Detection and Isolation of Novel Rhizopine-Catabolizing Bacteria from the Environment

Microbial rhizopine-catabolizing (Moc) activity was detected in serial dilutions of soil and rhizosphere washes. The activity observed generally ranged between 10⁶ and 10⁷ catabolic units per g, and the numbers of nonspecific culture-forming units were found to be approximately 10 times higher. A diverse set of 37 isolates was obtained by enrichment on scyllo-inosamine-containing media. However, none of the bacteria that were isolated were found to contain DNA sequences homologous to the known mocA, mocB, and mocC genes of Sinorhizobium meliloti L5-30. Twenty-one of the isolates could utilize an SI preparation as the sole carbon and nitrogen source for growth. Partial sequencing of 16S ribosomal DNAs (rDNAs) amplified from these strains indicated that five distinct bacterial genera (Arthrobacter, Sinorhizobium, Pseudomonas, Aeromonas, and Alcaligenes) were represented in this set. Only 6 of these 21 isolates could catabolize 3-O-methyl-scyllo-inosamine under standard assay conditions. Two of these, strains D1 and R3, were found to have 16S rDNA sequences very similar to those of Sinorhizobium meliloti. However, these strains are not symbiotically effective on Medicago sativa, and DNA sequences homologous to the nodB and nodC genes were not detected in strains D1 and R3 by Southern hybridization analysis.     

Seasonal occurrence and abundance of caridean shrimp larvae at Helgoland,German Bight

Plankton samples were collected from January 1985 to January 1986 three times per week at Helgoland to study seasonal occurrence and abundance of caridean shrimp larvae. A total of eleven species were obtained. Ninety-one % of all larvae collected during the sample period belonged toCrangon crangon L. andCrangon allmanni Kinahan, 6% toPhilocheras trispinosus Hailstone and 3% to the remaining eight species. Collections were generally dominated byC. crangon larvae. However,C. allmanni larvae were most abundant in June coinciding with hatching activities of the population near Helgoland.C. allmanni was observed to have the highest density of all species with approximately 8 larvae per m³. Larvae ofEualus occultus (Lebour),Eualus pusiolus (Kroyer),Hippolyte varians Leach andAthanas nitescens Leach were most likely released by populations inhabiting the rocky intertidal zone around Helgoland. The presence ofProcessa modica Williamson & Rochanaburanon andProcessa nouveli holthuisi Al-Adhub & Williamson in the German Bight was verified by observations of a series of different developmental stages. Larvae of the rare speciesCaridion steveni Lebour were also recorded. The observed shrimp species were placed into three different groups with respect to their seasonal occurrence. Possible advantages of the timing of larval dispersal relative to predation and food availability are given. The results on seasonal occurrence and relative abundance are discussed in relation to environmental factors (temperature, salinity) as well as to the geographical distribution of the species.     

Isolation and Characterization of Diuron-degrading Bacteria from Lotic Surface Water

The bacterial community structure of a diuron-degrading enrichment culture from lotic surface water samples was analyzed and the diuron-degrading strains were selected using a series of techniques combining temporal temperature gradient gel electrophoresis (TTGE) of 16 S rDNA gene V1–V3 variable regions, isolation of strains on agar plates, colony hybridization methods, and biodegradation assays. The TTGE fingerprints revealed that diuron had a strong impact on bacterial community structure and highlighted both diuron-sensitive and diuron-adapted bacterial strains. Two bacterial strains, designated IB78 and IB93 and identified as belonging to Pseudomonas sp. and Stenotrophomonas sp., were isolated and shown to degrade diuron in pure resting cells in a first-order kinetic reaction during the first 24 h of incubation with no 3,4-DCA detected. The percentages of degradation varied from 25% to 60% for IB78 and 20% to 65% for IB93 and for a diuron concentration range from 20 mg/L to 2 mg/L, respectively. It is interesting to note that diuron was less degraded by single isolates than by mixed resting cells, thereby underlining a cumulative effect between these two strains. To the best of our knowledge, this is the first report of diuron-degrading strains isolated from lotic surface water.     

Seasonal Community and Population Dynamics of Pelagic Bacteria and Archaea in a High Mountain Lake

The seasonal variations in community structure and cell morphology of pelagic procaryotes from a high mountain lake (Gossenköllesee, Austria) were studied by in situ hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes (FISH) and image-analyzed microscopy. Compositional changes and biomass fluctuations within the assemblage were observed both in summer and beneath the winter ice cover and are discussed in the context of physicochemical and biotic parameters. Proteobacteria of the beta subclass (beta-proteobacteria) formed a dominant fraction of the bacterioplankton (annual mean, 24% of the total counts), whereas alpha-proteobacteria were of similar relative importance only during spring (mean, 11%). Bacteria of the Cytophaga-Flavobacterium cluster, although less abundant, constituted the largest fraction of the filamentous morphotypes during most of the year, thus contributing significantly to the total microbial biomass. Successive peaks of threadlike and rod-shaped archaea were observed during autumn thermal mixing and the period of ice cover formation, respectively. A set of oligonucleotide probes targeted to single phylotypes was constructed from 16S rRNA-encoding gene clone sequences. Three distinct populations of uncultivated microbes, affiliated with the alpha- and beta-proteobacteria, were subsequently monitored by FISH. About one-quarter of all of the beta-proteobacteria (range, 6 to 53%) could be assigned to only two phylotypes. The bacterial populations studied were annually recurrent, seasonally variable, and vertically stratified, except during the periods of lake overturn. Their variability clearly exceeded the fluctuations of the total microbial assemblage, suggesting that the apparent stability of total bacterioplankton abundances may mask highly dynamic community fluctuations.Until recently, microbial ecologist studying aquatic bacteria faced a basic dilemma: they could either measure the abundance, biomass, growth rates, activity, etc. of the “average” bacterium under in situ conditions (e.g., see reference 13), ignoring the phylogenetic and physiological diversity of microbial communities, or they could isolate and ecophysiologically characterize individual bacterial strains (e.g., see reference 36) but were then not able to tell if these microorganisms were also common in the environment. Consequently, little knowledge has been gathered about the spatial and temporal abundance fluctuations of defined phylogenetic groups and of individual bacterial species in natural habitats. Molecular biological techniques used to identify microbes in environmental samples have recently provided new tools to study bacterioplankton biodiversity (e.g., see references 1, 9, 14, 15, and 19) and the in situ abundances of bacteria and archaea that could not be adequately distinguished before (2, 4, 5, 25). Microbiologists are now in a position to potentially elucidate the biogeography (24), population dynamics, and successions (28) not only of a few morphologically conspicuous microbes but of a large number of species, most of which might still be uncharacterized.Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes selectively visualizes bacterial cells with defined phylogenetic affiliations (3, 5). Based on a rapidly growing set of 16S (and, to a lesser extend, 23S) rRNA sequence data, it is probably the phylogenetically most sophisticated (22) approach for whole-cell in situ identification. On the other hand, FISH of plankton samples can be performed with minimal laboratory requirements (16), and evaluation relies on epifluorescence microscopy, which is a standard technique of aquatic microbial ecologists, e.g., for counting (30) and sizing (33) of picoplankton. In contrast to other identification approaches, FISH largely conserves the gestalt of the targeted microorganisms, i.e., their morphologies, cell sizes (26, 34), and cellular rRNA content (7, 32). So, despite the limitations of the method (as discussed in reference 5), its potential for the identification and cytometric analysis of planktonic microbes is just about to be recognized.Recent investigations have reported that various freshwater microbial communities are dominated by bacteria which are phylogenetically affiliated with the alpha and beta subclasses of the class Proteobacteria (alpha- and beta-proteobacteria, respectively) and with members of the Cytophaga-Flavobacterium cluster (2, 6, 16, 19). These observations were based on single or short-term sampling schemes. The instantaneous community composition of the bacterioplankton, however, may not be representative for different seasons, and the typical ranges of annual community variability remain to be established.The size distribution of planktonic bacteria, and particularly the appearance of filamentous cells, has come into the focus of aquatic microbial ecology in the context of studies of predator-prey interactions. It has been shown both in the laboratory (18, 37) and in field experiments (20) that the filamentous morphotype is a phenotypic adaptation of some microbes to protistan grazing, but there are probably numerous other causes for bacteria to elongate far beyond their typical sizes (e.g., see reference 23). Threadlike bacteria have been observed throughout the year in the plankton of a hypertrophic lake (41) but were also found in midwinter in an oligotropic alpine lake (31).In earlier studies, we demonstrated FISH to be an appropriate tool for the monitoring of spatial (2) and short-term temporal (26) dynamics of different phylogenetic groups of the planktonic microbial community in a high mountain lake. Here we report on the seasonal and vertical abundance distributions of pelagic members of Bacteria and Archaea in Gossenköllesee and analysis of the community structure at different levels of taxonomic resolution. We applied published domain- and group-specific oligonucleotide probes (5) but also used the sequence information from a 16S rRNA-encoding gene (rDNA) library obtained from Gossenköllesee bacterioplankton 1 year earlier to construct specific probes targeted at individual bacterial populations. Particular attention was paid to the changes in abundance and taxonomic composition of the filamentous bacterial morphotypes which were recognized as a permanently important fraction of the planktonic procaryotes in Gossenköllesee. Additionally, we monitored the seasonal changes in the biomass size distributions of the nonfilamentous fraction of the pelagic microbial community.     

Isolation and Identification of Novel Microcystin-Degrading Bacteria

Of 31 freshwater bacterial isolates screened using the Biolog MT2 assay to determine their metabolism of the microcystin LR, 10 were positive. Phylogenetic analysis (16S rRNA) identified them as Arthrobacter spp., Brevibacterium sp., and Rhodococcus sp. This is the first report of microcystin degraders that do not belong to the Proteobacteria.A number of studies have reported biological degradation of microcystin in samples from lakes and sediments (3, 4, 12, 15), but only a few bacterial strains with the ability to degrade microcystins have been isolated and characterized (6). Previously identified bacteria belonged to the Proteobacteria, and with the exception of one isolate (Sphingomonas sp. strain CB4), they were all shown to degrade microcystin LR (MC-LR) via the same degradation pathway: formation of linear MC-LR following cleavage at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg bond and hydrolysis at Ala-Leu to yield a tetrapeptide with Adda as the final product detected (1, 10, 11). Genes encoding enzymes involved in this pathway have been characterized and shown to have similarity in all bacterial isolates currently reported to degrade microcystins (2).Recent work indicated a greater diversity of microbes capable of degrading microcystins and nodularin, with the tentative identification of several novel degradation intermediates (5). In the present study, enrichment was used to isolate bacteria from three Scottish water bodies previously shown to contain microflora capable of microcystin degradation (5). The Biolog MT2 assay was used to screen the ability of the isolated bacteria to metabolize MC-LR, since this had previously been shown to be an effective means of demonstrating metabolism of microcystin by Paucibacter toxinivorans (6).The ability to metabolize MC-LR was determined in the Biolog MT screen, with 10 of the bacterial isolates giving a positive result. We subsequently confirmed that they could all degrade MC-LR in batch degradation studies, as evidenced by liquid chromatography-mass spectrometry (LC-MS) analysis. The microcystin-degrading bacteria were identified by using 16S rRNA gene analysis and investigated to determine the presence of mlrA, mlrB, mlrC, and mlrD, the genes previously reported to be involved in the degradation of MC-LR by Sphingomonas sp. strain ACM-3962 (2). We report here isolates identified as Arthrobacter spp., Brevibacterium sp., and Rhodococcus sp. which have the ability to degrade MC-LR, although none of the previously characterized mlr genes were detected.Surface water samples were collected in sterile Pyrex glass bottles on 26 September 2007 from Loch Rescobie (Ordinance Survey grid reference number NO 52505159), Forfar Loch (NO 293458), and the River Carron (NO 877857), Scotland, United Kingdom. Samples were stored at 4°C overnight and filtered as previously described (5). Aliquots from each water sample (2 × 500 ml) were processed and analyzed by high-performance LC to determine the presence of naturally occurring microcystins (13). Enrichment and shake flask die-away kinetics were monitored in triplicate for each water type (50 ml in sterile 100-ml Erlenmeyer flasks). To enrich bacteria with the ability to degrade a range of different microcystins, three microcystins, selected for their differing polarities, and the pentapeptide nodularin were added to each water sample. MC-LR, MC-RR, MC-LF, and nodularin (Enzo Life Sciences, Lausen, Switzerland) were resuspended in a small volume (100 μl) of methanol and diluted with Milli-Q to a total concentration of 0.4 mg ml⁻¹. The toxin cocktail was sterilized (0.2-μm Dynaguard filter; Fisher, United Kingdom) and added to each flask under aseptic conditions to give a final concentration of 1 μg ml⁻¹of each toxin (i.e., 4 μg ml⁻¹ total concentration). All flasks were incubated at 25°C ± 1°C with shaking at 100 rpm. Aliquots (2 ml) were removed from each flask under sterile conditions every 2 days, transferred into 4-ml glass vials, and frozen (−20°C) immediately. Die-away kinetics were monitored for 14 days. The frozen samples were freeze-dried, reconstituted in 200 μl of 50% aqueous methanol, and centrifuged at 15,000 × g for 10 minutes. The supernatant (100 μl) was removed for LC-MS analysis (5). Sterile controls (3 × 50 ml) were prepared, incubated, and sampled as described above to confirm whether loss of toxin was a result of microbial activity.After 14 days of enrichment, 1 ml of sample was removed aseptically from each flask, namely, the Loch Rescobie (R), Forfar Loch (F), and River Carron (C) samples. Serial dilutions (to 10⁻⁵) were made using Ringer''s solution (Oxoid Ltd., United Kingdom), and 1 ml of each dilution was removed and mixed with 20 to 25 ml of molten LB agar, poured onto sterile petri dishes, and incubated in the dark at 25°C for 5 days. Colonies with differing morphologies were resuspended in liquid LB medium, and pure cultures were obtained by repeated streaking onto LB agar plates. For the Biolog MT2 assay, a loop of each isolated bacterial strain was transferred to 5 ml of liquid LB medium and incubated overnight in the dark at 25°C. The exponentially growing cultures were then washed twice by centrifugation at 1,000 × g for 15 min, the bacterial pellets were resuspended in sterile 0.01 M phosphate-buffered saline, and the cultures were incubated at 25°C for 24 h to deplete residual carbon. The turbidity of all bacterial suspensions was an A₅₉₀ of 0.35. MC-LR was added to Biolog MT2 plates (Technopath, Limerick, Ireland) in triplicate to give final concentrations of 10, 1, 0.1, and 0 μg ml⁻¹. Wells were inoculated with bacterial suspension (150 μl), and plates were incubated at 25°C. Absorbance at 595 nm was recorded by using a Dynex microplate reader (Jencons, Leighton Buzzard, United Kingdom) immediately after inoculation (0 h) and at 3, 6, 15, 18, 24, and 48 h. Metabolism of MC-LR results in the reduction of tetrazolium violet, giving a color reaction that can be quantified spectroscopically (8). Bacterial isolates found by using the Biolog MT screen to metabolize MC-LR were evaluated for their ability to degrade MC-LR. Isolates C1, C3, and C6 (from the River Carron), F3, F7, and F10 (from Forfar Loch), and R1, R4, R6, and R9 (from Loch Rescobie) were grown overnight in LB liquid medium at 25°C. Bacterial isolates were washed and carbon depleted as described above (0.5 ml) and then added to glass universal bottles containing 9 ml of 0.2-μm-filter-sterilized water from their original locations. Aqueous MC-LR (0.5 ml) was added to each bottle under aseptic conditions at a final concentration of 5 μg ml⁻¹. Triplicate samples were prepared for each isolate and incubated at 25°C ± 1°C with shaking at 100 rpm. Aliquots (0.5 ml) were removed at 24-h intervals under sterile conditions, freeze-dried, reconstituted in 200 μl of 50% aqueous methanol, and centrifuged at 15,000 × g for 10 min. The supernatant (100 μl) was removed for LC-MS analysis performed as previously described (6). Experiments with sterile controls were performed for each water sample. Paucibacter toxinivorans DSMZ-16998 (Braunschweig, Germany) was used as a positive control as it has been reported to degrade MC-LR, MC-YR, and nodularin (16).To identify selected isolates, total DNA was extracted from the pellet by using an UltraClean DNA isolation kit (Mo Bio Laboratories, CA). Sequencing was performed with a BigDye Terminator cycle sequencing reaction kit (202 instrument; Applied Biosystems, United Kingdom) using 8F, 1492, and various other internal primers (518R and 1087R) on an automated DNA sequencer (ABI, United Kingdom) (7, 17). The quality of the sequence was checked by using the sequence analysis software (ABI), and the products of the forward and reverse primers were aligned using Kodon (Applied Maths, Saint-Martens-Latem, Belgium). The analyzed sequences were compared to DNA sequences in public databases using the BLAST function of NCBI (http://www.ncbi.nlm.nih.gov). Individual isolates were classified according to their similarity to sequences in the database. DNA sequences of all isolates, along with those of related bacteria and some known microcystin-degrading bacteria, were used to construct a phylogenetic tree using MEGA4 (18). The sequences were first aligned using Clustal W, and then a phylogenetic tree was constructed by performing neighbor-joining tree analysis with 1,000 bootstrap replicates. Each microcystin-degrading isolate was assayed to determine if mlr genes for the degradation of microcystin could be detected. The PCR method used primers specific for mlrA, mlrB, mlrC, and mlrD with conditions as described before (9). A positive control for these genes was used (Sphingopyxis sp. strain LH21).The Biolog MT2 plates, used to screen 31 isolates, were shown to be an effective means of rapidly identifying bacteria with the ability to metabolize MC-LR (Fig. ​(Fig.1).1). Ten isolates which demonstrated respiration in the presence of MC-LR using the Biolog format were subsequently proven to be microcystin-degrading bacteria in batch studies where MC-LR almost or totally disappeared after 3 days of incubation (Table ​(Table1).1). The results of LC-MS analysis indicated that MC-LR disappeared with no obvious biotransformation or intermediate degradation products. This may be because degradation resulted in only very low concentrations of these compounds and MC-LR is readily utilized by the isolates, as evidenced by respiration in the Biolog assay. Employing the Biolog MT plate enabled rapid (approximately 24 h) selection of bacteria in a high-throughput format (96-well plates) using considerably less microcystin or nodularin, i.e., 5 μg per isolate in the Biolog MT plate compared to 300 μg to follow degradation in die-away kinetics as described herein). Furthermore, following degradation by the latter method requires sample processing and high-performance LC analysis, increasing the time and cost. While the Biolog plates have been widely used for community profiling and bacterial identification, they have yet to be fully exploited in biodegradation studies, where they may facilitate rapid, cost-effective screening of many more bacterial isolates for the ability to utilize a wide range of environmental pollutants.Open in a separate windowFIG. 1.Results of Biolog screen for MC-LR metabolism by bacteria isolated from Loch Rescobie (A), Forfar Loch (B), and the River Carron (C) after 24 h of incubation. Control samples (black bars) contained no additional carbon source. MC-LR was added as the carbon source at 0.1 (open bars), 1 (hatched bars), and 10 (shaded bars) μg ml⁻¹. Error bars represent 1 standard deviation (n = 3).

TABLE 1.

Batch degradation of MC-LR by bacterial isolates in source

Contribution to the hydrographical structure of the eastern German Bight

Temperature, salinity, micronutrients, seston components and mesozooplankton were measured on a cruise in the eastern German Bight during November 1976. Three different water bodies and a mixing area which is divided into two subareas could be identified. The water masses differed significantly in regard to temperature, salinity, micronutrients and seston components. In some cases differences in the amounts of mesozooplankton could be found. Temperature and salinity of the water of the Elbe estuary and of the Wadden Sea were relatively low, but amounts of micronutrients and seston were high, whereas the water of the North Sea water body was of higher temperature and salinity with lower amounts of micronutrients and seston. The North Frisian coastal water and a southern mixing area can be regarded as mixing areas between these water bodies.     

Culturability and In Situ Abundance of Pelagic Bacteria from the North Sea

The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.     

Seasonal variability and inter-specific differences in hatching of calanoid copepod resting eggs from sediments of the German Bight (North Sea)

Hatching experiments were conducted on sediment samples collectedon seven cruises between March 2002 and February 2003, at fourstations near Helgoland (54°11'N, 7°53'E). Samples wereincubated for up to 12 months and examined weekly for calanoidcopepod nauplii. 12807 nauplii hatched in total. Of these 36.43%were stage N1 (pooled), 44.08% were Temora longicornis (stageN2 and older), and 17.96% were Centropages hamatus (stage N2and older). Some Acartia spp. and an unidentified species werefound, too. Hatching varied significantly between sampling dates.At three stations, counts of all nauplii peaked in samples takenin April, while at one station the maximum was recorded in thesample collected in October. The seasonal pattern of hatchingwas similar in T. longicornis and C. hamatus. In March, April,October and February numbers of all nauplii were initially low,but increased over the following 2–3 weeks. In June, Augustand December, however, average numbers were comparatively highat first, but declined thereafter. In three of the four samplesthat were incubated for 12 months nauplii hatched from the sedimentfor the entire experimental period. Temora longicornis and C.hamatus displayed clearly distinct patterns of hatching in theselong-term incubations.     

大蒜内生细菌的分离及拮抗菌筛选与鉴定

利用常规分离方法对大蒜鳞茎进行内生细菌的分离,采用对峙法和平板涂布法对分离的内生菌进行拮抗试验研究,并对菌株DSP6进行16S rDNA全序列鉴定。结果表明:分离得到19株内生细菌,其中10株菌对2种以上植物病原真菌有不同程度的抑制作用,占分离菌总数的52.6%,DSN7对番茄早疫病的抑菌圈半径最大,为13mm;17株菌对5种病原细菌中至少1种有抑制作用,占分离菌总数的89.5%,其中菌株DSP3对大肠杆菌的抑菌圈半径最大,达到10 mm;菌株DSP6对供试的9种病原菌有较强的抑菌作用,且抑菌圈平均半径最大,为6.88mm;16S rDNA全序列鉴定显示,菌株DSP6与芽孢杆菌属Bacillus axarquiensis相似性为100%,表明菌株DSP6为Bacillus axarquiensis。     

Seasonal Variations in Survival of Indicator Bacteria in Soil and Their Contribution to Storm-water Pollution

Survival of a fecal coliform (Escherichia coli) and a fecal streptococcus (Streptococcus faecalis var. liquifaciens) was studied through several years at shaded and exposed outdoor soil plots. Death rates for both organisms were calculated for the different seasons at both sites. The 90% reduction times for the fecal coliform ranged from 3.3 days in summer to 13.4 days in autumn. For the fecal streptococcus, 90% reduction times were from 2.7 days in summer to 20.1 days in winter. During summer, the fecal coliform survived slightly longer than the fecal streptococcus; during autumn, survival was the same; and in spring and winter the fecal streptococcus survived much longer than the fecal coliform. Both organisms were isolated from storm-water runoff collected below a sampling site when counts were sufficiently high in soil. Isolation was more frequent during prolonged rains, lasting up to 10 days, than during short rain storms. There was evidence of aftergrowth of nonfecal coliforms in the soil as a result of temperature and rainfall variations. Such aftergrowth may contribute to variations in bacterial count of storm-water runoff which have no relation to the sanitary history of the drainage area.     

Enumeration of Viable Bacteria in the Marine Pelagic Environment

The low percentage of living bacteria commonly obtained when comparing viable counts with total direct counts in seawater could be due more to inappropriate techniques for appreciating the growth ability of living cells than to unadapted culture conditions. The most-probable-number counts in filtered seawater cultures and the microscopic counts of 4(prm1),6-diamidino-2-phenylindole (DAPI)-stained aggregate-forming units grown on black polycarbonate filters appeared significantly correlated to the direct counts. Both these techniques show that in the superficial and intermediate water masses, the living cells may constitute an important (frequently higher than 20%) but highly variable part of the total populations. These viable counts appear more realistic than the conventional CFU counts, which provide only 0.001 to 0.2% of the total counts.     

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.     

Comparison of trace heavy-metal levels from monitoring in the German Bight and the southwestern Baltic Sea

Since 1973, monitoring for selected trace heavy metals has been performed in the German Bight (North Sea) and the western Baltic Sea. This paper deals with a set of filtered and unfiltered samples from a network of 22 stations covering the whole German Bight based on a cruise with RV “Gauss” in May 1974, and a similar series of samples taken at different depths at 18 stations in the Baltic Sea in October 1974. The metals Cd, Cu, Fe, Mn, and Ni were determined by flameless atomic absorption spectrometry. Data comparison is facilitated since sampling and analytical methods were identical. Distributions of metal-content data have been calculated from unfiltered and filtered samples. Qualitative and semi-quantitative aspects of the major differences or similarities between the histograms obtained from both sea areas are discussed.     

Distribution Pattern of Photosynthetic Picoplankton and Heterotrophic Bacteria in the Northern South China Sea

The environmental regulation of plcoplankton distribution in the northern South China Sea was examined In winter and summer of 2004. The average abundance of Synechococcus, Prochlorococcus, and heterotrophlc bacteria was lower In winter （30, 21, and 780×10^3 cells/cm^3, respectively） than In summer （53, 85, and 1 090×10^3 cells/cm^3, respectively）, but the seasonal pattern was opposite for plcoeukaryotlc phytoplankton （4 500 and 3 200 cells/cm^3 In winter and summer, respectively）. Synechococcus, picoeukaryotes, and bacteria were most abundant in the nutrient-rich coastal zone and continental shelf, but Prochlorococcus was most abundant In the continental slope and open ocean. The vertical distribution of each photosynthetic group and heterotrophlc bacteria changed between the two seasons. Synechococcus populations with apparently different phycoerythrobilin content occurred at many stations In the summer. In addition, two different populations of Prochlorococcus were found： （i） small, weakly fluorescing cells in the surface layer; and （ii） larger, strongly fluorescent cells In the deep layer. The distribution pattern of photosynthetic plcoplankton and heterotrophlc bacteria depends on environmental effects and their ecophyslologlcal differences. The distribution of Synechococcus appeared to be related to nutrient availability, whereas the distribution of Prochlorococcus appeared to be limited by temperature. Synechococcus was the only plcophytoplankton with a consistent strong relationship with bacteria.     

Linking Sequence to Function in Soil Bacteria: Sequence-Directed Isolation of Novel Bacteria Contributing to Soilborne Plant Disease Suppression

Microbial community profiling of samples differing in a specific ecological function, i.e., soilborne plant disease suppression, can be used to mark, recover, and ultimately identify the bacteria responsible for that specific function. Previously, several terminal restriction fragments (TRF) of 16S rRNA genes were statistically associated with damping-off disease suppression. This work presents the development of sequence-based TRF length polymorphism (T-RFLP)-derived molecular markers to direct the identification and isolation of novel bacteria involved in damping-off pathogen suppression. Multiple sequences matching TRF M139 and M141 were cloned and displayed identity to multiple database entries in the genera incertae sedis of the Burkholderiales. Sequences matching TRF M148, in contrast, displayed greater sequence diversity. A sequence-directed culturing strategy was developed using M139- and M141-derived markers and media reported to be selective for the genera identified within this group. Using this approach, we isolated and identified novel Mitsuaria and Burkholderia species with high levels of sequence similarity to the targeted M139 and M141 TRF, respectively. As predicted, these Mitsuaria and Burkholderia isolates displayed the targeted function by reducing fungal and oomycete plant pathogen growth in vitro and reducing disease severity in infected tomato and soybean seedlings. This work represents the first successful example of the use of T-RFLP-derived markers to direct the isolation of microbes with pathogen-suppressing activities, and it establishes the power of low-cost molecular screening to identify and direct the recovery of functionally important microbes, such as these novel biocontrol strains.     

Isolation and Identification of Three Potassium-Solubilizing Bacteria from Rape Rhizospheric Soil and Their Effects on Ryegrass

More than 90% of potassium (K) in soil exists in forms of insoluble silicate minerals and cannot be directly utilized by plants. K-solubilizing bacteria (KSB) can improve soil fertility and plant growth as biofertilizers by decomposing silicate minerals and releasing insoluble K into soluble forms. The objectives of this study were to isolate and characterize KSB from rape rhizospheric soil and to evaluate their effects on ryegrass growth. In this study, 16 cultivable potential KSB were isolated from rape rhizosphere at first. Then, quantitative analysis revealed that three KSB strains, named S-15, S-17 and S-18, showed the best K mineral solubilizing ability and they were identified as Mesorhizobium sp., Paenibacillus sp. and Arthrobacter sp. Inoculating the three strains into available K limit soil increased available K content significantly. The result of the pot experiment revealed that the three strains increased ryegrass growth vigor, biomass yield and K uptake to different degrees in available K deficient soil. S-17 showed the most pronounced ryegrass growth promotion ability. Further studies are required to determine the effects of the three KSB on mobilization of K-bearing minerals under field conditions.     

Isolation and Characterization of Bacteria from the Baltic Sea

A bacteriological examination was done on samples of water and sediment from three localities in the Baltic. The highest numbers of bacteria were recovered from areas subjected to pollution. The isolates included members of the family Enterobacteria-ceae, the genus Pseudomonas and strains of Aeromonas hydrophila, Alteromonas putrefaciens and some Gram positive bacteria. It is suggested tentatively that H2S production in the black sediments was caused by Alt. putrefaciens. None of the isolates had an absolute requirement for NaCl, although all of them were salt-tolerant to varying degrees, and most were able to grow aerobically at salinities comparable with those found in seawater. Isolates belonging to the family Enterobacteriaceae were, however, unable to grow anaerobically under comparable conditions. Freshwater strains of several genera of the family Enterobacteriaceae and of Aeromonas hydrophila and Aer. sobria displayed salt tolerance identical with that of the Baltic isolates. One strain each of Escherichia coli, Klebsiella pneumoniae and Yersinia enterocolitica survived well during three weeks at 17°C in artificial seawater lacking both carbon and nitrogen sources. These results suggest the need for a re-evaluation of the persistence of potentially pathogenic bacteria in the sea.     

固碳微生物菌株的分离鉴定及其固碳能力测定

利用微生物回收和固定CO2气体的生物固碳方法,成为解决"温室效应"这一重大环境问题的焦点,本研究目的是分离筛选固碳微生物菌株。利用无碳源无机培养基从活性污泥、沼液和设施土壤中分离筛选出以CO2为碳源的菌株24株,选取其中生长较快的8株菌进行cbbL基因、形态观察、生理生化反应测定和16S rDNA测序分析,将测序结果在BlAST数据库中比对,进行固碳菌株的分子鉴定,并对其菌体含量和RubisCO酶活性进行比较。选取RubisCO酶活性最高的菌株C2-8R,进行土壤施用试验,通过土壤RubisCO酶活性的测定,确定分离筛选的固碳菌的固碳能力。研究表明,可通过无碳源培养基进行固碳微生物菌株的筛选,筛选的固碳菌分别隶属于假单胞菌属和嗜甲基菌属,并可通过RubisCO酶活性来反映微生物的固碳能力。     

利用一碳化合物(C1)细菌的分离及基因组文库的构建

从污水样品中筛选到能利用甲醇的菌株B,经16SrDNA测序分析鉴定为肺炎克氏杆菌(Klebsiella pneumo-niae)。甲醛耐受能力的测试表明该菌对甲醛具有较强耐受能力,能在含有8￣15mmol/L甲醛的LB培养基上生长。Southern杂交分析说明这菌株的基因组中有甲基营养菌6-磷酸己酮糖合成酶(HPS)和6-磷酸果糖异构酶(PHI)基因的同源序列。本研究以pUC118为载体构建了基因组文库,进一步检测结果说明所构建的基因组文库质量符合要求。