The Family Phyllobacteriaceae

Abstract

The family Phyllobacteriaceae belongs to the order Rhizobiales in the Alphaproteobacteria and currently comprises the 72 species in 13 genera: Ahrensia, Aliihoeflea, Aminobacter (including Chelatobacter), Aquamicrobium (including Defluvibacter), Chelativorans, Hoeflea, Lentilitoribacter, Mesorhizobium, Nitratireductor, Phyllobacterium, Pseudahrensia, Pseudaminobacter, and Thermovum. They form a single cluster within the 16S rRNA gene phylogeny. The family consists of environmental (soil, water) and plant-associated bacteria that have a heterotrophic respiratory metabolism with oxygen as terminal electron acceptor. One Aquamicrobium species can use nitrate as an alternative terminal electron acceptor. One Mesorhizobium species is facultatively chemolithotrophic using thiosulfate or elemental sulfur as sole energy source. Candidatus Liberibacter, a group of uncultivated phloem-inhabiting bacteria that are associated with various plant diseases in citrus and Solanaceae or are endophytic in pear plants, is also associated with the family. However, comprehensive phylogenetic analyses indicate the position of this group as a member of the Phyllobacteriaceae is uncertain.

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

Short Description of the Family

Phyl.lo.bac.te.ri.a’ce.ae. N.L. neut. n. Phyllobacterium, type genus of the family; suff. -aceae, suffix to denote a family; N.L. fem. pl. n. Phyllobacteriaceae, the Phyllobacterium family.

The family Phyllobacteriaceae belongs to the Rhizobiales order in the Alphaproteobacteria class of the phylum Proteobacteria. It was proposed by Mergaert and Swings (2005a) in Bergey’s Manual of Systematic Bacteriology and was validated in 2006. At the time the family comprised six genera and one Candidatus genus: Phyllobacterium, Aminobacter, Aquamicrobium, Defluvibacter, Candidatus Liberibacter, Mesorhizobium, and Pseudaminobacter. Defluvibacter has since been transferred to Aquamicrobium (Kämpfer et al. 2009). Aminobacter includes Chelatobacter heintzii which is regarded as a later subjective synonym of Aminobacter aminovorans (Kämpfer et al. 2002). The basis for the proposal of this family was that these genera form a cluster in the 16S rRNA gene phylogeny. The description of the family (Mergaert and Swings 2005a) is rather brief: “Rod-shaped, ovoid, or reniform cells when cultured in vitro. Nonsporeforming. Gram negative. Aerobic. Cells cultured in vitro are motile by means of polar, subpolar, or lateral flagella. Strains grow well on complex solid media at 28 °C. Occur in leaf nodules and the rhizosphere of higher plants. The mol % G+C of the DNA is 60–62”. With the inclusion of the additional genera Ahrensia, Chelativorans, Hoeflea, Lentilitoribacter, Nitratireductor, Pseudahrensia, and Thermovum to the Phyllobacteriaceae cluster, most of this definition still applies except that cells can also be nonmotile and members of the family can also occur in seawater, marine sediments, activated sludge, and soil and thermophilic members are found in compost. The range of the G+C content of DNA is 48–65 %. Comprehensive phylogenetic analysis reveals that the position of Candidatus Liberibacter as a member of the Phyllobacteriaceae cluster is uncertain (see below).

Phylogenetic Structure of the Family and Its Genera

In the 16S rRNA gene phylogeny, the Phyllobacteriaceae family forms a single cluster in the phylum Alphaproteobacteria, and inside this large cluster, the different species generally group together per genus, in support of the current taxonomy (Fig. 18.1 ).

Fig. 18.1

Phylogenetic reconstruction of the family Phyllobacteriaceae based on 16S rRNA and created using the neighbor-joining algorithm with the Jukes-Cantor correction. The sequence datasets and alignments 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

An important exception is the genus Mesorhizobium: these species make up several groups and separate lineages grouping in between the other genus clusters (Fig. 18.1 ). The type species Mesorhizobium loti forms tight subcluster with Mesorhizobium ciceri, Mesorhizobium australicum, Mesorhizobium shangrilense, Mesorhizobium sangaii, and Mesorhizobium qingshengii. Mesorhizobium chacoense forms a separate but related lineage, and the Aminobacter cluster is their nearest neighbor. Nineteen species form the largest subcluster: Mesorhizobium metallidurans, Mesorhizobium temperatum, Mesorhizobium mediterraneum, Mesorhizobium gobiense, Mesorhizobium tarimense, Mesorhizobium caraganae, Mesorhizobium robiniae, Mesorhizobium muleiense, Mesorhizobium tianshanense, Mesorhizobium tamadayense, Mesorhizobium amorphae, Mesorhizobium septentrionale, Mesorhizobium huakuii, Mesorhizobium plurifarium, Mesorhizobium silamurunense, Mesorhizobium opportunistum, Mesorhizobium abyssinicae, Mesorhizobium hawassense, and Mesorhizobium shonense. Mesorhizobium albiziae groups at the periphery of this subcluster as does Mesorhizobium thiogangeticum. The position of the two latter species, however, varied depending on the filter applied. In most trees, it constitutes a separate lineage at some distance from other Mesorhizobium subclusters or other genera of the family. Mesorhizobium thiogangeticum was not recovered from legume nodules but was isolated from soil by enrichment using reduced sulfur compounds as sole electron sources; it is the only Mesorhizobium species reported to be facultatively chemolithoautotrophic. Two other species, Mesorhizobium camelthorni and Mesorhizobium alhagi, make up a further subcluster that groups most closely to the Chelativorans cluster. The 16S rRNA gene phylogeny of Mesorhizobium is thus polyphyletic, and the genus may in the future require taxonomic rearrangements if further evidence would support these observations.

Ahrensia and Lentilitoribacter group together with Hoeflea species, but, according to branch length, are clearly distinct.

Nitratireductor species form a single cluster, except for Nitratireductor basaltis which is located separately.

Candidatus Liberibacter, consisting of psyllid-transmitted, as yet uncultured, phloem-limited bacteria associated with greening disease or huanglongbing disease of citrus and yellows disease of various Solanaceae plants or endophytic in pear plants, was initially placed inside the Phyllobacteriaceae based on a limited phylogenetic indications (Mergaert and Swings 2005a; Garnier 2005). A more comprehensive analysis performed for this chapter revealed that the position of Candidatus Liberibacter as a member of the Phyllobacteriaceae cluster is uncertain. Although it does group on a long branch inside the Phyllobacteriaceae cluster in Fig. 18.1 , in most other trees calculated using other filters and including other neighboring taxa, Candidatus Liberibacter grouped outside the family and occupied a separate position in the Alphaproteobacteria. Its membership of the family therefore seems not strongly supported by 16S rRNA phylogeny.

Comments on the Membership of the Family

Although the genus Ahrensia is was classified in the Rhodobacteraceae in Bergey’s Manual of Systematic Bacteriology (Garrity et al. 2005) based on the phylogenetic analysis of 16S rRNA genes, since then more taxa have been described in the Rhizobiales, and current 16S rRNA gene phylogeny places Ahrensia in the Phyllobacteriaceae (Living Tree Project, release 111). It is therefore included in this chapter.

“Aliihoeflea aestuarii” gen. nov., sp. nov. was described for a bacterium isolated from tidal flat sediments (Roh et al. 2008). Its 16S rRNA gene sequence was reported to cluster with members of the Phyllobacteriaceae. Fatty acid data, quinone, and DNA G+C content data are also in agreement with the family characteristics; therefore, although Aliihoeflea aestuarii has as yet not been included in a validation list, it is included here in the chapter on Phyllobacteriaceae.

Molecular Analyses

DNA-DNA Hybridization Studies

In all multispecies genera of the family, DNA-DNA hybridizations with existing species have been performed to justify proposals of new species.

Other Sequence Analyses

Genes other than the 16S rRNA gene have been reported in Mesorhizobium where recA sequences are available for all species and a number of other genes including atpD, gyrB, dnaK, and rpoB have also been reported for several of the Mesorhizobium species. However, for other Phyllobacteriaceae genera, only in a few cases have other genes been reported and used for phylogenetic purposes: for three of six Aminovorans species sequences are available for atpD, dnaK, and recA (Maynaud et al. 2012); for four of eight Phyllobacterium species, an atpD sequence has been reported, as well as a recA sequence for one species (Mantelin et al. 2006b), and for two of six Nitratireductor species, an rpoD sequence is available (unpublished data available through NCBI dataportal). Given this lack of data for all genera, it is currently not possible to comprehensively assess the phylogeny of the family using a housekeeping gene other than the 16S rRNA gene. At present most data are available for recA where 6 of the 13 genera are represented (three of these are extracted from total genome information). Given that Mesorhizobium is represented with 30 species versus only 8 species from other genera, this tree (Fig. 18.2 ) does not permit a comprehensive comparison with the 16S rRNA gene phylogeny (Fig. 18.1 ). It thus remains to be established whether the subclusters of Mesorhizobium in the latter phylogeny are confirmed by recA or other gene phylogenies.

Fig. 18.2

Phylogenetic reconstruction of the family Phyllobacteriaceae based recA sequences. The tree was constructed using the maximum likelihood method and general time-reversible model in MEGA (Tamura et al. 2011). A bootstrap analysis with 500 replicates was performed to assess the support of the clusters; values above 50 % are shown at the nodes

Genome Comparisons

Only in recent years have some complete genome sequences been reported or drafts made available. An overview of the type strains and a few other strains is given in Table 18.1 . Unpublished draft genomes are available in public database online for Ahrensia kielensis, Chelativorans sp., Hoeflea phototrophica, Mesorhizobium australicum, Mesorhizobium ciceri bv. biserrulae, and Mesorhizobium opportunistum (Table 18.1 ).

Table 18.1 Overview of genome sequences available for type strains of the Phyllobacteriaceae

The genome of Mesorhizobium loti MAFF303099 was reported more than 10 years ago (Kaneko et al. 2000); however, later this strain was shown to be a representative of another Lotus symbiont, Mesorhizobium huakuii bv. loti (Turner et al. 2002). Its genome consists of one chromosome (7 Mb) and two megaplasmids (352 kb and 208 kb); a transmissible symbiotic island containing 580 protein-encoding genes including genes for nodulation and nitrogen fixation was identified on the chromosome, inserted into the phe-tRNA gene as in other Mesorhizobium loti strains (Kaneko et al. 2000). The genomes of several other Mesorhizobium strains have been sequenced and had a similar size between 6.2 and 7.6 Mb with one circular chromosome and either no, one or two megaplasmids (Table 18.1 ). Mesorhizobium amorphae CCNWGS0123 is a copper-resistant rhizobium that contributes to survival of the host plant in copper-, zinc-, and chromium-containing environments. Its genome was found to harbor numerous genes involved in copper resistance including a copper efflux system and multicopper oxidases, as well as various genes for plant growth promotion that most rhizobia share. In addition genes involved in the biosynthesis of a number of antibiotics and in chloramphenicol resistance were present (Hao et al. 2012). Mesorhizobium alhagi CCNWXJ12-2^T is very resistant to salt (0.8 M) and alkali (pH 12). Its genome was found to encode various systems contributing to salt resistance and osmoregulation including multiple membrane transport system (Zhou et al. 2012).

The genomes have been reported for three Nitratireductor strains. For the type species, Nitratireductor aquibiodomus strain RA22 from a marine water sample in India has been sequenced; its 16S rRNA gene was reported as 100 % identical to that of the type strain. Annotation revealed genes for iron acquisition, ammonia and sulfur assimilation, biosynthesis of ectoine and betaine, and uptake of choline and betaine, indicative of its marine habitat requiring osmotic stress tolerance. Genes for catabolism of aromatic compounds, including genes for the chloroaromatic degradation pathway, correspond with the observation that many Nitratireductor strains were obtained from sources contaminated with pyrene, crude oil, or pesticide (Singh et al. 2012). Nitratireductor pacificus pht-3B^T, although isolated from a pyrene-degrading consortium from deep-sea sediments, is unable to utilize pyrene, and this was confirmed by the absence of polycyclic aromatic hydrocarbon (PAH)-degrading dioxygenase in its genome (Lai et al. 2012a). Nitratireductor indicus C115^T originates from a crude oil-degrading consortium from deep seawater; however, it cannot degrade n-alkanes or PAHs as sole carbon source. Also here, the genome sequence confirmed the absence of any alkane-degrading monooxygenase or PAH-degrading dioxygenase (Lai et al. 2012b).

Two complete genomes have been reported representing Candidatus Liberibacter, a group of uncultured bacteria associated with citrus and Solanaceae plant diseases; genomes were obtained from DNA isolated from the phloem-feeding psyllid vectors that transmit the pathogen (Duan et al. 2009; Lin et al. 2011). As can be expected for obligate intracellular endophytes, the genomes are small and have a low GC content: 0.99 Mb and 36.5 mol % and 1.26 Mb and 35.2 mol % for Candidatus Liberibacter asiaticus psy62 and Candidatus Liberibacter solanacearum CLso-ZC1, respectively. Candidatus Liberibacter asiaticus psy62 harbored few genes for the biosynthesis of compounds that can be obtained from the host and more genes for motility such as type IV pili and flagellar genes; it had no transposons or insertion elements but did have some phage-related genes (Duan et al. 2009). Its genome also revealed the absence of several key components required for oxidative phosphorylation and several terminal oxidases, pointing to a limited potential for aerobic respiration; genome analysis suggests that the organism cannot reduce sulfur compound, but instead anaerobic respiration is coupled to nitrogen metabolism. The presence of an active TCA cycle suggests that a range of amino acids (present in phloem fluid) may serve as energy sources (Duan et al. 2009). Candidatus Liberibacter solanacearum shares 884 protein-encoding genes with Candidatus Liberibacter asiaticus. Comparison of both genomes revealed many rearrangements and gene losses/gains (Lin et al. 2011). Candidatus Liberibacter solanacearum also contained several small and two large phage-derived segments, one of which was similar to a segment in Candidatus Liberibacter asiaticus. The analysis of its gene repertoire suggests it can take up glucose but not sucrose or fructose and has limited capacity for aerobic respiration and for the biosynthesis of amino acids and lacks a complete restriction-modification system. It has several transport systems for amino acids and a system (NttA) for the uptake of ATP and ADP from the host. The comparison further revealed that Candidatus Liberibacter solanacearum has reduced capacity for nucleic acid modification, increased potential for amino acid and vitamin biosynthesis, and a high-affinity iron transport system (Lin et al. 2011). Lin et al. (2011) point out that the approach of extracting bacterial genome information from the vector does not exclude that other genetic components such as plasmids or linear chromosomes could be present.

Based on the complete genome (Duan et al. 2009; Tyler et al. 2009), a computational analysis of the Candidatus Liberibacter asiaticus proteome has been performed, and the results predicting 3D structure, function, cellular localization, and potential virulence factors are publically available (http://prodata.swmed.edu/liberibacter_asiaticus/curated/) as a tool for further study of this pathogen (Cong et al. 2012).

Phenotypic Analyses

A comparison of some general features of the members of the Phyllobacteriaceae is given in Table 18.2 .

Table 18.2 Morphological and chemotaxonomic characteristics of the genera of Phyllobacteriaceae

Ahrensia Uchino et al. 1999, 1^VL

Ah.ren’si.a. N.L. fem. n. Ahrensia, named in honor of R. Ahrens, a German microbiologist, for his contribution to the taxonomy of marine species of Agrobacterium.

The genus Ahrensia comprises rod-shaped cells that do not form spores. They are motile with polar flagella. Aerobic and oxidase and catalase positive. The major quinone is ubiquinone Q10; the major fatty acid is C18:1; the main hydroxy fatty acid is C12:0 3-OH. No 2-hydroxy fatty acids are present. The G+C content of the DNA is 48 mol %. The type species is Ahrensia kielensis.

The following description of Ahrensia kielensis is based on those from Uchino et al. (1998) and from Rüger and Höfle (1992). The species is able to grow at 5 °C, but not at 37 °C. Na⁺ is required. Cells are motile rods, 0.6–1.0 × 2.0–4.0 μm. Hardly any carbon sources are used: the type strain tested negative for 12 carbohydrates, 11 carboxylic acids, 3 alcohols, 7 amino acids, and putrescine (Rüger and Höfle 1992). H₂S is produced from cysteine; hydrolysis of gelatin and starch is negative. Nitrate is not reduced to nitrite or gas. Acids are produced from fructose, maltose, xylose, and glycerol after 4–6 weeks of incubation. Negative in the following tests: indole production, methyl red, Voges-Proskauer, lysine and ornithine decarboxylase, and hydrolysis of casein, chitin, and alginate.

The major fatty acid is C18:1 ω7c; C12:0 3-OH and iso-C13:0 3-OH are present, but 2-hydroxy fatty acids are absent (Uchino et al. 1998; Park et al. 2013). The G+C content of the DNA is 48 mol %.

The type strain IAM 12618^T was isolated from seawater of the Baltic Sea.

Aliihoeflea Roh et al. 2008

A.li.i.ho.e.fle’a, L. adj. and pronoun alius, other, another, different; N.L. fem. n. Hoeflea, a bacterial genus name; N.L. fem. n. Aliihoeflea, the other Hoeflea.

Aliihoeflea comprises rod-shaped cells that are catalase and oxidase positive. The major quinone is ubiquinone Q10; the major fatty acids are C18:1 ω7c and C19:0 cyclo ω8c. G+C content is approximately 53 mol %. The type species is Aliihoeflea aestuarii.

The following description of the phenotype is based on the description of the strain N8^T, thus far the only strain of Aliihoeflea aestuarii (Roh et al. 2008). Cells are rod shaped (0.50–0.75 μm × 1.25–1.50 μm). Colonies on MA are circular with entire margin, convex, shiny, and cream colored. Growth is also possible on Trypticase soy agar, SA, LA, and yeast mannitol agar, but not on R2A. Temperature range for growth is 17–37 °C; the optimal growth temperature is 30 °C. Optimal NaCl concentration is 1 % (w/v), although NaCl is not required and up to 8 % is tolerated during growth. Nitrates are not reduced to nitrites or nitrogen. Indole is not produced. Glucose is not fermented, and hydrolysis of starch, esculin, gelatin, and PNPG (p-nitrophenyl-β-d-galactopyranoside) is negative. Urease positive and arginine dihydrolase negative. Glycogen, Tween 80, l-arabinose, d-fructose, pyruvic acid methyl ester, succinic acid monomethyl ester, acetic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, α-ketobutyric acid, α-ketoglutaric acid, α-ketovaleric acid, d,l-lactic acid, succinic acid, succinamic acid, l-alaninamide, d-alanine, l-alanine, l-glutamic acid, glycyl-l-glutamic acid, l-leucine, l-serine, inosine, uridine, and thymidine can be used as sole carbon sources. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase. Negative for lipase (C14), acid phosphatase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. The genomic DNA G+C content is 53.4 mol %. The type strain KCTC 22052^T was isolated from tidal flat sediment in Yeosu (34°47′26″ N 127°34′01″ E), Republic of Korea.

Aminobacter Urakami et al. 1992, 90^VP

Am.i.no.bac’ter, N.L. n. aminum, amine; N.L. masc. n. bacter, rod; N.L. masc.n. Aminobacter, amine rod.

The genus Aminobacter comprises non-spore-forming rod-shaped cells that can utilize methylamine. Cells are motile by means of subpolar flagella. They multiply by budding. Poly-β-hydroxybutyrate granules are accumulated in the cells. Good growth in nutrient broth and PYG broth. No water-soluble fluorescent pigment is produced. No growth factors are required. Oxidase and catalase positive and urease negative. Aerobic respiratory metabolism, not fermentative (Urakami et al. 1992).

The following tests are negative: methyl red, Voges-Proskauer, indole production, hydrogen sulfide production, hydrolysis of gelatin and starch, denitrification, litmus milk, and fermentation of sugars.

Ammonia is produced. Acids are produced from sugars oxidatively. Monomethylamine, trimethylamine, trimethylamine-N-oxide, and sugars are utilized. Methanol, methane, and hydrogen are not utilized. Ammonia, nitrate, urea, peptone, and methylamine are utilized as nitrogen sources.

Good growth occurs at pH 6.0–8.0 and at 30–37 °C. No growth above pH 9.0 and below pH 5.0 at 42 °C and in the presence of 3 % NaCl (Urakami et al. 1992).

The type strains of all species can utilize l-arabinose and l-alanine; none can use adonitol (McDonald et al. 2005; Maynaud et al. 2012). McDonald et al. (2005) performed a biochemical characterization of all Aminobacter species except Aminobacter anthyllidis and found that all type strains could utilize N-acetyl-d-glucosamine, d-cellobiose, d-fructose, d-galactose, d-glucose, d-mannose, d-maltose, d-ribose, d-xylose, i-inositol, d-mannitol, d-sorbitol, acetate, 4-aminobutyrate, dl-3-hydroxybutyrate, dl-lactate, oxoglutarate, l-histidine, l-leucine, l-ornithine, and l-proline and hydrolyze bis-para-nitrophenyl (pNP)-phosphate, pNP-phenyl-phosphonate, l-alanine-para-nitroanilide (pNA), and l-proline-pNA. None of the strains could utilize p-arbutin, α-d-melibiose, salicin, maltitol, putrescine, cis-aconitate, trans-aconitate, adipate, azelate, citrate, fumarate, itaconate, mesaconate, suberate, l-phenylalanine, 3-hydroxybenzoate, and phenylacetate. All type strains used sucrose. None of the strains could hydrolyze esculin, pNP-β-d-galactopyranoside, pNP-β-d-glucuronide, 2-deoxythymidine-5′-pNP-phosphate, and l-glutamate-γ-3-carboxy-pNA and none produced acid from lactose, adonitol, rhamnose, methyl d-glucoside, erythritol, and melibiose. Additional features and differentiating characteristics of the species are shown in Table 18.3 .

Table 18.3 Comparison of selected characters of the species of Aminobacter

Only strains of Aminobacter ciceronei and Aminobacter lissarensis utilize several methyl halides as sole carbon sources (Table 18.3 ). Of all Aminobacter strains tested, two strains of Aminobacter ciceronei (ER2 and C147; not the type strain) were the sole Aminobacter strains that could degrade atrazine and carbofuran (McDonald et al. 2005). Aminobacter anthyllidis, which is capable of nodulation and was isolated from a Zn-Pb mining site through trapping with Anthyllis vulneraria, can tolerate 1–2 mM of Zn and 0.3–1 mM of Cd in YEM broth after 1 week (Maynaud et al. 2012).

The DNA base composition ranges from 62 to 64 mol % G + C. The main cellular fatty acids include C18:1, and the main hydroxy fatty acids include C12:0 3-OH. The ubiquinone system is ubiquinone Q10.

The type species is Aminobacter aminovorans, originally described as Pseudomonas aminovorans (Urakami et al. 1992).

Aquamicrobium Bambauer et al. 1998, 631^VL emend. Lipski and Kämpfer 2012

A.qua.mi.cro’bi.um, L. n. aqua, water; N.L. neut. n. microbium, a microbe; N.L. neut. n. Aquamicrobium, a bacterium living in water/wastewater.

This description is based on the emended description of Lipski and Kämpfer (2012). Aquamicrobium consists of pleomorphic or regularly shaped short rods that are mesophilic and grow best at pH 6–9. They can tolerate up to 7 % NaCl (w/v) and utilize sugars, carbonic acids, amino acids, and alcohols for growth. Major quinone is Q10, major fatty acid is C18:1 cis-11, major polyamine is spermidine, and main polar lipids are phosphatidylglycerol, phosphatidylcholine, and phosphatidylethanolamine. G+C content of the DNA is 57–65 mol %. The type species is Aquamicrobium defluvii.

A number of phenotypic and other characteristics of the Aquamicrobium type strains are listed in Table 18.4 . All species are oxidase and catalase positive. Aquamicrobium defluvii is able to utilize thiophene-2-carboxylate as sole carbon source in the presence of molybdate (Bambauer et al. 1998). In addition, acetate, propionate, butyrate, crotonate, glucose, fructose, mannose, xylose, mannitol, and sorbitol are used for growth with oxygen or nitrate as electron acceptors. Nitrate is reduced to nitrite. No growth was observed with thiophene-2-acetate, thiophene-3-carboxylate, thiophene-3-acetate, thiophene-2-carbaldehyde, thiophene-2-methanol, thiophene-2-mandelate, thiophene-2-acrylate, thiophene, benzothiophene, dibenzothiophene, pyrrole-2-carboxylate, furan-2-carboxylate, pyridine, nicotinate, benzoate, phenylacetate, phthalate, galactose, ribose, sorbose, maltose, saccharose, cellobiose, and lactose. Hydrolysis of gelatin, arginine dihydrolase, lysine decarboxylase, and urease is negative (Bambauer et al. 1998). Aquamicrobium lusatiense is able to degrade 4-chlorophenol, 2,4-dichlorophenol, and phenol, and this capacity was not lost over repeated transfers and attempts at curing. Indeed, genes for chlorocatechol 1,2-dioxygenase and 2,4-dichlorophenol hydroxylase were shown to be located on the chromosome rather than on a megaplasmid (Fritsche et al. 1999). Hydrolysis of urea, starch, gelatin, casein, DNA, Tween 80, and esculin is negative (Fritsche et al. 1999). Aquamicrobium aerolatum is positive for phosphatase and l-alanine aminopeptidase (Kämpfer et al. 2009).

Table 18.4 Comparison of selected characters of the species of Aquamicrobium

Small amounts of 12:0 3-OH were reported for Aquamicrobium defluvii and Aquamicrobium lusatiense and iso-15:0 3-OH for Aquamicrobium aerolatum by Kämpfer et al. (2009), but a later study comprising all species did not find hydroxy fatty acids (Lipski and Kämpfer 2012).

Chelativorans Doronina et al. 2010, 1047^VP

Che.la’ti.vo.rans. N.L. n. chelatum, a chelate; L. part. adj. vorans, devouring; N.L. masc. n. Chelativorans, a bacterium digesting metal chelates.

Chelativorans strains are non-spore-forming rods. The genus was described as nonmotile (Doronina et al. 2010), although flagella were later reported for Chelativorans multitrophicus DSM 9103^T and several Chelativorans sp. strains (Kaparullina et al. 2011). They often occur as pairs and multiply by binary fission. They form small white colonies on EDTA/mineral salt agar (diameter 0.1–0.3 mm after 7 days at 30 °C). Optimal NaCl concentration for growth is 1.5 %. No PHB inclusions; electron dense inclusions are thought to consist of calcium and magnesium phosphates and are absent in cells grown on fumarate. Oxidase and catalase positive; indole is produced; no nitrate reduction to nitrite; no nitrogen fixation. Optimal temperature and pH for growth are 25–35 °C and 6.5–7.5. Aerobic respiratory metabolism; able to use EDTA as carbon, nitrogen and energy source, either facultatively (Chelativorans multivorans) or obligately (Chelativorans oligotrophicus). No autotrophic or methylotrophic growth; unable to use methanol or methylated amines as carbon, nitrogen, or energy source (Doronina et al. 2010). Unable to use alcohols, amines, malate, pyruvate, l-alanine, and l-serine as carbon and energy sources (Kaparullina et al. 2011). Chelativorans oligotrophicus has several defective or missing enzymes in the central carbon metabolism. The tricarboxylic acid cycle lacks α-ketoglutarate dehydrogenase activity, and 6-phosphofructokinase (ATP/PPi) is also absent (Doronina et al. 2010). The major cellular fatty acids are summed feature 7 (C18:1 ω7c, C18:1 ω9t and/or C18:1 ω12t) and C19:0 cyclo ω8c. Hydroxy fatty acids C12:0 3-OH, C13:0 3-OH, and C15:0 iso 3-OH are absent. The major ubiquinone is Q10. Predominant polar lipids are phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidyldimethylethanolamine, phosphatidylmonomethylethanolamine, and diphosphatidylglycerol. Mesorhizobium-specific ornithine lipid is absent. sym-Homospermidine is the main polyamine with small amounts of spermidine and putrescine present. The DNA G+C content is 60–64 mol %. The type species is Chelativorans multitrophicus (Doronina et al. 2010).

Additional characters and differentiating features of both species are shown in Table 18.5 .

Table 18.5 Characteristics of the species of Chelativorans

Hoeflea Peix et al. 2005, 1165^VP

Hoef.le.a’. N.L. fem. n. Hoeflea honoring Manfred Höfle, German microbiologist, in recognition of his contribution to the taxonomy of marine bacteria.

Cells are non-spore-forming, motile short rods. They are aerobic chemoorganotrophs and are oxidase and catalase positive except for Hoeflea alexandrii which was described as oxidase negative. Cells do not require NaCl; however, they can grow in the presence of up to 5 % NaCl. Growth is possible at a temperature of 18–33°C although some species can grow at higher and lower temperatures (Table 18.6 ). pH range for growth is 6–8 or 9, and Hoeflea suaedae has a more wide pH range of 5–10. No nitrate reduction to nitrite or nitrogen except for Hoeflea suaedae which was reported to reduce nitrate to nitrite.

Table 18.6 Characteristics of Hoeflea species

The main fatty acid is C18:1 ω7c, and other important fatty acids (>3 %) are C16:0, 11-Me C18:1 ω7c, and C19:0 cyclo ω8c. Only small amounts of hydroxy fatty acid are present. Ubiquinone Q10 is the major quinone; the main polar lipids are phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and sulfoquinovosyldiacylglyceride, although the latter polar lipid was not reported from Hoeflea anabaenae. The G+C content of the DNA ranges from 53 to 60 mol %. The type species is Hoeflea marina.

Additional characters and differentiating features of the five Hoeflea species are shown in Table 18.6 .

Hoeflea anabaenae cells attach to Anabaena heterocysts; of the other Hoeflea species, only Hoeflea phototrophica has been observed to do this, although at a much lower frequency (Stevenson et al. 2011). Hoeflea siderophila is the only species reported to be iron oxidizing, using FeS, FeSO₄, or FeCO₃ for lithotrophic growth while depositing iron oxides on the cell surface. It also has a facultative anaerobic metabolism. In anaerobic, iron-oxidizing conditions, it uses nitrate or N₂O as terminal electron acceptor. Nitrate is converted to nitrite which inhibits growth as it accumulates. In those conditions, nitrite can chemically oxidize up to 20 % of Fe(II). When N₂O is given as an electron acceptor, there is virtually no chemical Fe(II) oxidation, and N₂ is formed. The organism is also able to grow mixotrophically or organotrophically in microaerobic or anaerobic condition, using nitrate or N₂O as electron acceptors. Nitrite, ClO₄⁻, S⁰, thiosulfate, and Fe(OH)₃ are not used as electron acceptors, and H₂ oxidation is not possible (Sorokina et al. 2012).

Lentilitoribacter Park et al. 2013, 2365^VL

Len.ti.li.to.ri.bac’ter. L. masc. adj. lentus, slow, delayed; L. n. litus-oris, the seashore, coast; N.L. masc. n. bacter, rod; N.L. masc. n. Lentilitoribacter, slowly growing rod from the coast).

Lentilitoribacter cells are non-spore-forming, nonmotile, and rods to short rods. They are catalase and oxidase positive and do not reduce nitrate to nitrite. Aerobic. The predominant ubiquinone is Q10. The major fatty acids are C18:1 ω7c, 11-methyl-C18:1 ω7c, and summed feature 3 (iso-C15:0 2-OH and/or C16:1 ω7c). The major polar lipids are phosphatidylglycerol and phosphatidylmonomethylethanolamine. The DNA G+C content is 49.3 mol %. The type species is Lentilitoribacter donghaensis.

Lentilitoribacter donghaensis cells are rods, 0.3–0.6 × 0.6–4.0 μm. Colonies on marine agar are circular, slightly convex, smooth, whitish yellow and less than 0.5 mm in diameter after 10 days at 25 °C. Optimal growth temperature is 25 °C; growth occurs at 4 and 30 °C, but not at 35 °C. Optimal pH is between 7.0 and 7.5; growth occurs at pH 5.5, but not at pH 5.0. Grows in the presence of 1.0–5.0 % NaCl (bstl growth with 2.0 % NaCl). Requires Mg²⁺ ions for growth. Hydrolyzes Tween 20, 40, 60, and 80, but not esculin, casein, gelatin, hypoxanthine, l-tyrosine, starch, and xanthine. Acid is produced from d-xylose, but not from l-arabinose, d-cellobiose, d-fructose, d-galactose, d-glucose, myo-inositol, lactose, maltose, d-mannitol, d-mannose, d-melezitose, melibiose, d-raffinose, l-rhamnose, d-ribose, d-sorbitol, sucrose, and d-trehalose. In the API ZYM tests, alkaline phosphatase, esterase lipase (C8), and leucine arylamidase are positive, while esterase (C4), trypsin, and acid phosphatase activities are weakly present, and lipase (C14), valine arylamidase, cysteine arylamidase, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, or α-fucosidase activities are negative. The major fatty acids (>10 %) are C18:1 ω7c, 11-methyl-C18:1 ω7c, and summed feature 3 (iso-C15:0 2-OH and/or C16:1 ω7c). C10:0 3-OH is the only hydroxy fatty acid detected.

The type strain CCUG 62792^T was isolated from seawater from the coast around Baekdo harbor in the East Sea, South Korea. Its DNA G+C content is 49.3 mol %.

Mesorhizobium Jarvis et al. 1997, 897^VP

Me.so.rhi.zo’bi.um. Gr. adj. mesos, middle; N.L. neut. n. Rhizobium, bacterial genus name; N.L. neut.n. Mesorhizobium, rhizobia, phylogenetically intermediate between the genera Bradyrhizobium and Rhizobium. This etymology is given in the original description (Jarvis et al. 1997); alternatively in the List of Prokaryotic names with Standing in Nomenclature (www.bacterio.cict.fr), the name Mesorhizobium is said to refer to the growth rate of the bacteria which is intermediate between that of the genera Rhizobium and Bradyrhizobium.

The genus Mesorhizobium comprises 30 species, most occurring as nitrogen-fixing endosymbionts in root nodules of various legume plants. The species Mesorhizobium thiogangeticum was isolated from the soil adjacent to the roots of the legume Clitoria ternatea, by enrichment using reduced sulfur compounds as sole carbon and energy source (Ghosh and Roy 2006).

All species comprise rod-shaped cells that form creamy, white, or colorless colonies on agar media. They are aerobic organotrophs; only Mesorhizobium thiogangeticum is capable of facultative chemolithotrophic growth using thiosulfate or elemental sulfur as energy source (Ghosh and Roy 2006). Optimal temperature for growth is around 28 °C, and optimal pH is about 7. Three species have been reported to grow at 4°C: Mesorhizobium ciceri, Mesorhizobium sangaii, and Mesorhizobium shonense (Zhou et al. 2013); Mesorhizobium ciceri is the only species reported to grow at 40 °C (Jarvis et al. 1997; Zhou et al. 2013). Several species can grow in the presence of 1 or 2 % NaCl (Table 18.7 ); for Mesorhizobium shangrilense, even growth with 3 % NaCl was reported (Lu et al. 2009).

Table 18.7 Characteristics of the type strains of species of Mesorhizobium

The fatty acid C18:1 ω7c is present in all species in large amounts (at least 10 % detected in itself or as part of a summed feature), while C16:0, 11-Me C18:1 ω7c, and C19:0 cyclo ω8c are also important (at least 5 %) in more than two thirds to half of the species. Hydroxy fatty acids have been reported at more than 1 % in 7 of the 30 species and comprise mostly C12:0 3-OH and/or iso-C13:0 3-OH; only in Mesorhizobium albiziae and Mesorhizobium temperatum has iso-C15:0 3-OH been reported at more than 1 % (Wang et al. 2007). Polar lipids have been reported for six of the species (Table 18.7 ): all comprised phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine, while five also contained diphosphatidylglycerol and several unidentified phospholipids (Zhang et al. 2012; Zheng et al. 2013). The DNA G+C content ranges from 57.9 % to 65.1 %.

The type species is Mesorhizobium loti. Additional characters and differentiating features of Mesorhizobium species are shown in Table 18.7 .

Nitratireductor Labbé et al. 2004, 54^VP

Ni.tra.ti.re.duc’tor. N.L. masc. n. nitras, nitrate; L. v. reducere, to bring back, to reduce; N.L. masc. n. nitratireductor, nitrate-reducing bacterium.

The genus Nitratireductor comprises six species that occur in various marine habitats. All species are aerobic chemoorganotrophs; nitrate reduction varies between strains. Cells are rods, short rods, or coccoid. Motility is variable. Optimum temperature for growth is 25–35 °C. No growth below 10 °C. pH range is 5–12. All species are oxidase and catalase positive. Major quinone is ubiquinone Q10. The main fatty acid is C18:1 ω7c/ω6c. DNA G+C content is 56.7–63 mol %. The type species is Nitratireductor aquibiodomus.

All species are positive for leucine arylamidase (API ZYM tests) and for the use of d-glucose and N-acetyl-glucosamine (API 20NE); all are negative for indole production (API 20NE). Further characteristics and differentiating features of the species are given in Table 18.8 .

Table 18.8 Characteristics of the type strains of Nitratireductor species

Phyllobacterium (ex Knösel 1962) Knösel 1984, 356^VP

Phyl.lo.bac.te’ri.um. Gr. neut. n. phyllon, leaf; L. neut. n. bacterium, rod; N.L. neut. n. phyllobacterium, leaf bacterium (occurring in leaf nodules of higher plants).

Cells are rods and motile by means of polar, subpolar, or lateral flagella. The optimal growth temperature is 28 °C, and there is no growth at 40 °C. Growth occurs in 1 % NaCl. Glucose metabolism is oxidative. Oxidase positive; urease is positive except for Phyllobacterium endophyticum. Indole production, β-galactosidase, and gelatinase are negative for all species. Some other enzyme activities that were originally included in the genus description, however, have not been reported for all species. These include DNase (negative, but no data for P. endophyticum and P. trifolii), hydrolysis of Tween 80 (negative, but not tested for P. catacumbae, P. endophyticum, and P. trifolii), starch (negative for P. myrsinacearum and P. catacumbae), pectin and cellulose (both only reported as negative for P. myrsinacearum), nitrate reduction (positive for P. myrsinacearum, negative for P. catacumbae, P. endophyticum and P. trifolii). Esculin is hydrolyzed (weak reaction for P. trifolii). 3-Ketolactose test is negative (no data for P. endophyticum and P. trifolii). Assimilation of d-glucose, d-mannose, l-arabinose, d-mannitol, and N-acetylglucosamine is positive for all species. Maltose is used by all species except P. endophyticum. Quinones have only been reported for Phyllobacterium endophyticum and comprised Q10 (88 %) and Q9 (12 %) (Flores-Felix et al. 2013). Additional characteristics of the Phyllobacterium species are given in Table 18.9 .

Table 18.9 Comparison of selected characters of the species of Phyllobacterium

The G+C content of the DNA ranges from 51 to 61 mol % (Tm). The type species is Phyllobacterium myrsinacearum.

Pseudahrensia Jung et al. 2012, 2059^VP

Pseu.dah.ren’si.a. Gr. adj. pseudes, false; N.L. fem. n. Ahrensia, a bacterial genus name; N.L. fem. n. Pseudahrensia, the false Ahrensia.

Pseudahrensia cells are aerobic, non-spore-forming, nonmotile, and ovoid to rod shaped. Catalase, oxidase, and nitrate reduction are positive. The predominant ubiquinone is Q10. The major fatty acid is C18:1 ω7c. The major polar lipids are phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine. The DNA G+C content of the type strain of the type species is 60.1 mol %. The type species is Pseudahrensia aquimaris.

Pseudahrensia aquimaris cells are nonmotile, ovoid to rod shaped, and 0.5–1.0 × 1.0–7.0 μm. Colonies on MA are circular, convex, smooth, glistening, cream colored and 1.0–1.5 mm in diameter after 5 days at 30 °C. Temperature range for growth is 4–32 °C; optimal growth at 30 °C, pH 7–8, and 2–3 % NaCl. Grows at pH 5.5, but not pH 5. Can grow in 10 % NaCl, but not in 11 % or without NaCl. Na⁺ and Mg²⁺ ions are required for growth. No anaerobic growth on marine agar. Nitrate is reduced to nitrite. Gelatin is hydrolyzed. H₂S is not produced. Esculin; casein; hypoxanthine; starch; Tween 20, 40, 60, and 80; l-tyrosine; urea; and xanthine are not hydrolyzed. Acid is positive from d-fructose, d-galactose, d-glucose, lactose, maltose, d-mannose, d-ribose, and sucrose; no acid production from l-arabinose, cellobiose, myo-inositol, d-mannitol, melezitose, melibiose, raffinose, l-rhamnose, d-sorbitol, trehalose, or d-xylose. Susceptible to ampicillin, cephalothin, chloramphenicol, gentamicin, kanamycin, neomycin, novobiocin, penicillin G, polymyxin B, and streptomycin; resistant to carbenicillin, lincomycin, oleandomycin, and tetracycline. The following enzymes are present (API ZYM): alkaline phosphatase, esterase (C4), leucine arylamidase, acid phosphatase, esterase lipase (C8) (weak), and trypsin (weak); the following enzymes are absent: lipase (C14), valine arylamidase, cystine arylamidase, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, α- and β-galactosidase, β-glucuronidase, α- and β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase (Jung et al. 2012).

Major fatty acid (>10 %) is C18:1 ω7c; no other fatty acids are present at more than 5 %; the only hydroxy fatty acid detected is C18:0 3-OH, apart from the possible presence of C14:0 3-OH as part of summed feature 2 (Jung et al. 2012). However, Park et al. (2013) report the presence of iso-C13:0 3-OH and the absence of C18:0 3-OH.

The type strain (CCUG 60023^T) was isolated from seawater, Yellow Sea of the island of Hwang-do, Korea.

Pseudaminobacter Kämpfer et al. 1999, 894^VP

Pseud.ami.no.bac’ter. Gr. adj. pseudos, false; N.L. masc.n. Aminobacter, bacterial genus name; N.L. masc. n. Pseudaminobacter, false Aminobacter.

Pseudaminobacter cells are rod shaped and motile. Obligate aerobic heterotrophs. They have an oxidative metabolism and can use d-glucose, d-ribose, d-xylose, acetate, propionate, pyruvate, β-alanine, N-acetyl-d-glucosamine, 4-aminobutyrate, dl-3-hydroxybutyrat, dl-lactate, oxoglutarate, l-alanine, l-histidine, l-leucine, and l-proline as sole carbon source. Growth occurs on nutrient agar (Oxoid), Caso agar, R2A agar (Oxoid), and TSB agar (BBL). Colonies are circular, entire, slightly convex and smooth, glistening, and pale beige on nutrient agar at 25 °C. Oxidase and catalase positive. Main ubiquinone is Q10. The major polyamines are spermidine, sym-homospermidine, and putrescine. Polar lipids include phosphatidylcholine, phosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, and diphosphatidylglycerol in nearly the same amounts. Main fatty acids are C18:1 and C19:0 cyclo ω8c. The only hydroxy fatty acid is C15:O iso 3-OH. The G+C content of the DNA is 62.9–63.9 mol %. The type species is Pseudaminobacter salicylatoxidans.

One additional species was described, Pseudaminobacter defluvii. Both species produce acid weakly from glucose, but not from lactose, sucrose, salicin, inositol, sorbitol, l-arabinose, raffinose, maltose, d-xylose, trehalose, cellobiose, d-arabitol, mannose, adonitol, rhamnose, methyl d-glucoside and erythritol. Both species hydrolyze bis-para-nitrophenyl (pNP)-phosphate, pNP-phenyl-phosphonate, l-alanine-para-nitroanilide (pNA), and l-proline-pNA, but not pNP-α-d-glucopyranoside, pNP-β-d-glucopyranoside, pNP-phosphorylcholine, esculin, pNP-β-d-galactopyranoside, pNP-β-d-glucuronide, 2-deoxythymidine-5′-pNP-phosphate, and l-glutamate-γ-3-carboxy-pNA. They do not assimilate p-arbutin, d-melibiose, salicin, maltitol, putrescine, trans-aconitate, adipate, azelate, fumarate, itaconate, mesaconate, suberate, l-tryptophan, 3-hydroxybenzoate, and phenylacetate. Additional characteristics of the Psedaminobacter species are given in Table 18.10 .

Table 18.10 Characteristics of the two Pseudaminobacter species. Data taken from Kämpfer et al. (1999)

Thermovum Yabe et al. 2012, 2994^VP

Ther.mo’vum. Gr. n. thermê, heat; L. neut. n. ovum, egg, oval; N.L. neut. n. Thermovum, a heat(-loving) oval-shaped organism.

Thermovum comprises Gram-positive ovoid cells that do not form spores. Thermophilic. Major fatty acids (>10 %) are C18:1 ω7c, C19:0 ω8c, and C18:0. Polar lipids comprise phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol, phosphatidylmonomethylethanolamine, an unknown glycolipid, and a ninhydrin-positive phospholipid. The main quinone is ubiquinone Q10. The type species is Thermovum composti.

Thermovum composti cells are nonmotile, ovoid shaped, and 0.9 μm × 1.4 μm (after 2 days at 50 °C). Catalase and oxidase positive. Growth occurs at 23–57 °C, with optimal growth at 50 °C, at pH 5.9–8.8 (optimum, pH 7.0) and in the presence of 0–4 % (w/v) NaCl. In addition to the major fatty acids listed above in the genus description, C16:0 is a further important fatty acid (5–10 %) in Thermovum composti, while no hydroxy fatty acids were reported. Negative for gelatinase, urease, and indole production. Positive for nitrate reduction and for the utilization of d-arabinose, l-arabinose, d-ribose, d-xylose, d-galactose, d-glucose, cellobiose, lactose, melibiose, gentiobiose, d-fucose, and potassium 5-ketogluconate; negative for the utilization of glycerol, erythritol, l-xylose, d-adonitol, methyl β-d-xylopyranoside, d-fructose, d-mannose, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl α-d-mannopyranoside, methyl α-d-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, esculin ferric citrate, salicin, maltose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, turanose, d-lyxose, d-tagatose, l-fucose, dl-arabitol, potassium gluconate, and potassium 2-ketogluconate. The following enzyme activities were present (API ZYM): esterase C4, esterase C8, leucine arylamidase, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase, trypsin, and valine arylamidase; the following were absent: alkaline phosphatase, lipase C14, cystine arylamidase, acid phosphatase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, mannosidase, and α-fucosidase. The type strain JCM 17863^T was isolated from compost. The G+C content of its DNA is 63.4 mol %.

If appropriate, the description of metabolic pathways and/or physiology may deserve an individual heading.

Isolation, Enrichment, and Maintenance Procedures

The genera and species of the family have an aerobic respiratory metabolism and originate from a wide range of habitats. No single isolation or enrichment procedure is available to selectively obtain all or most members of the family, and therefore the genera are discussed separately below.

The only species of the genus Ahrensia, Ahrensia kielensis, was isolated from the Baltic Sea during studies of star-shaped-aggregate-forming bacteria and was originally named Agrobacterium kielense (Ahrens 1968). Because Dr. Renata Ahrens later withdrew the proposal, these species were not documented elsewhere in the following years, and no details on specific isolation conditions are available in recent literature (Rüger and Höfle 1992). With the recent sequencing of the genome of this organism, it may become possible in future to propose suitable isolation or enrichment strategies. The organism can be cultivated on regular marine media (e.g., Difco Marine Broth) at 26 °C and can be freeze-dried for long-term preservation.

Aliihoeflea was isolated from tidal flat sediment samples by plating on marine agar 2216 (Difco). Circular colonies—convex with entire margin, shiny, and cream colored—were 0.5–1.0 mm in diameter after 2 days incubation at 30 °C. Growth also occurs on trypticase soy agar (TSA, Difco), Luria agar (Difco), and yeast extract mannitol agar (YMA, per liter, 10 g d-mannitol, 0.5 g KH₂PO₄, 0.2 g MgSO₄ · 7H₂O, 0.1 g NaCl, 4 g CaCO₃, 0.4 g yeast extract, 15 g agar; pH 6.8–7.0) (Roh et al. 2008).

All Aminobacter species were isolated from the soil using various enrichment or trapping methods. Aminobacter anthyllidis was isolated from the nodules of Anthyllis vulneraria that was used as a trapping plant and was grown in soil from a zinc and lead mining site (Maynaud et al. 2012). The surface-sterilized nodules were crushed in sterile water, and the bacteria were isolated by streaking the suspension on YMA (Vincent 1970; recipe as listed above) and incubating at 28 °C.

Aminobacter aganoensis, Aminobacter aminovorans, and Aminobacter niigatensis were isolated from soil by enrichment using methylamine compounds (mono-, di-, tri-, or tetramethylamine, trimethylamine-N-oxide, or tetramethylammonium hydroxide) or methylformamide compounds (N-methylformamide or N,N-dimethylformamide) (Urakami 2005). For routine growth of PYG medium, pH 7.0 can be used at 30 °C (Urakami et al. 1992).

Aminobacter ciceronei and Aminobacter lissarensis are methylotrophic species. Aminobacter ciceronei was isolated from CH₃Br-fumigated soil in the USA by enrichment on a mineral salt medium under a modified atmosphere of air plus CH₃Br (Miller et al. 1997). Aminobacter lissarensis strain CC495 was isolated from the top 5 cm of soil in a beech wood in County Down, Northern Ireland, by enrichment with CH₃Cl as the sole carbon and energy source. One gram of soil was added to 100 ml of minimal medium in 500-ml flasks containing 0.125 g of CH₃Cl. The minimal medium had the following composition (in grams per liter): KH₂PO₄ (4.5), K₂HPO₄ (10.5), MgSO₄ · 7H₂O (0.15), and NH₄NO₃ (1.5), pH adjusted to 7.2 with 6 M NaOH; a trace element solution was added (10 ml. l⁻¹) containing (in mg.l⁻¹) H₃BO₃(500), CuSO₄ · 5H₂O (40), KI (100), FeSO₄ · 7H₂O (200), MnSO₄ · 7H₂O (400), (NH₄)6Mo₇O₂₄ · 4H₂O (200), and ZnSO₄ (400). In the pure cultures, the medium was additionally supplemented with a vitamin solution (5 ml.l⁻¹) containing (in milligrams per liter) folic acid (4), p-aminobenzoic acid (200), and cyanocobalamin (200). CH₃Cl (0.15 g) was added as an aqueous solution to give a concentration in the culture medium, after equilibration of the gaseous and aqueous phases, of 11.8 mM (30 mM if partitioning is neglected and the total CH₃Cl present is expressed as a concentration in the aqueous phase) (Coulter et al. 1999).

All Aminobacter species can be stored in broth medium plus 20 % glycerol at −80 °C or can be lyophilized and stored at 4 °C.

Two Aquamicrobium species were isolated from activated sludge, Aquamicrobium defluvii and Aquamicrobium lusatiense (Bambauer et al. 1998; Fritsche et al. 1999; Kämpfer et al. 2009). The former species originated from a municipal wastewater plant and was obtained on a mineral medium with thiophene-2-carboxylate as the sole source of carbon and nitrate as the electron acceptor. The mineral salt medium (Bambauer et al. 1998), also used for cultivation, contained per l 3.56 g Na₂HPO₄ · 2H₂O, 0.4 g NH₄Cl, and 0.07 g K₂SO₄. After autoclaving, 1 l of medium was supplemented with 2 ml of a sterile solution containing per l 100 g MgCl2, 25 g CaCl2, 10 ml vitamin solution (Balch et al. 1979), and 1 ml trace element solution (Widdel et al. 1983). Thiophene-2-carboxylate (2–30 mM final concentration) was added from a sterile, tenfold concentrated stock solution. For anaerobic growth, the medium was supplemented with 5–20 mM KNO₃ (Bambauer et al. 1998).

Three other species were isolated from air or waste gas in a duck shed and an animal rendering plant: Aquamicrobium aerolatum, Aquamicrobium ahrensii, and Aquamicrobium segne (Kämpfer et al. 2009; Lipski and Kämpfer 2012). The latter two species were isolated on Antibiotic Sulfonamide Sensitivity-test agar (Merck 1.05392) (Ahrens et al. 1997). Aquamicrobium aerolatum was isolated collecting bioaerosol samples by filtration over gelatin filters and isolation on trypticase soy agar incubated at 26 °C. The organism can also be grown on nutrient agar (Kämpfer et al. 2009). Aquamicrobium aestuarii was isolated from crude oil-contaminated sediments of a tidal flat (Jin et al. 2013) by incubating approximately 10 g of sediment with 100 ml of 0.2 μm filtered seawater containing 3 ml crude oil in 500-ml Erlenmeyer flask at 25 °C. The enrichment was aerated (180 rpm) and was transferred (1:20) four times every 2 weeks. For isolation, the enrichment was plated on marine agar 2216 (BD) plates and incubated under aerobic conditions at 25 °C for 5 days. In addition, the species grows well on R2A agar (BD), Luria-Bertani agar, trypticase soy agar, and marine agar (Jin et al. 2013).

Chelativorans strains were obtained from sludge samples. Chelativorans multitrophicus was isolated from a mixed microbial culture enriched in a column packed with activated carbon that was continuously fed with a mineral medium containing EDTA as sole source of carbon, nitrogen, and energy. The original inoculum of the column was activated sludge from various industrial wastewater treatment plants and soil extracts. For the isolation, further aerobic enrichment in continuous culture on a column packed with glass beads and fed with mineral medium (per liter, 1.0 g MgSO4 · 7H₂O, 0.2 g CaCl₂ · 2H₂O, 0.13 g KH₂PO₄, and 0.615 g Na₂HPO₄, 2 ml of Widdel trace element solution (Pfennig et al. 1981) and 1 ml of a vitamin solution (Egli et al. 1988)) containing 200–300 mg.l⁻¹ EDTA as well as batch cultures to establish optimal growth conditions were used (Weilenmann et al. 2004). The best conditions for growth were 30 °C, initial EDTA concentration in the range of 1–1.5 g.l⁻¹, CaCl₂ · 2H₂O concentration in the mineral medium of 0.4 g.l⁻¹ and an initial pH of 7.0. Pure cultures were obtained by successive plating on Plate Count Agar and liquid culture in the mineral medium (Weilenmann et al. 2004). Chelativorans multitrophicus was obtained by enrichment from municipal sludge samples: 10 g of sample was suspended in 100 ml of medium (per liter, 1.0 g EDTA, 1.0 g MgSO4 · 7H₂O, 0.4 g CaCl₂ · 2H₂O, 0.26 g KH₂PO₄, and 0.83 g Na₂HPO₄ · 12H₂O and trace elements and vitamins (Egli et al. 1988), pH 7.0). The medium was incubated in a 750-ml flask on a shaker (150–200 rpm) at 28 °C for 2 weeks. Five milliliters of this enrichment then inoculated into a 750-ml flask with 100 ml of fresh medium and cultivated for 2 weeks. After five such transfers, pure colonies were picked from plates of the same medium plus agar (Chistyakova et al. 2005).

Hoeflea species have been isolated from different aquatic environments. Hoeflea marina comprises one strain, LMG 128^T, that was originally classified as Agrobacterium ferrugineum (other strains of this species have been renamed as Pseudorhodobacter ferrugineus, a member of the Rhodobacteraceae). Hoeflea marina was isolated from water from the Baltic Sea, off the coast of Germany, during a study of star-forming bacteria (Ahrens 1968; Peix et al. 2005). Hoeflea phototrophica was isolated from cultures of the marine dinoflagellates Alexandrium lusitanicum and Prorocentrum lima. Wine red colonies were obtained by plating washed single dinoflagellate cells onto 1/10-strength Difco marine agar. Pigmentation was found to depend on the salt concentration with cultures with 3, 6, or 9 g.l⁻¹ sea salts being very pink, while at 35 g.l⁻¹ cultures were colorless (Biebl et al. 2006). Hoeflea alexandrii was purified from cultures of another marine dinoflagellate, Alexandrium minutum. In this case, the washed dinoflagellate cells were sonicated prior to plating on full- and half-strength Difco marine agar and incubation during 7 days at 15 °C. Brown-pigmented colonies were obtained. Marine agar or broth was used for routine cultivation at 30 °C (Palacios et al. 2006). Hoeflea anabaenae was isolated from a culture of the cyanobacterium Anabaena under heterotrophic conditions in the brackish marine purity liquid medium (per liter, 20 g NaCl, 17 g AC broth (Difco), 8 g MgSO₄ · 7H₂0, 1.5 g CaCl₂ · 2H₂0 (Stevenson and Waterbury 2006)) from a culture in which it was attached almost exclusively to Anabaena heterocysts. It is also able to grow aerobically at 30 °C in full- and half-strength marine broth (Difco) and marine agar and liquid or solid PY medium (20 g sea salts, 3 g peptone, and 0.5 g yeast extract per liter (Biebl et al. 2005)). Hoeflea suaedae was isolated from the root surface of the halophyte Suaeda maritima. Surface-sterilized and dried root pieces (1 g) were ground in 9 ml of autoclaved filtered seawater (AFS) with a sterile mortar and pestle. Dilution series were plated in triplicate on one-tenth-strength R2A (1/10 R2A) medium in filtered seawater and supplemented with 50 μg/ml cycloheximide. Plates were incubated at 28 °C for 2–3 weeks. Routine maintenance is on 1/10 R2A medium in filtered seawater, and the organism can be stored with 15 % glycerol at −70 °C (Bibi et al. 2012).

Hoeflea siderophila was isolated from fresh ochreous sediments collected near the outlet of an iron-rich brackish spring using dilution plating on the following medium (g per liter): NaCl, 20; NH₄Cl, 0.3; CaCl₂⋅6H₂O, 0.3; MgCl₂⋅7H₂O, 3; NaHCO₃, 0.5; 10 % phosphate buffer (pH 7.0), 0.1; Hepes buffer (pH 7.2), 3.0; KNO₃, 0.3; CH₃COONa, 0.15; vitamins and trace elements (Pfennig and Lippert 1966); Difco agar, 5.0; and pH 7.0. Before inoculation, the medium was supplemented with fresh sterile FeS suspension (Hanert 1981) (0.2 mL per 10 mL of medium). Inoculated media were incubated for 2–3 weeks at 28 °C. Growth consisted of dense spherical colonies, orange-colored due to the formation of iron oxides. In liquid medium, iron oxidation results in an ochreous precipitate (Sorokina et al. 2012).

Hoeflea hydrophila was isolated from marine sediments by serial dilution in filter-sterilized natural seawater containing 0.1 % yeast extract. After aerobic incubation at 25 °C for 2 weeks, a sample from the lowest dilution showing growth was plated on the same medium, and after incubation at 25 °C for 2 weeks, single colonies that were beige, circular, and convex with regular edges were purified on marine agar 2216 (Difco). This species can be routinely grown on marine broth or marine agar. Marine broth cultures plus 20 % glycerol can be stored at −80 °C (Jung et al. 2013).

Lentilitoribacter was isolated from coastal seawater by dilution plating on marine agar 2216 (Becton–Dickinson) at 25 °C. These conditions were also used for routine cultivation. For short-term preservation, marine agar cultures can be stored at 4 °C, while for long-term preservation, glycerol suspensions (20 %) can be stored at −80 °C (Park et al. 2013).

Mesorhizobium comprises soil bacteria that can live endosymbiotically in root nodules on various legume plants where they can fix atmospheric nitrogen contributing to plant nutrition. A widely used approach to isolate rhizobia is through the use of legume plants to trap the bacteria from a particular soil. Surface-sterilized seeds are allowed to germinate in the soil, and after the plants develop, nodules are harvested for isolation of the bacteria (Vincent 1970). A similar approach is also used to verify the nodulation capacity of a strain with a particular host species. Isolation from surface-sterilized and crushed nodules is performed using yeast mannitol agar (YMA, per liter, 10 g d-mannitol, 0.5 g KH₂PO₄, 0.2 g MgSO₄ · 7H₂O, 0.1 g NaCl, 4 g CaCO₃, 0.4 g yeast extract, 15 g agar, pH 6.8–7.0). Incubation at 28 °C will result in the formation of creamy, entire, and convex mucoid colonies after 3–4 days. This procedure can result in growth of not only Mesorhizobium strains but also other rhizobia. Phenotypic distinctions are not straightforward: Mesorhizobium members can in some cases be distinguished in that they have a moderately fast growth rate (generation time 4–15 h) compared to Rhizobium (<6 h) and Bradyrhizobium (>6 h). Also they produce acid on YMA (as do Rhizobium strains), while Bradyrhizobium strains produce alkali (Chen et al. 2005). More certain genus assignment, however, requires verification of the partial 16S rRNA gene sequence.

All Mesorhizobium species have been isolated using the trap legume method except for one species, Mesorhizobium thiogangeticum, which was obtained from soil adjacent to the root of the legume Clitoria ternatea through enrichment using reduced sulfur compounds as sole carbon and energy source. Soil was supplemented with Na₂S₂O₃ · 5H₂O (5 %), Na₂S (1 %), and elemental sulfur powder (5 %) and incubated at 30°C for 2 weeks with intermittent sprinkling of sterile water. After this enrichment, soil samples (1 %, w/v) were incubated on a rotary shaker at 30 °C in mineral salt thiosulfate yeast extract liquid medium (20 mM Na₂S₂O₃ · 5H₂O supplemented with 5 g yeast extract per liter, pH 7.0–7.5) in mineral salt solution that contained (per liter of distilled water) 1 g NH₄Cl, 4 g K₂HPO₄, 1.5 g KH₂PO₄, 0.5 g MgSO₄ · 7H₂O, and 5.0 ml trace metal solution (Vishniac and Santer 1957). When the color of the phenol red indicator had changed to yellow, serial dilutions were plated on the same agar medium for the isolation of pure cultures (Ghosh and Roy 2006).

Nitratireductor strains have been isolated from diverse marine sources, often by standard dilution plating techniques. Nitratireductor aquibiodomus was isolated from a marine aquarium denitrification system fitted with cellulose carriers. For the isolation, cellulose carriers were homogenized, and a dilution series was plated onto trypticase soy agar and R2A and incubated at room temperature for 3 weeks. Nitratireductor aquibiodomus was one of the several organisms that were picked up (Labbé et al. 2003); its colonies were white, smooth, circular, and convex (Labbé et al. 2004). Nitratireductor basaltis was isolated from black sand from Soesoggak beach, Jeju Island, Korea, by dilution plating onto marine agar 2216 (Difco) and incubating at 30–37 °C. It is not reported whether other organisms were able to grow in these conditions; the colonies of Nitratireductor basaltis were creamy, circular, convex, and smooth (Kim et al. 2009). Nitratireductor kimnyeongensis was isolated from a dried seaweed sample from Kimnyeong beach in Jeju, Republic of Korea (Kang et al. 2009), by transferring a piece of dried seaweed directly transferred onto isolation medium (WAT-SW agar) consisted of 0.05 % MgSO₄.7H₂O, 0.05 % CaCl₂.2H₂O, and 1.5 % agar in 60 % natural seawater and 40 % distilled water (pH 7.3) (Lee 2006). The organism can also be conveniently grown on trypticase soy agar where, after 5 days of incubation, colonies are small (0.5–1 mm in diameter), light yellow, circular, convex, smooth, and entire (Kang et al. 2009). Nitratireductor aquimarinus was isolated from exponential cultures of the marine diatom Skeletonema costatum by plating a 10-μl sample on marine agar 2216 (Difco) and incubating aerobically for 1 week. Colonies are creamy, smooth, circular, and convex. Growth is also good on trypticase soy agar at 35 °C. Strains can be preserved in trypticase soy broth supplemented with 30 % glycerol and −80 °C (Jang et al. 2011). Nitratireductor indicus was isolated from a deep-sea water sample taken at a depth of 2,488 m taken with Niskin bottles attached to a CTD (conductivity, temperature, and depth) sampler at 25.3217°S 70.0405°E in the southwestern part of the Indian Ridge. The seawater was enriched with 1 % sterilized crude oil, and after two months, bacteria were isolated by using the plating on 216L medium (per liter seawater: 1.0 g CH₃COONa, 10.0 g tryptone, 2.0 g yeast extract, 0.5 g sodium citrate, and 0.2 g NH₄NO₃; pH 7.5) (Lai et al. 2009; Lai et al. 2011b). On marine agar, colonies are unpigmented, smooth gray, and slightly raised in the center and have a regular margin (Lai et al. 2011a). Nitratireductor pacificus was also isolated from deep-sea water samples, this time from a pyrene-degrading enrichment (described in Wang et al. 2008) by using phthalate as sole carbon source in mineral medium (MM, comprising per liter: 3.5 g MgSO₄ · 7H₂O, 0.05 g CaCl₂, 24 g NaCl, 0.35 g KCl, 1.0 g NH₄NO₃, 1.0 KH₂PO₄, 1.0 g K₂HPO₄, 0.01 g FeCl₃, 0.0001 g ZnSO₄ · 7H₂O, 24 mg SrCl₂ · 6H₂O, and 0.08 g KBr, adjusted to pH 7.4) (Wang et al. 2008; Lai et al. 2011a). On marine agar, colonies are smooth gray, nonpigmented with a regular margin, and slightly raised in the center (Lai et al. 2011a).

Most recently, a new species, Nitratireductor lucknowense, was published, though not yet validated. It was isolated from pesticide-contaminated soil from a γHCH (lindane) manufacturing site in India. Five-gram soil samples collected from three different locations were mixed together in 50 ml of sterile mineral medium (Senoo and Wada 1989). After the slurry had settled, the liquid phase was used to enrich for bacteria on 0.34 M lindane. On trypticase soy agar, the new species produces colonies that are straw yellow, smooth, circular, glistening, opaque, and convex with an entire margin (Manickam et al. 2012). NaCl is tolerated up to 2 % (Manickam et al. 2012), considerably less than most other Nitratireductor species which can tolerate up to 7 or 8 % (Table 18.8 ).

Most Phyllobacterium species are plant associated, and while the first species were isolated from tropical ornamental plants, these bacteria have since also been isolated from other plants elsewhere and from non-plant sources such as volcanic rock used for construction. Different isolation procedures have been used and are summarized in the following overview. The selectivity in most cases is not documented.

Phyllobacterium myrsinacearum and its junior subjective synonym Phyllobacterium rubiacearum (Mergaert et al. 2002) have been isolated from leaf nodules of members of the plant families Rubiaceae (Pavetta zimmermanniana) and Myrsinaceae (Ardisia crispa, Ardisia crenata). Washed leaf pieces carrying nodules were macerated by rubbing and placed in saline. After shaking, dilutions were plated onto carrot juice agar containing yeast extract (fresh carrot juice, 500 ml; water, 500 ml; FeSO₄ · 7H₂O, 0.1 g; MnSO₄ · H₂O, 0.1 g; agar, 15 g; pH 7.2; the medium is sterilized by fractional sterilization). After incubation at 28 °C, typical nonpigmented to beige, slimy, and circular colonies that are translucent to opaque in the center are transferred into liquid carrot juice medium. After 24- to 48-h phase, contrast microscopy can be used to verify the formation of star-shaped clusters. Stock cultures can be kept on trypticase soy agar at 5 °C for 1–2 months, and cultures can be lyophilized for long-term preservation (Knösel 1984). Isolates of this species have also been obtained from the root surface of sugar beet by using trypticase soy broth agar as a nonselective medium (Lambert et al. 1990; Mergaert et al. 2002).

Phyllobacterium trifolii was isolated from the nodules of Trifolium pratense as described above for Mesorhizobium species. Colonies on YMA are white, mucoid, translucent, and convex. The growth rate of this Phyllobacterium species (generation time 2 h) is faster than most mesorhizobia. Growth is also possible on nutrient agar (Valverde et al. 2005).

A further five species were also obtained using the same procedure, this time from roots of Brassica napus cv. Eurol (Phyllobacterium bourgognense, Phyllobacterium brassicaearum), root nodules of Lathyrus numidicus and Astragalus algerianus (Phyllobacterium ifriqiyense), root nodules of Astragalus algerianus and Argyrolobium uniflorum (Phyllobacterium leguminum) (Mantelin et al. 2006b), and root nodules of Phaseolus vulgaris (Flores-Felix et al. 2013). Colonies are circular, white or cream colored with regular margins, and in most strains highly mucoid (Mantelin et al. 2006b).

Phyllobacterium catacumbae does not originate from a plant-associated source. It was isolated from tuff, volcanic rock used in the walls of the Roman catacombs of Saint Callixtus, Rome, Italy. The B-4 medium used for isolation contained (per liter) 2.5 g calcium acetate, 4 g yeast extract and 15 g agar, pH 8, and incubation was at 28 °C. Colonies are circular, smooth, and beige. Growth is also good on trypticase soy agar (Jurado et al. 2005).

Phyllobacterium species can be stored at −80 °C in broth medium plus 20 % glycerol or at 4 °C lyophilized.

Pseudahrensia was isolated from seawater from the Yellow Sea, Korea, by the standard dilution plating on marine agar 2216 (Difco) and incubating at 25 °C. Colonies are circular, convex, smooth, glistening, and cream colored. This organism can be routinely cultivated on marine agar at 30 °C (Jung et al. 2012).

Although no precise isolation media have been published, Pseudaminobacter strains have been isolated by exploiting specific degradation capacities. The Pseudaminobacter salicylatoxidans type strain was isolated as a degrader 6-aminonaphthalene-2-sulfonate from a microbial consortium degrading this substrate and originating from the river Elbe, Germany (Nortemann et al. 1986; Kämpfer et al. 1999). The type strain of Pseudaminobacter defluvii was isolated from activated sludge which was enriched with thiocyanate (Katayama-Fujimura et al. 1983; Kämpfer et al. 1999). Growth is possible on several media including nutrient agar, trypticase soy agar, trypticase soy broth plus 1.5 % agar, and R2A (Oxoid) (Kämpfer 2005).

Thermovum composti was isolated from mature compost produced by a field-scale composter used for the treatment of livestock excreta. Dilution series of 1 g of compost in saline solution were plated onto isolation medium composed of (per liter distilled water) 1 g yeast extract, 2 g tryptone, 1 g NaCl, 1 g MgSO₄ · 7H₂O, 20 g agar, 20 mg trimethoprim, 10 mg nalidixic acid, and 20 mg kanamycin, pH 7.0. After incubation at 50 °C for 7 days, colonies were picked and repeatedly transferred for purification. Colonies on nutrient agar are cream colored. Stock cultures in trypticase soy broth, grown for 2 days at 50 °C, can be supplemented with 20 % glycerol and stored at −80 °C (Yabe et al. 2012).

Ecology

Members of the Phyllobacteriaceae are versatile environmental bacteria that occur in diverse habitats that often are polluted or nutritionally rather rich. These habitats can be marine (Aliihoeflea, Ahrensia, Aquamicrobium, Hoeflea, Lentilitoribacter, Nitratireductor, Pseudahrensia) or polluted freshwater (Pseudaminobacter, Aquamicrobium, Chelativorans) systems, soil (Aminobacter, Chelativorans, Mesorhizobium, Nitratireductor, Phyllobacterium, Thermovum), or air (Aquamicrobium). Several genera are plant associated or associated with dinoflagellates or cyanobacteria (Hoeflea, Mesorhizobium, Phyllobacterium).

Aquamicrobium species have been isolated from diverse polluted environments (activated sludge, oil-polluted sediments, air/waste gas from poultry/animal rearing) and have degradative capabilities that may significantly contribute to the breakdown of pollutants: Aquamicrobium defluvii is able to degrade thiopene-2-carboxylate (Bambauer et al. 1998), Aquamicrobium lusatiense can utilize phenol and chlorophenols such as 4-chloro-2-methylphenol, 2,4-dichlorophenol, and 4-chlorophenol (Fritsche et al. 1999), and Aquamicrobium aestuarii was enriched from marine sediments using crude oil (Jin et al. 2013).

Chelativorans species have been found in municipal and industrial sludge samples and are able to degrade EDTA, a chelating agent with many applications that is generally recalcitrant to biodegradation. Chelativorans multitrophicus and Chelativorans oligotrophicus are able to use EDTA as sole carbon, nitrogen, and energy source, facultatively and obligately, respectively. They may have a significant role in the clearing of EDTA pollution in surface waters (Doronina et al. 2010).

Hoeflea phototrophica contains bacteriochlorophyll at reduced salt concentrations, but not at the concentration seawater (3.5 %); it also contains a carotenoid pigment thought to be spheroidenone and was found to contain pufL and pufM genes coding for proteins of the photosynthetic reaction center. However, Hoeflea phototrophica does not grow anaerobically in light or dark conditions, and conditions under which it may live phototrophically have not been described in detail (Biebl et al. 2006).

Hoeflea siderophila is able to grow mixotrophically and organoheterotrophically. It is the only species of the genus that is capable of fac. chemolithotrophic growth through iron oxidation at neutral pH, in anaerobic conditions with nitrate or N₂O as terminal electron acceptor, and microaerobically with oxygen. It is one of the rare species that is capable of neutrophilic lithotrophic iron oxidation (Sorokina et al. 2012).

Mesorhizobium strains are soil bacteria that can, in the vicinity of a compatible legume host species, enter into a molecular dialogue with the plant, resulting in the formation of root nodules on the plant that can be occupied by the bacteria. The bacteria receive a safe habitat and food, while they in turn can fix atmospheric nitrogen and thus contribute to plant nutrition. Most Mesorhizobium species (29 of 30) have been described as symbiotic with various legume species (Table 18.11 ). The only species that was not obtained from nodules, Mesorhizobium thiogangeticum obtained through enrichment from the soil adjacent to the legume Clitoria ternatea, was not able to nodulate this host nor Pisum sativum or Cicer arietinum (Ghosh and Roy 2006). Mesorhizobium has occasionally also been reported from marine systems (Sfanos et al. 2005; Krick et al. 2007) and from aquatic microbial mats in Antarctica (Peeters et al. 2012).

Table 18.11 Host plants nodulated by Mesorhizobium species

Several Phyllobacterium species have been isolated from leaf or root nodules of plants and contribute to plant growth promotion (Table 18.12 ). For Phyllobacterium myrsinacearum, there is no direct evidence that it actively induces nodule formation in leaves. Nodulation has been confirmed for Phyllobacterium trifolii (Valverde et al. 2005),

Table 18.12 Sources of isolation and nodulation capacity reported for the different Phyllobacterium species

Unspecified Phyllobacterium strains have also been reported from the rhizosphere of Lotus spp. (Oger et al. 2004), associated with roots in Brassica napus (Bertrand et al. 2001), as endophytes in Ipomea batatas (Khan and Doty 2009), and in root nodules of many legumes including Dalbergia louvelii (Rasolomampianina et al. 2005), Lathyrus gmelinii (Baymiev et al. 2011), Acacia sp. (Hoque et al. 2011), Sophora alopecuroides (Zhao et al. 2010), Vicia sp. (Lei et al. 2008), and Ononis tridentata (Rincon et al. 2008). They have also been found as free-living bacteria in water (Mergaert et al. 2001) and associated with unicellular organisms (Gonzalez-Bashan et al. 2000).

The possible symbiotic function of phyllobacteria is reported to be the production of plant growth hormones, protective antibacterial and antifungal activity (Lambert et al. 1990), phosphate solubilization (Chen et al. 2006), root hair elongation (Galland et al. 2012), and nitrogen fixation (Valverde et al. 2005).

Both Pseudaminobacter species have been isolated from polluted aquatic environments (Kämpfer et al. 1999). Pseudaminobacter salicylatoxidans can degrade substituted naphthalenesulfonates and substituted salicylates. One strain has also shown to be a facultative sulfur chemolithotroph that can oxidize S₂O₃²⁻, S₄O₆²⁻, SO₃²⁻, S₂²⁻, and S⁰ directly to SO₄²⁻ without any intermediate formation (Ghosh and Dam 2009). In polluted oligotrophic aquatic systems, these bacteria may play an important role in biodegradation.

Thermovum is the only genus of the family that is thermophilic (maximum growth temperature 60 °C). It was isolated from mature compost; its role in the compost ecosystem is not documented (Yabe et al. 2012).

Pathogenicity, Clinical Relevance

Mesorhizobium amorphae has been reported as an amoeba-associated bacterium that may be involved in nosocomial pneumonia through contaminated water supplies (La Scola et al. 2003; Berger et al. 2006). As no recent reports confirming these observations were found, the significance of Mesorhizobium amorphae as a nosocomial pathogen is not clear. No other animal or human pathogens are among the current members of the Phyllobacteriaceae.

Many species, particularly of Mesorhizobium and Phyllobacterium, are plant endophytes, rhizoplane or rhizosphere bacteria that have plant beneficial effects (see next section below).

No plant pathogenic effects have been reported for most members of the family Phyllobacteriaceae. Candidatus Liberibacter is, however, a serious plant pathogen. It is included here, although the membership of this group is currently uncertain (see above under Sect. 1.2, “Phylogenetic Structure of the Family and Its Genera”). The trivial name “liberobacter” (sic) (from the Latin liber [bark] and bacter [bacteria]) was proposed in 1994 for a phloem-limited bacterium-like organism associated with citrus greening disease, also known as huanglongbing disease or yellow dragon disease, a severe and widespread citrus disease that is transmitted by the Asian citrus psyllid (Diaphorina citri) and the African citrus psyllid (Trioza erytreae) (Jagoueix et al. 1994). The disease causes yellowing and blotchy mottling of the leaves, production of bitter, small and misshapen fruits, and ultimately death of the tree (http://www.aphis.usda.gov/plant_health/plant_pest_info/). Two species, Candidatus Liberibacter asiaticus (originally Liberobacter asiaticum) and Candidatus Liberibacter africanus (originally Liberobacter africanum), were proposed for the Indian and South African liberibacters, respectively, which can be distinguished based on temperature sensitivity (in Africa symptoms occur only in cooler regions), serology, and genomic properties (Jagoueix et al. 1994). Garnier et al. (2000) corrected the spelling of the genus name and proposed a separate subspecies, Candidatus Liberibacter africanus subsp. capensis, for a South African liberibacter in the ornamental rutaceous tree, Calodendrum capense. The citrus disease was later also reported from Brazil, and the pathogen recognized as a new species, Candidatus Liberibacter americanus, spread by the vector Diaphorina citri (Texeira et al. 2005). Although the species epithet originally referred to the geographic occurrence of the group, Candidatus Liberibacter asiaticus has also been found in the Americas (Raddadi et al. 2011). In the USA, the Asian citrus psyllid, Diaphorina citri, has been present in Florida since 1998, and Candidatus Liberibacter asiaticus was found in Florida in early September 2005. In 2010, the USDA imposed a plant quarantine in several states and territories in the USA to stop the spread of citrus greening (http://www.aphis.usda.gov/plant_health/plant_pest_info/). The disease was reported in South California in 2012 (www.californiacitrusthreat.com). The European and Mediterranean Plant Protection Organization (EPPO) has placed Liberibacter africanus, Liberibacter asiaticus, and Liberibacter americanus and the vector Diaphorina citri on its A1 list of pests recommended for regulation as quarantine pests. This list comprises pests regarded as absent from the EPPO region. The vector Trioza erytreae was included on the A2 List of pests recommended for regulation as quarantine pests that are locally present in the EPPO region.

Citrus huanglongbing disease is regarded as a pest of urgent phytosanitary concern for southern parts of the EPPO region where citrus is grown. Several PCR and real-time PCR tests have been developed for the detection of these Candidatus Liberibacter sp. in plants and in the psyllid vectors (Morgan et al. 2012 and references therein). To eliminate or suppress Candidatus Liberibacter asiaticus, a combination of penicillin and streptomycin administered by trunk injection or root soaking was shown to be effective in citrus plants (Zhang et al. 2011).

Several additional species have more recently been reported to affect Solanaceae plants. Candidatus Liberibacter psyllaurous is associated with psyllid yellows disease of potato (Solanum tuberosum L.) and tomato (Solanum lycopersicum L.) in North America and is transmitted by Bactericera cockerelli (Hansen et al. 2008). Candidatus Liberibacter solanacearum was reported from tomato, capsicum (Capsicum annuum), potato, tamarillo (Solanum betaceum), cape gooseberry (Physalis peruviana), and chilli (Capsicum sp.) from New Zealand; it is transmitted by the psyllid Bactericera cockerelli. It has also been detected in the USA in potatoes affected by zebra chip disease (Liefting et al. 2009). Candidatus Liberibacter solanacearum and its vector Bactericera cockerelli have been placed on the EPPO A1 and A2 lists, respectively, and are considered of particular concern for southern and central parts of the EPPO region and for areas with mild winters in the northern part (http://www.eppo.int/QUARANTINE/quarantine.htm).

Candidatus Liberibacter europaeus, the most recently described species, is the only one presently considered to be an endophyte rather than a plant pathogen. It was reported from Italy and is found in the midgut lumen, salivary glands, and Malpighian tubules of the pear psyllid pest Cacopsylla pyri and has been detected in pear plant tissue, both in laboratory-inoculated plants and in field-collected samples. However, the plants remained free of disease symptoms (Raddadi et al. 2011).

As the family Phyllobacteriaceae comprises many soil bacteria and other free-living bacteria, it is not surprising that many species show diverse resistance patterns to a number of antibiotics which they can be expected to encounter in their natural habitat. These characters can often be used for phenotypic differentiation and have therefore been included above in the Sect 3, “Phenotypic Analyses”.

Application

Most strains of the genus Mesorhizobium are capable of nitrogen-fixing symbiosis in root nodules of legume plants, allowing the host plant to grow in soils with lower nitrogen content than other plants. Several agricultural crops, including chickpea (Cicer arietinum), alfalfa or lucerne (Medicago sativa), and several Lotus species (Table 18.11 ), are nodulated by mesorhizobia, and these bacteria thus make an important contribution to the success of these crops. Interest to introduce new Lotus forage species in Australia has stimulated research into compatible Mesorhizobium strains (Howieson et al. 2011). Mesorhizobium strains are also important as symbionts of legume trees such as Acacia and Prosopis spp. used in tropical agroforestry systems (de Lajudie et al. 1998; Bala et al. 2003; Degefu et al. 2013).

In addition to symbiotic nitrogen fixation, several strains of Mesorhizobium and Phyllobacterium are recognized for other plant growth-promoting effects. Mesorhizobia can exert these effects by producing the plant hormone indole acetic acid or ammonia (Ahmad et al. 2008). Mesorhizobium mediterraneum PECA21 was shown to solubilize phosphorus from tricalcium phosphate and improve growth and phosphorus content in chickpea (Peix et al. 2001). Mesorhizobium loti MP6 was shown to increase yield and resistance to white rot (Sclerotinia sclerotiorum) in Brassica campestris in India (Chandra et al. 2007). Other strains are resistant to heavy metals (Maynaud et al. 2013). Phyllobacterium brassicacearum STM 196 was shown to promote the growth of oilseed rape and Arabidopsis and to stimulate root hair elongation possibly through activation of the ethylene signalling pathway (Mantelin et al. 2006a; Galland et al. 2012).

A strategy for the regeneration of disused mining site is through revegetation to allow sustainable plant cover to stabilize the site and limit wider environmental impact. In France, Anthyllis vulneraria is one of the legume species tolerant of these contaminated sites (Mahieu et al. 2011). It is nodulated by two members of the Phyllobacteriaceae: Aminobacter anthyllidis and Mesorhizobium metallidurans. Both species are tolerant to high concentrations of heavy metals and contribute through this symbiosis to the success of the legume species in the process of revegetation of contaminated sites (Vidal et al. 2009; Maynaud et al. 2012). The combined inoculation of legumes with plant growth-promoting rhizobacteria and Mesorhizobium loti strains that have the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase which hydrolyzes the precursor to the plant hormone ethylene was shown to have a synergistic positive effect on plant growth, nutrition, and adaptation to metal-polluted soils. The ACC-hydrolyzing bacteria eliminate the plant growth inhibition caused by increased ethylene production and can thus be valuable participants in the phytostabilization of contaminated mining sites (Safronova et al. 2012). In an application of the same mechanism, a strain of Mesorhizobium ciceri transformed to express exogenous ACC deaminase was shown to improve yields of chickpea and reduce susceptibility to root rot disease (Nascimento et al. 2012). One highly chromium-tolerant Mesorhizobium strain was shown to improve the yield and decrease chromium uptake in chickpea (Wani et al. 2008).

Aminobacter strains were reported to be responsible for the degradation of 2,6-dichlorobenzamide, a frequent groundwater pollutant that is a degradation product of the herbicide 2,6-dichlorobenzonitrile (dichlobenil). A specific PCR test targeting the 16S rRNA genes was designed to monitor the distribution of Aminobacter strains (Sjoholm et al. 2010). Aminobacter aminovorans strains previously classified as Chelatobacter heintzii are able to degrade nitrilotriacetate and EDTA (Auling et al. 1993; Nortemann 1999). Chelativorans species are also able to degrade EDTA and particularly Chelativorans oligotrophicus has been applied in biofilters to remove EDTA and EDTA-metal complexes (Kuvichkina et al. 2012; Kaparullina et al. 2012).

Pseudaminobacter strains have been investigated for use in bioremediation of soil contaminated with atrazine and one strain was reported to use as sole carbon and nitrogen source (Topp 2001). They have also been implicated in the degradation of methyl parathion (Zhang et al. 2005).

The salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans BN12T has been characterized extensively (Hintner et al. 2004; Matera et al. 2008; Ferraroni et al. 2012). A soil isolate very similar to Pseudaminobacter salicylatoxidans was shown to be a sulfur chemolithotroph (Bagchi et al. 2005; Mandal et al. 2007).