M. Gillis

Acetobacter diazotrophicus sp. nov., a Nitrogen-Fixing Acetic Acid Bacterium Associated with Sugarcane

Results of deoxyribonucleic acid (DNA)-ribosomal ribonucleic acid and DNA-DNA hybridizations, together with a phenotypic and chemotaxonomic analysis, revealed that nitrogen-fixing bacteria isolated from roots and stems of sugarcane belong to a new species in the genus Acetobacter, for which the name Acetobacter diazotrophicus sp. nov. is proposed. Strain LMG 7603 (= Dobereiner PAI 5 = ATCC 49037) is the type strain.

Azoarcus gen. nov., Nitrogen-Fixing Proteobacteria Associated with Roots of Kallar Grass (Leptochloa fusca (L.) Kunth), and Description of Two Species, Azoarcus indigens sp. nov. and Azoarcus communis sp. nov.

Among the nitrogen-fixing bacteria associated with roots of Leptochloa fusca (L.) Kunth in saline-sodic soils in the Punjab of Pakistan, we repeatedly found yellow-pigmented, straight to curved, gram-negative rods. To group and identify these organisms, we examined morphological, nutritional, and biochemical features and performed polyacrylamide gel electrophoretic analyses of cellular proteins, gas chromatographic analyses of fatty acids, DNA-rRNA hybridizations, and DNA-DNA hybridizations. Our results showed that 11 isolates formed five groups distinct at the species level, with each group containing one to three members. These bacteria constituted a separate rRNA branch in rRNA superfamily III (corresponding to the beta subclass of the Proteobacteria) at a branching Tm(e)nlevel of 67.7°C [Tm(e)nis the temperature at which 50% of a hybrid is denatured under standard conditions]. On this branch, the five groups were located in two clusters with Tm(e)nvalues of 79.4 to 80.4°C and around 71.5°C. We propose a new genus, the genus Azoarcus, for these strains. Azoarcus indigens is the type species and has a growth factor requirement; its type strain is strain VB32 (= LMG 9092). A second named species, Azoarcus communis, includes a strain obtained from French refinery oily sludge, strain LMG 5514. Bacteria of this genus have a strictly aerobic type of metabolism, fix nitrogen microaerobically, and grow well on salts of organic acids but not on carbohydrates. Swedish isolates obtained from human sources (E. Falsen group 15 strains LMG 6115 and LMG 6116), as well as “[Pseudomonas] gasotropha” LMG 7583T, were also located on this rRNA branch at a lower Tm(e)nlevel (70.4 to 71.2°C).

A New Species of Devosia That Forms a Unique Nitrogen-Fixing Root-Nodule Symbiosis with the Aquatic Legume Neptunia natans (L.f.) Druce

ABSTRACT Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or Rhizobium. The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.

Characterization of tropical tree rhizobia and description of Mesorhizobium plurifarium sp. nov.

A collection of strains isolated from root nodules of Acacia species in Senegal was analysed previously by electrophoresis of total cell protein, auxanographic tests, rRNA-DNA hydridization, 16S rRNA gene sequencing, DNA base composition and DNA-DNA hybridization [de Lajudie, P., Willems, A., Pot, B. & 7 other authors (1994). Int J Syst Bacteriol 44, 715-733]. Strains from Acacia were shown to belong to two groups, Sinorhizobium terangae, and a so-called gel electrophoretic cluster U, which also included some reference strains from Brazil. Further taxonomic characterization of this group using the same techniques plus repetitive extragenic palindromic-PCR and nodulation tests is presented in this paper. Reference strains from Sudan and a number of new rhizobia isolated from nodules of Acacia senegal, Acacia tortilis subsp. raddiana and Prosopis juliflora in Senegal were included. As a result of this polyphasic approach, the creation of a new species, Mesorhizobium plurifarium, is proposed for a genotypically and phenotypically distinct group corresponding to the former cluster U and containing strains isolated from Acacia, Leucaena, Prosopis and Chamaecrista in West Africa (Senegal), East Africa (Sudan) and South America (Brazil). The type strain of Mesorhizobium plurifarium ORS 1032 has been deposited in the LMG collection as LMG 11892.

Transfer of several phytopathogenic Pseudomonas species to Acidovorax as Acidovorax avenae subsp. avenae subsp. nov., comb. nov., Acidovorax avenae subsp. citrulli, Acidovorax avenae subsp. cattleyae, and Acidovorax konjaci.

DNA-rRNA hybridizations, DNA-DNA hybridizations, polyacrylamide gel electrophoresis of whole-cell proteins, and a numerical analysis of carbon assimilation tests were carried out to determine the relationships among the phylogenetically misnamed phytopathogenic taxa Pseudomonas avenae, Pseudomonas rubrilineans, Pseudomonas setariae, Pseudomonas cattleyae, Pseudomonas pseudoalcaligenes subsp. citrulli, and Pseudomonas pseudoalcaligenes subsp. konjaci. These organisms are all members of the family Comamonadaceae, within which they constitute a separate rRNA branch. Only P. pseudoalcaligenes subsp. konjaci is situated on the lower part of this rRNA branch; all of the other taxa cluster very closely around the type strain of P. avenae. When they are compared phenotypically, all of the members of this rRNA branch can be differentiated from each other, and they are, as a group, most closely related to the genus Acidovorax. DNA-DNA hybridization experiments showed that these organisms constitute two genotypic groups. We propose that the generically misnamed phytopathogenic Pseudomonas species should be transferred to the genus Acidovorax as Acidovorax avenae and Acidovorax konjaci. Within Acidovorax avenae we distinguished the following three subspecies: Acidovorax avenae subsp. avenae, Acidovorax avenae subsp. cattleyae, and Acidovorax avenae subsp. citrulli. Emended descriptions of the new taxa are presented.

Hydrogenophaga, a new genus of hydrogen-oxidizing bacteria that includes Hydrogenophaga flava comb. nov. (formerly Pseudomonas flava), Hydrogenophaga palleronii (formerly Pseudomonas palleronii), Hydrogenophaga pseudoflava (formerly Pseudomonas pseudoflava and Pseudomonas carboxydoflava), and Hydrogenophaga taeniospiralis (formerly Pseudomonas taeniospiralis)

The relationships of the yellow-pigmented hydrogen-oxidizing species Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas palleronii, Pseudomonas taeniospiralis, and “Pseudomonas carboxydoflava,” which are all members of the acidovorans ribosomal ribonucleic acid (rRNA) complex in rRNA superfamily III, were studied by using deoxyribonucleic acid (DNA):rRNA hybridization, immunotyping, numerical analysis of biochemical and auxanographic features, polyacrylamide gel electrophoresis of cellular proteins, numerical analysis of fatty acid patterns, and DNA:DNA hybridization. Our results show that these five yellow-pigmented hydrogen-oxidizing Pseudomonas species are more closely related to each other than to other taxa belonging to the acidovorans rRNA complex. We propose the transfer of these species to a new genus, Hydrogenophaga, with the following four species: Hydrogenophaga flava (formerly Pseudomonas flava), Hydrogenophaga pseudoflava (to accommodate both Pseudomonas pseudoflava and “Pseudomonas carboxydoflava”), Hydrogenophaga taeniospiralis (formerly Pseudomonas taeniospiralis), and Hydrogenophaga palleronii (formerly Pseudomonas palleronii). The type species is H. flava, with monotype strain DSM 619 (= LMG 2185 = CCUG 1658). Because H. flava grows slowly and unreliably, but is genotypically and protein electrophoretically very similar to H. pseudoflava, the latter species can be used as an alternative reference taxon for the new genus. The type strains of H. pseudoflava, H. taeniospiralis, and H. palleronii are strains GA3 (= LMG 5945 = CCUG 13799). DSM 2082 (= LMG 7170 = CCUG 15921), and Stanier 362t1 (= LMG 2366t1 = CCUG 20334), respectively.

Intra- and Intergeneric Similarities of the Ribosomal Ribonucleic Acid Cistrons of Acetobacter and Gluconobacter

Hybrids between 14C-labeled ribosomal ribonucleic acid (rRNA) from either Gluconobacter oxydans subsp. oxydans NCIB 9013, Acetobacter aceti subsp. aceti NCIB 8621t1, or Zymomonas mobilis subsp. mobilis ATCC 29191 and deoxyribonucleic acid (DNA) from acetic acid bacteria and representative strains of possibly related and other gram-negative bacteria were prepared. Each hybrid was described by two parameters: T m(e) , the temperature at which 50% of the hybrid was denatured, and the percentage of rRNA. binding, the amount of 14C-labeled rRNA (in micrograms) duplexed under stringent conditions per 100 μg of filter-fixed homologous or heterologous DNA. Each taxon occupied a definite area on the rRNA similarity maps. Parameters of hybrids formed with rRNA from G. oxydans subsp. oxydans NCIB 9013 showed that the acetic acid bacteria consist of two separate but closely related groups corresponding to the genera Acetobacter and Gluconobacter. When compared with rRNA from A. aceti subsp. aceti NCIB 8621t1, both genera were indistinguishable, showing that there were many strains of. Acetobacter whose rRNA cistrons are as different from the reference Acetobacter rRNA as from Gluconobacter rRNA. The rRNA cistrons of Acetobacter were more heterogeneous than these of Gluconobacter. The great similarities among the T m(e) s of the heterologous hybrids and among the numerous phenotypic features stress that both genera are more closely related to each other than to any other genus. The parameters of the DNA:rRNA hybrids located the acetic acid bacteria as a separate branch in an rRNA superfamily consisting of Rhodopseudomonas, Beijerinckia, Agrobacterium, Rhizobium, some Spirillum species, and Paracoccus denitrificans. Parameters of hybrids formed with rRNA from Z. mobilis subsp. mobilis ATCC 29191 showed that the genus Zymomonas forms a separate branch in the same rRNA superfamily. We detected a number of misnamed organisms. “Acetobacter” aceti subsp. xylinum NCIB 4112, “Acetobacter” aceti subsp. orleanensis NCIB 6426, and “Acetobacter” lermae NRRL B-1810 belong in the genus Gluconobacter. “Gluconobacter” industrius IFO 3261, “Gluconobacter” dioxyacetonicus IAM 1814, “Gluconobacter” sp. strains A4.1 and M28, “Pseudomonas” melophthora NCPPB 461 and 462, and “Pseudomonas” pomi NCPPB 463 are all regular members of Acetobacter. Our evidence is against the maintenance of “intermediate” strains of acetic acid bacteria. “Gluconobacter” liquefaciens NCIB 9505 and IAM 1834 and “G.” melanogenus IAM 1835 and IAM 1836 are genetically regular members of the genus Acetobacter. “Acetobacter” aurantius IFO 3246 is a Gluconobacter. “A.” aurantius IFO 3249, 3247, 13330, and 13333 are not acetic acid bacteria at all. We propose to unite Acetobacter and Gluconobacter in the family Acetobacteraceae. The ranges of the moles percent guanine plus cytosine of the DNAs have been determined for the different taxa in both genera.

NOTES: Comamonadaceae, a New Family Encompassing the Acidovorans rRNA Complex, Including Variovorax paradoxus gen. nov., comb. nov., for Alcaligenes paradoxus (Davis 1969)

A new family, the Comamonadaceae, is proposed for the organisms belonging to the acidovorans rRNA complex in the beta subclass of the Proteobacteria. This family includes the genera Comamonas, Acidovorax, Hydrogenophaga, Xylophilus, and Variovorax (formerly Alcaligenes paradoxus), as well as a number of phylogenetically misnamed Aquaspirillum and phytopathogenic Pseudomonas species.

Acidovorax, a new genus for Pseudomonas facilis, Pseudomonas delafieldii, E. Falsen (EF) group 13, EF group 16, and several clinical isolates, with the species Acidovorax facilis comb. nov., Acidovorax delafieldii comb. nov., and Acidovorax temperans sp. nov.

Pseudomonas facilis and Pseudomonas delafieldii are inappropriately assigned to the genus Pseudomonas. They belong to the acidovorans rRNA complex in rRNA superfamily III (i.e., the beta subclass of the Proteobacteria). The taxonomic relationships of both of these species, two groups of clinical isolates (E. Falsen [EF] group 13 and EF group 16), and several unidentified or presently misnamed strains were examined by using DNA:rRNA hybridization, numerical analyses of biochemical and auxanographic features and of fatty acid patterns, polyacrylamide gel electrophoresis of cellular proteins, and DNA:DNA hybridization. These organisms form a separate group within the acidovorans rRNA complex, and we propose to transfer them to a new genus, Acidovorax. We describe the following three species in this genus: the type species, Acidovorax facilis (formerly Pseudomonas facilis), with type strain LMG 2193 (= CCUG 2113 = ATCC 11228); Acidovorax delafieldii (for the former Pseudomonas delafieldii and most of the EF group 13 strains), with type strain LMG 5943 (= CCUG 1779 = ATCC 17505); and Acidovorax temperans (for several former Pseudomonas and Alcaligenes strains and most of the EF group 16 strains), with type strain CCUG 11779 (= LMG 7169).

Reclassification of the Causal Agents of Bacterial Blight (Xanthomonas campestris pv. oryzae) and Bacterial Leaf Streak (Xanthomonas campestris pv. oryzicola) of Rice as Pathovars of Xanthomonas oryzae (ex Ishiyama 1922) sp. nov., nom. rev.

On the basis of phenotypic, genotypic, and chemotaxonomic data, Xanthomonas oryzae (ex Ishiyama 1922) sp. nov., nom. rev. is proposed; this species comprises Xanthomonas oryzae pv. oryzae (Ishiyama 1922) comb. nov. and Xanthomonas oryzae pv. oryzicola (Fang, Ren, Chen, Chu, Faan, and Wu 1957) comb. nov., the causal agents of bacterial blight and bacterial leaf streak of rice, respectively.