Endophytic Bacterial Communities in Ginseng and their Antifungal Activity Against Pathogens

Plant roots are associated with diverse communities of endophytic bacteria which do not exert adverse effects. The diversity of bacterial endophytes associated with ginseng roots cultivated in three different areas in Korea was investigated. Sixty-three colonies were isolated from the interior of ginseng roots. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates belonged to three major phylogenetic groups: the high G+C Gram-positive bacteria (HGCGPB), low G+C Gram-positive bacteria (LGCGPB), and the Proteobacteria. The dominant species at the three different ginseng growing areas were: HGCGPB at Ganghwa (55.0%), LGCGPB at Geumsan (45.5%), and Proteobacteria at Jinan (61.9%). Most cellulase-, xylanase-, and pectinase-producing colonies among the isolates belong to the LGCGPB group, except for Pectobacterium carotovora which belonged to the Proteobacteria. The 13 isolates belonging to LGCGPB and Proteobacteria were assessed for their antifungal activity against phytopathogenic fungi such as Rhizoctonia solani. Among them, Paenibacillus polymyxa GS01, Bacillus sp. GS07, and Pseudomonas poae JA01 show potential activity as biocontrol agents against phytopathogenic fungi. Finally, most of the low G+C Gram-positive bacteria with antifungal activity against phytopathogenic microorganisms showed cellulolytic enzyme activity while some Proteobacteria with the antifungal activity and the high G+C Gram-positive bacteria did not show any cellulolytic activity.     

NAD+-glycohydrolase productivity of haemolytic streptococci assayed by a simple fluorescent method and its relation to T serotype

Abstract The ability of haemolytic streptococci to produce NAD⁺-glycohydrolase was investigated by a fluorescent assay. Enzyme production was found in 31 (91%) of 34 group A, 17 (61%) of 28 group C and eight (27%) of 30 group G isolates. The high producers were found in 22 (65%) of group A, one (4%) of group C and none of group G isolates. The high producers of the group A isolates belonged to T-1, T-3, T-4 or T-12 serotype. These results suggest that NAD+-glycohydrolase productivity of streptococci is closely related to specific Lancefield's groups or T serotypes.     

Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern

Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously. These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods. Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers). Clone libraries focused on ponds with salinity of 8%, 22% and 32%. Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences. Thus, the use of two sets of primers recovered a higher number of phylotypes. Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences. For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin. Thus, representatives of the alpha-, beta-, gamma- and epsilon-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group (CFB), high-G+C Gram-positive bacteria and cyanobacteria were found. In the 22% salt pond, alpha- and gamma-Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria. From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium. A decrease in the richness of bacterial genera was therefore apparent along the gradient. Archaea behaved quite similarly. In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota. In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae. Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT. Crenarchaeaota were not detected. Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend. This was because diversity in the crystallizers can be considered as 'microdiversity', the co-existence of several closely related clones of Bacteria (the S. ruber cluster) and Archaea (the SPhT cluster). Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant. Both clusters, however, showed a considerable degree of microdiversity. The meaning of such microdiversity remains to be determined.     

Phylogenetic diversity of bacteria isolates from the Qinghai-Tibet Plateau permafrost region

The Qinghai-Tibet Plateau in east Asia is a unique and important permafrost environment. However, its microbiology remains largely unexplored to date. In this study, sediment samples were collected from the Qinghai-Tibet Plateau permafrost region, bacteria isolation procedures were performed 8 times, and the samples incubated at 4 degrees C for nearly 3 months. The number of colony forming units (cfu) ranged from 0 to 10(7)/(g dry soil). The quantity of culturable bacteria grew exponentially within the first few weeks, and then slowed gradually to a plateau. Phylogenetic analyses indicated that all the isolates fell into 6 categories: high G+C Gram-positive bacteria, low G+C Gram-positive bacteria, alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria, and Cytophaga-Flavobacterium-Bacteroides group bacteria. The isolates belong to 19 genera, but the genera Arthrobacter and Pseudomonas were predominant. With the increase in incubation time, the isolated populations changed in terms of both species and their respective quantities. Of the 33 analyzed isolates, 9 isolates related to 8 genera might be new taxa. These results suggest that the Qinghai-Tibet Plateau permafrost region is a specific ecologic niche that accommodates an original microbial assemblage.     

Diversity of Culturable Bacteria Isolated from Highland Barley Cultivation Soil in Qamdo,Tibet Autonomous Region

The soil bacterial communities have been widely investigated. However, there has been little study of the bacteria in Qinghai-Tibet Plateau, especially about the culturable bacteria in highland barley cultivation soil. Here, a total of 830 individual strains were obtained at 4°C and 25°C from a highland barley cultivation soil in Qamdo, Tibet Autonomous Region, using fifteen kinds of media. Seventy-seven species were obtained, which belonged to 42 genera and four phyla; the predominant phylum was Actinobacteria (68.82%), followed by Proteobacteria (15.59%), Firmicutes (14.29%), and Bacteroidetes (1.30%). The predominant genus was Streptomyces (22.08%, 17 species), followed by Bacillus (6.49%, five species), Micromonospora (5.19%, four species), Microbacterium (5.19%, four species), and Kribbella (3.90%, three species). The most diverse isolates belonged to a high G+C Gram-positive group; in particular, the Streptomyces genus is a dominant genus in the high G+C Gram-positive group. There were 62 species and 33 genera bacteria isolated at 25°C (80.52%), 23 species, and 18 genera bacteria isolated at 4°C (29.87%). Meanwhile, only eight species and six genera bacteria could be isolated at 25°C and 4°C. Of the 77 species, six isolates related to six genera might be novel taxa. The results showed abundant bacterial species diversity in the soil sample from the Qamdo, Tibet Autonomous Region.     

Description of an unusual gram-negative anaerobic rod isolated from periodontal pockets

A Gram-negative rod which grew with an unusual colonial "water-drop" form was isolated from periodontal pocket samples from 12 patients. Six strains were characterized by biochemical tests, cell wall analyses, malate dehydrogenase mobilities, protein profiles, and serology. By these criteria, the organisms formed a group of similar strains which were anaerobic, nonmotile, nonsporing, Gram-negative rods resembling Bacteroides. Comparison of the isolates to American Type Culture Collection strains of Bacteroides showed that they represented a closely related group, distinct from the described species of oral Bacteroides. Initial results on the DNA of the isolates suggested a base ratio of 54-57% G + C. Despite the DNA G + C base ratios currently accepted for the Bacteroides (28-61 mol% G + C), many species fall into a narrower range of 40-52 mol% G + C. This range would exclude the organisms described here and suggests that placing them into the genus Bacteroides may be inappropriate.     

Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola

The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola. The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes. The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas). One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria. The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.     

Comparative 16S rDNA sequence analysis of some alkaliphilic bacilli and the establishment of a sixth rRNA group within the genus Bacillus

Abstract Comparative sequence analysis of the 16S rDNA of 14 alkaliphilic or alkalitolerant, Gram-positive, aerobic, endo-spore forming bacterial strains was performed. Bacillus alcalophilus DSM 485^T and Bacillus cohnii DSM 6307^T were included to represent the two validly described alkaliphiles assigned to the genus Bacillus . The majority of isolates (8 strains) clustered with B. alcalophilus DSM 485^T forming a distinct phylogenetic group (rRNA group 6) within the radiation of the genus Bacillus and related taxa. Bacillus cohnii DSM 6307^T and two of the isolates, DSM 8719 and DSM 8723, grouped with B. fastidiosus and B. megaterium and are allocated to rRNA group 1. The remaining two strains DSM 8720 and DSM 8721 show an equidistant relationship to both groups.     

Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.     

Natronoincola histidinovorans gen. nov., sp. nov., a New Alkaliphilic Acetogenic Anaerobe

Two strains, asporogenous Z-7940 and sporogenous Z-7939, of a moderately haloalkaliphilic, obligately anaerobic, fermentative bacteria, motile, with Gram-positive cell wall structure, were isolated from soda deposits in Lake Magadi, Kenya. Both strains are mesophilic and utilize only two amino acids, histidine and glutamate, with formation of acetate and ammonium as the main end products. Strain Z-7939 in addition is able to utilize pyruvate. DNA-DNA homology between strains Z-7940 and Z-7939 was 94%, indicating that in spite of phenotypic differences they belong to the same species. They are true alkaliphiles with a pH range for growth of the type strain Z-7940 from pH 8.0 to pH 10.5, optimum at pH 9.4. Both strains obligately depend on sodium and bicarbonate ions. The optimum salt concentration for growth of the type strain is 8–10% wt/vol and the range from 4% to 16%. The G+C content of strain Z-7940 is 31.9 mol% and the strain Z-7939 is 32.3 mol%. Analysis of 16S rDNA sequence of the type strain shows it to belong to cluster XI of the low G+C Gram-positive bacteria. On the basis of its distinct phylogenetic position and physiological properties, we propose a new genus and new species Natronoincola histidinovorans for these strains. The type strain is Z-7940 (=DSM 11416). Received: 5 March 1998 / Accepted: 3 April 1998     

A PCR fingerprinting technique to distinguish isolates of Lactococcus lactis

A fingerprinting technique similar to repetitive extragenic palindromic PCR was developed to identify strains of Lactococcus lactis. The method distinguishes closely related strains and discriminates among some with identical ldh sequences. The fingerprinting primer LL-Rep1 complements a moderately repeated sequence found in low G+C Gram-positive bacteria and may therefore prove useful for discriminating among strains of other low G+C Gram-positive species.     

Characterization of a cryptic plasmid from a Greenland ice core Arthrobacter isolate and construction of a shuttle vector that replicates in psychrophilic high G+C Gram-positive recipients

Over 60 Greenland glacial isolates were screened for plasmids and antibiotic resistance/sensitivity as the first step in establishing a genetic system. Sequence analysis of a small, cryptic, 1,950 bp plasmid, p54, from isolate GIC54, related to Arthrobacter agilis, showed a region similar to that found in theta replicating Rhodococcus plasmids. A 6,002 bp shuttle vector, pSVJ21, was constructed by ligating p54 and pUC18 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. Candidate Gram-positive recipients were chosen among glacial isolates based on phylogenetic relatedness, relatively short doubling times at low temperatures, sensitivity to antibiotics, and absence of indigenous plasmids. We developed an electroporation protocol and transformed seven isolates related to members of the Arthrobacter, Microbacterium, Curtobacterium, and Rhodoglobus genera with pSVJ21. Plasmid stability was demonstrated by successive transformation into Escherichia coli and four Gram-positive isolates, growth without antibiotic, and plasmid re-isolation. This shuttle vector and our transformation protocol provide the basis for genetic experiments with different high G+C Gram-positive hosts to study cold adaptation and expression of cold-active enzymes at low temperatures.     

Globicatella sanguis gen.nov., sp.nov., a new gram-positive catalase-negative bacterium from human sources.

Phylogenetic studies were performed on some Gram-positive catalase-negative cocci from human clinical sources of uncertain taxonomic position. 16S rRNA sequence analysis demonstrated that the isolates represent a hitherto unknown line of descent within the low G+C Gram-positive bacteria for which the name Globicatella sanguis gen.nov., sp.nov. is proposed.     

Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes.

An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G + C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G + C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.     

Globicatella sanguis gen.nov., sp.nov., a new Gram-positive catalase-negative bacterium from human sources

Phylogenetic studies were performed on some Gram-positive catalase-negative cocci from human clinical sources of uncertain taxonomic position. 16S rRNA sequence analysis demonstrated that the isolates represent a hitherto unknown line of descent within the low G + C Gram-positive bacteria for which the name Globicatella sanguis gen.nov., sp.nov. is proposed.     

A phylogenetic analysis of micro-organisms isolated from subsurface environments

Three methods were used to provide information on the identity and phylogenetic relatedness of 19 aerobic, chemoheterotrophic bacteria isolated from topsoil and deep subsurface sediments at a site in South Carolina. These methods were (i) analysis of selected physiological traits, (ii) restriction endonuclease analysis (REA) of genomic DNA, and (iii) analysis of 16S ribosomal RNA sequences. When the 16S rRNA sequences were compared with those for 12 standard strains, two topsoil isolates and six subsurface strains formed a tight group with the high-G+C Gram-positive bacteria and appeared to be most closely related to Arthrobacter globiformis— a coryneform-actinomycete bacterium with unusually effective survival capabilities. The rest of the subsurface isolates were scattered among the standard strains from the Proteobacteria— including the pseudomonads and Agrobacterium tumefaciens— or the low-G+C Gram-positive bacteria.     

Gram-positive, catalase-positive cocci from dry cured Iberian ham and their enterotoxigenic potential.

Iberian ham is an uncooked, cured meat product ripened under natural uncontrolled conditions for 18 to 24 months. Gram-positive, catalase-positive cocci are the main microbial population in Iberian ham for most of the ripening time. Since some of these organisms are able to produce enterotoxins, adequate characterization and toxicological study are needed. For this, 1,327 gram-positive, catalase-positive cocci, isolated from Iberian hams at different stages and locations, were characterized by physiological and biochemical tests. Selected isolates were further characterized by guanine-cytosine (G+C) content and restriction enzyme analysis of genes coding for 16S rRNA. The toxigenic potential of these organisms was tested with specific DNA gene probes for staphylococcal enterotoxins A, B, C, and D and confirmed by semiquantitative sandwich enzyme immunoassay. The majority of the isolates were identified as Staphylococcus spp. and Micrococcus spp. Non-identified gram-positive, catalase-positive cocci which were moderately halophilic and showed a 42 to 52% G+C content were detected. A great variety of staphylococcal strains were found within the different species at any sampling time. Two strains of Staphylococcus xylosus, one Staphylococcus cohnii strain, and four of the non-identified organisms with 42 to 52% G+C contents hybridized with some of the DNA probes for C and D staphylococcal enterotoxin genes. S. xylosus hybridizing with C-enterotoxin probe reacted with both C and D enterotoxins in the immunological test. In addition, enterotoxin D was confirmed in the nonidentified strains. Some toxigenic organisms were isolated from the final product, posing a health hazard for the consumer.     

Microbial diversity in ikaite tufa columns: an alkaline,cold ecological niche in Greenland

Ikaite tufa columns from the Ikka Fjord in south-western Greenland constitute a natural, stable environment at low temperature and with a pH ranging from neutral at the exterior to very alkaline (pH 10.4) at the interior of the column. Phylogenetic analysis of culturable organisms revealed ten different isolates representing three of the major bacterial divisions. Nine of the isolates showed 94-99% similarity to known sequences, whereas one isolate displayed a low degree of similarity (less than 90%) to a Cyclobacterium species. Seven of the isolates were shown to be cold active alkaliphiles, whereas three isolates showed optimal growth at neutral pH. Phylogenetic analysis of DNA isolated directly from the ikaite material demonstrated the presence of a microbial flora more diverse than the culturable isolates. Whereas approximately half of the phylotypes showed 90-99% similarity to known meso- or thermophilic alkaliphiles, the rest of the sequences displayed less than 90% similarity when compared to known 16S rRNA gene sequences in databases. Thus, in the present paper, we demonstrate that ikaite columns that host a specialized macroscopic flora and fauna also contain a unique, cold active, alkaliphilic microflora.     

Occurrence of two structural types of mercury reductases among Gram-positive bacteria

Structural variants of mercury reductase containing the N-terminal domain, which is easily cleaved by trypsin, have been found in Gram-positive bacteria with a low genomic G + C content (Bacillus, Staphylococcus and, possibly, some other genera). Mercury reductases without the N-terminal domain and relatively resistant to limited proteolysis are typical for Gram-positive bacteria with a high genomic G + C content (Arthrobacter, Citreobacterium, Micrococcus, Mycobacterium, Rhodococcus). Both types of mercury reductase genes may be located on plasmids.     

Bacterial endophytes of the wildflower Crocus albiflorus analyzed by characterization of isolates and by a cultivation-independent approach

The presence and taxonomy of endophytic bacteria of the entire aerial parts of crocus (Crocus albiflorus), a wildflower native in the Alps, were investigated. A combination of plating of plant macerates, isolation and sequence identification of isolates, and direct 16S rDNA PCR amplification followed by whole-community fingerprinting (T-RFLP) and by construction of a bacterial clone library was used. The results clearly indicated that a wide range of bacteria from diverse phylogenetic affiliation, mainly gamma-Proteobacteria and Firmicutes, live in association with plants of C. albiflorus. The community composition of the culturable component of the microflora was remarkably different from that of the clone library. Only three bacterial divisions were found in the culture collection, which represented 17 phylotypes, whereas six divisions were identified in the clonal analysis comprising 38 phylotypes. The predominant group in the culture collection was the low G+C Gram-positive group, whereas in the clone library, the gamma-Proteobacteria predominated. Interestingly, the most prominent bacterium within the uncultured bacterial community was a pseudo monad closely related to a cold-tolerant Pseudomonas marginalis strain. The results suggest that Crocus supports a diverse bacterial microflora resembling the microbial communities that have been described for other plants and containing species that have not been described in association with plants.