabolites such as siphonazole. As part of a study isolating myxobacterial wolf pack predators, we serendipitously isolated a novel environmental strain (CA052B) from the edge of a stream at Llansteffan, United Kingdom, which was identified as a member of the Herpetosiphon genus. A lawn culture method was utilized to analyze the predatory activity of CA052B against 10 prey organisms of clinical relevance. CA052B was found to prey on Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Enterococcus faecalis, Bacillus subtilis, and Candida albicans Purified CA052B outer membrane vesicles also exhibited killing activity against the prey organisms when tested by flow cytometry. 16S rRNA sequencing of CA052B showed 98 to 99% identity with other Herpetosiphon species members. Comparing the genome of CA052B with the publicly available genomes of H. aurantiacus and H. geysericola revealed average nucleotide identities of only 84% and 91%, respectively, whereas the genome-to-genome distance calculation showed sequence identities of 28.2% and 46.6%, respectively. Biochemical characterization also revealed distinctions between CA052B and both H. gulosus and H. giganteus Thus, strain CA052B^T (= DSM 107618^T = NBRC 113495^T) is proposed to be the type strain of a novel species, Herpetosiphon llansteffanense sp. nov. The genome sequence of CA052B also revealed diverse secondary metabolite biosynthetic clusters, encouraging further exploration of its antibiotic production potential.IMPORTANCE Predatory bacteria are able to kill and consume other microbes and are therefore of interest as potential sources of new antimicrobial substances for applications in the clinic. "Wolf pack" predators kill prey by secreting antimicrobial substances into their surroundings, and those substances can kill prey organisms independently of the predatory cells. The genus Herpetosiphon exhibits wolf pack predation, yet its members are poorly described compared to other wolf pack predators, such as the myxobacteria. By providing a thorough characterization of a novel Herpetosiphon species, including its predatory, biochemical, and genomic features, this study increases our understanding of genomic variation within the Herpetosiphon genus and how that variation affects predatory activity. This will facilitate future rational exploitation of genus members (and other wolf pack predators) as sources of novel antimicrobials.

Keywords: antimicrobials; biological control; comparative genomics; myxobacteria; predator; prey.

FIG 1

A spreading colony of CA052B on VY-2 agar after 1 week of growth. VY-2 medium contains yeast cells, which are lysed by CA052B as it grows outwards. Note the orange pigmentation of older cells in the center of the plate due to the accumulation of carotenoids.

FIG 2

(A) Scanning electron microscope (SEM) images of CA052B cells (left) and aggregates (right). (B) Transmission electron microscope (TEM) images of CA052B. Constrictions between adjoining cells are indicated with arrows. (C) TEM (left) and SEM (right) images of CA052B/Escherichia coli cocultures. Arrows indicate “ghost” prey cells with degraded cytoplasmic contents (left).

FIG 3

Flow cytometric analysis of prey cell killing by CA052B OMVs and supernatant. (A) Live P. mirabilis cells. (B) P. mirabilis cells treated with ethanol to produce a mixture of live and dead cells. (C) Live P. mirabilis cells treated with CA052B OMVs. (D) Live P. mirabilis cells treated with CA052B supernatant. Percentages of events exceeding the propidium iodide fluorescence intensity (PI-A-Compensated) thresholds, shown as vertical lines, are presented in panels C and D.