Gluconacetobacter kakiaceti sp. nov., an acetic acid bacterium isolated from a traditional Japanese fruit vinegar.

Two novel acetic acid bacteria, strains G5-1(T) and I5-1, were isolated from traditional kaki vinegar (produced from fruits of kaki, Diospyros kaki Thunb.), collected in Kumamoto Prefecture, Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains G5-1(T) and I5-1 formed a distinct subline in the genus Gluconacetobacter and were closely related to Gluconacetobacter swingsii DST GL01(T) (99.3% 16S rRNA gene sequence similarity). The isolates showed 96-100% DNA-DNA relatedness with each other, but <53% DNA-DNA relatedness with closely related members of the genus Gluconacetobacter. The isolates could be distinguished from closely related members of the genus Gluconacetobacter by not producing 2- and 5-ketogluconic acids from glucose, producing cellulose, growing without acetic acid and with 30% (w/v) d-glucose, and producing acid from sugars and alcohols. Furthermore, the genomic DNA G+C contents of strains G5-1(T) and I5-1 were a little higher than those of their closest phylogenetic neighbours. On the basis of the phenotypic characteristics and phylogenetic position, strains G5-1(T) and I5-1 are assigned to a novel species, for which the name Gluconacetobacter kakiaceti sp. nov. is proposed; the type strain is G5-1(T) (=JCM 25156(T)=NRIC 0798(T)=LMG 26206(T)).

Comparison of components and synthesis genes of cell wall teichoic acid among Lactobacillus plantarum strains.

The contents, components, and synthesis genes of cell wall teichoic acid (WTA) in 18 strains of Lactobacillus plantarum were compared. The WTA of each strain was classified by its components as being either the glycerol- or the ribitol-type. The different strains in the WTA type showed marked differences also in two gene regions, tagD1-tagF2 and lp_1816-tagB2, as for the presence or absence, nucleotide sequences, and transcriptional activities. Our results clearly showed that the tagD1-tagF2 and lp_1816-tagB2 regions contained the synthesis genes of the WTA backbone of L. plantarum. We verified that the genes in the tagD1-tagF2 region were involved in the synthesis of the glycerol-type backbone. Furthermore, we propose that the genes in the lp_1816-tagB2 region were tarI, tarJ, tarK, and tarL, which are involved in the synthesis of the ribitol-type backbone.

Structures of two monomeric units of teichoic acid prepared from the cell wall of Lactobacillus plantarum NRIC 1068.

The cell wall of Lactobacillus plantarum contains large amounts of cell wall teichoic acid (WTA). WTA was isolated from the cell wall of L. plantarum NRIC 1068 (= ATCC 8014 and 17-5) by extraction with trichloroacetic acid, and two monomeric units (F1 and F2) were prepared from the alkaline hydrolysate of WTA. Componential analysis by HPLC showed that these monomers were composed of ribitol, glucose, and phosphoric acid. Structural analyses of the monomers were performed by NMR spectroscopy with comparison to chemically synthesized monomers. The structures of F1 and F2 were determined to be 3,4-alpha-D-diglucosyl-2-phosphoryl ribitol and 3,4-alpha-D-diglucosyl-1-phosphoryl ribitol respectively. The unique structure of WTA in L. plantarum results from modification of the main chain with multiple glucose residues.

MxaF gene, a gene encoding alpha subunit of methanol dehydrogenase in and false growth of acetic acid bacteria on methanol.

MxaF gene, a gene encoding alpha subunit of methanol dehydrogenase, was investigated for acetic acid bacteria, and growth on methanol was examined for the bacteria by using various media. Of 21 strains of acetic acid bacteria studied, Acidomonas methanolica strains showed the presence of mxaF gene exclusively, and grew on a defined medium containing methanol. Further, none of the strains tested of which the growth on methanol had been previously reported, except for Acidomonas methanolica, showed the presence of mxaF gene or the growth on methanol. Precautions were taken against false growth on compounds used for identification of bacteria.

Different substrate specificities of two triazine hydrolases (TrzNs) from Nocardioides species.

Nocardioides sp. strain MTD22 degraded atrazine, ametryn and atraton, as did Arthrobacter aurescens strain TC1 and Nocardioides sp. strain C190. These strains contain trzN, a gene coding for TrzN, triazine hydrolase showing a broad substrate range. However, Nocardioides sp. strain AN3 degraded only atrazine despite containing trzN. These differences in s-triazine degradation are presumed to be due to differences in the amino acid sequences of TrzNs. Consequently, 1371 nucleotides of the trzN coding sequences of strains AN3 and MTD22 were determined. Comparisons of the amino acid sequences of TrzNs indicated that three residues of strain AN3 (Thr(214), His(215) and Gln(241)) were distinct from those of the other three strains (Pro(214), Tyr(215) and Glu(241)). To confirm the relationships between these amino acid sequences and the substrate specificities of TrzNs, wild and chimera trzN genes were constructed and expressed in Escherichia coli cells. Cells expressing wild MTD22 trzN (Pro(214)Tyr(215)Glu(241)) and chimera AN3-MTD22 trzN (Thr(214)His(215)Glu(241)) degraded all s-triazines, but the degradation rate was markedly decreased in AN3-MTD22 trzN. Wild AN3 trzN (Thr(214)His(215)Gln(241)) and chimera MTD22-AN3 trzN (Pro(214)Tyr(215)Gln(241)) degraded only atrazine. These results suggest that the substitution of Glu(241) for Gln(241) significantly decreases enzyme affinity for ametryn and atraton.

The effect of sodium acetate on the activity of L- and D-lactate dehydrogenases in Lactobacillus sakei NRIC 1071(T) and other lactic acid bacteria.

The effect of sodium acetate on the production of stereoisomers of lactic acid produced by Lactobacillus sakei NRIC 1071(T) and other lactic acid bacteria was studied. L. sakei NRIC 1071(T) started producing L-lactic acid at the early logarithmic phase and d-lactic acid at the late logarithmic phase. The activity of L-lactate dehydrogenase [EC 1.1.1.27, L-LDH] from the resting cells of L. sakei NRIC 1071(T) appeared at the early stage of the logarithmic phase during the growth, and the activity of D-lactate dehydrogenase [EC 1.1.1.28, D-LDH] at the late stage of the logarithmic phase. The resting cells and cell-free extracts of L. sakei NRIC 1071(T) did not produce DL-lactic acid from L- or D-lactic acid. Stained bands of L-LDH and D-LDH appeared in the cell-free extracts from the cells of L. sakei NRIC 1071(T). Consequently, L. sakei conclusively produced L- and D-lactic acid by the action of L-LDH and D-LDH. This finding leads to the conclusion that lactate racemase [EC 5.1.2.1] does not exist in this strain. When the specific activity of LDHs (the total activity of L-LDH plus D-LDH) from the cells cultivated in the presence of sodium acetate is compared with that cultivated in its absence, the ratio of the activity between the cells cultivated in the former condition and those in the latter fell from 1.7 on the cell-free extracts to 1.3 on the preparation of the QAE-Toyopearl 550c chromatography. This result indicates that the amount of LDHs in the cells of L. sake NRIC 1071(T) cultivated in the presence of 50 mM sodium acetate was much more than that in the cells cultivated in the absence of sodium acetate. The shift of the type of stereoisomers of lactic acid from the DL-type to the L-type is discussed in the case of L. sakei strains.

Characterization of Lactobacillus sakei by the type of stereoisomers of lactic acid produced.

Lactobacillus sakei strains were characterized by the shift of the type of stereoisomers of lactic acid produced in the presence of 50 mM sodium acetate in a medium. Of 27 Lactobacillus sakei strains studied, 20 strains showed high levels of DNA-DNA similarity with L. sakei NRIC 1071(T), and were confirmed as L. sakei. The three remaining strains were identified as Lactobacillus curvatus by DNA-DNA similarity, and three other strains were included in the cluster of Lactobacillus plantarum/Lactobacillus pentosus/Lactobacillus paraplantarum and one strain in the cluster of Lactobacillus paracasei on the basis of 16S rRNA gene sequences. Of the 20 L. sakei strains, 19 strains shifted the type of stereoisomers of lactic acid produced from the DL-type to the L-type in the presence of 50 mM sodium acetate. L. curvatus strains and strains included in the cluster of L. plantarum/L. pentosus/L. paraplantarum and in the cluster of L. paracasei did not shift the type of stereoisomers of lactic acid produced. The change of the type of stereoisomers of lactic acid from the DL-type to the L-type in the presence of sodium acetate was concluded to be species-specific for L. sakei and useful for identification of strains in this species.

The effect of sodium acetate on the growth yield, the production of L- and D-lactic acid, and the activity of some enzymes of the glycolytic pathway of Lactobacillus sakei NRIC 1071(T) and Lactobacillus plantarum NRIC 1067(T).

The effect of sodium acetate was studied on the change of the growth yield, the production of L- and D-lactic acid, and the activity of lactate dehydrogenases (LDHs; L-lactate dehydrogenase [EC 1.1.1.27, L-LDH] plus D-lactate dehydrogenase [EC 1.1.1.28, D-LDH]), fructose-1, 6-bisphosphate aldolase [EC 4.1.2.13, FBP-aldolase], and phosphofructokinase [EC 2.7.1.11, PFK] of Lactobacillus sakei NRIC 1071(T) and Lactobacillus plantarum NRIC 1067(T). The growth yield of L. sakei NRIC 1071(T) was increased 1.6 times in the presence of sodium acetate compared with its absence. The activity of LDHs in L. sakei NRIC 1071(T) and L. plantarum NRIC 1067(T) was retained longer under the addition of sodium acetate in the reaction mixture. As a result, these strains produced much more lactic acid in the presence of sodium acetate compared with its absence. Furthermore, the activity of L-LDH in L. sakei NRIC 1071(T) cultivated in the presence of sodium acetate increased three times or more compared with the activity of the cells cultivated in its absence. Consequently, the type of stereoisomers of lactic acid produced by L. sakei shifted from the DL-type to the L-type because the ratio of L-lactic acid to D-lactic acid produced became larger with the addition of sodium acetate to culture media. This phenomenon was not observed in L. plantarum NRIC 1067(T). Further, the participation of lactate racemase is discussed from the viewpoint of the production of D-lactic acid by L. sakei.

Acetobacter okinawensis sp. nov., Acetobacter papayae sp. nov., and Acetobacter persicus sp. nov.; novel acetic acid bacteria isolated from stems of sugarcane, fruits, and a flower in Japan.

Eleven strains of acetic acid bacteria were isolated from stems of sugarcane, fruits, and a flower in Japan. The isolates were separated into three groups, Groups I, II, and III, in the genus Acetobacter according to phylogenetic analysis based on 16S rRNA sequences. The isolates had sequence similarities of 99.8-100% within the Group, 99.3-99.6% to those of the type strains of each related Acetobacter species, and less than 98.4% to those of the type strains of other Acetobacter species. Genomic DNA G+C contents of Groups I, II, and III were 59.2-59.4, 60.5-60.7, and 58.7-58.9 mol%, respectively. The isolates in the Group showed high values of DNA-DNA relatedness to each other, but low values less than 46% to the type strains of related Acetobacter species. A good correlation was found between the three Groups and groups based on DNA G+C contents and DNA-DNA relatedness. All the strains had Q-9 as the main component, and Q-8 and Q-10 as minor components. The isolates in the three Groups did not completely match with any Acetobacter species on catalase reaction, the production of ketogluconic acids from D-glucose, growth on ammoniac nitrogen with ethanol (Hoyer-Frateur medium and Frateur modified Hoyer medium), growth on 30% (w/v) D-glucose, growth in 10% (v/v) ethanol, or DNA G+C contents. On the basis of phylogenetic relationships in the genus Acetobacter and chemosystematic and phenotypic characteristics, the three Groups were regarded as novel species in the genus Acetobacter. Acetobacter okinawensis sp. nov. is proposed for Group I, Acetobacter papayae sp. nov. for Group II, and Acetobacter persicus sp. nov. for Group III.

A mucus adhesion promoting protein, MapA, mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinal epithelial cells.

Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.

Reclassification of Gluconacetobacter hansenii strains and proposals of Gluconacetobacter saccharivorans sp. nov. and Gluconacetobacter nataicola sp. nov.

Ten strains previously assigned to Acetobacter hansenii (=Gluconacetobacter hansenii), Acetobacter pasteurianus LMG 1584 and eight reference strains of the genus Gluconacetobacter were reclassified by 16S rRNA gene sequencing, DNA-DNA similarity, DNA base composition and phenotypic characteristics. The A. hansenii strains and A. pasteurianus LMG 1584 were included in the cluster of acetic acid bacteria (family Acetobacteraceae) by 16S rRNA gene sequences. Further, they were separated into seven distinct groups by DNA-DNA similarity. DNA-DNA similarity group I was identified as G. hansenii. DNA-DNA similarity group II was retained as Gluconacetobacter sp., because DNA-DNA similarity between the strain and Gluconacetobacter entanii LTH 4560(T) could not be determined. This was due to a lack of availability of the type strain from any source. DNA-DNA similarity group III was regarded as a novel species, for which the name Gluconacetobacter saccharivorans sp. nov. (type strain, LMG 1582(T)=NRIC 0614(T)) is proposed. DNA-DNA similarity group IV included the type strains of Gluconacetobacter oboediens and Gluconacetobacter intermedius, and three A. hansenii strains. This group was identified as G. oboediens because high values of DNA-DNA similarity were obtained between the type strains and G. oboediens has priority over G. intermedius. DNA-DNA similarity group V was identified as Gluconacetobacter europaeus. DNA-DNA similarity group VI was regarded as a novel species, for which the name Gluconacetobacter nataicola sp. nov. (type strain, LMG 1536(T)=NRIC 0616(T)) is proposed. DNA-DNA similarity group VII was reclassified as Gluconacetobacter xylinus. The description of G. hansenii is emended.

Cystite formation of Arthrobacter ureafaciens NRIC 0157(T).

The cystite formation of Arthrobacter ureafaciens NRIC 0157(T) was studied by the use of a newly designed CT medium composed of 2.0% D-glucose, 0.28% NH(4)H(2)PO(4), 0.136% K(2)HPO(4) 0.136% KH(2)PO(4), 0.0005% MgSO(4).7H(2)O, and 0.0007% CaCl(2).2H(2)O. Cystites are drumstick-shaped and oval cells and are larger than vegetative cells. Cystites are Gram-negative, whereas vegetative cells are Gram-positive by the KOH reaction. The concentration and ratio of K(+) and Mg(2+) in CT medium mainly affected the cystite formation. Cystites are considered aberrant forms produced by nutritional imbalance.

The formation and structures of cystites of Arthrobacter ureafaciens NRIC 0157T induced by antibiotics.

The cystite formation of Arthrobacter ureafaciens NRIC 0157(T) was induced by some antibiotics, and the addition of tetracycline at a lag phase was effective for cystite formation in YPM liquid medium. Cystites differed from vegetative cells in the cell wall structure, protein content, and water content. Furthermore, the characteristics and structures of cystites induced by tetracycline were similar to those of cystites produced by nutritional imbalance in CT medium. Consequently, various triggers would induce cystite formation. It is interesting that cystite formation was found in part of the Arthrobacter strains and seemed to correlate with the type of peptidoglycan.

Emendation of the genus Acidomonas Urakami, Tamaoka, Suzuki and Komagata 1989.

The genus Acidomonas and the species Acidomonas methanolica were recharacterized by using the type strain (NRIC 0498(T)), three reference strains and 10 methanol-utilizing bacteria that were isolated from activated sludge from three different sewage-treatment plants in Tokyo. Based on 16S rDNA sequences, all strains formed a single cluster within the Acetobacteraceae that was clearly different from the genera Acetobacter, Gluconobacter, Gluconacetobacter, Asaia and KOZAKIA: The 14 strains were identified as a single species, Acidomonas methanolica, by DNA-DNA similarities, showed DNA G+C contents that ranged from 62 to 63 mol% and had Q-10 as the major quinone, accounting for >87 % of total ubiquinones. Cells of Acidomonas methanolica had a single polar flagellum (or occasionally polar tuft flagella); this differs from a previous study that described peritrichous flagella. Oxidation of acetate was positive for all strains, but oxidation of lactate was weakly positive and varied with strains. Dihydroxyacetone was not produced from glycerol. Pantothenic acid was an essential requirement for growth. The strains tested grew at mostly the same extent at pH 3.0-8.0. Therefore, Acidomonas methanolica should be regarded as acidotolerant, not acidophilic. The descriptions of the genus Acidomonas and the species Acidomonas methanolica Urakami, Tamaoka, Suzuki and Komagata 1989 are emended with newly obtained data.

The ratio of L-form to D-form of lactic acid as a criteria for the identification of lactic acid bacteria.

Stereoisomers of lactic acid produced by lactic acid bacteria were determined by HPLC by using an enantiomeric resolution column. In general, the ratios of L-form to D-form (the type of stereoisomer) obtained were compared with those in references. Values of the type of stereoisomer of lactic acid were discussed from the viewpoint of identification of lactic acid bacteria.

Taxonomic heterogeneity of strains comprising Gluconacetobacter hansenii.

The taxonomic standing of Gluconacetobacter hansenii was clarified through phenotypic characteristics, quinones, DNA base composition, DNA relatedness, and the production of gluconic and ketogluconic acids from glucose. All strains that Gosselé et al. (Syst. Appl. Microbiol., 4, 338-368, 1983) employed in the establishment of Acetobacter hansenii (=G. hansenii) were used in this study. Phenotypic differences were shown among the strains of G. hansenii, suggesting heterogeneity within the species. The major ubiquinone was Q-10 for all strains of G. hansenii, except for strain IFO 3296, which was characterized by Q-9. This excluded IFO 3296 from the species G. hansenii and placed it in the genus Acetobacter. DNA relatedness revealed four distinct homology groups (I, II, III, and IV) among strains of the species. Group I was distinguished from the other genomic groups by a lower G1C range from 58.9 to 59.2 mol%. Groups II, III, and IV showed higher G+C contents of 60.4 to 62.2, 60.8, and 61.7 mol%, respectively. Groups I and IV produced both 2- and 5-ketogluconic acids from glucose, and Group III produced only 2-ketogluconic acid. Group II included strains that produced both 2- and 5-ketogluconic acids and strains that produced only 2-ketogluconic acid. It is clear that G. hansenii consists of genotypically heterogeneous strains comprising four homology groups (I, II, III, and IV). Since group I contains the type strain (IFO 14820(T)=LMG 1527(T)) of the species, this group is designated as the species G. hansenii.

Gluconobacter asaii Mason and Claus 1989 is a junior subjective synonym of Gluconobacter cerinus Yamada and Akita 1984.

Five strains received as Gluconobacter cerinus and Gluconobacter asaii were examined for DNA base composition, DNA-DNA similarity, 16S rRNA gene sequences and phenotypic characteristics, including acid production from ethanol, growth on L-arabitol and meso-ribitol and requirement for nicotinic acid. The five strains showed DNA base compositions ranging from 54 to 56 mol% G+C. G. cerinus IFO 3267T and IAM 1832 and G. asaii IFO 3276T and IFO 3275 showed high levels of DNA-DNA similarity (70-100%) between each other and low values of DNA-DNA similarity (16-35%) to Gluconobacter frateurii IFO 3264T and Gluconobacter oxydans IFO 14819T. G. cerinus IFO 3267T and G. asaii IFO 3276T were located at an identical position in a phylogenetic tree deduced from 16S rRNA gene sequences. Two G. cerinus strains and two G. asaii strains did not require nicotinic acid for growth and did not grow on L-arabitol or meso-ribitol. G. cerinus IAM 1832 did not produce acid and required nicotinic acid and/or other growth factors. G. asaii IFO 3265 showed a high degree of DNA-DNA similarity (97%) to G. frateurii IFO 3264T and low similarity values (each 32%) to G. cerinus IFO 3267T and G. asaii IFO 3276T. This strain did not require nicotinic acid and grew well on L-arabitol and meso-ribitol. Therefore, G. asaii IFO 3265 was reclassified as G. frateurii. The results obtained revealed a synonymous relationship between G. cerinus and G. asaii. G. asaii is a junior subjective synonym of G. cerinus because G. cerinus has priority over G. asaii.

Recharacterization of Pseudomonas fulva Iizuka and Komagata 1963, and proposals of Pseudomonas parafulva sp. nov. and Pseudomonas cremoricolorata sp. nov.

Seven Pseudomonas fulva strains obtained from culture collections were taxonomically studied. The seven strains were separated into three clusters (Clusters I to III) on the basis of 16S rRNA gene sequences, and located phylogenetically in the genus Pseudomonas sensu stricto. Further, the strains were classified into 4 groups (Groups I to IV) on the basis of DNA-DNA similarity. As a result, Cluster I was split into Groups I and II. Group I included the type strain of P. fulva and two strains, and levels of DNA-DNA similarity ranged from 88 to 100% among the strains. Group II contained two strains, and the level between the two strains ranged from 91 to 100%. Group III consisted of one strain. Group IV included one strain, and this strain showed a high level of DNA-DNA similarity with the type strain of Pseudomonas straminea NRIC 0164(T). Clusters II and III corresponded to Groups III and IV, respectively. The four groups were separated from one another and from related Pseudomonas species at the level from 3 to 45% of DNA-DNA similarity. The strains of Groups I, II, and III had ubiquinone 9 as the major quinone. According to numerical analysis by the use of 133 phenotypic characteristics, the seven P. fulva strains were split into four phenons (Phenons I to IV). The groups by DNA-DNA similarity corresponded well with the phenons produced by numerical taxonomy, and differential characteristics were recognized. Consequently, Group I was regarded as P. fulva because the type strain (NRIC 0180(T)) of this species was included in this group. Strains in Group II were identified as a new species, Pseudomonas parafulva sp. nov., and the type strain is AJ 2129 (=IFO 16636=JCM 11244=NRIC 0501). NRIC 0181 in Group III was identified as a new species, Pseudomonas cremoricolorata sp. nov., and the type strain is NRIC 0181 (=IFO 16634=JCM 11246). NRIC 0182 in Group IV was identified as P. straminea on the basis of the high level of DNA-DNA similarity with the type strain of this species.