Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Taxonomy, Historical and Current

Family Xanthobacteraceae Lee, Liu, Anzai, Kim, Aono, and Oyaizu 2005, 1916VP

Xan.tho.bac.te.ra’ce.ae. N.L. masc. n. Xanthobacter, type genus of the family; suff. –aceae, ending to denote a family; N.L. fem. pl. n. Xanthobacteraceae, the Xanthobacter family.

Gram-negative, rod-shaped chemoorganotrophic or facultatively chemolithoautotrophic bacteria, motile or nonmotile. Do not form spores. Some species fix N2. The major isoprenoid quinone is Q-10. The family Xanthobacteraceae was circumscribed on the basis of phylogenetic analysis of 16S rRNA sequences. The family is phenotypically, metabolically, and ecologically diverse.

Type genus: Xanthobacter.

The mol% G+C of the DNA varies between 61 and 69.

The family Xanthobacteraceae was created in 2005, based on 16S rRNA sequence comparisons (Lee et al. 2005). At the time of writing (May 2012), the family contained 7 genera with 28 species (Tables 26.1 – 26.7 ): Xanthobacter [type genus] (8 species, the names of 2 of which were thus far effectively but not validly published), Ancylobacter (7 species, including one whose name was effectively but not yet validly published), Azorhizobium (2 species), Labrys (7 species), Pseudolabrys (1 species), Pseudoxanthobacter (1 species), and Starkeya (2 species).

Table 26.1 The genera classified within the family Xanthobacteraceae, as of May 2012
Full size table

The genus Xanthobacter was established by Wiegel et al. (1978) based on numerical taxonomy comparisons of organisms assigned at the time to the genus Corynebacterium. The species Corynebacterium autotrophicum (Baumgarten et al. 1974) was renamed Xanthobacter autotrophicus, and proposed as the type species of the new genus. A comparative study showed that the nitrogen-fixing facultative chemolithotrophic isolate known as Mycobacterium flavum strain 301 (Federov and Kalininskaya 1961) can be classified within the new genus as Xanthobacter flavus (Malik and Claus 1979). Later 16S rRNA sequence comparison confirmed the phylogenetic relationship. Based on 16S rRNA phylogeny, the species Blastobacter viscosus 7d (Loginova and Trotsenko 1980) and Blastobacter aminooxidans 14a (Doronina et al. 1984) were moved from the genus Blastobacter (Bradyrhizobiaceae) to the genus Xanthobacter as X. viscosus and X. aminoxidans (Doronina and Trotsenko 2003).

The genus name Ancylobacter was proposed by Raj (1983) as a substitute for the genus name Microcyclus (Ørskov 1928). The name Microcyclus was earlier used for a fungus in 1904, and therefore the name was considered illegitimate, even though it appeared in the Approved Lists of Bacterial Names of 1980. The strain described by Ørskov as the type of Microcyclus aquaticus has been lost. Subsequently, new similar strains were isolated by Ørskov, and one of these strains (ATCC 25396) was proposed as the neotype strain of M. aquaticus, now A. aquaticus comb. nov. (Ørskov 1957; Larkin and Borrall 1979; Staley et al. 2005). Ancylobacter vacuolatus (Xin et al. 2006) was earlier described as Renobacter vacuolatum (Nikitin 1971).

Taxonomic rearrangement of obligately or facultatively chemolithoautotrophic organisms that oxidize reduced sulfur compounds, earlier classified as members of the genus Thiobacillus, led to the removal of T. novellus (Starkey 1934) (a member of the Alphaproteobacteria) from the genus Thiobacillus (Betaproteobacteria) and its reclassification as Starkeya novella gen. nov., comb. nov. (Kelly et al. 2000).

Phylogenetic Structure of the Family and Its Genera

Figure 26.1 shows a Neighbor Joining tree of the type strains of the 28 species of the family Xanthobacteraceae. The family is associated with the Alphaproteobacteria, close relatives being the families Hyphomicrobiaceae, Bradyrhizobiaceae, Beijerinckiaceae, Methylocystaceae, and Acetobacteraceae.

Fig. 26.1
figure 1figure 1

Phylogenetic reconstruction of the family Xanthobacteraceae based on 16S rRNA and created using the neighbor-joining algorithm with the Jukes-Cantor correction. The sequence dataset and alignment were used according to the All-Species Living Tree Project (LTP) database (Yarza et al. 2010; http://www.arb-silva.de/projects/living-tree). The tree topology was stabilized with the use of a representative set of nearly 750 high-quality type strain sequences proportionally distributed among the different bacterial and archaeal phyla. In addition, a 40 % maximum frequency filter was applied in order to remove hypervariable positions and potentially misplaced bases from the alignment. Scale bar indicates estimated sequence divergence

Full size image

The family Xanthobacteraceae, originally with five genera (Xanthobacter, Azorhizobium, Ancylobacter, Labrys, and Starkeya) was proposed by Lee et al. (2005) based on 16S rRNA comparisons of the members of the Alphaproteobacteria. It is interesting to note that in an earlier phylogenetic analysis that did not include Xanthobacter spp., Labrys monachus did not cluster with Starkeya novella (Thiobacillus novellus) and with Ancylobacter aquaticus (Fritz et al. 2004). The close phylogenetic relationship between Xanthobacter and Azorhizobium was first reported in 1996 (Rainey and Wiegel 1996; Wiegel 2005). Already then, it was noted that members of Xanthobacter and Azorhizobium are intermixed in the tree, as seen also in Fig. 26.1 . A Maximum Likelihood (RAxML) tree constructed (not shown) placed the two species of Azorhizobium on a single branch surrounded by members of the genus Xanthobacter. Both the Neighbor Joining and the Maximum Likelihood trees place the two Starkeya species between the Ancylobacter branches.

Genome Analysis

At the time of writing (May 2012), three genome sequences of members of the Xanthobacteraceae had been published (Table 26.8 ): the type strain of Azorhizobium caulinodans (Lee et al. 2008), the type strain of Starkeya novella, and Xanthobacter autotrophicus Py2, a strain that can grow on alkenes (van Ginkel and de Bont 1986) and has many interesting physiological and biochemical features as described below. The chromosomes are 4.77–5.37 Mbp in length and contain 4,483–4,847 predicted genes. X. autotrophicus Py2 in addition contains a 316-kb plasmid encoding 308 predicted proteins. Of the 4,717 predicted proteins encoded by the 5.37 Mbp genome of A. caulinodans, 3.7 % are unique for this organism. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct “symbiosis region” (Lee et al. 2008).

Phages

Wilke and Schlegel (1979) described three phages infecting Xanthobacter autotrophicus strain GZ29. Two lytic phages CA1 and CA2 have heads of 61–68 nm diameter and tails of 98–100 and 166–175 nm length, respectively. A third phage designated CA3, with a head diameter of 37–43 nm, a 43–50 nm tail, and a small DNA molecule of 3.3 kDa, did not form plaques and was detected only by its transducing activity and by electron microscopy.

Forty-three plaque-forming phages against the stem-nodulating Azorhizobium caulinodans were isolated from rhizosphere soil of different leguminous plant species. They all had a head and short (14–18 nm) non-contractible and non-flexible tails and were assigned to the Podoviridae (Sharma et al. 2008).

Phenotypic Analyses

The Properties of the Genera and Species of Xanthobacteraceae

Phenotypically the members of the family Xanthobacteraceae are quite diverse. With the other members of the Alphaproteobacteria, they share a Gram-negative type of cell wall, presence of ubiquinone Q-10 as the major respiratory quinone (with Q-9, Q-8, and Q-11 sometimes found in minor amounts), and other chemotaxonomic traits such as the types of fatty acids present. Predominant polyamines are putrescine and sym-homospermidine, as characterized in Xanthobacter autotrophicus 7c and CB2, Azorhizobium caulinodans, and Labrys wisconsinensis (Hamana et al. 1990; Wiegel 2006; Albert et al. 2010). They are all aerobes, although Labrys wisconsinensis was described as a facultative anaerobe as growth was obtained anaerobically on plate count broth supplemented with 0.075 % agar (Albert et al. 2010). Chemolithoautotrophic growth is widespread within the family, with hydrogen and/or reduced sulfur compounds serving as electron donor and energy source. Another property found in many of the genera is the ability to fix N2.

Genus Xanthobacter Wiegel, Wilke, Baumgarten, Opitz, and Schlegel 1978, 580AL

Xan.tho.bac’ter Gr. adj. xanthos, yellow; N.L. masc. n. bacter, rod, staff; N.L. masc. n. Xanthobacter, yellow rod.

Rod-shaped, sometimes twisted cells, 0.4–1.0 × 0.8–6.0 μm, nonmotile or motile by peritrichous flagella. Gram-negative type of cell wall. Pleomorphic cells are sometimes produced on media containing succinate and other tricarboxylic acid cycle intermediates, whereas coccoid cells as well as cells up to 10 μm long are produced on media containing an alcohol as the sole carbon source. Refractile (polyphosphate) and lipid (poly-β-hydroxybutyrate) bodies are evenly distributed in the cells. Due to polyphosphate granules, the Gram reaction frequently appears falsely to be positive or variable. Aerobic, with a strictly respiratory type of metabolism. Neutrophilic and mesophilic. Colonies are opaque and generally slimy, yellow due to the presence of zeaxanthin dirhamnoside. Most strains can grow chemolithoautotrophically in mineral media under an atmosphere of H2, O2, and CO2, as well as chemoorganoheterotrophically on methanol, ethanol, n-propanol, n-butanol, and various organic acids as carbon sources. The carbohydrate utilization spectrum is limited. Some strains can use substituted thiophenes as sole carbon, energy, and sulfur sources. When degrading aliphatic epoxides, tested strains contain coenzyme M, which otherwise is a typical coenzyme of the obligate anaerobic methanogenic archaea. N2 is fixed under a decreased O2 pressure.

The mol% G+C of the DNA is 65–70.

Type species: Xanthobacter autotrophicus.

The genus Xanthobacter currently contains eight species: X. autotrophicus, X. agilis, X. aminoxidans, X. flavus, X. polyaromativorans (a name effectively but not yet validly published), X. tagetidis, X. viscosus, and X. xylophilus (a name effectively but not yet validly published). The main features of the members of the genus Xanthobacter are summarized in Table 26.2 .

Table 26.2 Comparison of selected characteristics of the members of the genus Xanthobacter
Full size table

Additional comments:

  • Irregular twisted cells (X. tagetidis) and branched cells (X. autotrophicus, X. flavus) are commonly found during growth on tricarboxylic acid cycle intermediates. Branching cells do not show septa at the branching points (Wiegel 2005).

  • Most members of the genus multiply by symmetric division, but X. viscosus and X. aminoxidans reproduce by budding (Doronina and Trotsenko 2003). X. polyaromaticivorans, a species that degrades polycyclic and heterocyclic aromatic compounds, is atypical as it shows no autotrophic growth (Hirano et al. 2004).

  • X. flavus was originally described as nonmotile. However, reexamination of the type strain showed peritrichous flagella in exponential cultures grown on methanol, ethanol, n-propanol, isopropanol, butanol, or gluconate, but not in media containing citrate, fumarate, malate, succinate, glutamate, glutamine, yeast extract, or in cells growing autotrophically on H2 + CO2 (Reding et al. 1992).

  • Cellular fatty acids are high in C18:1 and include C18:0, 3-OH C16:0, and (in X. agilis) C19:0 cyclo ω7c (Urakami et al. 1995).

  • X. polyaromaticivorans is slightly pinkish-orange, probably due to a zeaxanthin with terminal substitutions different from zeaxanthin dirhamnoside (Wiegel 2006).

  • X. xylophilus, isolated from acidic low-salinity dystrophic water with decaying spruce wood, is moderately acidophilic, and uses organic acids (citrate, oxalate, succinate, gluconate), as well as xylose and xylan (Zaichikova et al. 2010a).

  • X. tagetidis is associated with the roots of marigold (Tagetes) plants, which accumulate high concentrations of thiophenes. It grows on substituted thiophenes, and also is a facultative autotroph that can grow on H2, thiosulfate, or sulfide as electron donors. It fixes CO2 using ribulose bisphosphate carboxylase/oxygenase (Padden et al. 1997).

  • All strains of X. autotrophicus and X. flavus produce an α-polyglutamine capsule located between the cell wall and the slime (Wiegel 2005).

  • Two methanol degrading isolates 25P and 32P, classified as strains of X. autotrophicus, were described by Doronina et al. (1996).

Genus Ancylobacter Raj 1983, 397VP

An.cy.lo.bac’ter. Gr. adj. ankulos, crooked, curved; N.L. masc. n. bacter, rod; N.L. masc. n. Ancylobacter, a curved rod.

Curved rods, 0.3–1.0 × 1.0–3.0 μm. Rings (0.9–3.0 μm outer diameter) are occasionally formed prior to cell separation. Cells are encapsulated. Some strains produce gas vesicles. Gram-negative. Generally nonmotile or motile by means of a single polar flagellum. Obligately aerobic with a strictly respiratory type of metabolism. Colonies are white to cream colored. Oxidase and catalase positive. Chemoorganotrophic, using a variety of sugars or salts of organic acids as carbon sources. Chemolithotrophic growth has been reported on molecular hydrogen. Some strains are facultatively methylotrophic, using methanol and formate.

The mol% G+C of the DNA is 66–69.

Type species: Ancylobacter aquaticus.

The genus Ancylobacter currently contains seven species: A. aquaticus, A. abiegnus (a name effectively but not yet validly published), A. dichloromethanicus, A. oerskovii, A. polymorphus, A. rudongensis, and A. vacuolatus. The main features of the members of the genus Ancylobacter are summarized in Table 26.3 .

Table 26.3 Comparison of selected characteristics of the members of the genus Ancylobacter
Full size table

Additional comments:

  • Overviews of the biology of Ancylobacter were published by Raj (1989) and by Staley et al. (2005).

  • Staley et al. (2005) consider the type strain of A. aquaticus to be avacuolate, despite a claim to the contrary (Raj 1977).

  • Autotrophic growth of several strains was reported with hydrogen (Namsaraev and Nozhevnikova 1978; Malik and Schlegel 1981) or with thiosulfate as energy source (Stubner et al. 1998). CO2 fixation is mediated by ribulose bisphosphate carboxylase/oxygenase (Loginova et al. 1978; Firsova et al. 2009).

  • A. dichloromethanicus, isolated from contaminated soil, uses dichloromethane, methanol, formate, formaldehyde, and a range of larger carbon sources (Firsova et al. 2009).

  • The major fatty acid of A. aquaticus is C18:1; further present C16:0, C18:0 C19:0 cyclo. The major respiratory quinone is Q-10, with minor amounts of Q-9 and Q-11 (Urakami and Komagata 1986).

Genus Azorhizobium Dreyfus, Garcia, and Gillis 1988, 97VP

A.zo.rhi.zo’bi.um. N.L. n. azotum [from Fr. n. azote (from Gr. prep. a, not; Gr. n. zôê, life; N.Gr. n. azôê, not sustaining life)], nitrogen; N.L. pref. azo-, pertaining to nitrogen; N.L. neut. n. Rhizobium, a bacterial generic name; N.L. neut. n. Azorhizobium, a nitrogen (using) Rhizobium.

Motile, short, rod-shaped Gram-negative cells, showing peritrichous flagella on solid medium and one or more lateral flagella in liquid medium. Obligately aerobic. N2 is fixed under microaerobic conditions when nicotinic acid is provided. Oxidase- and catalase-positive; urease-negative. Among sugars, only glucose is oxidized; the favorite carbon substrates are organic acids such as lactate or succinate. Grow also on malonate and on proline. Starch is not hydrolyzed. Denitrification is not observed. Nodulate the stems and roots of leguminous plants of the genus Sesbania.

The mol% G+C of the DNA of the type species is 66.

Type species: Azorhizobium caulinodans.

The genus Azorhizobium currently contains two species: A. caulinodans and A. doebereinerae. The main features of the members of the genus Azorhizobium are summarized in Table 26.4 .

Table 26.4 Comparison of selected characteristics of the members of the genus Azorhizobium
Full size table

Additional comments:

  • Overviews of the properties of nitrogen-fixing stem nodules of Sesbania and the biology of Azorhizobium were published by Dreyfus and Dommergues (1981), Dreyfus et al. (1984), Goormachtig et al. (1998), and Kuykendall (2005).

  • A. doebereinerae was isolated from root nodules of Brazilian woody species Sesbania virgata. Its colonies on YMA agar are similar to those of A. caulinodans: scant extracellular polysaccharide, fast to intermediate growth rate, and causing alkalinization of the medium. Neither A. caulinodans nor A. doebereinerae use mannitol or sucrose, compounds used by most Rhizobium, Sinorhizobium, Mesorhizobium, and Bradyrhizobium spp. However, they can use dl-lactate. A. doebereinerae differs from A. caulinodans as it does not use either l-leucine or d-glucose (de Souza Moreira et al. 2006).

Genus Labrys Vasilyeva and Semenov 1985, 375VP (Effective Publication: Vasilyeva and Semenov 1984, p. 92 (Russian edition)); Emended Islam, Kawasaki, Nakagawa, Hattori and Seki, 2007, 556; Emended Albert, Waas, Langer, Pavlons, Feldner, Rosselló-Mora and Busse, 2010, 1757)

La’brys. N.L. masc. n. Labrys (from Gr. n. labrus), double-headed ax, an organism resembling a double-headed ax by the shape of the cell.

Motile or nonmotile cells; can be rod-shaped, and may or may not possess triangular radial symmetry. They may have two to three short prosthecae and can be aerobes or facultative anaerobes. Cells divide by budding. Buds are produced directly from the mother cell at the tip of the triangle that lacks prosthecae. In this stage, the cell resembles a double-headed ax. Most strains are aerobic, chemoorganotrophic. Use carbohydrates and some organic acids as sole carbon and energy sources. Oxidase and catalase positive. May be facultative methylotrophs. The primary polar lipids are diphosphatidylglycerol, phosphatidylmonomethylethanolamine, and phosphatidylcholine. The predominant fatty acids are C19:0 cyclo ω8c, C16:0, C18:0, and C18:1ω7c. The major ubiquinone is Q-10, and the major polyamine is sym-homospermidine.

The mol% G+C of the DNA is 61–68.

Type species: Labrys monachus.

The genus Labrys currently contains seven species: L. monachus, L. methylaminiphilus, L. miyagiensis, L. neptuniae, L. okinawensis, L. portucalensis, and L. wisconsinensis. The main features of the members of the genus Labrys are summarized in Table 26.5 .

Table 26.5 Comparison of selected characteristics of the members of the genus Labrys
Full size table

Additional comments:

  • L. neptuniae was isolated from root nodules of the pan-tropical aquatic legume Neptunia oleracea (Chou et al. 2007).

  • L. portucalensis is a fluorobenzene-degrading bacterium obtained from industrially contaminated sediment in northern Portugal. Fluorobenzene is used as a solvent in the pharmaceutical industry, as an insecticide, and as a reagent for plastic and resin polymers production (Carvalho et al. 2005, 2008).

  • L. okinawensis and L. miyagiensis are budding bacteria isolated from rhizosphere habitats in Japan (Islam et al. 2007).

  • Labrys methylaminiphilus was isolated from freshwater Lake Washington sediment following enrichment on methylamine. It also uses a variety of mono- and disaccharides, organic acids, aromatic compounds, and alcohols. Methanol is not used. It also grows on polymers such as agarose and humic acid (Miller et al. 2005).

  • Some species accumulate considerable amounts of poly-β-hydroxybutyrate (Vasilyeva 2005).

Genus Pseudolabrys Kämpfer, Young, Arun, Shen, Jäckel, Rosselló-Mora, Lai, and Rekha 2006, 2470VP

Pseu.do.la’brys. Gr. adj. pseudês false; N.L. masc. n. Labrys a bacterial genus name; N.L. masc. n. Pseudolabrys the false Labrys.

Nonmotile, short, rod-shaped Gram-negative cells that multiply by division and not by budding. Methanol, methylamine, formaldehyde, and formamide are not used as sole carbon sources. The major fatty acids are C16:0, C18:1ω7c, and C19:0 cyclo ω8c.

The mol% G+C of the DNA of the type species and only species described is 67.

Type species: Pseudolabrys taiwanensis. The main features of the single member of the genus Pseudolabrys are summarized in Table 26.6 .

Table 26.6 Comparison of selected characteristics of the members of the monospecific genera Pseudolabrys and Pseudoxanthobacter
Full size table

Genus Pseudoxanthobacter Arun, Schumann, Chu, Tan, Chen, Lai, Kämpfer, Shen, Rekha, Hung, Chou, and Young 2008, 1573VP

Pseu.do.xan.tho.bac’ter. Gr. adj. pseudês false; N.L. masc. n. Xanthobacter a bacterial genus name; N.L. masc. n. Pseudoxanthobacter the false Xanthobacter.

Motile, short, rod-shaped Gram-negative or Gram-variable cells that accumulate poly-β-hydroxybutyrate as polar inclusion bodies. The major quinone is Q-10. The characteristic diamino acid of the peptidoglycan is meso-diaminopimelic acid. Polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatyidyldimethylethanolamine, and an unknown aminolipid. The predominant fatty acids are C16:0, C18:1ω7c and C19:0 cyclo.

The mol% G+C of the DNA of the type species and only species described is 68.

Type species: Pseudoxanthobacter soli. The main features of the single member of the genus Pseudoxanthobacter are summarized in Table 26.6 .

Additional comments:

  • P. soli is a nitrogen-fixing species isolated from soil. Its major respiratory quinone is Q-10, with minor amounts of Q-9 and Q-8. The organism can fix N2 and multiplies by budding (Arun et al. 2008).

Genus Starkeya Kelly, McDonald, and Wood 2000, 1800VP

Star.key’a. N.L. fem. n. Starkeya, named after Robert L. Starkey, who made important contributions to the study of soil microbiology and sulfur biochemistry.

Nonmotile, short, rod-shaped Gram-negative cells. Colonies grow on thiosulfate agar. Biotin is required. Strictly aerobic and facultative chemolithoautotrophic. Oxidize and grow on thiosulfate and tetrathionate but not on elemental sulfur or thiocyanate. Neutrophilic and mesophilic. Contain ubiquinone Q-10. Major cellular fatty acids are C18:1 and C19:0 cyclo. Growth is also observed with formate. Some strains may degrade methylated sulfides. Isolated from soils and presumably widely distributed.

The mol% G+C of the DNA is 67–69.

Type species: Starkeya novella.

The genus Starkeya currently contains two species: S. novella and S. koreensis. The main features of the members of the genus Starkeya are summarized in Table 26.7 .

Table 26.7 Comparison of selected characteristics of the members of the genus Starkeya
Full size table
Table 26.8 Properties of the sequenced genomes of members of the Xanthobacteraceae (as of May 2012)
Full size table

Additional comments:

  • For optimal autotrophic development, biotin is required. When grown on formate, high levels of ribulose bisphosphate carboxylase/oxygenase are expressed (Kelly and Wood 2005).

  • S. novella uses a variety of sugar alcohols, amino acids, carboxylic acids, and fatty acids for heterotrophic growth; reduced sulfur compounds serving as electron donors include thiosulfate, tetrathionate, dimethylsulfide, and dimethylsulfoxide (Kelly et al. 2000).

  • A S. novella isolate from sewage was shown to oxidize methanethiol, dimethylsulfide, and dimethyldisulfide (Cha et al. 1999).

Isolation, Enrichment, and Maintenance Procedures

Strategies for the enrichment and isolation of Xanthobacter species can be based on the ability to fix N2 under chemolithoautotrophic growth conditions under a gas mixture of H2 (10 %), N2 (70–75 %), CO2 (10 %), and air (5–10 %), and on the specific yellow color and the characteristic “fried-egg” appearance of colonies caused by slime production on media containing 1 % succinate and nutrient broth, and the appearance of branched cells in such colonies (Wiegel 2005, 2006). Some isolates were obtained by chance, such as X. autotrophicus strain 7c, which was isolated from an enrichment for propane-oxidizing bacteria, using black mud of a pond in Germany as inoculum; the Xanthobacter obtained did not use propane. X. tagetidis can be reproducibly enriched from Tagetes (marigold) roots using thiophene-2-carboxylate or thiophene-2-acetate as substrates (Padden et al. 1997).

For enrichment and isolation of gas-vacuolated strains of Ancylobacter, 100 ml of a freshwater source can be added to a sterile aluminum foil-covered beaker containing 10 mg Bacto peptone (Difco). After 2 weeks incubation, at room temperature the culture is plated onto a hydrolysate medium containing glucose (Van Ert and Staley 1971; Staley et al. 2005). For the isolation of motile variants of gas-vacuolate strains of Ancylobacter aquaticus, selection on soft agar plates can be used: Growth radiating from the center of the colony consists of motile cells (Lara and Konopka 1987).

Ancylobacter oerskovii and two strains of Ancylobacter polymorphus were isolated from soil after enrichment with oxalate. All known species of the genus appear to use oxalate as the sole source of carbon (Lang et al. 2008), a finding that can probably be used as the basis for selective isolation procedures. Enrichment on dichloromethane as carbon and energy source, amended with a low concentration of yeast autolysate, was successfully applied for the isolation of A. dichloromethanicus (Firsova et al. 2009).

Azorhizobium grows well on nitrogen-free agar media, distinguishing the genus from other legume-nodulating bacteria (Bradyrhizobium, Mesorhizobium, Rhizobium) (Dreyfus et al. 1988). Lactate and succinate are preferred substrates for growth (Kuykendall 2005). Selective inhibitors are also useful in studies of Azorhizobium and related plant-associated nitrogen-fixing bacteria: Azorhizobium is resistant to carbencillin; nalidixic acid inhibits Azorhizobium but allows growth of Rhizobium (Robertson et al. 1995).

Low nutrient concentrations may favor growth of Labrys spp. in enrichment cultures. This, L. miyagiensis, isolated from grassland soil in Japan, grows in 1/100 diluted nutrient broth but is inhibited by full-strength nutrient broth medium (Islam et al. 2007). L. methylaminiphilus, a facultatively methylotrophic bacterium obtained from sediment of Lake Washington, WA, USA, was enriched using mineral medium with 0.01 % methylamine (Miller et al. 2005). The type strain of L. monachus was isolated from silt samples from Lake Mustjarv (Estonia) using horse manure extract, obtained by heating 1 % (w/v) dry manure in distilled water. The sediment is left to settle and a liquid medium is prepared from the supernatant (Vasilyeva 2005). Some other Labrys species were obtained on nonselective media. L. neptuniae was isolated from root nodules of the tropical aquatic legume Neptunia oleracea on yeast extract —mannitol agar (Chou et al. 2007). Finally, L. wisconsinensis was isolated from Lake Michigan water as a colony that grew on Difco Plate Count Agar—a nonselective general medium for heterotrophic bacteria (Albert et al. 2010).

The type strains (and thus far only isolates) of the soil bacteria Pseudoxanthobacter soli and Pseudolabrys taiwanensis were both isolated as colonies on nutrient agar, without any prior enrichment or selection procedure (Arun et al. 2008; Kämpfer et al. 2006).

No selective isolation procedures for Starkeya spp. have yet been tested. Vitamins may be important in determining the outcome of enrichment and isolation experiments: For optimal autotrophic growth, S. novella requires biotin, and for optimal heterotrophic growth yeast extract, biotin, or other vitamins such as pantothenate may be required, depending on the substrate (Kelly et al. 2000). S. koreensis was isolated from rice straw using R2A agar, a nonselective general low-nutrient plating medium (Im et al. 2006).

Maintenance

Cultures can be maintained if they are refrigerated at 4 °C for a number of weeks. For long-term preservation, lyophilization is recommended.

Physiological and Biochemical Features

Physiologically and biochemically there are a number of noteworthy features within in the family Xanthobacteraceae: the ability of some strains of Xanthobacter, Ancylobacter, and Labrys to degrade chlorinated and brominated alkanes, alkenes, and aromatic compounds; the degradation of thiophenes by X. tagetidis; the ability of Xanthobacter spp. to grow on polyaromatic compounds; and the growth of some X. autotrophicus strains to grow on ethylene, propylene, and other alkenes.

Xanthobacter autotrophicus GJ10 was isolated on 1,2-dichloroethane as the sole carbon and energy source. It possesses a hydrolytic haloalkane dehydrogenase with a broad substrate specificity that degrades dichloroethane to 2-chloroethanol. This intermediate is further metabolized via chloroacetaldehyde to chloroacetic acid, which is dechlorinated by a second dehalogenase to glycolate. A similar strain GJ11 was isolated from sediment of the River Rhine (Janssen et al. 1984, 1985). Xanthobacter sp. strain TM1, isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent in Portugal, degrades dichloromethane, chloroacetic acid, dichloroethane, 2-chloroethanol, 2-fluorobenzoate, 3-fluorobenzoate, 4-fluorobenzoate, 2-chlorobenzoate, 4-chlorobenzoate, and methanol (Emanuelsson et al. 2009). Xanthobacter sp. strain ENV481, isolated from a microcosm model with aquifer soil and groundwater from a landfill, New Jersey, can degrade bis(2-chloroethyl) ether, a compound used as a solvent for fats and greases, a cleaning fluid for textiles, a constituent of paints and varnishes, and an insecticide (McClay et al. 2007). Xanthobacter flavus strain 14p1, isolated from river sludge in Germany, degrades 1,4-dichlorobenzene but no other aromatic or chloroaromatic compounds. The degradation pathway starts with dioxygenation, followed by ring opening via ortho cleavage of dichlorocatechol to 2,5-dichloro-cis,cis-muconic acid (Spiess et al. 1995; Spiess and Görisch 1996; Sommer and Gorisch 1997). X. autotrophicus strain GJ10 grows on 1,2-dichloroethane, bromochloromethane, dibromomethane, and 1-bromo-2-chloroethane. A novel pathway of degradation of dihalomethanes to formaldehyde was proposed. Cells growing on 1,2-dichloroethane converted 2-fluoroethanol and 1-chloro-2-fluoroethane to 2-fluoroacetate (Torz et al. 2007). Labrys portucalensis can grow on fluorobenzene (Carvalho et al. 2008). Co-metabolism of chlorinated compounds may also occur: X. autotrophicus strains GJ10 and Py2 degrade trichloroethylene during growth on propene (Reij et al. 1995; Inguva and Schreve 1999). When grown on propylene, Xanthobacter strain Py2 can degrade trichloroethylene, 1-chloroethylene (vinyl chloride), 1,3-dichloropropylene, 2,3-dichloropropylene, and other related compounds. Addition of propylene oxide, propionaldehyde, and glucose enhanced the rate of degradation of chlorinated alkenes (Ensign et al. 1992).

Several isolates of the genus Ancylobacter degrade chlorinated aliphatic compounds. A. dichloromethanicus uses dichloromethane (Firsova et al. 2009). A. aquaticus strain GJ10 (Janssen et al. 1984, 1985) and strains AD25 and AD27, isolated from slurries of brackish water sediment and activated sludge, respectively (van den Wijngaard et al. 1992), degrade 1,2-dichloroethane. Strains AD25 and AD27 also use 2-chloroethylvinylether as sole carbon and energy source. Such chlorinated ethers are synthesized for the production of anesthetics, sedatives, and cellulose ethers (van den Wijngaard et al. 1993). Other chlorinated compounds broken down by Ancylobacter strains include 2-chloroethanol, chloroacetate, and 2-chloropropionate (Staley et al. 2005; van den Wijngaard et al. 1992). The organochlorine fungicide pentachloronitrobenzene (PCNB) could be degraded by Labrys portucalensis strain pcnb-21, isolated from a PCNB-polluted soil in China (Li et al. 2011). Labrys sp. strain Wy1, isolated from soil in a rubber estate in Malaysia, can use the herbicide 2,2-dichloropropionate (2,2-DCP) as sole source of carbon (Wong and Huyop 2011).

Xanthobacter tagetidis was isolated from the roots of marigold (Tagetes) plants. Tagetes species accumulate thiophenes in the roots at concentrations up to 1 % of the root mass. X. tagetidis grows on thiophenes such as thiophene-2-carboxylate, thiophene-3-carboxylate, on analogs of these compounds (pyrrole-2-carboxylate, furan-2-carboxylate), and on the condensed thiophene dibenzothiophene (Padden et al. 1997).

Xanthobacter polyaromaticivorans 127 W grows on a range of polycyclic and heterocyclic aromatic compounds under extremely low oxygen concentrations. Polycyclic aromatic hydrocarbons used include anthracene, fluorene, naphthalene, phenanthrene, dibenzothiophene, dibenzofuran, and biphenyl. It also degrades dibenzothiophene (Hirano et al. 2004). Another Xanthobacter strain, isolated from forest soil in the UK by enrichment with cyclohexane vapor as the carbon source, metabolizes cyclohexane via cyclohexanol, cyclohexanone, and 1-oxa-2-oxocycloheptane (ε-caprolactone) to adipic acid (Trower et al. 1985).

Biochemically one of the most interesting processes performed by some Xanthobacter isolates is the degradation of alkenes. Alkene-utilizing Xanthobacter strains were obtained from enrichment cultures with propene and 1-butene. A monooxygenase was found to be involved, forming 1,2-epoxyalkanes as intermediate (van Ginkel and de Bont 1986). Alkene degradation and the metabolism of the epoxide intermediates was studied in-depth in X. autotrophicus strain Py2, which grows on ethylene, propylene, and butylene (Small and Ensign 1997). The involvement of coenzyme M (2-mercaptoethanesulfonate) in the process came as a big surprise. Coenzyme M has been known for many decades as a central component of the biochemical pathway of methanogenesis in archaea, but it was never before found in any other organisms. Xanthobacter strain Py2 possesses a linear megaplasmid that encodes enzymes of aliphatic alkene and epoxide metabolism and coenzyme M biosynthesis. Epoxidation of propylene to epoxypropane is followed by a sequence of three reactions resulting in epoxide ring opening and carboxylation to form acetoacetate. Coenzyme M plays a central role in epoxide carboxylation by serving as the nucleophile for epoxy ring opening and as the carrier of the C3 unit that is finally carboxylated to acetoacetate, releasing the coenzyme (Sluis and Ensign 1997; Krum and Ensign 2001; Krishnakumar et al. 2008; Pandey et al. 2011). Shotgun proteomics revealed proteins specific to growth on propylene, including the enzymes necessary for the biosynthesis of coenzyme M (Broberg and Clark 2010).

Another substrate metabolized via an epoxide intermediate is the terpene limonene, used by Xanthobacter sp. C20. This strain was isolated from the River Rhine using cyclohexane as the sole carbon and energy source. It possesses a novel bioconversion pathway in which limonene is converted to limonene-8,9-epoxide in a reaction that involves cytochrome P-450 (van der Werf et al. 2000).

Ecology

Members of the genus Xanthobacter may be ubiquitous in wet soil and sediments. They can be found in freshwater (X. agilis), wet soil containing decaying organic material (X. autotrophus, X. flavus), compost of root balls of Tagetes (X. tagetidis) (Padden et al. 1997), and they are associated with plant roots including wetland rice (Oyaizu-Masuchi and Komagata 1988; Reding et al. 1991; Wiegel 2005, 2006). One X. flavus originated from marine sediment (Lidstrom-O’Connor et al. 1983; Meijer et al. 1990). X. xylophilus is moderately acidophilic (opt. 5.5, range 4.8–6.8) and was found in acidic low-salinity dystrophic water with decaying spruce wood (Zaichikova et al. 2010a). Xanthobacter species may play an important role in the degradation of toxic organic compounds in polluted environments. X. viscosus and X. aminoxidans were found in activated sludge of a water treatment plant processing paper mill pulp (Loginova and Trotsenko 1980; Doronina et al. 1984; Doronina and Trotsenko 2003). X. autotrophicus was suggested to be involved in the biodegradation of toluene in a freshwater stream in Delaware (Tay et al. 1999).

Ancylobacter aquaticus is found in freshwater habitats, including ponds, creeks, and lakes (Van Ert and Staley 1971; Konopka et al. 1976) and in rice paddies and soil environments (Stubner et al. 1998). A. abiegnus was isolated from dystrophic waters with decaying spruce wood (Zaichikova et al. 2010b). Ancylobacter species may also be involved in the degradation of toxic compounds. A. dichloromethanicus, a facultatively methylotrophic bacterium that can grow on dichloromethane, was isolated from contaminated soil in Russia (Firsova et al. 2009). Ancylobacter sp. strain XJ-412-1 degrades the herbicide metsulfuron-methyl. It was isolated from an agricultural soil in China, which had been exposed to sulfonylurea herbicides for many years (Lu et al. 2011).

Azorhizobium is associated with stem- or root nodules of Sesbania and some other leguminous plants (Dreyfus and Dommergues 1981; Dreyfus et al. 1984, 1988; Kuykendall 2005; de Souza Moreira et al. 2006). A comparative study of rhizosphere and non-rhizosphere soils in four vegetation zones of Senegal showed Azorhizobium to be more abundant on leaves and stems than Rhizobium in three out of the four vegetation zones. Approximately 90 % of the stem nodules and 39–48 % of the root nodules on S. rostrata were formed by Azorhizobium (Robertson et al. 1995).

Little is known about the distribution of Labrys species in nature. They have been recovered from unpolluted freshwater environments (Vasilyeva 2005; Albert et al. 2010) as well as from polluted sites (e.g., the fluorobenzene-degrading L. portucalensis (Carvalho et al. 2008). L. neptuniae was isolated from root nodules of the aquatic pan-tropical legume Neptunia oleracea, but it probably is not the dominant bacterium there: Over 95 % of the colonies of Neptunia-associated bacteria in Taiwan that developed on yeast extract—mannitol agar belonged to Allorhizobium undicola (Chou et al. 2007).

Hardly anything is known about the distribution and the ecological role of the genera Pseudolabrys and Pseudoxanthobacter. It is also known little about the ecological niches where Starkeya species may be important. The type strains of S. novella and S. koreensis were isolated from soil and from rice straw, respectively (Starkey 1934; Im et al. 2006), not from environments rich in sulfide.

Pathogenicity, Clinical Relevance

No pathogenic bacteria are known within the Xanthobacteraceae. The plant-associated species of the genus Azorhizobium that colonize the stems of Sesbania and some other leguminous plants live in symbiosis with their host, and they were never shown to cause harm to their plant hosts (Dreyfus et al. 1984; Kuykendall 2005).

Table 26.9 summarizes data on the sensitivity of members of the Xanthobacteraceae to different antibiotics. Such data are not available for all members of the family, and the information present does not show any clear patterns, possibly except for the fact that all species tested were sensitive to erythromycin. Wiegel (2006) commented that the available data do not suggest antibiotic typing as a valid method for identification of Xanthobacter. This may be the case for other genera of the family as well. The resistance of Azorhizobium to carbencillin was mentioned above and allows the selective isolation with the exclusion of Rhizobium (Robertson et al. 1995).

Table 26.9 Sensitivity of species of the family Xanthobacteraceae to selected antibiotics
Full size table

Application

As shown above, many representatives of the genera Xanthobacter, Ancylobacter, and Labrys degrade toxic compounds: chlorinated alkanes, alkenes, polyaromatic compounds, thiophenes, etc., and the bacteria may be involved in the biodegradation of such compounds in polluted environments. Many such stains were isolated from sites polluted with such chemicals. However, no applications based on the use of such bacteria in bioremediation operations are known.

The polysaccharide slime produced by Xanthobacter species may have interesting biotechnological applications. Wiegel (2006) mentioned the possible use of these polysaccharides as drag-reducing substances for minimizing friction in turbulent flows in pipelines and water turbines or as viscosifiers in oil fields.

An intriguing application of gas-vacuolated Anyclobacter aquaticus was proposed: Mosquitocidal toxin genes from Bacillus sphaericus and Bacillus thuringiensis var. israelensis were introduced into Ancylobacter by electroporation. The transformed cells exhibited significant toxicity toward mosquito larvae. Such transgenic Ancylobacter could be released in water bodies infested with mosquito larvae, and due to the buoyancy of the cells conferred by the gas vesicles the toxin will accumulate at the water surface (Yap et al. 1994). No information could be found whether this interesting idea has ever been developed into field applications.

Starkeya novella, due to its potential to oxidize sulfide and methylated sulfur compounds, and its adaptability to a broad pH range (5–10), could be applied in biofilters for the removal of bad-smelling sulfur compounds, for example, from piggery wastewater (Chung et al. 1997).