J. De Ley

Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov.

Hybridization experiments were carried out between DNAs from more than 70 strains of Campylobacter spp. and related taxa and either 3H-labeled 23S rRNAs from reference strains belonging to Campylobacter fetus, Campylobacter concisus, Campylobacter sputorum, Campylobacter coli, and Campylobacter nitrofigilis, an unnamed Campylobacter sp. strain, and a Wolinella succinogenes strain or 3H- or 14C-labeled 23S rRNAs from 13 gram-negative reference strains. An immunotyping analysis of 130 antigens versus 34 antisera of campylobacters and related taxa was also performed. We found that all of the named campylobacters and related taxa belong to the same phylogenetic group, which we name rRNA superfamily VI and which is far removed from the gram-negative bacteria allocated to the five rRNA superfamilies sensu De Ley. There is a high degree of heterogeneity within this rRNA superfamily. Organisms belonging to rRNA superfamily VI should be reclassified in several genera. We propose that the emended genus Campylobacter should be limited to Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis. Wolinella curva and Wolinella recta are transferred to the genus Campylobacter as Campylobacter curvus comb. nov. and Campylobacter rectus comb. nov., respectively. Bacteroides gracilis and Bacteroides ureolyticus are generically misnamed and are closely related to the genus Campylobacter. Campylobacter nitrofigilis, Campylobacter cryaerophila, and an unnamed Campylobacter sp. strain constitute a new genus, for which the name Arcobacter is proposed; this genus contains two species, Arcobacter nitrofigilis comb. nov. (type species) and Arcobacter cryaerophilus comb. nov. Wolinella succinogenes so far is the only species of the genus Wolinella. The genus Helicobacter is also emended; Campylobacter cinaedi and Campylobacter fennelliae are included in this genus as Helicobacter cinaedi comb. nov. and Helicobacter fennelliae comb. nov., respectively. The genus Flexispira, with Flexispira rappini as the only species, is closely related to the genus Helicobacter. The free-living, sulfur-reducing campylobacters do not belong to any of these genera; they probably constitute a distinct genus within rRNA superfamily VI.

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).

Proposal for a New Family, Campylobacteraceae

The genera Campylobacter, Arcobacter, Helicobacter, Wolinella, and “Flexispira” constitute, within the class Proteobacteria, a separate eubacterial lineage identified as rRNA superfamily VI. A considerable number of common genotypic and phenotypic features differentiate the genera Campylobacter and Arcobacter from the other members of this group. Therefore, we propose that the genera Campylobacter and Arcobacter should be included in a separate family, for which the name Campylobacteraceae is proposed.

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).

Genotypic relationships and taxonomic localization of unclassified Pseudomonas and Pseudomonas-like strains by deoxyribonucleic acid: ribosomal ribonucleic acid hybridizations

The deoxyribonucleic acid (DNA):ribosomal ribonucleic acid (rRNA) hybridization technique was used to reveal the relationships and taxonomic positions of an additional 83 strains belonging to 43 saprophytic or pathogenic Pseudomonas species and 29 named and unnamed Pseudomonas-like strains. The DNA:rRNA hybrids were characterized by the following two parameters: (i) the temperature at which one-half of the hybrid was eluted and (ii) the percentage of rRNA binding (the amount of rRNA bound per 100 g of filter-fixed DNA). We also used, for a limited number of strains, numerical analysis of carbon assimilation tests to delineate the finer taxonomic relationships of organisms. Of the 83 strains examined, 78 could be definitely assigned either to an rRNA branch or to an rRNA superfamily within the Proteobacteria. Only 25 of our strains belong in the genus Pseudomonas sensu stricto (our Pseudomonas fluorescens rRNA branch). In general, about two-thirds of the named Pseudomonas species have been misclassified and are distributed over at least seven genera all through the Proteobacteria. These organisms need to be reclassified and generically renamed according to their phylogenetic relationships. However, more detailed phenotypic and genotypic studies are necessary before definite nomenclatural proposals can be made. A comprehensive list of the phylogenetic affiliations of the Pseudomonas species is included.

Inter- and intrafamilial similarities of rRNA cistrons of the Pasteurellaceae.

We performed hybridizations between labeled rRNAs from seven representative members of the family Pasteurellaceae and from three other taxa on the one hand and DNAs from 53 strains known or presumed to belong to the Pasteurellaceae on the other hand. The members of the Pasteurellaceae are most closely related to members of the Enterobacteriaceae, the Vibrionaceae, the Aeromonadaceae, and the genus Alteromonas. The family Pasteurellaceae is very heterogeneous. There are at least seven rRNA branches. Several organisms with the same genus name are dispersed over the entire dendrogram. The Histophilus ovis, [Haemophilus] ducreyi, [Actinobacillus] actinomycetemcomitans, and [Haemophilus] aphrophilus rRNA branches are separate and quite remote from the three authentic genera in this family; this might justify eventual later separate generic status. DNA-rRNA hybridization with suitable, labeled rRNA probes is an excellent method to establish whether an organism belongs in the Pasteurellaceae; e.g., some strains of Bisgaards taxa 7, 13, and 16 and of the gas-producing SP group certainly belong in this family, whereas three bovine lymphangitis organisms (strains NCTC 10547, NCTC 10549, and NCTC 10553), [Haemophilus] piscium ATCC 10801T (T = type strain), and [Pasteurella] piscicida ATCC 17911 belong in the Enterobacteriaceae, the Aeromonadaceae, and the Vibrionaceae, respectively.

Effect of various external factors on the fermentative production of hydrogen gas from glucose by Clostridium butyricum strains in batch culture

Summary The fermentation of glucose by Clostridium butyricum strains NCTC 7423, IFO 3315t1, 3847, 3858 and IAM 19001 was investigated in batch cultures at the defined pH values of 5.5, 6, 7, 7.5 or 8. Acetate, butyrate, lactate, formate, CO 2 and H 2 formations were determined quantitatively. The pH range for growth and fermentation is strain-linked and varies between 5.5–6 (for strain IFO 3847) and 5.5–8 (for strain NCTC 7423). The higher the pH of the fermentation medium, the more the H2 production differs from the one, theoretically expected according to the acetate and butyrate production. For at least one strain (NCTC 7423) formate then serves as an additional electron sink. With increasing incubation pH the acetate/butyrate ratio increases above 1 for strains NCTC 7423, IFO 3315t1, 3858 and IAM 19001. No change in the fermentation pattern occurred with increasing acetate or butyrate concentrations up to 200 mM, except for the maximum velocity of gas production, which slowed down gradually with added butyrate. Between 400 and 500 mM acetate or butyrate added the glucose consumption was completely or almost completely inhibited.

Hydrogen gas production from continuous fermentation of glucose in a minimal medium with Clostridium butyricum LMG 1213t1

Summary We investigated the formation of hydrogen gas by Clostridium butyricum strain LMG 1213t1 in a chemostat in glucose limitation. At pH 5.1 the fermentation did not reach a steady-state. Within the pH range 5.5 to 7.1 in the culture vessel, and at constant dilution rate D no marked change in product spectrum was found. The produced gas consisted of approximately 60% H 2 and 40% CO 2 . About 1.75 mol H 2 was produced per mol glucose consumed. Hydrogen gas production rate increased proportionally with dilution rate D. At the highest dilution rate investigated 21.7 mmol H 2 was produced per hour and per liter reactor. Slightly higher efficiencies of hydrogen gas formation were obtained with acetate or butyrate (sodium salts) in the inflow medium. Acetate and butyrate were produced in a ratio of approximately 1.8 to 3 in all conditions tested. With increasing dilution rate D and at constant pH, slightly less fatty acids and slightly more cell material were formed. Y glucose max and Y ATP max values were 28.56 and 12.30 g dry weight/mol glucose or ATP, respectively. No change in fermentation pattern was observed with increasing concentrations of acetate or butyrate (up to 102.7 mM and 77.2 mM respectively) in the inflow medium.

Numerical Analysis of Phenotypic Features and Protein Gel Electrophoregrams of a Wide Variety of Acetobacter strains. Proposal for the Improvement of the Taxonomy of the Genus Acetobacter Beijerinck 1898, 215

Ninety-eight strains, representing all Acetobacter species and subspecies from the Approved Lists of Bacterial Names (Skerman et al., 1980), were examined in a numerical analysis of 177 phenotypic features and compared to ninety-eight Gluconobacter and seven Frateuria strains. Four phenons could be delineated, corresponding to Frateuria (phenon 1), A. aceti subsp. liquefaciens (phenon 2), Gluconobacter (phenon 3) and Acetobacter minus A. aceti subsp. liquefaciens (phenon 4). Acetobacter, Frateuria and Gluconobacter are well- could be distinguished. Comparison of the protein electrophoregrams of Acetobacter strains revealed a fairly high internal homogeneity within phenon 2, subphenons C and D. Strains of the subphenon E gave very divergent protein patterns. The following classificatory changes are proposed within the genus Acetobacter: (1) Acetobacter liquefaciens sp. nov. is proposed for the homogeneous phenon 2, containing all 12 A. aceti subsp. liquefaciens strains (% G + C range of 62.3 to 64.6; IAM 1834 as type strain); (2) for the homogeneous subphenon D containing 8 A. aceti subsp. aceti strains, the name Acetobacter aceti emend, should be retained (% G + C range of 55.9 to 59.5; NCIB 8621 as type strain); (3) for subphenon E, a heterogeneous group, containing a variety of Acetobacter subspecies (all with their type strain) the species name Acetobacter pasteurianus emend, is preserved with LMD 22.1 as type strain; this species has the broad % G + C range of 52.8 to 62.5; (4) for subphenon C, a new species, Acetobacter hansenii sp. nov. is proposed (% G + C range of 58.1 to 62.6, NCIB 8746 as type strain). Minimal descriptions and differentiating keys are provided.