Phage specificity and lipopolysaccarides of stem- and root-nodulating bacteria (Azorhizobium caulinodans, Sinorhizobium spp., and Rhizobium spp.) of Sesbania spp

Phage susceptibility pattern and its correlation with lipopolysaccharide (LPS) and plasmid profiles may help in understanding the phenotypic and genotypic diversity among highly promiscuous group of rhizobia nodulating Sesbania spp.; 43 phages were from two stem-nodulating bacteria of S. rostrata and 16 phages were from root-nodulating bacteria of S. sesban, S. aegyptica and S. rostrata. Phage susceptibility pattern of 38 Sesbania nodulating bacteria was correlated with their LPS rather than plasmid profiles. Different species of bacteria (A. caulinodans- ORS571, SRS1-3 and Sinorhizobium saheli- SRR907, SRR912) showing distinct LPS subtypes were susceptible to different group of phages. Phages could also discriminate the strains of Si. saheli (SSR312, SAR610) possessing distinct LPS subtypes. Phages of Si. meliloti (SSR302) were strain-specific. All the strains of R. huautlense having incomplete LPS (insignificant O-chain) were phage-resistant. In in vitro assay, 100% of the phages were adsorbed to LPS of indicator bacterium or its closely related strain(s) only. These observations suggest the significance of LPS in phage specificity of Sesbania nodulating rhizobia. Highly specific phages may serve as biological marker for monitoring the susceptible bacterial strains in culture collections and environment.     

Pyrolysis mass spectrometry characterisation and numerical taxonomy ofAeromonas spp.

Reference strains (2) and 29 isolates ofAeromonas spp. from clinical material and environmental specimens were characterised in traditional biochemical tests, and in pyrolysis mass spectrometry, which gives data reflecting whole-cell composition. Numerical taxonomic analyses of the data sets were compared with conventional identification at species level, and pathogenic potential, as inferred from the origin of the isolates. Clustering with conventional test reaction patterns showed, for each of the species represented, a clearly defined core group of typical isolates, surrounded by a halo of aberrant strains. One further cluster comprised strains intermediate betweenA. caviae andA. hydrophila, and one strain was grossly atypical in both analyses. Clustering from pyrolysis data corresponded less well with species identification. Broadly, the biochemical division between core and halo strains was supported in pyrolysis forA. caviae andA. sobria, but the main group ofA. hydrophila in pyrolysis comprised strains clustering in the core and halo groups of this species, and three strains intermediate betweenA. hydrophila andA. caviae in biochemical tests. Two further pyrolysis clusters comprised core and halo strains ofA. hydrophila. However, pyrolysis clustering correlated well with inferred pathogenicity, showing four clusters of probable pathogens, six clusters of probable nonpathogens, and one two member cluster of doubtful status. Most strains that clustered in the species haloes, or in species-intermediate groups in biochemical tests, were non-human isolates, or were isolated in the absence of symptomatic infection. The correlation of inferred pathogenicity with biochemical clustering was poorer than that with pyrolysis clustering.Abbreviations CTRP conventional test reaction pattern - PyMS pyrolysis mass spectrometry     

Species delimitation in the genusLipomyces by nuclear genome comparison

Species delimitation inLipomyces was attempted by nuclear genome comparison in conjuction with the re-evaluation of 48 physiological characters of 65 strains.High intraspecific (>75%) and low interspecific (<28%) similarity values established thatL. japonicus, L. lipofer andL. tetrasporus are genetically isolated, and also distinct fromL. kononenkoae andL. starkeyi.Ambiguous similarity values were obtained withL. kononenkoae andL. starkeyi. Strains previously assigned toL. kononenkoae constitute two related clusters. While similarity values within each cluster range from 76–99%, representatives of the two clusters reassociate for only 47%. Since these clusters are differentiated by their ecologically relevant maximum growth temperature,L. kononenkoae is subdivided. Strains previously assigned toL. starkeyi resolve into four closely related clusters. While similarity values within each cluster range from 78–100%, representatives of the four clusters reassociate for only 59–69%. Since these four clusters are poorly differentiated, the subdivision ofL. starkeyi does not appear possible without recourse to other criteria.Four unassigned strains constitute a further two clusters. Reassociation within these clusters is of the order of 91–100%, while reassociation between them occurs only at 59%. Reassociation of representatives of these clusters with those of theL. kononenkoae andL. starkeyi complexes is around 40% and 31%, respectively. These two clusters consequently appear to be intermediate betweenL. kononenkoae andL. starkeyi, and will, as such, have to be considered in any delimitation of these two species. A key to the taxa ofLipomyces and related genera of the Lipomycetaceae is given.     

Actinomycete diversity associated with foaming in activated sludge plants

Large numbers of mycolic acid-containing actinomycetes were isolated from foam and scum samples taken from three activated-sludge sewage-treatment plants using several selective isolation media. Organisms presumptively identified as gordonae formed the dominant population in all of the samples. A representative set of these strains have chemical properties consistent with their classification in the genusGordona. Forty-eight of theGordona strains were compared through 165 unit characters with the type strains of validly described species ofGordona. The resultant data were examined using the Jaccard and simple matching coefficients and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. The numerical classification was only marginally affected by the statistics used or by test error, estimated as 3.92%. The isolates were assigned to five multi-membered and 28 single-membered clusters defined by the simple matching coefficient at the 89% similarity level. With few exceptions, the isolates were sharply separated from theGordona marker strains. Essentially the same classification was obtained when the test strains were examined using a Curie-point pyrolysis mass spectrometric procedure. It can be concluded that the gordonae form a heterogeneous taxonomic group, the members of which can be distinguished from representatives of validly described species ofGordona.     

Systematics of theLactobacillus population on rat intestinal mucosa with special reference toLactobacillus reuteri

The systematics of theLactobacillus population of the intestines of 88 different rats was studied; 80 rats had been fed on fermented oat-meal soup (Molin et al. 1992). One-hundred-twenty-twoLactobacillus strains from the intestinal mucosa were phenotypically classified together with twenty-eight reference strains ofLactobacillus andLeuconostoc, using 49 unit characters. Data were examined using Jaccard coefficient, and unweighted pair group algorithm with arithmetic averages. Two major and eleven minor clusters were defined at the 76% S_J-similarity level: Cluster 1 included thirty isolates which could not be identified further, but had resemblance to the type strains ofL. jensenii, L. gasseri, L. crispatus, and to some extent toL. acidophilus. Cluster 12 including fifty-four intestinal isolates was identified asL. reuteri; and so was cluster 13 (five isolates). Isolates of the major clusters were found in all parts of the intestines. The genomic homogeneity of theL. reuteri isolates was scrutinized by endonuclease restriction analysis of the chromosomal DNA, and the isolates could be divided into six genomic strains.     

Restriction polymorphisms in the internal transcribed spacers and 5.8S rDNA ofSaccharomyces

Polymorphism in enzymatically amplified ribosomal DNA (rDNA) were examined in 18 strains ofSaccharomyces. Restriction patterns generated from the region spanning the internal transcribed spacers (ITS) and the 5.8S rDNA produced two clusters corresponding toS. bayanus andS. cerevisiae. The type culture ofS. carlsbergensis (ATCC 76529), which could not be separated from theS. cerevisiae group by small subunit (SSU) rDNA patterns, showed aScrfI profile that was distinct from all the other strains. The type culture ofs. intermedius var.turicensis (ATCC 76519) is assigned toS. bayanus on the basis of the combined results of SSU and ITS restriction analyses.S. kluyveri occurred at a separate branch of the distance tree and is unrelated to any of the strains. Results were in general agreement with reported DNA homologies and are discussed in relation to other molecular and genetic data.     

The application of whole-cell protein electrophoresis for the classification and identification of basidiomycetous yeast species

The relationships among 65 basidiomycetous yeast strains were determined by one-dimensional electrophoresis of SDS-solubilized whole-cell proteins. Protein profiles were compared by the Pearson product moment correlation coefficient (r). The strains investigated represented species from the generaCystofilobasidium, Filobasidium, Filobasidiella, Kondoa, Leucosporidium, Mrakia andRhodosporidium. Except for the genusMrakia, all species constituted separate protein electrophoretic clusters. The species of the genusMrakia (M. frigida, M. gelida, M. nivalis andM. stokesii) show highly similar protein patterns, suggesting that these four species may be synonymous. Strains of two varieties ofFilobasidiella neoformans, F. neoformans var.neoformans andF. neoformans var.bacillispora, could not be differentiated by protein electrophoresis.For the delineation of the protein electrophoretic clusters of the yeasts studied, literature data relying on other criteria, such as DNA base composition, carbon source utilization patterns, enzymatic protein electrophoregrams, ubiquinone systems, DNA-DNA homology and rRNA sequence data were used. It was demonstrated that a database of SDS-protein patterns provides a valuable tool for the identification of yeasts.     

Mapping of the MouseLy-6, Xp-14, andGdc-1 loci to chromosome 15

TheLy-6 locus is now regarded as a gene complex consisting of at least five closely linked loci (Ly-6A-Ly-6E) whose polymorphic products are identified by monoclonal antibodies and distinguished by different tissue distributions.Ly-6 has been assigned by other investigators to chromosome (Chr) 9 (linked toThy-1 or to Chr 2. We report that theLy-6 gene complex, together with theXp-14 andGdc -1 loci, is situated on Chr 15 linked toGpt1. These new linkage data are derived from four sources: (1) three separate crosses that failed to demonstrate linkage ofLy-6 to eitherThy-4 on Chr 9 or to any of five genes present on Chr 2; (2) the NXSM recombinant inbred strains, which suggested the linkage ofLy-6 andXp-14 toGpt-1 on Chr 15; (3) severalGpt-1 andGdc-1 congenic strains that confirmed the assignment ofLy-6 andXp-14 to Chr 15; and (4) backcrosses that further confirmed the linkage ofLy-6, Gpt-1, Gdc-4, andXp-14, the probable gene order beingGpt-11/Ly-6 Xp-14-Gdc-1.     

Differentiation of species in the Bacillus brevis group and the Bacillus aneurinolyticus group based on the electrophoretic whole-cell protein pattern

Ninety strains of eleven Bacillus species in the Bacillus brevis group and the Bacillus aneurinolyticus group were compared with the electrophoretic whole-cell protein pattern. The strains were separated into two clusters at the similarity of 55%. One cluster (cluster 1) was consisted of strains from the B. brevis group, and another cluster (cluster 2) was composed of strains from the B. aneurinolyticus group. The cluster 1 was separated into eight subclusters. Out of eight subclusters, seven subclusters contain strains from B. brevis, B. laterosporus, B. agri, B. reuszeri, B. choshinensis, B. formosus, and B. borstelensis. Another subcluster was further separated into two related clusters, which corresponded to B. centrosporus and B. parabrevis, and they were fused at the similarity of 76%. Cluster 2 was separated into two subclusters, which corresponded to B. aneurinolyticus and B. migulanus. The above eleven species showed characteristic patterns distinct from one another, and this correlated well with the published DNA relatedness data. The comparison of the electrophoretic whole-cell protein pattern proved to be useful for evaluation of taxonomic relationships among these taxa.     

Taxonomy and phylogeny of the tribePlucheeae (Asteraceae)

The tribePlucheeae (Benth.)A. Anderb., has been analysed cladistically by means of a computerized parsimony program (Hennig 86), using theArctotideae as outgroup. The results of the analysis are presented in a consensus tree and one cladogram. Four major monophyletic subgroups can be recognized: TheColeocoma group (3 genera), thePterocaulon group (3 genera), theLaggera group (6 genera), and thePluchea group (12 genera). All recognized genera are described and most genera are supplied with taxonomical notes including comments on their taxonomic status. Genera such asBlumea, Pluchea, andEpaltes are demonstrated to be unnatural assemblages.Monarrhenus andTessaria are both closely related to thePluchea complex. The old generic nameLitogyne Harv. has been taken up for one species ofEpaltes, the genusRhodogeron is reduced to a synonym ofSachsia, and the following new combinations are made;Litogyne gariepina (DC.)A. Anderb., andSachsia coronopifolia (Griseb.)A. Anderb.     

Taxonomic relationships ofThiobacillus halophilus,T. aquaesulis,and other species ofThiobacillus,as determined using 16S rDNA sequencing

Total base sequences of the 16S rRNA genes ofThiobacillus halophilus andThiobacillus aquaesulis show that these bacteria fall into the gamma- and beta-subdivisions, respectively of the Proteobacteria. The closest relative ofT. halophilus isThiobacillus hydrothermalis (with 98.7% similarity), and the closest relative ofT. aquaesulis isThiobacillus thioparus (93.2% similarity). Physiological properties and mol% G+C content of their DNA serve to confirm that these four organisms are all distinct species. It is reiterated that the species currently assigned to the genusThiobacillus are clearly so diverse that they need reclassification into several genera. The type species,T. thioparus, is unequivocally placed in the beta-subdivision of the Proteobacteria, thus requiring that the use of the genus nameThiobacillus be restricted to the chemolithoautotrophic species falling into that group.T. aquaesulis andT. thioparus may thus be regarded as true species ofThiobacillus. The relatively large number of obligately chemolithoautotrophicThiobacillus species falling in the gamma-subdivision of the Proteobacteria need further study in order to assess the case for reclassification into one or more new or different genera.     

A rapid identification method for aflatoxin-producing strains ofAspergillus flavus andA. parasiticus by ammonia vapor

The colony reverse of aflatoxin (AF)-producing strains ofAspergillus flavus andA. parasiticus turned pink when their cultures were exposed to ammonia vapor. The color change was visible for colonies grown on media suitable for AF production such as potato dextrose, coconut, and yeast extract sucrose agars after 2 d incubation at 25°C. Of the 120 strains ofA. flavus, A. parasiticus, and two related species inA. flavus group:A. oryzae andA. sojae tested in this study, only the AF-producing strains ofA. flavus andA. parasiticus showed the pink pigmentation. The color change occurred immediately after the colony was contacted with ammonia vapor. This method was useful for rapid screening the AF-producing strains ofA. flavus andA. parasiticus.     

Intraspecific metabolic diversity among strains ofBurkholderia cepacia isolated from decayed onions,soils, and the clinical environment

A collection of 218 strains ofBurkholderia cepacia (including 18% strain replicates) was assembled from organic soils, decayed onions, and clinical sources. Each strain was characterized for virulence to onion, catabolic ability using the Biolog GN microtiter plate, and several other behaviors. Overall test reproducibility was estimated at 98%. The results obtained using the Biolog GN system corresponded well to those obtained using standard methods. Three coefficients of resemblance (Gower similarity, pattern difference, and Jaccard similarity) were calculated and clustered by the group-average method. The sorted matrices and phenograms, while giving evidence of an underlying phenetic structure to theB. cepacia nomenspecies, gave little evidence of sorting by broad source of isolation. Strains isolated from within fields or samples were frequently found to be similar, however, strains isolated from fields with similar cropping histories were not. The Gower-transformed centroids of ordained clusters were projected in a principal coordinate system and estimates of disjunction were calculated. Strains ofB. cepacia were shown to be non-uniformly distributed in taxonomic space. Strains isolated by serial dilution on onion slices formed a tight phenetic cluster which includes the type strain of the nomenspecies and that of a synonymous group (Pseudomonas multivorans); the strains in this phenon were generally virulent to onion and were partially differentiated from others by pectolytic behavior and by the production of diffusible pigment on King's medium A. Further characterization should better resolve the taxonomy of the nomenspecies.     

The karyotypes ofMicroseris bigelovii,M. douglasii,andM. pygmaea (Asteraceae)

The karyotypes of the three annuals,Microseris bigelovii, M. douglasii andM. pygmaea, consist of 2n = 18, small, submetacentric chromosomes. Length, centromere position, C-banding pattern, silver staining of NOR's, and the use of base specific fluorochromes, allow the identification of four of the nine chromosome pairs. The banding pattern ofM. bigelovii andM. pygmaea is identical, but intraspecific differences are found between strains ofM. douglasii.     

Nutritional physiology of type isolates of currently accepted species ofExophiala andPhaeococcomyces

Nutritional physiological and tolerance tests were performed for all type strains of species currently classified in the black yeast generaExophiala andPhaeococcomyces, including some additional type strains of taxa recently reidentified asExophiala species. Most describedExophiala species can be distinguished by physiological characters.Exophiala jeanselmei with its varieties, andE. castellanii should all be retained as separate taxa. The pairs of strainsMycotorula schawii/Exophiala dermatitidis, Hormodendrum negronii/Exophiala jeanselmei var.lecaniicorni andSporotrichum gougerotii/Torula bergeri were found to be conspecific. Phenetic analyses of physiological data support the identity ofPhaeococcomyces exophialae as a yeast-like synanamorph ofExophiala spinifera. The taxonomic positions of the generaNadsoniella, Phaeoannellomyces andWangiella are discussed. The generaExophiala andPhaeococcomyces are unrelated.     

Active MHC class Ib genes in rat are pseudogenes in the mouse

The most telomeric class I region of the MHC in rat and mouse is the M region, which contains about 20 class I genes or gene fragments. The central part carries three class I genes—M4, M5, and M6—which are orthologous between the two species. M4 and M6 are pseudogenes in the mouse but transcribed, intact genes in the rat. To analyze the pseudogene status for the mouse genes in more detail, we have sequenced the respective exons in multiple representative haplotypes. The stop codons are conserved in all mouse strains analyzed, and, consistent with the pseudogene status, all strains show additional insertions and deletions, taking the genes further away from functionality. Thus, M4 and M6 indeed have a split status. They are silent in the mouse but intact in the closely related rodent, the rat.GenBank accession numbers: AF057065 to AF057072 (exon 3 of H2-M4 of reported mouse strains), AF057976 to AF057985 (exon 3 of RT1.M4 of reported rat strains), AF058923 and AF058924 (exon 2 of RT1.M4 of strains PVG and BN), AY286080 to AY286092 (exon 4 of H2-M6 of reported mouse stains), and AY303772 (full-length genomic sequence of RT1.M6-1^l)     

Rapid identification ofRhizobium species based on cellular fatty acid analysis

As understanding of the evolutionary relationships between strains and species of root nodule bacteria increases the need for a rapid identification method that correlates well with phylogenetic relationships is clear. We have examined 123 strains ofRhizobium: R. fredii (19),R. galegae (20),R. leguminosarum (22),R. loti (17),R. meliloti (21), andR. tropici (18) and six unknowns. All strains were grown on modified tryptone yeast-extract (TY) agar, as log phase cultures, scraped from the agar, lysed, and the released fatty acids derivatized to their corresponding methyl esters. The methyl esters were analysed by gas-chromatography using the MIDI/Hewlett-Packard Microbial Identification System. All species studied contained 16:0, 17:0, 18:0 and 19cyclo_w9C fatty acids but onlyR loti andR tropici produced 12:0 3 OH,13:0 iso 3 OH,18:1_w9C and 15:0 iso 3 OH,17:0 iso 3 OH and 20:2_w6,9C fatty acids respectively. Principal component analysis was used to show that strains could be divided into clusters corresponding to the six species. Fatty acid profiles for each species were developed and these correctly identified at least 95% of the strains belonging to each species. A dendrogram is presented showing the relationships betweenRhizobium species based on fatty acid composition. The data base was used to identify unknown soil isolates as strains ofRhizobium lacking a symbiotic plasmid and a bacterium capable of expressing a symbiotic plasmid fromR. leguminosarum asSphingobacterium spiritovorum.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

Comparison of four media for the isolation ofAspergillus flavus group fungi

Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in theAspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States. The four media wereAspergillus flavus andparasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB). M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number ofA. flavus group strains and growth of the fewest competing fungi. M-RB supported an average of 12% moreA. flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively. M-RB was successfully employed to isolate all three aflatoxin producing species,A. flavus, A. parasiticus andA. nomius, and both the S and L strains ofA. flavus. M-RB is a defined medium without complex nitrogen and carbon sources (e.g. peptone and yeast extract) present in BC-RB. M-RB should be useful for studies on the population biology of theA. flavus group.Abbreviations M-RB Modified Rose Bengal Agar - CZ-RB Czapeks Rose Bengal Agar - BC-RB Bell and Crawford's Rose Bengal Agar - AFPA Aspergillus flavus andparasiticus agar     

Preliminary analysis of numerical taxonomy of the genusSaguinus based on dental measurements

Although several investigations have been made from different viewpoints, the classification or interspecific relationships ofSaguinus still remain uncertain. In the present study, we applied multivariate analysis methods to dental measurements of part ofSaguinus populations of sufficient sample size and obtained the following conclusions.Saguinus can be classified into two main groups: one consists ofS. oedipus andS. leucopus, and the other ofS. fuscicollis, S. nigricollis, S. labiatus, andS. mystax. Concerning the former group, the two subspecies ofS. oedipus, S. o. oedipus, andS. o. geoffroyi, show a close affinity with each other and also a close relationship toS. leucopus, while the latter group consists of two subgroups of species, one includingS. fuscicollis andS. nigricollis, and the other includingS. labiatus andS. mystax. The biological distance betweenS. oedipus oedipus andS. o. geoffroyi is slightly larger than that between the pairs ofS. fuscicollis andS. nigricollis and ofS. labiatus andS. mystax. Factor analysis revealed significant factors which could explain the differences among the seven maleSaguinus populations. Taking all the results into account, it seems necessary to reconsider the phylogenetic relationships within the genusSaguinus.