Phylogeny of Culturable Estuarine Bacteria Catabolizing Riverine Organic Matter in the Northern Baltic Sea

The objective of our study was to isolate and determine the phylogenetic affiliation of culturable estuarine bacteria capable of catabolizing riverine dissolved organic matter (RDOM) under laboratory conditions. Additions of RDOM consistently promoted the growth of estuarine bacteria in carbon-limited dilution cultures, with seasonal variation in growth rates and yields. At least 42 different taxa were culturable on solid agar media and, according to quantitative DNA-DNA hybridizations, constituted 32 to 89% of the total bacterial number in the enriched treatments. Five species in the Cytophaga-Flexibacter-Bacteroides group and one in the γ-proteobacteria phylogenetic group (Marinomonas sp.) were numerically dominant during the stationary phase of the RDOM-enriched dilution cultures but not in the control cultures. Four of the isolates in Cytophaga-Flexibacter-Bacteroides group were putatively affiliated with the genus Flavobacterium. All dominating isolates were determined to be new species based on comparison to the current databases. The same group of species dominated independently of the season investigated, suggesting a low diversity of bacteria catabolizing RDOM in the estuary. It also suggested a broad tolerance of the dominating species to seasonal variation in hydrography, chemistry, and competition with other species. Taken together, our results suggest that a limited group of bacteria, mainly in the Flavobacterium genus, played an important role in introducing new energy and carbon to the marine system in the northern Baltic Sea.     

Combining culture-dependent and -independent methodologies for estimation of richness of estuarine bacterioplankton consuming riverine dissolved organic matter

Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated beta-proteobacteria to be RDOM consumers.     

Bacterial community associated with Pfiesteria-like dinoflagellate cultures

Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment. Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins. As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four ' Pfiesteria -like' dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay. A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria. The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures. The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species. Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth. Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells. These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the α-3 or SAR83 cluster.     

Combining Culture-Dependent and -Independent Methodologies for Estimation of Richness of Estuarine Bacterioplankton Consuming Riverine Dissolved Organic Matter

Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated β-proteobacteria to be RDOM consumers.     

Tributyltin-resistant bacteria from estuarine and freshwater sediments.

Resistance to tributyltin (TBT) was examined in populations from TBT-polluted sediments and nonpolluted sediments from an estuary and from fresh water as well as in pure cultures isolated from those sediments. The 50% effective concentrations (EC50s) for populations were higher at a TBT-polluted freshwater site than at a site without TBT, suggesting that TBT selected for a TBT-resistant population. In contrast, EC50s were significantly lower for populations from a TBT-contaminated estuarine site than for those from a site without TBT, suggesting that other factors in addition to TBT determine whether populations become resistant. EC50s for populations from TBT-contaminated freshwater sediments were nearly 30 times higher than those for populations from TBT-contaminated estuarine sediments. We defined a TBT-resistant bacterium as one which grows on trypticase soy agar containing 8.4 microM TBT, a concentration which prevented the growth of 90% of the culturable bacteria from these sediments. The toxicity of TBT in laboratory media was influenced markedly by the composition of the medium and whether it was liquid or solid. Ten TBT-resistant isolates from estuarine sediments and 19 from freshwater sediments were identified to the genus level. Two isolates, each a Bacillus sp., may be the first gram-positive bacteria isolated from fresh water in the presence of a high concentration of TBT. There was a high incidence of resistance to heavy metals: metal resistance indices were 0.76 for estuarine isolates and 0.68 for freshwater isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tributyltin-resistant bacteria from estuarine and freshwater sediments.

Resistance to tributyltin (TBT) was examined in populations from TBT-polluted sediments and nonpolluted sediments from an estuary and from fresh water as well as in pure cultures isolated from those sediments. The 50% effective concentrations (EC50s) for populations were higher at a TBT-polluted freshwater site than at a site without TBT, suggesting that TBT selected for a TBT-resistant population. In contrast, EC50s were significantly lower for populations from a TBT-contaminated estuarine site than for those from a site without TBT, suggesting that other factors in addition to TBT determine whether populations become resistant. EC50s for populations from TBT-contaminated freshwater sediments were nearly 30 times higher than those for populations from TBT-contaminated estuarine sediments. We defined a TBT-resistant bacterium as one which grows on trypticase soy agar containing 8.4 microM TBT, a concentration which prevented the growth of 90% of the culturable bacteria from these sediments. The toxicity of TBT in laboratory media was influenced markedly by the composition of the medium and whether it was liquid or solid. Ten TBT-resistant isolates from estuarine sediments and 19 from freshwater sediments were identified to the genus level. Two isolates, each a Bacillus sp., may be the first gram-positive bacteria isolated from fresh water in the presence of a high concentration of TBT. There was a high incidence of resistance to heavy metals: metal resistance indices were 0.76 for estuarine isolates and 0.68 for freshwater isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of surfactant-resistant pseudomonads from the estuarine surface microlayer

Bioremediation efforts often rely on the application of surfactants to enhance hydrocarbon bioavailability. However, synthetic surfactants can sometimes be toxic to degrading microorganisms, thus reducing the clearance rate of the pollutant. Therefore, surfactant-resistant bacteria can be an important tool for bioremediation efforts of hydrophobic pollutants, circumventing the toxicity of synthetic surfactants that often delay microbial bioremediation of these contaminants. In this study, we screened a natural surfactant-rich compartment, the estuarine surface microlayer (SML), for cultivable surfactant-resistant bacteria using selective cultures of sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB). Resistance to surfactants was evaluated by colony counts in solid media amended with critical micelle concentrations (CMC) of either surfactants, in comparison with non-amended controls. Selective cultures for surfactant-resistant bacteria were prepared in mineral medium also containing CMC concentrations of either CTAB or SDS. The surfactantresistant isolates obtained were tested by PCR for the Pseudomonas genus marker gacA gene and for the naphthalene-dioxygenase-encoding gene ndo. Isolates were also screened for biosurfactant production by the atomized oil assay. A high proportion of culturable bacterioneuston was tolerant to CMC concentrations of SDS or CTAB. The gacA-targeted PCR revealed that 64% of the isolates were Pseudomonads. Biosurfactant production in solid medium was detected in 9.4% of tested isolates, all affiliated with genus Pseudomonas. This study shows that the SML is a potential source of surfactant-resistant and biosurfactant-producing bacteria in which Pseudomonads emerge as a relevant group.     

Specificity of Pseudomonas Isolates on Healthy and Fusarium Head Blight-Infected Spikelets of Wheat Heads

The specificity of culturable bacteria on healthy and Fusarium head blight (FHB)-infected spikelets of wheat heads was investigated to find a candidate of biocontrol agents against FHB. The bacterial genus Pseudomonas was commonly isolated from the tissues, and phylogenetic analysis using 16S ribosomal RNA gene sequences of isolates of the genera revealed that particular phylogenetic groups in the genus specifically inhabited either healthy or infected spikelet tissues. The specificity of each group was suggested to be due to differences in the ability to form biofilms and colonize spikelet tissues; isolates originated from healthy spikelets formed biofilms on polyvinyl chloride microplate wells and highly colonized the spikelet tissues. Other bacterial groups obtained from FHB-infected spikelets less formed biofilms and attached with low densities on the spikelet tissues. Their colonization on the tissues, however, was promoted when co-inoculated with the causal pathogenic fungus, Fusarium graminearum, and several isolates were observed to smash the mycelia in vivo. Moreover, based on results of in vitro mycelial growth inhibition activity, the diseased tissue-originated isolates were verified to have a negative effect on the fungal growth. These results suggest that Pseudomonas isolates obtained from infected spikelet tissues were highly associated with the FHB pathogen and have potential as candidates for biological control against FHB.     

Isolation of previously uncultured rumen bacteria by dilution to extinction using a new liquid culture medium

A new anaerobic medium that mimics the salts composition of rumen fluid was used in conjunction with a dilution method of liquid culture to isolate fermentative bacteria from the rumen of a grass-fed sheep. The aim was to inoculate a large number of culture tubes each with a mean of < 1 culturable cell, which should maximize the number of cultures that develop from a single bacterium. This minimizes the effort that has to be put into purifying the resultant cultures. Of 1000 tubes, 139 were growth positive. Of the 93 that were able to be subcultured, 54 (58%) appeared to be pure cultures. The phylogenetic placements of these 54 cultures, together with another 6 cultures obtained from a preliminary study, were determined. Using a criterion of < 93% 16S rRNA gene sequence identity to a previously named bacterium as a proxy for defining a new genus, 27 (45%) of the 60 cultures belonged to 14 potentially novel genera. Many of these had 16S rRNA genes that shared > 97% sequence identity to genes of uncultured bacteria detected in various gastrointestinal environments. This strategy has therefore allowed us to cultivate many novel rumen bacteria, opening the way to overcoming the lack of cultures of many of the groups detected using cultivation-independent methods.     

Soil microbial counts and identification of culturable bacteria in an extreme by arid zone

Sixteen samples of two soil cores (about 550 and 180 cm in depth) were drilled at intervals in the lower reach of Heihe river basin (northwest of China) in order to illustrate soil microbial characteristics and diversity of culturable bacteria in an extreme by arid environment. Soil water content, organic matter, total nitrogen, pH, direct cell counts, and culturable microorganism counts were evaluated. The total cell concentration was 19-1120/microg (i.e. 0.19-11.2 x 10(8) per g) soil, the culturable bacteria count being 0.2-10.9 per microg (i.e. 2 x 10(5)-10.9 x 10(6) CFU/g) soil. The number of direct cell counts obtained by 4',6-diamidino-2-phenylindole-staining or the cound of culturable microbes after enrichment with different media were statistically significantly correlated with soil organic matters, total nitrogen content, soil water content and surface vegetation; this partly explained the larger number in the deeper first core than in the shallower one. As part of identification of 228 colonies isolated from the two cores, thirty-two were selected for 16S rDNA amplification, sequencing and molecular identification. These 32 isolates were affiliated to 5 major groups of bacteria: alpha-Proteobacteria, 5-Proteobacteria, gamma-Proteobacteria, the high-G+C G+-bacteria, the low-G+C G- -bacteria, and the Cytophaga-Flexibacter-Bacteroides group. Twenty-eight were rod- or short-rod shaped, which accounted for >87.5% of all species; only 4 of 32 species were cocci (<12.5%).     

Phylogenetic diversity of culturable bacteria from Antarctic sandy intertidal sediments

The diversity of culturable bacteria associated with sandy intertidal sediments from the coastal regions of the Chinese Antarctic Zhongshan Station on the Larsemann Hills (Princess Elizabeth Land, East Antarctica) was investigated. A total of 65 aerobic heterotrophic bacterial strains were isolated at 4°C. Microscopy and 16S rRNA gene sequence analysis indicated that the isolates were dominated by Gram-negative bacteria, while only 16 Gram-positive strains were isolated. The bacterial isolates fell in five phylogenetic groups: Alpha- and Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. Based on phylogenetic trees, all the 65 isolates were sorted into 29 main clusters, corresponding to at least 29 different genera. Based on sequence analysis (<98% sequence similarity), the Antarctic isolates belonged to at least 37 different bacterial species, and 14 of the 37 bacterial species (37.8%) represented potentially novel taxa. These results indicated a high culturable diversity within the bacterial community of the Antarctic sandy intertidal sediments.     

Diversity of substrate utilization and growth characteristics of sulfate-reducing bacteria isolated from estuarine sediment in Japan

Two different isolation methods, the dilution colony-counting method (colony-isolation) and enrichment culture, were used to isolate sulfate-reducing bacteria (SRBs) from estuarine sediment in Japan. Lactate was used as an electron donor for colony-isolation, and lactate or propionate was used for enrichment culture. All isolates were classified into six different phylogenetic groups according to the 16S rRNA gene-based analysis. The closest relatives of the colony-isolates (12 strains) were species in the genera of Desulfobacterium, Desulfofrigus, Desulfovibrio and Desulfomicrobium. The closest known relative of the lactate-enrichment isolates was Desulfovibrio acrylicus and that of the propionate-enrichment isolates was Desulfobulbus mediterraneus. All isolates were incompletely-oxidizing SRBs. Overall patterns of utilization of electron donors and acceptors, as well as fermentative substrates, differed depending on the affiliation of the strain. Furthermore, even if several strains used the same substrate, the growth rates were often significantly different depending on the strain. It was strongly suggested that various species of SRBs could coexist in the sediment by competing for common substrates as well as taking priority in favorable or specific substrates for each species and the community of SRBs should be able to oxidize almost all major intermediates of anaerobic decomposition of organic matter such as lower fatty acids, alcohols and H2 as well as amino acids. Thus, it was indicated by the phylogenetic and physiological analyses of the isolates that the SRB community composed of diverse lineages of bacteria living in anoxic estuarine sediment should be able to play an extensive role in the carbon cycle as well as the sulfur cycle of the earth.     

Phylogenetic diversity of culturable fungi associated with the Hawaiian Sponges Suberites zeteki and Gelliodes fibrosa

Sponges are well documented to harbor large amounts of microbes. Both culture-dependent and molecular approaches have revealed remarkable bacterial diversity in marine sponges. Fungi are commonly isolated from marine sponges, yet no reports on phylogenetic diversity of sponge-inhabiting fungi exist. In this report, we investigated the phylogenetic diversity of culturable fungi from the Hawaiian alien marine sponges Suberites zeteki and Gelliodes fibrosa. A total of 44 independent isolates were recovered from these two sponge species, representing 7 orders and 22 genera of Ascomycota. The majority (58%) of fungal isolates from S. zeteki resided in the Pleosporales group, while the predominant isolates (52%) from G. fibrosa were members of the Hypocreales group. Though differing in fungal species composition and structure, culturable communities of these two sponges displayed similar phylogenetic diversity. At the genus level, only two genera Penicillium and Trichoderma in the Eurotiales and Hypocreales orders, respectively, were present in both sponge species. The other genera of the fungal isolates were associated with either S. zeteki or G. fibrosa. Some of these fungal genera had been isolated from sponges collected in other marine habitats, but more than half of these genera were identified for the first time in these two marine sponges. Overall, the diversity of culturable fungal communities from these two sponge species is much higher than that observed in studies of marine sponges from other areas. This is the first report of phylogenetic diversity of marine sponge-associated fungi and adds one more dimension to our current understanding of the phylogenetic diversity of sponge-symbiotic microbes.     

Cellulose-degrading potentials and phylogenetic classification of carboxymethyl-cellulose decomposing bacteria isolated from soil

In a previous study, culturable carboxymethyl-cellulose (CMC) decomposing soil bacteria isolated from different sampling positions across an agricultural encatchment have been classified into 31 pattern groups by digestion of amplified 16S rDNA using a single restriction enzyme (Ulrich and Wirth: Microb. Ecol. 37, 238-247, 1999). In order to reveal relationships between phylogenetic diversity and phenotypic functions, a further differentiation of two selected site-specific pattern groups (I and H) was performed, resulting in a sub-classification of four and three ARDRA groups, respectively. Based on sequencing a representative isolate of each ARDRA group, the isolates were assigned to the genus Streptomyces. The ARDRA groups were dispersed across various clades of the genus with a direct affiliation to species known for cellulolytic activity in one group, only. The isolates differed in potentials to degrade colloidal, native or highly crystalline cellulose derivatives. Out of 39 isolates, 11 were capable of degrading all substrates, 17 were restricted to degrade CMC only, and 11 were active decomposers of exclusively both CMC and colloidal cellulose. In most cases, the genetic classification of the isolates corresponded with groupings based on cellulose degrading capabilities. Thus, isolates of four ARDRA groups were restricted to the degradation of CMC, while two further isolates which efficiently degraded all cellulose derivatives formed two separate ARDRA groups. The major ARDRA group, however; displayed a high variability of degradation capabilities. The study of additional phenotypic features revealed a broad potential to decompose a set of various carbon substrates, which matched the phylogenetic classification in several cases.     

Chemolithotrophic haloalkaliphiles from soda lakes

This paper summarizes recent data on the occurrence and properties of lithotrophic prokaryotes found in extremely alkaline, saline (soda) lakes. Among the chemolithotrophs found in these lakes the obligately autotrophic sulfur-oxidizing bacteria were the dominant, most diverse group, best adapted to haloalkaline conditions. The culturable forms are represented by three new genera, Thioalkalimicrobium, Thioalkalivibrio and Thioalkalispira in the Gammaproteobacteria. Among them, the genus Thioalkalivibrio was most metabolically diverse, including denitrifying, thiocyanate-oxidizing and facultatively alkaliphilic species. Culturable methane-oxidizing populations in the soda lakes belong to the type I methanotroph group in the Gammaproteobacteria, mostly in the genus Methylomicrobium. The nitrifying bacteria in hyposaline soda lakes were represented by a new species Nitrobacter alkalicus (Alphaproteobacteria), and by an alkaliphilic subspecies of Nitrosomonas halophila (Betaproteobacteria). Both belonged to the low salt-tolerant alkaliphiles. The facultatively autotrophic haloalkaliphilic isolates able to grow with hydrogen as electron donor were identified as representatives of the alpha-3 subclass of the Proteobacteria (aerobic) and of the Natronolimnicola - Alkalispirillum group in the gammaproteobacteria (nitrate-reducing). While all chemolithotrophic isolates from soda lakes belong to the alkaliphiles with a pH optimum for growth around 10, only the sulfur-oxidizing group included species able to grow under hypersaline conditions. This indicates that carbon and nitrogen cycles in the hypersaline alkaline lakes might not be closed.     

Culturable diversity of lithotrophic haloalkaliphilic sulfate-reducing bacteria in soda lakes and the description of Desulfonatronum thioautotrophicum sp. nov., Desulfonatronum thiosulfatophilum sp. nov., Desulfonatronovibrio thiodismutans sp. nov., and Desulfonatronovibrio magnus sp. nov

Soda lake sediments usually contain high concentrations of sulfide indicating active sulfate reduction. Monitoring of sulfate-reducing bacteria (SRB) in soda lakes demonstrated a dominance of two groups of culturable SRB belonging to the order Desulfovibrionales specialized in utilization of inorganic electron donors, such as formate, H₂ and thiosulfate. The most interesting physiological trait of the novel haloalkaliphilic SRB isolates was their ability to grow lithotrophically by dismutation of thiosulfate and sulfite. All isolates were obligately alkaliphilic with a pH optimum at 9.5–10 and moderately salt tolerant. Among the fifteen newly isolated strains, four belonged to the genus Desulfonatronum and the others to the genus Desulfonatronovibrio. None of the isolates were closely related to previously described species of these genera. On the basis of phylogenetic, genotypic and phenotypic characterization of the novel soda lake SRB isolates, two novel species each in the genera Desulfonatronum and Desulfonatronovibrio are proposed.     

Impact of cultivation on characterisation of species composition of soil bacterial communities

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere. The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.     

Phylogenetic Diversity of Numerically Important Arctic Sea-Ice Bacteria Cultured at Subzero Temperature

Heterotrophic bacteria in sea ice play a key role in carbon cycling, but little is known about the predominant players at the phylogenetic level. In a study of both algal bands and clear ice habitats within summertime Arctic pack ice from the Chukchi Sea, we determined the abundance of total bacteria and actively respiring cells in melted ice samples using epifluorescence microscopy and the stains 4', 6'-diamidino-2-phenylindole 2HCl (DAPI) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), respectively. Organic-rich and -poor culturing media were used to determine culturable members by plating (at 0 degrees C and 5 degrees C) and most-probable-number (MPN) analyses (at -1 degrees C). Total bacterial counts ranged from 5.44 x 10(4) ml(-1) in clear ice to 2.41 x 10(6) ml(-1) in algal-band ice samples, with 2-27% metabolically active by CTC stain. Plating and MPN results revealed a high degree of culturability in both types of media, but greater success in oligotrophic media (to 62% of total abundance) and from clear ice samples. The bacterial enumeration anomaly, commonly held to mean 相似文献   

Cultivation and growth characteristics of a diverse group of oligotrophic marine Gammaproteobacteria

Forty-four novel strains of Gammaproteobacteria were cultivated from coastal and pelagic regions of the Pacific Ocean using high-throughput culturing methods that rely on dilution to extinction in very low nutrient media. Phylogenetic analysis showed that the isolates fell into five rRNA clades, all of which contained rRNA gene sequences reported previously from seawater environmental gene clone libraries (SAR92, OM60, OM182, BD1-7, and KI89A). Bootstrap analyses of phylogenetic reliability did not support collapsing these five clades into a single clade, and they were therefore named the oligotrophic marine Gammaproteobacteria (OMG) group. Twelve cultures chosen to represent the five clades were successively purified in liquid culture, and their growth characteristics were determined at different temperatures and dissolved organic carbon concentrations. The isolates in the OMG group were physiologically diverse heterotrophs, and their physiological properties generally followed their phylogenetic relationships. None of the isolates in the OMG group formed colonies on low- or high-nutrient agar upon their first isolation from seawater, while 7 of 12 isolates that were propagated for laboratory testing eventually produced colonies on 1/10 R2A agar. The isolates grew relatively slowly in natural seawater media (1.23 to 2.63 day(-1)), and none of them grew in high-nutrient media (>351 mg of C liter(-1)). The isolates were psychro- to mesophilic and obligately oligotrophic; many of them were of ultramicrobial size (<0.1 micro m(3)). This cultivation study revealed that sporadically detected Gammaproteobacteria gene clones from seawater are part of a phylogenetically diverse constellation of organisms mainly composed of oligotrophic and ultramicrobial lineages that are culturable under specific cultivation conditions.     

Diagnosis of flavobacteriosis by direct amplification of rRNA genes

A broad-range bacterial PCR method with universal 16S rDNA targeting primers and bacterial cultivation was used to identify the putative pathogen in flavobacterial outbreaks. Restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing of the partial 16S rDNA PCR products of 10 skin samples and 10 representative isolates derived from the same fish specimens revealed differences between direct molecular and cultivation-based analysis. Flavobacterium columnare-like sequences dominated in the direct molecular analysis in most cases, whereas most of the isolates belonged to a phylogenetically heterogeneous group of flavobacteria clustering with F. hibernum. F. columnare was isolated in only 1 outbreak. The possible explanations for the different results may be attributable to difficulties in the plate cultivation procedure of external flavobacterial samples. During plate cultivation, the dominating Flavobacterium species can be masked by saprophytic species of the same genus or other genera, or the growth of flavobacteria can be completely inhibited by antagonistic bacteria such as Pseudomonas. Direct analysis of the prevailing 16S rDNA sequences avoids the problems with cultivation and may thus be preferable for the diagnosis of flavobacterial diseases. When isolating flavobacteria from external samples, serial dilution of the sample before plating can improve the results.