Strain Z-7288^T (= DSM 5501 = ATCC 49924) is the type strain of the species Acetohalobium arabaticum, which is the type species of the genus Acetohalobium [1,2]. The genus name derives from the Latin word ‘acetum’, meaning vinegar, and the Greek words ‘halos’ and ‘bios’, meaning salt and life, respectively, in order to indicate an acetate-producing organism living in salt [3]. The species name derives from Arabat, a peninsula between the Sea of Azov and Sivash [3], since the strain was isolated from lagoons of the Arabat spit (East Crimea) which separates Sivash lake from the Sea of Azov [2]. Currently, this is the only known strain in the genus Acetohalobium. A. arabaticum participates together with other halophilic bacteria and the genera Methanohalophilus and Methanohalobium in the C₁-trophic chain in hypersaline environments [2]. Here we present a summary classification and a set of features for A. arabaticum Z-7288^T, together with the description of the complete genomic sequencing and annotation.

The cells of A. arabaticum are bent rods, motile by one to two subterminal flagella (Table 1) [2]. The flagella are stated in the original description [2], though they are not visible in our study (Figure 1). The cells are single, in pairs or form short chains, being 0.7-1 µm in diameter and 1-5 µm in length [2]. Other typical cell aggregates are palisades and ribbons, which are formed by adhesion of cells having intimate contact (Figure 1) [2]. The multiplication is by binary fission. The outer membrane is typical of a Gram-negative organism [2]. Growth is completely inhibited by 100 µM/ml streptomycin, benzylpenicillin, bacitracin, erythromycin, gentamycin, kanamycin, vancomycin or tetracyclin [2]. Strain Z-7288^T is obligately anaerobic, tolerating up to 12 mM H₂S. Neither O₂, S₂O₃^2-, SO₄^2-, nor S⁰ can serve as electron acceptors. Strain Z-7288^T requires a salt concentration of 10-25% NaCl, the optimum is 15-18% NaCl [2]. The optimal pH is between 7.6 and 8.0 [2].

Figure 1 Scanning electron micrograph of A. arabaticum Z-7288T

A. arabaticum exhibits three modes of nutrition [2]: It is chemolithoautotrophic using H₂ together with CO₂ or CO; it is methylotrophic using trimethylamine (TMA); and it is organotrophic using betaine, lactate, pyruvate or histidine. Carbohydrates are not utilized. No growth occurs on methanol, monomethylamine (MMA), dimethylamine (DMA), dimethylglycine, choline or sarcosine [2]. When grown on TMA, an equimolar amount of acetate is formed along with lesser amounts of DMA and MMA [2]. Betaine is degraded mainly to acetate and minor amounts of methylamines [2].

Carbonic anhydrase (CA; carbonate hydrolyase, EC 4.2.1.1) has been studied in strain Z-7288^T and in other acetogenic bacteria [16]. This zinc-containing enzyme is found in animals, plants, bacteria and archaea and catalyzes the following reaction: CO₂ + H₂O ↔ HCO₃^- and H⁺ [16]. Further biochemical details of CA are described elsewhere [16]. Strain Z-7288^T displayed CA activities similar to those of other CA-containing bacteria [16-18]. With lactate as cultivation substrate the specific activity of CA in strain Z-7288^T has been determined to be 2.1± 0.4 units per mg or protein [16]. It has been suggested that one physiological function for CA in acetogens is to increase intracellular CO₂ levels [16].

The 16S rRNA genes of the other type strains in the family Halobacteroidaceae share between 85.9% (Orenia sivashensis [19]) and 95.1% (Sporohalobacter lortetii [20]) sequence identity with strain Z-7288^T [21]. Uncultured clone sequences from environmental samples and metagenomic surveys do not surpass 84-86% sequence similarity to the 16S rRNA gene sequence of strain Z-7288^T, indicating a lack of further members of the genus Acetohalobium in the habitats screened thus far (status June 2010).

Figure 2 shows the phylogenetic neighborhood of A. arabaticum Z-7288^T in a 16S rRNA based tree. The sequences of the five 16S rRNA gene copies in the genome of Acetohalobium arabaticum Z-7288^T differ from each other by up to one nucleotide, and differ by up to three nucleotides from the previously published 16S rRNA sequence generated from DSM 5501 (X89077).

Figure 2 Phylogenetic tree highlighting the position of A. arabaticum Z-7288T relative to the type strains of the other genera within the order Halanaerobiales. The trees were inferred from 1,308 aligned characters [22,23] of the 16S rRNA gene sequence under the maximum likelihood criterion [24] and rooted in accordance with the current taxonomy [25]. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 300 bootstrap replicates [26] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [27] are shown in blue, published genomes [28] in bold.

The genome consists of a 2,469,596 bp long chromosome with a 36.6% GC content (Table 3 and Figure 3). Of the 2,443 genes predicted, 2,353 were protein-coding genes, and 90 RNAs; Seventy-one pseudogenes were also identified. The majority of the protein-coding genes (76.4%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Figure 3 Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.