Reclassification of Bacteriovorax marinus as Halobacteriovorax marinus gen. nov., comb. nov. and Bacteriovorax litoralis as Halobacteriovorax litoralis comb. nov.; description of Halobacteriovoraceae fam. nov. in the class Deltaproteobacteria

Abstract

The taxonomic status of saltwater Bdellovibrio-like prokaryotic predators has been revised to assign species to Halobacteriovorax gen. nov. A reclassification of Bacteriovorax marinus as Halobacteriovorax marinus comb. nov. (type strain ATCC BAA-682^T = DSM 15412^T) and Bacteriovorax litoralis as Halobacteriovorax litoralis comb. nov. (type strain ATCC BAA-684^T = DSM 15409^T) is proposed. This revision is necessary because a previous proposal to retain saltwater isolates as species of Bacteriovorax and reclassify Bacteriovorax stolpii as Bacteriolyticum stolpii was not approved. The type species of a genus cannot be reassigned to another genus. Bacteriovorax stolpii is thus retained as the type species of Bacteriovorax and Halobacteriovorax marinus is the type species of Halobacteriovorax and of Halobacteriovoraceae fam. nov.

The taxonomy of the group of prokaryotic predators known as Bdellovibrio and like organisms (BALOs) has undergone significant changes since 2000. BALOs are ubiquitous in nature, and are found in a variety of habitats including soil, sewage, freshwater and marine environments. Baer et al. (2000) demonstrated that there was only a distant relationship between Bdellovibrio bacteriovorus 100^T and two other terrestrial bacteria, Bdellovibrio stolpii UKi2^T and Bdellovibrio starrii A3.12^T. They therefore proposed to assign the latter two species to a new genus, Bacteriovorax, and proposed that the type species should be Bacteriovorax stolpiicomb. nov. Further studies by Snyder et al. (2002) of 26 freshwater/terrestrial and saltwater isolates, based on 16S rRNA gene sequence analysis, highlighted the fact that the marine isolates clustered with Bacteriovorax, not Bdellovibrio. This result led to a further report (Baer et al., 2004) which then classified two marine isolates as species of Bacteriovorax: Bacteriovorax marinus sp. nov. and Bacteriovorax litoralis sp. nov.

At the same time, a subsequent, more comprehensive, phylogenetic analysis by Davidov & Jurkevitch (2004) of a large number (71) of terrestrial strains of BALOs from various habitats revealed the considerable diversity among these predatory bacteria. Of note was the fact that none of the Bdellovibriostrains was of marine origin, and Bacteriovoraxstrains could be separated into soil/freshwater and marine/saltwater groups. They proposed that Bacteriovorax starrii be assigned to a new genus, Peredibacter gen. nov., with Peredibacter starrii as the type species. Also proposed was a redefinition of taxonomic families, such that the Bdellovibriolineage remained in the existing family Bdellovibrionaceae, and the Bacteriovorax–Peredibacter groups were classified into a new family, Bacteriovoracaceae. The type genus is Bacteriovorax.

The need to review and revise the taxonomy of BALOs, as stated by Snyder et al. (2002), was especially necessary for saltwater isolates. Piñeiro et al. (2008) chose to use the gene coding for the β-subunit of RNA polymerase (rpoB) to permit greater differentiation of Bacteriovoraxisolates, based on comparative sequence analysis. Their results led to the proposal to reclassify the soil isolate Bacteriovorax stolpii as Bacteriolyticum stolpii gen. nov., comb. nov., as it showed significant diversity from the saltwater isolates. Unfortunately, the new genus name is illegitimate, because the type species of a genus cannot be reassigned to another genus (new or existing). This move would contradict Principle 1 of maintaining stability, Rule 15, Rule 37a (because the type species was removed from the genus Bacteriovorax, which can only be properly done if the other species in the genus were also renamed or moved with it) and Rule 51b (1) (whereby the name Bacteriovoraxshould have been adopted) of the Bacteriological Code. As this name is illegitimate, it casts doubt on the family Peredibacteraceaeproposed by Piñeiro et al. (2008), because it is possible the family should have remained Bacteriovoracaceae. The purpose of this report is to address these taxonomic errors.

Piñeiro et al. (2008) provided ample evidence that the differences in sequences of the 16S rRNA and rpoB genes were large enough to place the three genera, Bdellovibrio, Bacteriovorax and Peredibacter, each in its own family. We propose to reclassify the saltwater members of the genus Bacteriovorax as Halobacteriovorax gen. nov. in the family Halobacteriovoraceae fam. nov. Bacteriovorax stolpiiwould remain as the type (and only named) species of Bacteriovorax in the family Bacteriovoracaceaealong with six other isolates (Davidov & Jurkevitch, 2004). Peredibacter starriiwould remain as the type (and only named) species of Peredibacter in the family Peredibacteraceae (Piñeiro et al., 2008) along with four isolates from Davidov & Jurkevitch (2004).

The proposed families differ based on insertions and deletions in their 16S rRNA gene sequences. Insertions and deletions in this gene occur much less frequently than point mutations. Although the insertions and deletions in the 16S rRNA gene for each of these predatory bacterial families were presented previously (Piñeiro et al., 2008), they were misidentified and in Table 1 they appear with the corrected taxonomy. A phylogenetic tree was reconstructed based on 16S rRNA gene sequences (Fig. 1) using neighbour-joining and bootstrapping (1000 replicates) methods available in clustal w (Larkin et al., 2007). Sequence differences in the 16S rRNA gene between type strains of the BALOs are shown in Table 2. A maximum-likelihood tree calculated by phyml (Guindon et al. 2010) is presented in Fig. S1 (available in the online Supplementary Material) and a maximum-parsimony tree calculated by mega6 (Tamura et al. 2013) is presented in Fig. S2. Regardless of the method used to calculate the trees, Bdellovibrio and Halobacteriovorax are the most distant taxa with Peredibacter and Bacteriovorax as intermediates. Each named genus appears monophyletic.

Table 1. Characteristics of type species from BALOs.

	Family
	Bdellovibrionaceae		Bacteriovoracaceae	Peredibacteraceae	Halobacteriovoraceae
Type species/type strain	Bdellovibrio bacteriovorus 100T	Bdellovibrio exovorus JSST	Bacteriovorax stolpii UKi2T	Peredibacter starrii A3.12T	Halobacteriovorax marinus SJT	Halobacteriovorax litoralis JS5T
Source	Soil	Sewage	Soil	Soil	Saltwater	Saltwater
Internalized	Yes	No	Yes	Yes	Yes	Yes
NaCl required for growth (≥0.5 %)	No	No	No	No	Yes	Yes
Prey species*	E. coli	Caulobacter crescentus	E. coli	Pseudomonas putida	Vibrio parahaemolyticus	Vibrio parahaemolyticus
DNA G+C content (mol%)	51.5	46.1	41.8	43.5 %	37.7 %	37.8 %
Valine amino peptidase	−	Unknown	+	+	+	Unknown
Cystine amino peptidase	−	Unknown	+	+	+	Unknown
Chymotrypsin	+	Unknown	−	−	−	Unknown
Polymyxin B sensitivity (300 U)	−	Unknown	−	−	+	Unknown
C16 : 1ω9c fatty acid	−	Unknown	+	+	+	Unknown
iso-C13 : 1 3-OH fatty acid	+	Unknown	−	−	−	Unknown
C17 : 1ω6c fatty acid	−	Unknown	+	+	−	Unknown
16S insertions/deletions from Bdellovibrio†
ca. 181–196 of E. coli 16S	Baseline	Baseline	+12	−3	+15	+15
ca. 436 of E. coli 16S	Baseline	Baseline	+23	+22	+23	+23
ca. 1040 of E. coli 16S	Baseline	Baseline	+2	+2	+2	+2

^*For maintenance of cultures.

^†For type species only; other isolates within genera have additional insertions or deletions.

Fig. 1.

Neighbour-joining phylogram of BALOs. All BALO type species are included. Many isolates have not been described to species level and are listed as sp. plus the isolate name. Approximately full-length (~1400 bp) sequences are used except for Halobacteriovorax sp. Annapolis and Bdellovibrio sp. Tu-115. Bootstrapping was done with 1000 replicates in clustal x version 2.0 (Larkin et al., 2007). Horizontal lines are proportional to distances. Numbers along horizontal lines indicate the distance between nodes.

Table 2. Pairwise 16S rRNA gene sequence genetic distances between type strains of BALOs.

	Bacteriovorax stolpii UKi2T	Halobacteriovorax marinus SJT	Halobacteriovorax litoralis JS5T	Peredibacter starrii A3.12T	Bdellovibrio bacteriovorus 100T	Bdellovibrio exovorus JSST
Bacteriovorax stolpii	0.00	0.11	0.12	0.14	0.24	0.24
Halobacteriovorax marinus	0.11	0.00	0.08	0.16	0.24	0.24
Halobacteriovorax litoralis	0.12	0.08	0.00	0.17	0.24	0.24
Peredibacter starrii	0.14	0.16	0.17	0.00	0.25	0.25
Bdellovibrio bacteriovorus	0.24	0.24	0.24	0.25	0.00	0.08
Bdellovibrio exovorus	0.24	0.24	0.24	0.25	0.08	0.00

As a result of this proposal, the two saltwater species would be moved from the genus Bacteriovorax (Baer et al., 2004) and assigned to the genus Halobacteriovorax as Halobacteriovorax marinus gen. nov., comb. nov. and Halobacteriovorax litoralis comb. nov. For consistency with taxonomy (priority), we propose that Halobacteriovorax marinus be the type species of Halobacteriovorax. Numerous (111) additional isolates in several clusters would be moved as well (Piñeiro et al., 2007). In addition, Halobacteriovoraceae fam. nov. is described and the descriptions of the families Bacteriovoracaceae and Peredibacteraceae are revised.

A polyphasic approach to support the taxonomic proposals in this study would be useful. However, this taxonomic evidence has not been possible to obtain, as it is necessary to grow the predators in the presence of prey cells. Chemotaxonomic analyses could be performed on prey-independent mutants, but the results may not reflect the composition of wild-type strains (Schwudke et al., 2001; Koval et al., 2013). A limited set of data are presented in Table 1. The Halobacteriovoraceae are distinct from the other families of BALOs based on several properties (Table 1). It is the only family with members found in saltwater habitats, a phenotypic characteristic that is reflected in the requirement for NaCl for growth. The DNA G+C content (<38 mol%) of species is the lowest of any other BALOs. The only family that has been separated based on the detection of enzymes is the Bdellovibrionaceae. The presence or absence of three long chain fatty acids also distinguishes both the Halobacteriovoraceae and the Bdellovibrionaceae. Although classic phenotypic traits distinguish between the families, it is the 16S rRNA gene sequences that are the most useful and the only ones available for Halobacteriovorax litoralis and Bdellovibrio exovorus.

Description of Halobacteriovorax gen. nov.

Ha.lo.bac.te.ri.o.vo′rax. (Gr. n. hals, haloes salt; L. neut. n. bacterium a small rod; L. adj. vorax devouring, ravenous, voracious; N.L. masc. n. Halobacteriovorax devourer of bacteria in saltwater environments).

This genus consists of saltwater Gram-negative bacteria that prey upon other Gram-negative bacteria to complete a biphasic life cycle. Found in marine, estuarine and hypersaline lake environments. Low DNA G+C content (<39 mol%; Marbach et al., 1975; Baer et al., 2004), which distinguishes the genus from freshwater and terrestrial BALOs in which values range from 47 to 51 mol% for strains of Bdellovibrio bacteriovorus(Seidler et al., 1972; Torrella et al., 1978) and from 41 to 44 % for Bacteriovorax stolpii UKi2^T and Peredibacter starrii A3.12^T (Seidler et al., 1972). Prey more efficiently on saltwater bacteria than on freshwater bacteria. Optimal growth temperature range is typically 25–30 °C. The morphological description of the genus is the same as that of the type species, Halobacteriovorax marinus.

Description of Halobacteriovorax marinus comb. nov.

Halobacteriovorax marinus (ma.ri′nus. L. masc. adj. marinus of the sea, marine).

Basonym: Bacteriovorax marinus Baer et al. 2004

The cultural and biochemical characteristics of this species are described by Baer et al. (2004). The 16S rRNA gene sequence is characteristic of cluster III of the Halobacteriovoraceae, but differentiated from other bacteria. The whole genome sequence of Halobacteriovorax marinus SJ^T has been determined (Crossman et al., 2013).

The type strain is SJ^T ( = ATCC BAA-682^T = DSM 15412^T), which was isolated from water in St. John’s Island in the Caribbean using Vibrio parahaemolyticus P-5 as prey.

Description of Halobacteriovorax litoralis comb. nov.

Halobacteriovorax litoralis (li.to.ra′lis. L. masc. adj. litoralis pertaining to the coast).

Basonym: Bacteriovorax litoralis Baer et al. 2004

The cultural and biochemical characteristics of this species are described by Baer et al. (2004). The 16S rRNA gene sequence is characteristic of cluster IV of the Halobacteriovoraceae, but differentiated from other bacteria.

The type strain is JS5^T ( = ATCC BAA-684^T = DSM 15409^T), which was isolated from the Chesapeake Bay estuary using Vibrio parahaemolyticus P-5 as prey.

Description of Halobacteriovoraceae fam. nov.

Halobacteriovoraceae (Ha.lo.bac.te.ri.o.vo.ra.ce′ae. N.L. masc. n. Halobacteriovorax a bacterial genus name; -aceae ending denoting a family; N.L. fem. pl. n. Halobacteriovoraceae the Halobacteriovorax family).

The description of the family Halobacteriovoraceae is based on the description of the type genus Halobacteriovorax. Members of this family are vibroid bacteria, about 0.6–1.0 µm in length (Crossman et al., 2013). These species favour predation on Vibrio species and other saltwater prey. Different clusters within this genus favour estuarine or marine waters and some isolates have been found in salt lakes. There are no known freshwater isolates.

Emended description of Bacteriovoracaceae

The description of the family Bacteriovoracaceae is based on the description of the genus Bacteriovorax (Baer et al., 2004) and data from Piñeiro et al. (2007, 2008). This family is composed of Gram-negative, vibroid bacteria. They are obligate predators of many other Gram-negative bacteria. They exhibit a biphasic life cycle consisting of a motile attack phase and a phase that dwells in the periplasm of prey bacteria. They are a monophyletic offshoot of the family Bdellovibrionaceae. The only recognized species, Bacteriovorax stolpii, is found in freshwater and soil environments. The DNA G+C content is 41.8 mol% for the type strain of Bacteriovorax stolpii (Seidler et al., 1972). This species has distinctive insertions and deletions of nucleotides relative to the 16S rRNA gene sequence of Escherichia colithat differ from the families Halobacteriovoraceae, Peredibacteraceae and Bdellovibrionaceae. The type genus is Bacteriovorax. The presence of multiple clusters by 16S rRNA gene sequencing suggests the presence of additional species (Davidov & Jurkevitch 2004).

Emended description of Peredibacteraceae

The description of Peredibacteraceaeremains as given by Piñeiro et al. (2008) except for the removal of Bacteriovorax stolpii. The type genus is Peredibacter.

Abbreviations:

BALO

Bdellovibrio and like organism.