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JOURNAL OF SYSTEMATIC BACTERIOLOGY, J a n . 1990, p. 19-27 0020-7713/90/010019-09$02.00/0 Copyright 0 1990, International Union of Microbiological Societies INTERNATIONAL Vol. 40, No. 1 Taxonomic Study of Anaerobic, Gram-Negative, Rod-Shaped Bacteria from Breweries: Emended Description of Pectinatus cerevisiiphilus and Description of Pectinatus frisingensis sp. nov. , Selenomonas lacticllfex sp. nov. Zymophilus rafinosivorans gen. nov., sp. nov., and Zymophilus paucivorans sp. nov. KARL HEINZ SCHLEIFER,l* MONIKA LEUTERITZ,l NORBERT WEISS,2 WOLFGANG LUDWIG,l GUDRUN KIRCHHOF,l AND HELGA SEIDEL-RUFER3 Lehrstuhl fur Mikrobiologie, Technische Universitat Miinchen, 0-8000 Munich 2, Federal Republic of Germany'; Deutsche Sammlung von Mikroorganismen und Zellkulturen, 0-3300 Braunschweig-Stockheim, Federal Republic of Germany2; and Staatliche Brautechnische Prufi und Versuchsanstalt der Technische Universitat Miinchen, 8050 Freising- Weihenstephan, Federal Republic of Germany, A collection of 47 strains of obligately anaerobic, gram-negative, rod-shaped bacteria that were isolated mainly from spoiled beer and pitching yeast was studied to learn more about their taxonomic positions. A new species of the genus Pectinatus, PectinQtusfrisingensis, a new species of the genus Selenomonas, Selenomonas lacticifex, and a new genus comprising two species, Zymophilus raflnosivorans and Zyrnophilus paucivorans, are described. All of the strains contained directly cross-linked meso-diaminopimelic acid-containing peptidoglycan and in addition the diamine cadaverine or (rarely) putrescine. The diamine was covalently linked to the a-carboxyl group of D-glutamic acid in the peptide subunit of peptidoglycan. Lipid F was also found as a characteristic cellular compound. The phylogenetic relationships of members of these new species were examined by reverse transcriptase sequencing of 16s rRNA or by DNA-DNA hybridization studies or both. All of the organisms belong to the subdivision containing species with gram-negative cell walls within the phylum of gram-positive bacteria. This finding is in good agreement with the presence of a peptidoglycan that contains diamine. For a long time it was thought that gram-negative bacteria are not important as spoilage organisms of beer. However, more recently workers have described some anaerobic, gram-negative bacteria that cause turbidity and off-flavors in bottled beer. A gram-negative, anaerobic, rod-shaped bacterial contaminant of beer was isolated and described by Lee et al. (16) as Pectinatus cerevisiiphilus. Later, bacteria fitting the description of Lee et al. (16) were isolated from turbid and off-flavor beer samples in the Federal Republic of Germany (l), Scandinavia (9, lo), and Japan (22). Gramnegative, anaerobic beer spoilage cocci that were isolated by Weiss et al. (23) were named Megasphaera cerevisiae (7). Since the description of P. cerevisiiphilus was based on only one strain, we decided to carry out a more thorough taxonomic study of anaerobic, gram-negative bacteria isolated from spoiled beer or pitching yeast. MATERIALS AND METHODS Strains and cultivation. Strains that were isolated from spoiled beer are listed in Table 1, and strains that were isolated mainly from pitching yeast are shown in Table 2. P. cerevisiiphilus ATCC 29359T (= DSM 20467T) (T = type strain), Pectinatus sp. strain ATCC 33332T, Selenomonas ruminantium subsp. lactilytica DSM 2872T, and Selenomonas sputigena ATCC 3 5 M T were used as reference strains. Organisms were grown under anaerobic conditions at 30°C in a modified MRS medium containing (per liter) 10 g of peptone, 5 g of yeast extract, 2 g of meat extract, 10 g of glucose, 2 g of K2HP0,, 5 g of NaCl, 0.2 g of MgSO, 7H20, * Corresponding author 0.05 g of MnSO,, 1 ml of Tween 80, 40 ml of a salt solution (see below), 0.5 g of cysteine hydrochloride, and 1 mg of resazurin (pH 6.8). The salt solution contained (per liter) 2 g of CaCl,, 0.2 g of MgSO,, 1g of K,HPO,, 1g of KH2P04,10 g of NaHCO,, and 2 g of NaC1. For MRS agar medium 10 g of agar per liter of medium was added. Physiological tests. Sugar fermentation tests were carried out in microtiter plates. The wells were filled with 2 0 4 portions of filter sterilized 10% (wt/vol) solutions of sugars. A 10-ml portion of an overnight culture grown in MRS medium was centrifuged and suspended in 10 ml of sugarfree MRS medium. Wells containing the sugar solutions and a control well containing sugar-free medium were inoculated with 150-pl portions of the suspension and incubated at 30°C for 48 h under anaerobic conditions (GasPak; BBL Microbiology Systems, Cockeysville, Md.). To evaluate acid formation, 20 p,l of a solution of bromocresol purple (10 mg dissolved in 0.5 ml of ethanol and made up to 10 ml with distilled water) was added. The control well containing the sugar-free solution always showed a negative reaction. Utilization of esculin was determined in sugar-free modified MRS medium containing 0.25% esculin, 0.05% FeCl,, and 0.25% agar. Nitrate reduction was determined in sugar-free modified MRS medium also lacking resazurin and cysteine but containing 0.15% KNO,, 0.2% glucose, and 0.25% agar. Liquification of gelatin was tested in sugar- and resazurinfree modified MRS medium containing 4% gelatin and 1% agar. For production of H2S, we used the SiM medium of E. Merck AG (Darmstadt, Federal Republic of Germany) enriched with 5 g of yeast extract per liter, salt solution (see above), 0.05 g of cysteine hydrochloride per liter, and 1g of glucose per liter. 19 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 01:35:29 20 INT.J. SYST.BACTERIOL. SCHLEIFER ET AL. TABLE 1. Characteristics of strains of Pectinatus species isolated from spoiled beef ~~ Strain Group I ( P . cerevisiiphilus) ATCC 29359T WK1 WK2 WK3 WK4 BH1 BH2 Walc2 Walb Wa3l1 Wa312 Group I1 (P.frisingenesis) ATCC 33332T BB1 BB2 DS 1 DS2 DU1 DU2 MKKl MKK2 MKPl MU1 MU2 WL1 WL2 G+C content (mol%) 40 40 38 39 ND 40 40 40 41 40 40 39 41 40 40 ND 39 ND 40 38 40 39 39 ND 40 Production of acid from: Xylose Mannitol Dulcitol Adonitol Xylitol Inositol Cellobiose Melibiose g~~-$$e Gluconate Esculin - - - +2 - - + + + + - + + + + + + + + + + + + + + +W +W + +W + +W +W + + + +W +W + + All strains (except the strains indicated in parentheses) produce acid from ribose (strains DS1 and DS2), arabinose, rhamnose, glycerol, erythritol, sorbitol (strain ATCC 29359), fructose, mannose, glucose, and galactose (strain ATCC 33332); all strains (except the strains indicated in parentheses) fail to produce acid from sucrose, trehalose, melezitose, inulin, glycogen, lactose (strain Wa3/1), maltose (strains ATCC 33332T, DS1, DS2, and MU2), &nose (strains MKK 1 and MKP l), salicin (strains ATCC 29359T, MKK1, WL1, and WL2), and starch (strains MKKl and MKP1). +, Positive reaction; -, negative reaction; +w, weak positive reaction. Fermentation products. Fermentation products were determined as described by Holdeman et al. (11).A model 7620 gas chromatograph (Hewlett-Packard Co., Palo Alto, Calif.) equipped with a Chromosorb 101 column and a flame ionization detector was used for analysis of volatile and nonvolatile acids. Cell walls. Preparation of cell walls and determination of the peptidoglycan type were carried out as described by Schleifer and Kandler (20). Cadaverine and putrescine could be separated by gas chromatography, using a column filled with Chromosorb 103 (Sigma Chemical Co., Deisenhofen, Federal Republic of Germany). Quinones and quinonelike compounds. Analyses of quinones and quinonelike compounds were performed as described by Collins et al. (2). Determination of G+C contents of DNAs and DNA homology studies. DNA was isolated as described by Meyer and Schleifer (18). The melting point of the purified DNA was determined by using the method of Marmur and Doty (17), and the guanine-plus-cytosine (G + C) content was calculated by using the method of DeLey et al. (5). DNA from Escherichiu coli strain B (Sigma Chemical Co., St. Louis, Mo.), with a G + C content of 51.7 mol%, was used as the reference. DNA-DNA hybridization studies were carried out by spectrzphotometrically determining renaturation rates, as previously described (5, 13). 16s rRNA analysis. RNA was isolated as described previously (6). Sequencing of 16s rRNA by using reverse tran- scriptase was done as described by Lane et al. (15). Oligonucleotide primers were synthesized by standard methods, using a Biosearch Cyclone DNA synthesizer. The sequences of these primers are complementary to highly conserved regions of 16s rRNA. Sequence ambiguities were resolved by using terminal transferase (4). Alignment of the sequences was done with respect to conserved primary structures, as well as secondary structures. From this alignment binary dissimilarity values were calculated and subsequently transformed into phylogenetic distance (Knuc) values (12). Only those positions which were present in all sequences and were invariant in 30 to 90% of the aligned sequences were included. Thus, universal positions, as well as highly variable positions, were excluded. An unrooted phylogenetic tree was constructed from the distance values by using the method of Fitch and Margoliash (8). RESULTS Morphology. Group I and I1 strains were slightly curved, gram-negative, rod-shaped bacteria with rounded ends. They occurred predominantly as single cells or sometimes in pairs. They were 0.7 to 0.9 by 3 to 20 pm. The occurrence of longer, slightly helical filaments was characteristic for stationary-phase cultures. Younger cells were usually motile. Group I11 strains were straight to slightly curved, motile, gram-negative rod-shaped organisms with rounded ends. They were 0.7 to 0.9 by 3 to 15 pm. They occurred Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 01:35:29 VOL. 40, 1990 ROD-SHAPED BACTERIA FROM BREWERIES 21 TABLE 2. Physiological characteristics and G+C contents of gram-negative, anaerobic, slightly curved to helical or crescent-shaped bacteria isolated from pitching yeast and brewery wastea Strain Group 111 (Z. rafinosivorans) OW1311 OW1312 ow1111 ow1112 0w 10/1 ow1012 OW9 OW8 IB1 IB2 RG1 RG2 VB2 SH2T (= DSM 207GT) Group IIIa (Z. paucivorans) VB 1 VB3a AAIT (= DSM 20756*) AA2 Group IV (Selenomonas lacticifex) VB4bT (= DSM 20757T) VB6 VB7 VB8 G+C Production of acid from: content (mol%) Ribose Xylose Rhamnose Mannitol Adonitol Erythritol Xylitol Inositol Glycerol Salicin Amygdalin 41 41 41 39 38 41 40 38 39 40 40 40 39 41 39 41 40 40 51 51 52 52 +b + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + +W - +W +W +W +W +W +W - + +W + + - - - - - - + + + + + - - - + + + + + + + + + + + + + + + + + + - + + + + + + + +W +W +W +W +W +W +W +W +W +W +W +W + + - +- - +W - - +W +W - - + + + + + + + + + + + +W + + +W - +W +W + - + a All strains (except the strains indicated in parentheses) produce acid from arabinose (strain VBl), sorbitol (group IV strains and strain VB3a), dulcitol (group IIIa and group IV strains), fructose, rnannose, glucose, galactose (strains VB1 and VB3a), lactose (strains VB1, VB3a, and AAIT), maltose (strain VB4b), sucrose, cellobiose (strains VBS, VB7, and VB6), raffinose (strains OW8 and OW9 and group IIIa strains), and melibiose (strains OW8 and OW9 and group IIIa strains); all strains (except the strain indicated in parentheses) fail to produce acid from trehalose, rnelezitose, inulin, glycogen, or starch (strain OW10/2). +, Positive reaction; -, negative reaction; +w,weak positive reaction. predominantly as single cells or sometimes in pairs or short chains. Group IIIa strains were curved or helical, motile, gram-negative rod-shaped bacteria with rounded ends. They were 0.8 to 1.0 by 5 to 30 pm. They occurred singly or in pairs or short chains. Group IV strains were curved, crescent-shaped, motile, gram-negative rods with rounded ends. They were 0.6 to 0.9 by 5 to 15 km. They occurred predominantly as single cells or (rarely) in pairs. In some strains motility was lost after several subcultivations. Physiological and chemical properties. All of the strains which we studied were obligately anaerobic. The physiological properties of the strains belonging to the morphologically similar groups I and I1 are listed in Table 1, and the properties of group 111, IIIa, and IV strains are shown in Table 2. The G+C contents of the DNAs of group I and I1 strains were 38 to 41 mol% (Table I). Similar G+C contents were found for DNAs of representatives of groups I11 and IIIa (Table 2), whereas strains belonging to group IV had distinctly higher G+C contents (51 to 52 mol%) (Table 2). All of the strains which we studied contained the directly cross-linked meso-diaminopimelic acid peptidoglycan type. In addition, the a-carboxyl group of D-glutamic acid was substituted predominantly by cadaverine, and in some strains a minor amount of cadaverine was replaced by putrescine. Such a peptidoglycan type containing a diaminesubstituted glutamic acid was previously found in strains of Megasphaera, Selenomonas, and Sporornusa species (7, 14). Neither menaquinones nor ubiquinones were detected. However, as in Megasphaera, Selenomonas, and Sporomusa species, lipid F (21) was found in all of the strains studied. The major fermentation products in modified MRS medium were acetic and propionic acids. All group IV strains also produced major amounts of lactic acid, predominantly D-lactic acid. Distinct amounts of lactic acid were also formed by Selenomonas ruminantium subsp. lactilytica DSM 2872=, whereas all of the other strains formed little or no lactic acid. DNA-DNA hybridization studies. DNA-DNA hybridization studies showed clearly that group I, 11, 111, and IV strains form distinct genospecies. Within each group the DNA homology values were in the range of 70 to loo%, whereas no significant level of homology (<25%) was found between representatives of the different groups. Strains belonging to group IIIa not only exhibited high homology values among each other (>75%), but also exhibited a distinct relationship (homology values between 44 and 48%) to representatives of group 111. 16s rRNA sequences and phylogenetic relationships. The partial 16s rRNA sequences of Selenomonas ruminantium subsp. lactilytica, Selenomonas sputigena, Selenomonas lacticifex, Zymophilus paucivorans, P . cerevisiiphilus, and Pectinatus frisingensis are aligned in Fig. 1. Our sequences were compared with the 16s rRNA sequences of Megasphaera elsdenii, Sporornusa paucivorans (sequences kindly provided by C. R. Woese), Bacillus subtilis, Streptomyces coelicolor, and E . coli (3). An evolutionary distance analysis was performed as described above. An unrooted phylogenetic tree is shown in Fig. 2. DISCUSSION The comparison of the 16s rRNA sequences of the strains which we isolated with the sequences of reference strains revealed that our strains belong to the subdivision of species Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 01:35:29 22 INT. J. SYST.BACTERIOL. SCHLEIFER ET AL. 1 S.r. 72 s.l. TTaATGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTnnnnnnnnnnnnnnnnnnnnnnnn ..AACGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAAC ..ATTGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTnnnnnnnnnnnnnnnnnnnnnnnn Z.p. P.f. P.C. TTAATGGAGAGTTTGATCCTGGCTCAGGACAAACGCTGGCGGCGTGCTnnnnnnnnnnnnnnnnnnnnnnnn TTTATGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTnnnnnnnnnnnnnnnnnnnnnnnn S.S. ..aTTGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTnnnnnnnnnnnnnnnnnnnnnnnn 73 144 S.r. nnnnnnnnnnnnnnnnnnnnnnnnnnnnnACGGGTGAGTAACACGTAGGCAACCTGCCGCAAAGATGGGGAC S.S. CTTTATTTCGGTAAAGGTGAGAGTGGCAAACGGGTGAGTAACACGTAGGCAACCTGCCGACAGGATGGGGAC s . l . nnnnnnnnnnnnnnnnnnnnnaGTnGCaaaCGGGTGAGTAACnC~TAGGCAACCTGCCTTTGAGATGGGGAC z.p. nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnGGTGaGTAACaCGTAGGCAACCTGCCTTTTAGATGGGGAC P . f . nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnTGAGTAAcnnnTAggCAACCTGCCTCcAAGATGGGGAC P.C. nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnncaacctGCCTncaAGATGGGGAC 145 21 6 S.r. AACAGTCCGAAAGGACTGCTAATACCGAATGTTGTCaGaCTCCCGCATGGGAGCCTGATTAAAGATGGCCTC S . S . AACATTCCGAAAGGAATGCTAATACCGAATGGAGTCCGGGGATGGCATCATCCCCGGATAAAAGATGGCCTC s.l. AACAGTTCGAAAGGGCTGCTAATACCGaATGTTGTATATTTTCCACATGGAAGATATATTAAAGATGGCCTC z.p. AACATCGCGAAAGCGGTGCTAATACCGAATGTTGTGTCTTTCCCACATGGGAAAGCTAC~AAAGATGGCCTC P . f . AACATCtCGAAAGGGGTGCTAATACCGAATGTTGTAATGTTGTAAAGCgATTGCATAATGGCTTTACCAAAGGCGGC P.C. AACACTCCGAAAGGGGTgCTAATACCGAATGTTGTAATGCTGCTGCAnnnCAGTATTACCAAAGGtggC ... ... 21 7 288 S.r. TACTTGTAAgcTATCGCTTTGCGATGGGTnTGCGTCTGATTAGCTaGTTGGTG.GGGTAACGGCCTACCAAG S.S. TGAATAT..gCTATCGCCTGTCGATGGGCCTnCGTCTGATTAGCCAGTTGGCG.GGGTAACGGCCTACCAAA s . l . TATTTATAAgCTATCGCTCAAAGATGGGTnTGCGTCTGATTAGCTaGTTGGTG.GGGTAAcGGCCTACCAAG z.p. TATTTATAAGCTGTCACTAAAAGATGGGTCTGCGTCTGATTAGCTaGTTGGCG.GGGTAAATGCCCACCAAG P . f . .TTTTA...gCTGTCGCTTGGAGATGGGCCTaCGTCTGATTAGCTaGTTGGTGACGGTAACGGCGCACCAAG P.C. .TTTTA...gCTATCGCTTGGAGATGGGCCTaCGTCTGATTAGCTaGTTGGTGACGGTAACGGCGCACCAAG 289 S.r. S.S. 360 GCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGAACTGAGACACGGTCCaGACTCCTACGG GCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGG s.l. GCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACACATTGGGACTGAGACACGGCCCAGACTCCTACGG z.p. P.f. P.C. GCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGG GCAACGATCAGTAGCCGGTCTGAGAGGATGGACGGCCACATTGGGACTGAGACACGGCCCAaACTCCTACGG GCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAgACTCCTACGG S.r. GAGGCAGCAGTGGGGAATCTTcCGCAATGGGCGAAAGcCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGGT 361 S.S. s.l. z.p. P.f. P.C. 432 GAGGCAGCAGTGGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTC GAGGCAGCAGTGGGGAATCTTCCACAATGGACGACGAAAGTCTGATGGAGCAACG~CGCGTGAGTGAAGAAGGGT GAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGTT GAGGCAGCaGTGGGGAATCTTcCGCAATGGGCGAAAGcCTGACGGAGCAACGCCGCGTGAACGAGGAAGGTC GAGGCAGCAGTggGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAACGAGGAAGGTC 43 3 S.r. S.S. s.l. z.p. P.f. P.C. 504 TTCGGCTCGTaAAGCTCTGTTGACGGGGACGAACGTGCGTGCGAGATGCGAATAGTTTCTTGCAATGACGGTACCC TTCGGATCGTAAAGCTCTGTTGCACGGGACGAAAACCGAGCTTGAGAATATTGAGTTTGGGTGACGGTACCG TTCGGCTCGTAAAGCTCTGTTGTCGGGGACGAAtGTGCAGTATTTGAATAAAGTACTGCAATGACGGTACCT TTCGGATCGTtAAGCTCTGTTGTTTGAGACGAACGTGCAGTATACGAATAATGTGCTGTAATGACGGTATCA TTCGGATCGTAAAGTTCTGTTGCAGGGGACGAATGGCAGTAGTGTT~TACCACTATTGAATGACGGTACCC TTCGGATCGTtAAGTTCTGTTGCAGGGGACGAACGGCACTATAGCCAATAAGTATAGTGAATGACGGTACCC 505 S.r. S.S. s.l. z.p. P.f. P.C. 576 GTcGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCCAGCGTTGTCCGGAATT AGCGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCCAGCGTTGTCCGGAATT GACGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCaAGCGTTGTCCGGAATT AACTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATT TGTTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGCGGCAAGCGTTGTCCGGAATT TGTTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCgGTAATACGTAGGCGGCAAGCGTTGTCCGGAATt FIG. 1. Alignment of 16s rRNA partial sequences of Selenomonas ruminantium (S.r.), Sefenomonas sputigena ( S . S . ) , Sefenomonas lacticifex (S.l.), Zymophilus paucivorans (Z.P.), Pectinatus cerevisiiphifus (P.c.), and P . frisingensis (P.f.). The lower-case letters indicate nucleotides whose assignment is uncertain; n indicates a nucleotide of unknown composition. Dots were inserted for purposes of alignment. The 3’-terminalparts (about 55 bases) of the sequences were not determined. with gram-negative walls within the phylum of gram-positive eubacteria (24). This agrees very well with the occurrence of the diamine-substituted, directly cross-linked meso-diaminopimelic acid peptidoglycan type among this group of bacteria. The same peptidoglycan type has been found in Veilonella species and Centipedu periodontii (Weiss, unpublished data), indicating the relationship of these organisms to this subdivision within the gram-positive eubacteria. The 16s rRNA analysis showed that a representative of group I1 is closely related to P . cerevisiiphilus DSM 20467T (Fig. 2). Therefore, group I and I1 organisms should be placed in the genus Pectinatus. From 16s rRNA analyses it is also quite clear that the genus Pectinatus does not belong in the family Bacteriodaceae, as suggested by Lee et al. (16), since the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 01:35:29 VOL. 40, 1990 ROD-SHAPED BACTERIA FROM BREWERIES 577 23 648 ATTGGGCGTAAAGGGAGCGCAGGCGGGAAGGCAAGTCAGTCTTAAAAGTGCGGGgCTcAACCCCGTgATGGG S.S. ATTGGGCGTAAAGGGAGCGCAGGCGGACATATAAGTCCATCTTAAAAGTGCGGGGCTcAACCCCGTgAGGGG S.1. ATTGGGCGTAAAGGGAGCGCAGGCGGGAAGGTAAGTCGGTCTTAAAAGTGCGGGGCTcAACCCCGTgATGGG Z.p. ATTGGGCGTAAAGGGAGCGCAGGTGGGAATGTAAGTCAGTCAGTCTTAAAAGTGCGAGGCTcAACCTCGTGATGGG P . f . ATTGGGCGTAAAGGGAGCGCAGGCGGATGACTAAGCGGATCTTAAAAGTGCGGGGCTcaACCCCGTgATGGG P.c. ATTGGGCGTAAAGGGAGCGCAGGCGGAACATTAAGCGGATCTTAAAAGTGCGGGGCTcAACCCCGTGATGGG S.r. 649 720 4 . r . ATTGAAACTGTCTTTCTTGAGTGCAGGAGAGGAAAGCGGAATTCCTaGTGTAGCGGTGAAATGCGTAGATAT S.S. ATGGAAACTGTATGCCTTGAGTgCAGGAGAGGAAAGCGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATAT S.l. ATCGAAACTATCTTTCTTGAGTGCAGGAGAGGAAAGTGGAATTCCTaGTGTAGCGGTGAAATGCGTAGATAT Z.p. ACAGAAACTACATTTCTTGAGTGCAGGAGAGGAAAGTGGAATTCCTaGTGTAGCGGTGAAATGCGTAGATAT P.f. P.C. ATTCGAACTGGTCATCTTGAGTGCAGGAGAGGAAAGCGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATAT S.r. TgGGAGGAACACCaGTGGCGAAGGCGGCTTTCTGGACTGTaACTGACGCTGAGGCTCGAAAGCGTGGGGAGC TGGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCTCGAAAGCCAGGGGAGC TAGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGACTGTAACTGACGCTGAGGCTCGAAAGCAAGGGGAGC TAGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGACTGTAACTGACACTGAGGCTCGAAAGCTAGGGGAGC GTCCGAACTGAGGTTCTTGAGTGCAGGAGAGGAAAGCGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATAT 721 S.S. S.1. 2.p. P.f. P.c. 792 TGGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCTCGAAAGCCAGGGTAGC TGGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCTCGAAAGCCAGGGTAGC 793 864 S.r. GAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTAGGAGGTATTGACCCCTT S . S . GAACGGGATTAGATACCCCGGTAGTCCTnGCCGTAAACGATGAATGCTAGGTGTAGGAGGTATTGACCCCTC S.l. GAACGGGATTAGATACCCCGGTAGTCCTnGCCGTAAACGATGAATACTAGGTGTGGGAGGTATTGACCcTTT Z.p. GAACGGGATTAGATACCCCGGTAGTCCTnGCCGTAAACGATGAATACTAGGTGTAGGGGGTATTGACCCTTT P . f . GAACGGGATTAGATACCCCGGTAGTCCTnGCCGTAAACGATGGATACTAGGTGTAGGGGGTATTGACCCtnC P.c. GAACGGGATTAGATACCCCGGTAGTCCTnGCCGTAAACGATGGATACTAGGTGTAGGGGGTATTGACCcTTt 865 S.r. 936 CTGTGCCGGAGTTAACGCAATAAGCATTCCGCATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTG S.S. CTGTGCCGGAGTTAACGCAATAAGCATTCCGCCTGGGGAGTACGGTCGCAAGCATTCCGACTGAAACTCAAAGGAATTG S.1. CCGTGCCGGAGTTAACGCAATAAGTATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTG Z.p. CTGTGCCGGAGCTAACGCAATAAGTATTCCGCCTGGGGAGTACGGCCGCAAGGTTG~ACTCAAAGGAATTG P.f. P.c. CTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGCCGCAAGTCAGGCTGAAACTCAAAGGAATTG CTGTGCCGGAGCAAACGCAATAAGTATCCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTG S.r. ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGnnGCAACGCGAAGAACCTTACCAGGGCTTGA S.S. S.l. ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGaaGCAAGCATTCCCGCGAAGAACCTTACCAGGGCTTGA ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGnnGCAACGCGAAGAACCTTACCAGGGCTTGA 937 1008 Z.P. ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGanGCAACGCGAAGAACCTTACCAGGGCTTGA P.f. P.C. ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGnnGCAACGCGAAGAACCTTACCAGGGCTTGA S.r. S.S. CATTGAGTGAAAGctCTAGAGATAGAGCCC.TCtCTTCGGAGA.CAcGAAAACAGGTGGTGCATGGCTGTCG S.1. CATTGAGTGAAAGGTGCAGAGATGCATCCC.TCTCTTCGGAGA.CACGAAAACAGGTGGTGCATGGCTGTCG ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGAaGCAACGCGAAGAACCTTACCAGGGCTTGA 1009 1080 CATTGAGTGAAAGAGCTAGAGATAGCTTCc.nCTCTTCGGGGA.CACGAA~CAGGTGGTGCATGGCTGTCG Z.p. CATTGAGTGAAAGGCTAAGCATTCCGAGATTAGTCCCTCTTCTTCGGAAGACACAAAAACAGGTGGTGCATGGCTGTCG CATTGATTGaCGTATGCAGAGATGCATATTTTCTCTTCGGAGGACAAGAAAACAGGTGGTGCATGGCTGTCG CATTGATTGACGTATCCAGAGATGGATATTTTCTCTTCGGAGGACAAGAAAACAGGTGGTGCATGGCTGTCG P.f. P.c. 1 1 52 1081 S.r. S.S. TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCATTTGTTGCCAGCACGT TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGTCCTTTGTTGCCAGCGCGT S.l. TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTTTGTTGCCAGCACGT Z a p . TCAGCTCGTGTC~TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTATGTTGCCAGCACGT P . f . TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTAT&ATTTGTTGCCAGCACGT P.C. TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCATTTGTTGCCAGCACGT FIG. l-Continued genus Bacteroides and related genera represent a distinct phylum within the phylogenetic tree of eubacteria (24). A representative of group IV is distinctly related to Selenomonas ruminantium and Selenomonas sputigena and should therefore be assigned to the genus Selenomonas. A representative of group IIIa is only distantly related to any of the other anaerobic, gram-negative eubacteria. Group IIIa strains, together with group I11 strains (see below), form the basis for a new genus. DNA-DNA hybridization studies showed that all of the group I strains are closely related to P . cerevisiiphilus ATCC 29359T,whereas group I1 strains shared high DNA homology values with Pectinatus sp. strain ATCC 33332T. DNA-DNA hybridization studies between strains ATCC 29359T and ATCC 33332T showed a DNA homology value of 16%. The two Pectinatus species can be distinguished phenotypically. Differences were found in the degradation of xylose, cellobiose, melibiose, and N-acetylglucosamine. However, the phenotypic properties of P. cerevisiiphilus ATCC 29359Tare not in agreement with the original description (16). To be Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 01:35:29 24 INT. J. SYST.BACTERIOL. SCHLEIFER ET AL. 1 1 53 1224 S.r. CAAGGTGGGAACTCAAATGAGACTGCCGCGGACAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC S.S. AATGGCGGGAACTCAAAGGAGACTGCCGCGGAGAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC s.l. AATGGTGGGAACTCAAAAGAGACTGCCGCGGACAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC z . p . AATGGTGGGAACTCATGGGAGACTGCCGCGGATAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC P.f. CAAGGTGGGAACTCAAATGAGACTGCCGCGGATAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC P.C. AAAGGTGGGAACTCAAGTGAGACTGCCGCGGACAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC 1225 1296 S.r. S.S. CCCTTATGTCCTGGGCTACACACGTACTACAATGGGATGGACAGAGgGaAGCGAaCCCgcgAGGGCAAGCGA CCCTTATGTCCTGGGCTACACACGTACTACAATGGGATGGACAGAGGGAAGCGAAGGCGTGAGCCGGAGCGG s . l . CCCTTATGTCCTGGGCTACACACGTACTACAATGGGATGGACACgggGCAGCGAAGCCGCGAGGCCAAGCGA z . p . CCCTTACGTCCTGGGCTACACACGTACTACAATGGGACACACAGAGGGAAGCGAAGGAgTGATCTGGAGCGG P.f. CCCTTACGTCCTGGGCTACACACGTACTACAATGGGATACACAGAGGGAAGCGAAGGAGTGATCTgGAGCGG P.C. CCCTTATGTCCTGGGCTACACACGTACTACAATGGGATAAACAGAGGGAAGCGAGACCGCGAGGTGGAGCGA 1297 1368 S.r. ACCCCATtAACCATCTCCCAGTTCGGATTGCAGGCTGCAACCCGCCTGCATGAAGTCGGAATCGCTAGTAAT S.S. ACCCCACAAACCATCCCCCAGTTCGGATTGCAGGCTGCAACCCGCCTGCATGAAGTCGGAATCGCTAGTAAT s.l. ACCCCATAAACCATCCCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTTGGAATCGCTAGTAAT z . p . ACCCCAAAAAATGTCTCCCAGTTCGGATTGCAGGCTGCAACCCGCCTGCATGAAGTCGGAATCGCTAGTAAT P.f. AACCCAAAAAATATCCCCCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATG~GTCGGAATCGCTAGTAAT P.C. AACCCATAAATTATCTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATCGCTACTAAT 1369 1440 CGCTGGTCAGCATACAGCGGTGAATACGTTCCCGGGCCTTGTACACAccgccCGTCACACCACGGAAGTCAT S . S . CGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACACcgCcCGTCACACCACGGAAGTCAT s . l . CGCTGGTCAGCATACAGCGGTGAATACGTTCCCGgGCCTTGTACACACcgccCGTCACACCACGGAAGTCAT z . p . CGCAGGTCAGCATACTGCGGTGAATACGTTCCCggGTCTTGTACACACcgccCGTCACACCACGAAAGTCAT P.f. CGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACAccgccCGTCACACCACGAAAGTCAT P.C. CGCAGGTCAGCATACTGCGGTGAATACGTTCCCgggTCTTGTACACAccgccCGTCACACCACGAAAGTCAT S.r. 1441 1508 S.r. TCACACCCGAAGCCGG.TGGGTAAACCGCAAGGAtATAGGAtATAGCCGTCTAAGGTnGGGGCGATGACCGGGGn TCACGCCCAAAGCCGG.TGGGCtAACCGCAAGGAGGCAGCCGTCTAA~GCGGGGGCGATGACTGGGGn S.S. s. 1. TCACACCCGAAGCCGG.TGGGtAAACCGCAAGGACACAGCAAGGACACAGCCGTCtAAGGTnGGGGCGATGACCGGGGn z . p . TCACACCCGAAGCCGG.TGGGGTAACCGCAAGGAGCCAGCCGTCTAAGGTnGGGGCGATGACtGGGGT P.f. CCACACCCGAAGCCGGCTAAGGG..CCGCAAGGAACCGACCGTCTAAGGTnGGGGCGATGAccGGGGn P.C. CCACACCCGAAGCCGGCTAAGGG..cCGCAAGGCACCGACCGTCTAAGGTnGGGGCGATGAccGGGGn FIG. l-Continued Escherichia coli \ Selenomonas mmimntium Selenomonas sputigena Zymophilus paucivorans Pectinatus cerevisiiphilus Megasphaera elsdenii Sporomusa paucivorans Bacillus subtilis Streptomyces coelicolor FIG. 2. Unrooted phylogenetic tree showing the relationships among the organisms investigated in this study and reference organisms. Bar unit. = 0.1 K,,, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 01:35:29 VOL.40, 1990 ROD-SHAPED BACTERIA FROM BREWERIES sure that the present type strain of P. cerevisiiphilus is distinctly different from the original description of this species, we studied not only strain DSM 20467T but also strain ATCC 29359T. Both of these strains had the same phenotypic characteristics, had high levels of DNA homology (95%), and differed significantly from the original description of P. cerevisiiphilus. L. H . Moore has studied three different lyophilized cultures of strain ATCC 29359T (two in 1981 and one in 1989) and found the same characteristics as we did (L. H. Moore, personal communication). Therefore, the description of P. cerevisiiphilus is emended below, and strains belonging to group I1 are described as new species, P. frzsingensis. Groups 111 and IIIa were related to one another but were genetically distinct from other anaerobic gramnegative, rod-shaped bacteria (Fig. 2). They should be assigned to different species of a new genus, Zymophilus rafinosivorans and 2.paucivorans. Genetic, morphological, and physiological data indicate a distant relationship between group IV strains and Selenomonas ruminantium. Therefore, these strains should form a new species within the genus Selenomonas, namely Selenomonas lacticijkx. On the basis of the G+C content of its DNA (51 to 52 mol%), this organism can be clearly separated from other species of Selenomonas, such as Selenomonas sputigena, Selenomonas noxia, Selenomonas flueggei, Selenomonas infelix, Selenomonas dianae, and Selenomonas artemidis, that have significantly higher G+C contents (56 to 58 mol%) (19). Differential characteristics of the newly described species are given in Table 3. Emended description of Pectinadus cerevisiiphilus. The emended description below of Pectinatus cerevisiiphilus is based on the descriptions given by Lee et al. (16) and by Moore (personal communication) and our results. Cells are gram-negative, slightly curved rods that are 0.7 to 0.9 Frn in diameter and 2 to 30 p,m or more long. They occur singly, in pairs, and only rarely in short chains. The occurrence of longer, helical filaments is characteristic for older cells. The cells are usually motile. These organisms are obligately anaerobic, nonsporeforming mesophiles. Flagella emanate from only one side of the cell (comblike). They do not produce catalase and cytochromes. Colonies are circular, entire, beige to white, glistening, and opaque. The optimum temperature for growth is 30°C. Glucose is fermented to acetic and propionic acids. In contrasts to the original description given by Lee et al. (16), both Moore (for the type strain) and we (also for other strains) found that xylose and melibiose are fermented, whereas maltose and cellobiose are not. Cadaverine and putrescine are found as characteristic components of the cell wall peptidoglycan type, directly cross-linked meso-diaminopimelicacid. Lipid F is also found as a characteristic cellular compound. The G+C content of the DNA is 38 to 41 mol%. The type strain is strain ATCC 29359 (= DSM 20467). The phenotypic properties of the type strain are shown in Table 1. Description of Pectinutus fisingemis sp. nov. Pectinatus jbingensis (frisin-gen’sis. L. adj. fnsingensis, of Frisinga, the Latin name of Freising, a town in the Federal Republic of Germany where the organism was isolated). The description below is based on 11 strains which were isolated from spoiled beer. These organisms are obligately anaerobic, usually motile, nonsporeforming gram-negative rods. Cells are slightly curved and occur predominantly as single cells or sometimes in pairs. They are 0.7 to 0.9 by 3 to 20 pm. The occurrence of longer, slightly helical filaments is characteristic for older cultures. Colonies on modified MRS agar medium are shiny, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 01:35:29 25 26 SCHLEIFER ET AL. INT. J. SYST.BACTERIOL. TABLE 4. Characteristics for the differentiation of Selenomonas species Acid produced from: Species Cellobiose Lactose -a - - Selenomonas artemidis Selenomonas dianae Selenomonas flueggei Selenomonas infelix Selenomonas lacticifPx Selenomonas noxia Selenomonas ruminantium Selenomonas sputigena a + , Positive reaction; + + + - - - +- Mannitol Sucrose + + + + + + + + + + + - +- V + Lactic acid as a major fermentation product G+C content of DNA (mol%) - 58 53 56 58 51-52 57 48-53 57 - +V - -, negative reaction; v, variable reaction. opaque, circular, slightly yellow, and 1to 2 mm in diameter after 3 days at 30°C. The optimum temperature for growth is 30°C. Glucose is fermented to acetic and propionic acids, acetoin, and sometimes minor amounts of succinic acid. Phenotypic characteristics of the different group I1 strains are shown in Table 1. Cadaverine and rarely putrescine are found as characteristic components of the cell wall peptidoglycan. The peptidoglycan type is directly cross-linked meso-diaminopimelic acid. Lipid F is also found as a characteristic cellular compound. The G+C content of the DNA is 38 to 41 mol%. The type strain is strain ATCC 33332. The phenotypic properties of the type strain are shown in Table 1. P.frisingensis can be distinguished from P.cerevisiiphilus by its usual ability to ferment cellobiose, inositol, and N-acetyl-glucosamine and its inability to utilize xylose and melibiose. Descriptionof Selenomonas lacticifex sp. nov. Selenomonas lacticifex (1ac.ti’ci.fex. M. L. n. acidum lacticum, lactic acid; L. suffix fex, a maker; L.n. lacticifex, a maker of lactic acid). The description below is based on four strains that were isolated from pitching yeast. These organisms are obligately anaerobic, usually motile, nonsporeforming, gram-negative rods. The cells are curved and crescent shaped and occur predominantly as single cells or (rarely) in pairs. They are 0.6 to 0.9 by 5 to 15 pm. Colonies on modified MRS agar medium are smooth, opaque, circular, yellowish, and 2 to 3 mm in diameter after 3 days at 30°C. The optimum temperature for growth is 30°C. Glucose is fermented to acetic, lactic, and propionic acids. Phenotypic characteristics of the different group IV strains are shown in Table 2. Cadaverine and rarely putrescine are found as characteristic components of cell wall peptidoglycan. The peptidoglycan type is directly cross-linked meso-diaminopimelic acid. Lipid F is also found as a characteristic cellular compound. The G+C content,of the DNA is 51 to 52 mol%. The type strain is strain DSM 20757 (= VB4b). The phenotypic properties of the type strain are shown in Table 2. The differentiation of Selenomonas lacticifex from the previously described Selenomonas species is shown in Table 4. Selenomonas lacticifex is distinguished from most other Selenomonas species by its ability to ferment glucose not only to acetic and propionic acids but also to lactic acid as major products. Moreover, the G+C content of its DNA is distinctly lower. Only Selenomonas ruminantium subsp. lactilytica also produces major amounts of lactic acid and has a DNA G+C content similar to that of Selenomonas lacticifex. Selenomonas lacticifex can be distinguished from this subspecies by its inability to utilize mannitol and dulcitol. Description of Zymophilus gen. nov. Zymophilus (Zy.mo’phi.lus. Gr.n. zyme, leaven, yeast; Gr. adj. philus, lover; M.L. masc. n. Zyrnophilus, yeast lover). Slightly curved to helical rods, 0.7 to 1.0 by 3 to 30 pm, occurring singly, in pairs, or in short chains. No resting stages are known. Gram negative. Motile. Mobility can be lost after several subcultivations. Strictly anaerobic. Chemoorganotrophic, having a fermentative type of metabolism. Glucose is fermented to acetic and propionic acids. These organisms occur in pitching yeast. Cadaverine and rarely putrescine are found as characteristic components of cell wall peptidoglycan. The peptidoglycan type is directly cross-linked mesodiaminopimelic acid. Lipid F is found as a characteristic cellular compound. The G+C content of the DNA is 38 to 41 mol%. All of the organisms belonging to this new genus were isolated from pitching yeast or brewery waste. The type species is Zymophilus rafinosivorans. Major differences between Zymophilus and other similar genera are shown in Table 5. Description of Zymophilus racffinosivorans sp. nov. Zymophilus rafinosivorans (raf.fi.no.si’vo.rans . M.L. n. rafinosum, raffinose; L.v. vorare, to eat. M.L. part. adj. rafinosivorans, raffinose devouring). The description below is based on 10 strains that were isolated from pitching yeast and four strains (strains OWS, OW9, OW10/1, and OW10/2) that were isolated from brewery waste. These organisms are obligately anaerobic, motile, nonsporeforming, gram-nega- TABLE 5. Characteristics of the genus Zymophilus and other gram-negative, nonsporeforming, anaerobic genera Genus dr::‘ Zymophilus Pectinatus Selenomonas Megasphaera a +, Positive reaction; Products of glucose or fructose fermentationa G+C cocci + + +- - + Acetate Acetoin Butyrate + + + - + - ND - + (+> - Lactate - - or - + Succinate + + + - (+> ____ -, negative reaction; Propionate ________~ (+), minor products; ND, not determined. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 01:35:29 - Valerate caprateand - + ‘Ontent (mol%) 38-41 38-41 48-58 4453 VOL. 40, 1990 ROD-SHAPED BACTERIA FROM BREWERIES tive rods. Cells are straight to slightly curved and occur predominantly as single cells or sometimes in pairs or short chains. They are 0.7 to 0.9 by 3 to 15 pm. Colonies on modified MRS agar medium are circular, smooth, opaque, slightly yellow, and 1 to 2 mm in diameter after 3 days at 30°C. The optimum temperature for growth is 30°C. Glucose is fermented to acetic and propionic acids. Phenotypic characteristics of the different group I11 strains are shown in Table 2. The G+C content of the DNA is 38 to 41 mol%. The type strain is strain DSM 20765 (= SH2). The phenotypic properties of the type strain are shown in Table 2. Descriptionof Zymophilus paucivorans sp. nov. Zymophilus paucivorans (pau.ci’vo.rans. L. adj. paucus, little; L. v. vorare, to eat. ; M . L . adj. paucivorans, utilizing relatively few carbohydrates). The description below is based on four strains that were isolated from pitching yeast. These organisms are obligately anaerobic, motile, nonsporeforming, gram-negative rods. The cells are curved or helically shaped and occur singly, in pairs, or in short chains. They are 0.8 to 1.0 by 5 to 30 pm. In two of the strains motility disappeared after several subcultivations. Colonies on modified MRS agar medium are circular, smooth, slightly yellow, and 1 to 2 mm in diameter after 3 days at 30°C. Glucose is fermented to acetic and propionic acids and trace amounts of lactic acid. Phenotypic characteristics of the different group IIIa strains are shown in Table 2. Cadaverine and rarely putrescine are found as characteristic components of the cell wall peptidoglycan. The peptidoglycan type is directly cross-linked mesodiaminopimelic acid, Lipid F is also found as a characteristic cellular compound. The G+C content of the DNA is 39 to 41 mol%. The type strain is strain DSM 20756 (= AA1). The phenotypic properties of the type strain are shown in Table 2. 2. ra$nosivorans and 2. paucivorans are closely related species with homology values of 44 to 48%. However, these organisms are phenotypically so different that separation into two species is warranted. These organisms are not only morphologically quite different, but they also differ in the utilization of xylose, rhamnose, xylitol, raffinose, melibiose, and inositol (Tables 2 and 3). LITERATURE CITED Back, W., N. Weiss, and H. Seidel. 1979. Isolierung und systematische Zuordnung bierschadlicher Bakterien. 11. Gram-negative anaerobe Stabchen. Brauwissenschaft 32:233-238. Collins, M. D., T. Pirouz, M. Goodfellow, and D. E. Minnikin. 1977. Distribution of menaquinones in actinomycetes and Corynebacteria. J. Gen. Microbiol. 100:221-230. Dams, E., L. Hendriks, Y. van de Peer, J.-M. Neep, G. Smits, I. Vandenbempt, and R. de Wachter. 1988. Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res. 16: 1-87-199. De Borde, D. C., C. W. Neave, M. L. Herlocher, and H. F. Massab. 1986. Resolution of a common sequencing ambiguity by 27 terminal deoxynucleotidyl transferase. Anal. Biochem. 157: 275-282. 5 . De Ley, J., H. Cattoir, and A. Reynaerts. 1970. 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Presumed Pectinatus strains isolated from Japanese beer. Bull. Brew. Sci. 28:ll-14. 23. Weiss, N., H. Seidel, and W. Back. 1979. Isolierung und systematische Zuordnung bierschadlicher Bakterien. I. Gram-negative, strikt anaerobe Kokken. Brauwissenschaft 32:189-194. 24. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51: 22 1-27 1. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 01:35:29