- Volume 41, Issue 2, 1991

A total of 108 pectolytic, soft-rotting Erwinia strains were collected from 11 types of cacti growing in Arizona, Texas, northern Mexico, and Australia between 1958 and 1989. Four strains were collected from soils beneath or close to naturally rotting saguaro cacti. Collectively, these strains caused soft rots of saguaro, organ pipe, and senita cacti, Opuntia (cactus) fruits and pads, tomato fruits, and potato slices, but only occasionally caused soft rots of slices of carrot roots. A numerical cluster analysis showed that 98 of the 112 strains formed a uniform group (cluster 1A) that was distinguished from other pectolytic erwinias by an API 20E code of 1205131, by negative reactions in API 50CHE tests for l-arabinose, myo-inositol, d-cellobiose, melibiose, and d-raffinose, and, in supplemental tests, by positive reactions for malonate and growth at 43°C. The average levels of DNA relatedness of 22 cluster 1A strains to the proposed type strain (strain 1-12) as determined by the hydroxyapatite method were 88% in 60°C reactions (with 1% divergence within related sequences) and 87% in 75°C reactions. The levels of relatedness to the type strains of other Erwinia spp. were <38% in 75°C reactions. Cluster 1A strains also had a characteristic cellular fatty acid profile containing cyclo-(11,12)-nonadecanoic acid (C19:0 Cyclo C11-12) and missing tridecanoic acid (C13:0), heptadecanoic acid (C17:0), and cis-9-heptadecenoic acid (C17:1 CIS 9), which separated them from other pectolytic erwinias. Collectively, these data indicate that the members of cluster 1A are members of a new species, which we name Erwinia cacticida. Three cactus strains in cluster 1B appear to represent a second new species that is closely related to E. cacticida; these strains are designated E. cacticida-like pending the availability of additional strains for testing. The remaining cactus strains (in cluster 4) have the physiological, DNA, and fatty acid profiles of Erwinia carotovora.

Lipopolysaccharides (LPSs) from a number of bacteria belonging to the alpha-2 subgroup of the class Proteobacteria were screened for the presence of 27-hydroxy-octacosanoic acid (27-OH-28:0). With few exceptions, most of the bacteria contained 27-OH-28:0 in their lipid A fractions. In addition, some of the bacteria contained other n-2-hydroxylated long-chain fatty acids hitherto not reported. The distribution of 27-OH-28:0 was restricted to the alpha-2 subgroup. LPSs from members of the other subgroups (the beta and gamma subgroups), including some well-characterized enterobacterial LPSs, were devoid of 27-OH-28:0. Our results indicate that the presence of n-2-hydroxylated long-chain fatty acids in LPSs might be used as a chemophylogenetical marker.

Although the nonfermentative, asaccharolytic, putative anaerobes Wolinella curva, Wolinella recta, Bacteroides ureolyticus, and Bacteroides gracilis are phylogenetically related to the true campylobacters, the type strains of these species exhibited O2-dependent microaerophilic growth in brucella broth and on brucella agar. The optimum O2 levels for growth of these strains ranged from 4 to 14% in brucella broth and from 2 to 8% on brucella agar, when H2 was provided as the electron donor. No growth occurred under 21% O2, and scant or no growth occurred under anaerobic conditions unless fumarate or nitrate was provided as a terminal electron acceptor. Aspartate, asparagine, and malate also served as apparent electron acceptors. The organisms were catalase negative and, except for B. gracilis, oxidase positive. Catalase added to brucella broth enhanced growth. O2 uptake by all species was inhibited by cyanide and 2-heptyl-4-hydroxyquinoline N-oxide. We concluded that these organisms are not anaerobes but instead are microaerophiles, like their campylobacter relatives.

A total of 58 strains belonging to phylogenetically assigned or unassigned species of the class Proteobacteria, which are mostly phytopathogenic or might interact with plants, were analyzed for polyamines. The strains of the genus Xanthomonas contained spermidine as the main polyamine. Putrescine was the main polyamine of phytopathogenic strains belonging to the Pseudomonas fluorescens complex, which represents the phylogenetically defined nucleus of the genus Pseudomonas. The genera Azotobacter and Azomonas, which include free-living diazotrophic bacteria that also belong to the P. fluorescens complex, had a polyamine pattern like that of the strains belonging to the P. fluorescens complex. Xylophilus ampelinus (formerly called Xanthomonas ampelina) and unassigned phytopathogenic pseudomonads phylogenetically allocated to the beta subclass of the Proteobacteria contained 2-hydroxyputrescine, the specific polyamine of this subclass. The main polyamine in Rhizobium, Bradyrhizobium, and Phyllobacterium strains and misnamed pseudomonads belonging to the alpha subclass of the Proteobacteria was sym-homospermidine, which was also present in “Azotomonas fluorescens.”

A mycoplasma isolated from human spermatozoa and a human cervix was shown to be serologically distinct from 98 previously recognized Mycoplasma and Acholeplasma spp. Six mycoplasma colonies were cloned and examined in detail for morphology, growth, and biochemical characteristics; five of these were from sperm samples and one was from a cervix. These strains were closely related and had the following properties: Guanine-plus-cytosine content of 32 mol%, requirement for sterol, and anaerobic growth. Glucose was not metabolized, and arginine and urea were not hydrolyzed. Strain AH159 (= NCTC 11720) is the type strain of a new species, Mycoplasma spermatophilum.

Actinomycete ribosomal protein AT-L30 exhibits electrophoretic mobility that is specific for each genus. On the basis of this fact, we analyzed ribosomal AT-L30 proteins from 26 type strains of species belonging to the genera Actinomadura and Microtetraspora. The electrophoretic mobilities of AT-L30 preparations from these strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that they could be divided into two groups, one group with relative electrophoretic mobilities of 14.0 to 41.5 and another group with relative electrophoretic mobilities of -6.5 to 0. The first group corresponded to the genus Actinomadura, and the second group corresponded to the genus Microtetraspora. Partial amino acid sequencing of AT-L30 preparations from several strains proved that we were indeed dealing with the specified protein homologous to ribosomal protein L30 of Escherichia coli. Our results strongly supported the conclusions of previous work and thus proved the efficacy of ribosomal protein analysis as a novel approach for taxonomy of actinomycetes.

The phylogenetic interrelationships of members of the genus Listeria were investigated by using reverse transcriptase sequencing of 16S rRNA. The sequence data indicate that at the intrageneric level the genus Listeria consists of the following two closely related but distinct lines of descent: (i) the Listeria monocytogenes group of species (including Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) and (ii) the species Listeria grayi and Listeria murrayi. At the intergeneric level a specific phylogenetic relationship between the genera Listeria and Brochothrix was evident. The sequence data clearly demonstrated that the genus Listeria is phylogenetically remote from the genus Lactobacillus and should not be included in an extended family Lactobacillaceae.

The DNA base composition of Rickettsia tsutsugamushi was determined by reversed-phase high-performance liquid chromatography and compared with that of Rickettsia rickettsii. The G+C contents were 28.1 to 30.5 mol% for R. tsutsugamushi and 32.1 mol% for R. rickettsii.

The results of DNA-DNA hybridization and chemotaxonomic studies indicated that the glutamic acid producers Brevibacterium divaricatum DSM 20297T (T = type strain), “Brevibacterium flavum” DSM 20411, “Brevibacterium lactofermentum” DSM 1412 and DSM 20412, Corynebacterium lilium DSM 20137T, and Corynebacterium glutamicum DSM 20300T and DSM 20163 are members of the same species. It is proposed that all of these strains should be classified in the species Corynebacterium glutamicum. Another glutamic acid-producing strain, Corynebacterium callunae DSM 20147T, was not related at the species level to C. glutamicum and should retain its separate species status. A restriction fragment length polymorphism analysis in which oligonucleotides targeted against conserved regions of 16S and 23S rRNA genes were used as hybridizing probes distinguished the individual strains. This method may be a helpful tool for strain identification.

Lactobacillus uli sp. nov. and Lactobacillus rimae sp. nov. are described. These organisms are short, gram-positive, strictly anaerobic, rod-shaped bacteria that have DNA G+C contents of 53 and 45 mol%, respectively, produce major amounts of lactic acid, and have been isolated from human gingival crevices and periodontal pockets. The major cellular fatty acid derivatives for both species are C18:1 cis-9 fatty acid methyl ester and C18:1 cis-9 dimethylacetyl. The type strain of L. uli is strain VPI D76D-27C (= ATCC 49627), and the type strain of L. rimae is strain D140H-11A (= ATCC 49626). Emended descriptions of Lactobacillus minutus (based on selected strains) and Streptococcus parvulus (based on many additional strains) also are given.

Members of the genus Methanosarcina are recognized as major aceticlastic methanogens, and several species which thrive in low-salt, pH-neutral culture medium at mesophilic temperatures have been described. However, the environmental conditions which support the fastest growth of these species (Methanosarcina barkeri MST [T = type strain] and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T) have not been reported previously. Although the members of the genus Methanosarcina are widely assumed to grow best at pH values near neutrality, we found that some strains prefer acidic pH values. M. vacuolata and the two strains of M. barkeri which we tested were acidophilic when they were grown on H2 plus methanol, growing most rapidly at pH 5 and growing at pH values as low as 4.3. M. mazei grew best at pH values near neutrality. We found that all of the strains tested grew most rapidly at 37 to 42°C on all of the growth substrates which we tested. None of the strains was strongly halophilic, although the growth of some strains was slightly stimulated by small amounts of added NaCl. The catabolic substrates which supported most rapid growth were H2 plus methanol; this combination sometimes allowed growth of a strain under extreme environmental conditions which prevented growth on other substrates. The cell morphology of all strains was affected by growth conditions.

Nine bacterial strains isolated from root nodules of Astragalus sinicus were compared with 41 reference strains, including the type strains of the type species of the genera Rhizobium, Bradyrhizobium, and Agrobacterium, by performing a numerical analysis of 200 phenotype features. Representative strains belonging to different clusters were further compared with similar bacteria by using data from gel electrophoresis of whole-cell proteins, DNA G+C content data, and DNA-DNA hybridization data. The rhizobial strains isolated from nodules of A. sinicus constitute a distinct homology group that is quite different from previously described Rhizobium, Bradyrhizobium, and Agrobacterium species and from strains isolated from other Astragalus species. We propose the name Rhizobium huakuii sp. nov. for the strains isolated from A. sinicus. Type strain CCBAU 2609 (= 103) has been deposited in the Culture Collection of Beijing Agricultural University, Beijing, People’s Republic of China.

A new thermophilic, cellulolytic species of the genus Clostridium was isolated from riverside mud. The guanine-plus-cytosine content of its DNA is 34 mol%. The isolate ferments a wide range of carbohydrates. Its major fermentation products are ethanol, butanol, acetate, butyrate, lactate, carbon dioxide, and hydrogen. Hydrogen sulfide is also produced. The name Clostridium thermopapyrolyticum is proposed. The type strain has been deposited in the South American Biotechnology and Applied Microbiology microbiological resource center (United Nations Educational, Scientific, and Cultural Organization) culture collection, Tucumán, Argentina, as UBA 305.

Two major subspecies of Staphylococcus cohnii, namely S. cohnii subsp. cohnii, from humans, and S. cohnii subsp. urealyticum, from humans and other primates, are described on the basis of a study of 14 to 25 strains and 18 to 33 strains, respectively. DNA-DNA hybridization studies conducted in our laboratory in 1983 (W. E. Kloos and J. F. Wolfshohl, Curr. Microbiol. 8:115-121, 1983) demonstrated that strains representing the different subspecies were significantly divergent. S. cohnii subsp. urealyticum can be distinguished from S. cohnii subsp. cohnii on the basis of its greater colony size; pigmentation; positive urease, β-glucuronidase, and β-galactosidase activities; delayed alkaline phosphatase activity; ability to produce acid aerobically from α-lactose; and fatty acid profile. The type strain of S. cohnii subsp. cohnii is ATCC 29974, the designated type strain of S. cohnii Schleifer and Kloos 1975b, 55. The type strain of S. cohnii subsp. urealyticum is ATCC 49330.

A group of 11 strains, mostly isolated from sewage water in the Province of Navarra, Spain, were found to constitute a DNA relatedness group which is 2 to 39% related to 23 species of the genus Vibrio and 2 to 3% related to two Aeromonas species. Phenotypically, these strains have all of the properties that define the genus Vibrio. However, they differ from the previously described species by three or more properties. The strains are negative for arginine, ornithine, and lysine decarboxylase activities and the Voges-Proskauer test and are unable to utilize putrescine, gluconate, glucuronate, and histidine. They utilize and produce acid from sucrose and grow at 40°C. All strains grow in the presence of 0.5% (wt/vol) NaCl, and seven strains grow weakly in peptone water lacking NaCl. The group of strains which we studied can also be differentiated from other Vibrio species by fatty acid content. The G + C ratio of the DNA is 45 to 47 mol%. The name Vibrio navarrensis sp. nov. is proposed for these strains; strain 1397-6 (= CIP 103381) is the type strain.

The fatty acid profiles of 59 type strains representing genera, species, and serovars belonging to the family Leptospiraceae were investigated by using gas-liquid chromatography of fatty acid methyl ester (FAME) derivatives prepared from washed leptospires. The interstrain differences of the gas-liquid chromatography FAME profiles were quantified by performing a linear regression analysis in which we compared the FAME profiles of pairs of strains and expressed the results as correlation coefficients. Leptospiral strains could be differentiated into 17 FAME relatedness groups, which were characterized by both stringent similarities within a group and sharp differences between groups. In each group, a strain was selected as a FAME reference strain. FAME reference strains can be used as keys for a chemotaxonomic phenotypic classification of strains belonging to the family Leptospiraceae.

The DNAs of a variety of obligate methanotrophic bacteria were analyzed for base composition and nucleotide distribution. Genome molecular weights were determined for representative strains. Similarity maps attained by plotting DNA base composition versus nucleotide distribution and genome molecular weight showed that related species formed distinct clusters. Group I methanotrophs were found to form three clusters in both nucleotide distribution and genome size analyses. The first cluster consisted of five Methylomonas species, Methylomonas methanica, Methylomonas fodinarum, Methylomonas aurantiaca, “Methylomonas rubra,” and “Methylomonas agile.” The other clusters included both Methylomonas species and Methylococcus spcies, indicating the heterogeneity within these genera. One cluster contained low-G+C-content Methylococcus strains and included Methylococcus whittenburyi, Methylococcus bovis, Methylococcus vinelandii, Methylococcus luteus, and “Methylococcus ucraincus.” The type strains of Methylomonas pelagica and “Methylomonas alba” and a marine methanotrophic strain also clustered with the low-G+C-content Methylococcus group rather than with the genus Methylomonas. “Methylomonas gracilis” also appeared to be genetically distinct from the true Methylomonas species and clustered with the high-G+C-content Methylococcus strains. This cluster included Methylococcus capsulatus, Methylococcus thermophilus, and other moderately thermophilic group I methanotrophic strains. The group II methanotrophs belonging to the genera “Methylosinus” and “Methylocystis” formed separate generic clusters according to genome molecular weight data but not according to nucleotide distribution data.

Two cellulolytic clostridia, one thermophilic and the other mesophilic, were isolated and characterized. Cells of the thermophile are gram-negative rods that are motile with lophotrichous flagella and spherical terminal endospores which swell the cells. The optimum growth temperature is 55 to 60°C, with a range of 40 to 65°C. The deoxyribonucleic acid composition is 35 mol% G+C. The name Clostridium cellulosi sp. nov. is proposed. The type strain is AS 1.1777. Cells of the mesophile are gram negative and motile with peritrichous flagella and terminal oval or spherical spores which swell the cells. The deoxyribonucleic acid composition is 34 mol% G+C. The name Clostridium cellulofermentans sp. nov. is proposed. The type strain is AS 1.1775. Both C. cellulosi AS 1.1777 and C. cellulofermentans AS 1.1775 are deposited in the China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Academia Sinica, Beijing, People’s Republic of China.

DNA-rRNA hybridization results showed that members of the genus Moraxella, members of the genus Psychrobacter and their relatives, members of the genus Acinetobacter, the false neisseriae, and two misnamed Alysiella strains constitute a separate genotypic cluster. A new family, Moraxellaceae, is proposed to accommodate these organisms. The genus Moraxella is the type genus. Within the Moraxellaceae two main groups can be distinguished. One group includes the Acinetobacter species. The other group can be subdivided in four subgroups consisting of (i) the authentic moraxellae (Moraxella lacunata, Moraxella nonliquefaciens, Moraxella bovis, Moraxella catarrhalis, Moraxella caviae, Moraxella ovis, Moraxella cuniculi, and two misnamed Alysiella strains), (ii) the generically misnamed taxon Moraxella osloensis, (iii) the generically misnamed taxon Moraxella atlantae, and (iv) the generically misnamed taxon Moraxella phenylpyruvica, Psychrobacter immobilis, and allied organisms. The Moraxellaceae cluster belongs to the class Proteobacteria and is a member of superfamily II, which includes the authentic pseudomonads and related organisms. It is not related to the family Neisseriaceae.