Numerical survey of some bacterial taxa

Focht, D. D. (Iowa State University, Ames), and W. R. Lockhart. Numerical survey of some bacterial taxa. J. Bacteriol. 90:1314-1319. 1965.-A numerical analysis was made of 77 properties of each of 43 bacterial strains, representing 25 genera from 8 families in the orders Eubacteriales and Pseudomonadales. Four major groups were found, related to one another at approximately the same level of similarity: (1) a large cluster containing the subgroups (1a) Athiorhodaceae-Spirillaceae, (1b) Xanthomonas, and (1c) "inactive" Micrococcaceae-Achromobacteraceae; (2) a cluster containing the "active" Micrococcaceae and Lactobacillaceae; (3) the enterobacteria; and (4) Aeromonas. There was a sharp distinction between the branches of groups 1a, 1c, and 2. The composition of groups was essentially the same whether or not fermentation of carbohydrates (28 characters) was included in the analysis. Several individual strains, notably, Bacillus subtilis, Leuconostoc mesenteroides, Pseudomonas aeruginosa, and Erwinia amylovora, were related to none of the groups, and others (two species of Proteus, Flavobacterium devorans, and Lactobacillus casei) showed only minimal quantitative relationships with their groups. These results suggest that there may be significant variation in levels of similarity within microbial groups presently accorded equivalent taxonomic rank, and that some present distinctions among taxa, particularly at the generic level, cannot be confirmed on the basis of overall similarity.     

Antibiotic sensitivity and other properties of bacteria of the genus Aeromonas

Antibiotic sensitivity and other biological properties of Aeromonas and NAD-vibrios isolated from water of open waterbodies were studied comparatively. It was found that the organisms of these genera had a definite similarity with respect to antibiotic sensitivity, morphological, cultural and some antigenic properties. The differences in the antibiotic sensitivity of these organisms were not sufficient for definition of the taxonomic features.     

Phylogenetic relationship of the fish pathogenic Renibacterium salmoninarum to Arthrobacter, Micrococcus and related taxa

Abstract The phylogenetic position of the obligate fish pathogenic Gram-positive eubacterium Renibacterium salmoninarum was determined by the 16S ribosomal RNA cataloguing approach. Comparison of the catalogue to those of more than 165 Gram-positive organisms from 50 genera revealed that R. salmoninarum is a member of the actinomycetes subdivision. As derived from similarity coefficients (S_AB values), this organism is related to a subgroup harbouring morphologically and chemotaxonomically rather heterogeneous taxa, including Anthrobacter, Micrococcus, Cellulomonas, Jonesia, Promicromonospora, Stomatococcus and Brevibacterium . Thus, the inclusion of organisms characterized by regular rods increases the phenotypic variability of this phylogenetically coherent group.     

Pollution indicator bacteria associated with municipal raw and drinking water supplies.

A total of 3819 bacterial cultures isolated from municipal water samples were identified using a combination of Enterotubules and confirmatory media. Frequency distributions for the different genera or groups of bacteria were similar for raw water and drinking water isolations, except for Escherichia organisms which doubled their frequency in raw water. Differences between the membrane filter (MF) and presence-absence (P-A) test with regard to types of organisms isolated were limited to Klebsiella organisms which were preferentially cultured from MF plates. Members of the genus Enterobacter were isolated more than twice as frequently as any of the other coliform genera dealt with in this study. Aeromonas organisms were detected almost as often as such individual genera as Escherichia, Citrobacter, or Klebsiella. Although non-lactose fermenting colonies (false-negatives) of the coliform genera would not be detected by the MF technique, their lack of detection would likely be offset by the Aeromonas colonies (false-positives). At least 25% of the coliform isolates were either anaerogenic or non-lactose fermenters and would therefore go undetected by the most probably number (MPN) technique.     

Assessment of genetic variability and relatedness among atypical Aeromonas salmonicida from marine fishes,using AFLP-fingerprinting

Atypical strains of Aeromonas salmonicida are the causal agent of atypical furunculosis or ulcer disease in various fish species, including spotted wolffish Anarhichas minor, which is a promising species in the Norwegian fish-farming industry. Isolates of atypical A. salmonicida comprise a very heterogenous group showing large variety in biochemical, molecular and virulence characteristics. The genetic variability among atypical isolates from wolffish was characterised using amplified fragment length polymorphism analysis: AFLP-fingerprinting. Additional isolates from halibut Hippoglossus hippoglossus, turbot Scophthalmus maximus, cod Gadus morhua and several salmonid fishes were included for assessment of variability and relatedness among a total of 56 atypical isolates of A. salmonicida. They were compared to reference strains of A. salmonicida subspecies and to other Aeromonas species pathogenic in fishes. AFLP-fingerprints subjected to similarity analysis yielded a grouping of the isolates into several clusters, revealing genetic heterogeneity among the isolates. There seems to be a correlation between genetic similarity among isolates and the fish host. The Icelandic isolates, mainly from cod, formed a very homogeneous subcluster, which was closely related to the wolffish isolates. All atypical isolates from spotted and common wolffish grouped together in a large cluster and appear to be very homogeneous, even though they had been isolated over a period of 8 yr at different locations in Norway. On the other hand, most of the isolates from turbot and halibut grouped together into 2 different clusters, while the 9 atypical isolates from salmonids appeared in 4 different clusters. Thus, the atypical isolates of A. salmonicida from halibut, turbot and salmonid fishes seem to be more genetically diverse than those from wolffish and cod.     

Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria.

Strain CS1T (T = type strain) is a gram-negative, microaerophilic, urease-positive, spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CS1T were isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CS1T was 42.5 mol%. The 16S rRNA sequences of strain CS1T, strain DS3 (a spiral-shaped isolate from a dog), and Helicobacter mustelae were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16S rRNA sequences of Helicobacter pylori, "Flexispira rappini," Wolinella succinogenes, and 11 species of campylobacters. A dendrogram was constructed based upon sequence similarities. Strains CS1T and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed; one group consisted of strains CS1T and DS3, H. mustelae, H. pylori, "F. rappini," and W. succinogenes, and the other group contained the true campylobacters. The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CS1T and DS3, H. pylori, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5%. The phylogenetic positions of W. succinogenes and "F. rappini" were just outside this cluster. On the basis of the results of this study, we believe that strains CS1T (= ATCC 49179T) and DS3 represent a new species of the genus Helicobacter, for which we propose the name Helicobacter felis.     

Polymorphism of Aeromonas spp. tRNA intergenic spacers

We investigated the length polymorphism of the intergenic spacers lying between tRNA genes of Aeromonas spp. A total of 69 strains representing all known genomic species of Aeromonas were used in the study. tDNA-PCR patterns were examined by Dice coefficient (S(D)) and unweighted pair group method of clustering (UPGMA). The strains were allocated into 15 groups at a similarity level of 70%. The strains belonging to seven genomic species: A. hydrophila (HG 1), A. caviae (HG 4), A. sobria (HG 7), A. veronii (HG 8/10), A. encheleia (HG 16), A. popoffii (HG 17), and A. culicicola (HG 18) formed distinct clusters. Our study revealed a genetic heterogeneity of the following species: A. bestiarum, A. salmonicida, A. media, A. eucrenophila, A. jandaei, A. schubertii, and A. allosaccharophila.     

Phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas as determined by 16S ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridizations.

The phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas were investigated by using small-subunit ribosomal DNA (rDNA) sequencing. Members of the genus Aeromonas formed a distinct line within the gamma subclass of the Proteobacteria. Plesiomonas shigelloides also clustered within the confines of the gamma subclass of the Proteobacteria but exhibited a closer association with members of the family Enterobacteriaceae than with members of the family Aeromonadaceae. Species of the genus Aeromonas exhibited very high levels of overall sequence similarity (ca. 98 to 100%) with each other. Several of the relationships derived from an analysis of the rDNA sequence data were in marked disagreement with the results of chromosomal DNA-DNA pairing experiments. Diagnostic rDNA signatures that have possible value for differentiating most Aeromonas species were discerned.     

A Serological Approach to the Genus Pseudomonas

S ummary : Serological methods are described by which organisms of the genus Pseudomonas and related genera can be compared. More specific relationships are demonstrated by agglutination techniques or agar diffusion with trichloroacetic acid extracts; less specific relationships are shown by agar diffusion with disintegrated cell preparations. Up to three strain specific antigens may be found and at least six antigens are widely distributed amongst strains of the genera Pseudomonas, Achromobacter, Vibrio and Aeromonas .
The results obtained so far indicate little or no agreement with any accepted taxo-nomic divisions. Evidence is presented which suggests that most of the antigens detectable by agar diffusion techniques are polysaccharide in nature rather than protein.     

A Further Numerical Taxonomic Study of the Rhodochrous Group

A computer taxonomy was carried out as an aid to clarify the relationships between the genera Mycobacterium, and Nocardia as well as the ‘Rhodochrous’ group. The last group contained slightly acid-fast organisms isolated from sputa and soil and also included strains received as “Mycobacterium rhodochrous.” The group had been proposed as the genus Gordona by the present author. The rhodochrous group formed a cluster at the 87% similarity level and was clearly separated from all strains of the genera Mycobacterium and Nocardia. The cluster of the rhodochrous group was divided into seven subclusters at the 90% similarity level, and six of the seven corresponded to six species of the genus Gordona: G. bronchialis; G. rubropertincta (synonym G. rubra); G. terrae; G. aurantiaca; G. rhodochroa; G. rosea. If one accepts the concept that a phenetically well-defined cluster is a species, each of the subclusters of the rhodochrous group should be regarded as a species and the entire group as a genus. Discrepancies between the results of numerical classification and the results of lipid analyses remain as a problem to be solved in the future.     

Ribosomal ribonucleic acid similarities in the classification of Rhodococcus and related taxa

Duplexes were prepared between 14C-labelled rRNA from both Rhodococcus equi C7 and Rhodococcus rhodochrous N54 and DNA from 16 actinomycetes representing the genera Rhodococcus, Mycobacterium, Nocardia, Saccharopolyspora and Streptomyces. The relationships between the organisms were determined by plotting the temperature at which 50% of the duplex was denatured (Tm(e)) against the percentage of rRNA binding (microgram 14C-labelled rRNA duplexed per 100 micrograms filter-bound DNA). All of the strains formed stable duplexes but each organism occupied a definite area on the rRNA similarity map. All of the organisms share a close phylogenetic relationship but representatives of the genera Rhodococcus, Mycobacterium, Nocardia and Streptomyces fell into four recognizable clusters on the similarity map. These data support and extend current trends in the classification of Rhodococcus and allied taxa. The guanine plus cytosine content of the DNA from the test strains was within the range 69.3 to 76.9 mol %.     

Molecular phylogeny of the volvocine flagellates.

Phylogenetic studies of approximately 2,000 bases of sequence from the large and small nuclear-encoded ribosomal RNAs are used to investigate the origins of the genus Volvox. The colonial and multicellular genera currently placed in the family Volvocaceae form a monophyletic group that is significantly closer phylogenetically to Chlamydomonas reinhardtii than it is to the other unicellular green flagellates that were tested, including Chlamydomonas eugametos, Chlorella pyrenoidosa, and Haematococcus lacustris. Statistical analysis of 251 phylogenetically informative nucleotide positions rejects the "volvocine lineage" hypothesis, which postulates a monophyletic evolutionary progression from unicellular organisms (such as Chlamydomonas), through colonial organisms (e.g., Gonium, Pandorina, Eudorina, and Pleodorina) demonstrating increasing size, cell number, and tendency toward cellular differentiation, to multicellular organisms having fully differentiated somatic and reproductive cells (in the genus Volvox). The genus Volvox appears not to be monophyletic. Volvox capensis falls outside a lineage containing other representatives of Volvox (V. aureus, V. carteri, and V. obversus), and both of these Volvox lineages are more closely related to certain colonial genera than they are to each other. This implies either a diphyletic origin of Volvox from different colonial volvocacean ancestors, a phylogenetic derivation of some of the colonial genera from a multicellular (i.e., Volvox) ancestor, or both. Considered together with previously published observations, these results suggest that the different levels of organizational and developmental complexity found in the Volvocaceae represent alternative stable states, among which evolutionary transitions have occurred several times during the phylogenetic history of this group.     

Identification of the bacterial community of maple sap by using amplified ribosomal DNA (rDNA) restriction analysis and rDNA sequencing

The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the gamma- and beta-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). gamma-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control.     

Filamentous "Epsilonproteobacteria" dominate microbial mats from sulfidic cave springs

Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where microbial mats dominated by filamentous morphotypes are found. The full-cycle rRNA approach, including 16S rRNA gene retrieval and fluorescence in situ hybridization (FISH), was used to identify these filaments. The majority of the obtained 16S rRNA gene clones from the mats were affiliated with the "Epsilonproteobacteria" and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was closely related to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic springs, and sulfidic caves (97 to 99% sequence similarity), while group II formed a novel clade moderately related to deep-sea hydrothermal vent symbionts (90 to 94% sequence similarity). FISH with newly designed probes for both groups specifically stained filamentous bacteria within the mats. FISH-based quantification of the two filament groups in six different microbial mat samples from Lower Kane Cave showed that LKC group II dominated five of the six mat communities. This study further expands our perceptions of the diversity and geographic distribution of "Epsilonproteobacteria" in extreme environments and demonstrates their biogeochemical importance in subterranean ecosystems.     

Identification of Vibrio cholerae by Pyrolysis Gas-Liquid Chromatography

Cholera-like vibrios examined by pyrolysis gas-liquid chromatography could be distinguished from other common aerobic gram-negative bacilli, including oxidase-positive organisms, e.g., Aeromonas. Vibrios in Heiberg group I were subdivided into three types on the basis of differences in one complex in the chromatogram, and these closely corresponded with the identification as classical, El Tor, or "intermediate" biotypes of Vibrio cholerae by conventional methods.     

Regiocrella, a new entomopathogenic genus with a pycnidial anamorph and its phylogenetic placement in the Clavicipitaceae

A new genus, Regiocrella, is described with two species, R. camerunensis and R. sinensis, based on specimens collected in Cameroon and China. Both species are parasitic on scale insects (Coccidae, Homoptera). Morphological and molecular evidence place the new genus in the Clavicipitaceae (Hypocreales), despite its combination of characters that are atypical of that family; Regiocrella is characterized by having perithecia partly immersed in a subiculum, noncapitate asci, unicellular fusiform ascospores and pycnidial-acervular conidiomata. The two new species, R. camerunensis and R. sinensis, are distinguished based on ascospore and perithecium size. Morphological characters were evaluated and compared to other genera in the Clavicipitaceae, especially those parasitic on scale insects or with pycnidial-acervular anamorphs or synanamorphs (i.e. Aschersonia, Ephelis or Sphacelia): Atkinsonella, Balansia, Claviceps, Epichl?e, Hypocrella, Myriogenospora and Neoclaviceps. The phylogenetic relationships of Regiocrella were examined with three gene loci: large subunit nuclear ribosomal DNA (LSU), translation elongation factor 1-alpha (TEF), and RNA polymerase II subunit 1 (RPB1). The results of this study confirm that Regiocrella is distinct from other genera in the Clavicipitaceae and that its two species form a monophyletic group. Regiocrella is shown to be closely related to the scale insect pathogen Hypocrella and the plant-associated genera Balansia, Claviceps, Epichl?e, Myriogenospora and Neoclaviceps. This study also provides insights into the evolution of pycnidial-acervular conidiomata and scale insect parasitism within the Clavicipitaceae. Plant-associated genera form a monophyletic group correlated with Clavicipitaceae subfamily Clavicipitoideae sensu Diehl. We also demonstrate that scale insect parasites have multiple evolutionary origins within the family and genera with pycnidial-acervular anamorphs or synanamorphs have a single origin.     

TRADITIONAL GENERIC CONCEPTS VERSUS 18S rRNA GENE PHYLOGENY IN THE GREEN ALGAL FAMILY SELENASTRACEAE (CHLOROPHYCEAE, CHLOROPHYTA)

Coccoid green algae of the Selenastraceae were investigated by means of light microscopy, TEM, and 18S rRNA analyses to evaluate the generic concept in this family. Phylogenetic trees inferred from the 18S rRNA gene sequences showed that the studied species of autosporic Selenastraceae formed a well-resolved monophyletic clade within the DO group of Chlorophyceae. Several morphological characteristics that are traditionally used as generic features were investigated, especially the arrangement of autospores in the mother cells, colony formation, and pyrenoid structure. The parallel arrangement of autospores was confirmed for the genera Ankistrodesmus , Podohedriella , and Quadrigula. In mother cells of Monoraphidium and Kirchneriella , the autospores were arranged serially. Colony formation was either stable ( Quadrigula ) or variable ( Ankistrodesmus , Podohedriella ) within genera. All strains studied possessed naked or starch-covered pyrenoids within the chloroplast. The pyrenoid matrix was homogenous or penetrated by thylakoids. In contrast to considerations of traditional systematics, the present study showed that the presence and structure of pyrenoids are unsuitable for differentiation of genera in Selenastraceae. Furthermore, the molecular analyses showed that any morphological criterion considered so far is not significant for the systematics of the Selenastraceae on the generic level. Species assigned to different genera such as Ankistrodesmus and Monoraphidium were not monophyletic and therefore not distinguishable as separate genera. Species of Monoraphidium appeared in four different lineages of the Selenastraceae. Our phylogenetic analyses support earlier discussions to abandon the common practice of conceiving "small" genera (i.e. genera that are differentiated from other genera by only a few diacritic characteristics and that contain only a small number of species) and to reestablish "large" genera of Selenastraceae such as Ankistrodesmus.     

Antibiotic resistance of vibrio cholerae: special considerations of R-plasmids

Studies on the transmission of R plasmid by conjugation between enterobacteria and vibrio or related bacteria were reviewed. The majority of the reports confirmed successful transmission from enterobacteria to Vibrio cholerae and related species, although the transmission frequencies were extremely low and the transmitted R plasmid was very unstable except for thermosensitive kanamycin plasmid and usual R plasmid coexisting with P plasmid. Strains of V. cholerae and Aeromonas liquefaciens as well as A. salmonicida bearing R plasmid were detected in nature. R plasmid was relatively unstable in V. cholerae strains with which transmission of R plasmid to enterobacteria was confirmed. At present, only 3 R plasmids have been obtained from naturally occurring strains of V. cholerae. Although the 2 European plasmids belong to the C incompatibility group with 98 megadalton closed covalent circular DNA molecule, one plasmid belongs to the J group with more than 25 megadalton molecular weight, and no CCC of satelite DNA was detected in bacteria harboring this plasmid.     

Unusual sulfonolipids are characteristic of the Cytophaga-Flexibacter group

Capnocytophaga spp. contain a group of unusual sulfonolipids, called capnoids (W. Godchaux III and E. R. Leadbetter, J. Bacteriol. 144:592-602, 1980). One of these lipids, capnine, is 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid; the others are, apparently, N-acylated versions of capnine. The lipids were found, in amounts ranging from 2.5 to 16 mumol of capnoid sulfur per g of cells (wet weight), in two Cytophaga spp. and also in several closely related organisms: several Capnocytophaga spp., Sporocytophaga myxococcoides, two Flexibacter spp., and two Flavobacterium spp. With the exception of the flavobacteria, all of these bacteria have been shown to exhibit gliding motility. The two Flavobacterium spp. belong to a subset of that genus that shares many other characteristics with the cytophagas. Only the Capnocytophaga spp. contained large quantities of capnine as such; in all of the others, most (and possibly all) of the capnoids were present as N-acylcapnines. Capnoid-negative bacteria included some gliding organisms that may not be closely related to the cytophagas: two fruiting myxobacters, a gliding cyanobacterium (Plectonema sp.), Beggiatoa alba, Vitreoscilla stercoraria, Herpetosiphon aurantiacus, and Lysobacter enzymogenes. Nongliding bacteria representing nine genera were also tested, and all of these fell into the capnoid-negative group.     

Aeromonas proteolytica--a halophilic aeromonad?

Aeromonas proteolytica, a halophilic bacterium originally isolated from the intestine of a marine isopod, was compared with 93 strains of Aeromonas hydrophila and representative strains of five similar genera in a computer and serological analysis. Aeromonas proteolytica was peritrichate and non-reactive with six A. hydrohila antisera, and possessed a percentage guanine + cytosine ratio (49.5) outside the range for other aeromonads (55-62%). Moreover, in the computer analysis it shared a lower level of similarity with other aeromonads than with both strains of Vibrio anguillarum tested.