具细菌群体感应抑制活性海洋细菌的筛选鉴定

目的:从海洋环境中筛选对细菌群体感应有抑制作用的活性菌株,为以致病菌群体感应系统为靶点的新型疗法提供新的药用资源。方法:从海水中分离纯化细菌菌株,采用根癌农杆菌平板筛选模型筛选细菌群体感应抑制活性细菌,对筛选出的海洋细菌进行生理生化和16S rDNA序列测定,根据《伯杰氏手册》进行菌种分类鉴定。结果:从217株海洋细菌中筛选出1株能显著抑制细菌群体感应效应的海洋细菌Y2,该海洋细菌具有蜡样芽孢杆菌(Bacillus cereus)的典型特征,其16S rDNA序列与GenBank中蜡样芽孢杆菌16S rDNA的部分序列有100%的同源性。结论:海洋环境中也存在具有抑制细菌群体感应活性的微生物。     

Differential growth response of colony-forming alpha- and gamma-proteobacteria in dilution culture and nutrient addition experiments from Lake Kinneret (Israel), the eastern Mediterranean Sea,and the Gulf of Eilat

Even though it is widely accepted that bacterioplankton growth in lakes and marine ecosystems is determined by the trophic status of the systems, knowledge of the relationship between nutrient concentrations and growth of particular bacterial species is almost nonexistent. To address this question, we performed a series of culture experiments with water from Lake Kinneret (Israel), the eastern Mediterranean Sea, and the Gulf of Eilat (northern Red Sea). In the initial water samples, the proportion of CFU was typically <0.002% of the 4',6'-diamidino-2-phenylindole (DAPI) counts. During incubation until the early stationary phase, the proportion of CFU increased to 20% of the DAPI counts and to 2 to 15% of the DAPI counts in unenriched lake water and seawater dilution cultures, respectively. Sequencing of the 16S ribosomal DNA of colony-forming bacteria in these cultures consistently revealed an abundance of alpha-proteobacteria, but notable phylogenetic differences were found at the genus level. Marine dilution cultures were dominated by bacteria in the Roseobacter clade, while lake dilution cultures were dominated by bacteria affiliated with the genera Sphingomonas and CAULOBACTER: In nutrient (glucose, ammonium, phosphate) addition experiments the CFU comprised 20 to 83% of the newly grown cells. In these incubation experiments fast-growing gamma-proteobacteria dominated; in the marine experiments primarily different Vibrio and Alteromonas species appeared, while in the lake water experiments species of the genera Shewanella, Aeromonas, and Rheinheimera grew. These results suggest that major, but different, gamma-proteobacterial genera in both freshwater and marine environments have a preference for elevated concentrations of nutrients and easily assimilated organic carbon sources but are selectively outcompeted by alpha-proteobacteria in the presence of low nutrient concentrations.     

Differential Growth Response of Colony-Forming α- and γ-Proteobacteria in Dilution Culture and Nutrient Addition Experiments from Lake Kinneret (Israel), the Eastern Mediterranean Sea, and the Gulf of Eilat

Even though it is widely accepted that bacterioplankton growth in lakes and marine ecosystems is determined by the trophic status of the systems, knowledge of the relationship between nutrient concentrations and growth of particular bacterial species is almost nonexistent. To address this question, we performed a series of culture experiments with water from Lake Kinneret (Israel), the eastern Mediterranean Sea, and the Gulf of Eilat (northern Red Sea). In the initial water samples, the proportion of CFU was typically <0.002% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. During incubation until the early stationary phase, the proportion of CFU increased to 20% of the DAPI counts and to 2 to 15% of the DAPI counts in unenriched lake water and seawater dilution cultures, respectively. Sequencing of the 16S ribosomal DNA of colony-forming bacteria in these cultures consistently revealed an abundance of α-proteobacteria, but notable phylogenetic differences were found at the genus level. Marine dilution cultures were dominated by bacteria in the Roseobacter clade, while lake dilution cultures were dominated by bacteria affiliated with the genera Sphingomonas and Caulobacter. In nutrient (glucose, ammonium, phosphate) addition experiments the CFU comprised 20 to 83% of the newly grown cells. In these incubation experiments fast-growing γ-proteobacteria dominated; in the marine experiments primarily different Vibrio and Alteromonas species appeared, while in the lake water experiments species of the genera Shewanella, Aeromonas, and Rheinheimera grew. These results suggest that major, but different, γ-proteobacterial genera in both freshwater and marine environments have a preference for elevated concentrations of nutrients and easily assimilated organic carbon sources but are selectively outcompeted by α-proteobacteria in the presence of low nutrient concentrations.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment.

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Heterotrophic bacterial guild structure: Relationship to biodegradative populations

Numerical taxonomic analysis of a freshwater bacterial guild demonstrated that the bacteria capable of growth on phenanthrene and polychlorinated biphenyl media were representative of the taxa obtained from low nutrient oligotrophic media. The diversity of heterotrophic bacteria and members of new taxa recovered from the guild followed a poisson distribution relative to the number of isolation media used. Moderately high nutrient, yeast extract peptone and glucose agar was found to be the most selective isolation medium relative to the total number of taxa recovered whereas low nutrient, lake water agar was the least selective medium used. Carbon source utilization patterns of the isolated taxa indicated that taxa within the guild had broad niche ranges and could potentially occupy many niches within a dynamic environment. The structure of the bacterial guild was dominated by mesophilic oligotrophs. The results of this investigation demonstrate that potential biodegradative populations are representative of the diverse taxa found in uncontaminated freshwater environments.     

Study of the bacterial flora of a non-carbonated natural mineral water

Natural mineral water from a UK spring was monitored at various stages after it was pumped from the ground, through to bottling and during shelf life before consumption. Samples were collected in commercial PVC bottles, in PVC bottles previously sterilized and hand-filled and in glass bottles. The bacterial flora was counted on plate count agar (PCA) and on PCA diluted 10 times (PCA/10). The predominant bacteria were identified to genus level. Growth rates and nutrient types of isolates were determined by the nutrient-tolerance test (NT). The plate counts at the prebottling stage were low. During storage larger numbers of bacteria grew in glass than PVC bottles; the largest number grew in PVC bottles filled by hand. Most of the pigmented bacteria isolated were oligocarbotolerant.     

Study of the bacterial flora of a non-carbonated natural mineral water.

Natural mineral water from a UK spring was monitored at various stages after it was pumped from the ground, through to bottling and during shelf life before consumption. Samples were collected in commercial PVC bottles, in PVC bottles previously sterilized and hand-filled and in glass bottles. The bacterial flora was counted on plate count agar (PCA) and on PCA diluted 10 times (PCA/10). The predominant bacteria were identified to genus level. Growth rates and nutrient types of isolates were determined by the nutrient-tolerance test (NT). The plate counts at the pre-bottling stage were low. During storage larger numbers of bacteria grew in glass than PVC bottles; the largest number grew in PVC bottles filled by hand. Most of the pigmented bacteria isolated were oligocarbotolerant.     

Masking of antibiotic-resistance upon recovery of endophytic bacteria

During studies on internal plant colonization by rhizosphere bacteria and endophytic bacteria over several years, we frequently observed lack of growth of rifampicin-resistant mutants (rif+) on tryptic soy agar amended with rifampicin (RTSA). Following seed treatment of cucumber with 6 species of rif+ rhizosphere bacteria in one experiment, all strains were recoverable on RTSA when external root colonization was monitored. Following trituration of surface-disinfested roots, only one strain grew directly on RTSA; however colonies isolated on tryptic soy agar (TSA) grew within 18 h after transfer to RTSA. We term this temporary loss of the antibiotic-resistant phenotype ‘antibiotic masking’. Antibiotic masking was also observed with isolation of 7 rif+ endophytic bacterial strains from inside stems of cotton and with isolation of mutants of bacterial endophytes resistant to polymyxin B sulfate from cotton plants. Rifampicin-masking was not accounted for in vitro by inhibitory compounds from cotton plant extracts, by bacterial growth on low nutrient agar, or by competition with other bacteria. Collectively, these results suggest that expression of antibiotic-resistance may be altered in planta, although causes for this antibiotic-masking remain to be elucidated, methods for quantifying internal plant colonization by rif+ bacteria should account for this possibility. ei]Section editor: R O D Dixon     

Microcultural Study of Bacterial Size Changes and Microcolony and Ultramicrocolony Formation by Heterotrophic Bacteria in Seawater

With a microculture technique and time-lapse, phase-contrast photomicrography, it was possible to follow the division of individual cells and the development of microcolonies of bacteria in freshly collected marine water samples. A certain number of marine bacteria, upon inoculation onto a nutrient rich agar surface, displayed an increase in size as well as a high growth rate. Other bacteria were identified as very small marine bacteria (ultramicrobacteria). These had a very slow growth rate when inoculated onto a nutrient-rich agar surface. These latter cells formed very small microcolonies (ultramicrocolonies), and cell size did not increase significantly. These two types of marine heterotrophs could be described in terms of zymogenous and autochthonous bacteria, a concept used by Winogradsky for describing soil microorganisms.     

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.     

Manganese Oxidation by Bacterial Isolates from the Indian Ridge System

The abundance and activity of culturable manganese-oxidizing bacteria were assessed from near-bottom water samples of the tectonically active Carlsberg Ridge. Retrievable counts as colony forming units (CFU) on dilute nutrient agar medium (dilNA = 2 gm l⁻¹ nutrient broth+2% agar) and on dilNA supplemented with 1, 2 and 3 mM MnCl₂·4H₂O were in the order of 10⁶ CFU l⁻¹. Retrievability of heterotrophs ranged from non-detectable levels (ND) to 2.82 × 10⁶ CFU l⁻¹. The retrievable counts on Mn amended dilNA ranged from ND to 3.21× 10⁶, 1.47 × 10⁶ and 1.45 × 10⁶ CFU l⁻¹ on 1, 2 and 3 mM, respectively. About 87% of the Mn tolerant isolates (n = 39) showed taxonomic affinities to Pseudomonas I and II sp. Two representative strains CR35 and CR48 (CR–Carlsberg Ridge) isolated on manganese-supplemented media were tested for their ability to tolerate a range of Mn amendments from 1 nM to 100 mM in terms of growth and respiration. CR35 represents 66% of the total CFU (3.04 × 10⁶ CFU l⁻¹), while CR48 represented only 6% of the total CFU (1.05 × 10⁶ CFU l⁻¹). The colonies of these two isolates were dark brown in color suggesting precipitation of Mn as oxide. Tests for the effect on growth and respiration were conducted in media simulating heterotrophic (amended with 0.01% glucose) and lithotrophic (unamended) conditions. Maximum stimulation in growth and respiration of CR35 occurred at 100 μM Mn both in unamended and amended media. At levels of Mn greater than 100 μM the counts decreased steadily. Total respiring cells of CR48 were stimulated to a maximum at 1 μM Mn in unamended medium and 1 nM in amended medium. Total cells counts for the same decreased beyond 100 μM Mn in unamended and 1 nM in amended medium. The isolates were tested for their ability to oxidize Mn ammendments from 1 μM to 10 mM Mn. At the end of a 76-day incubation period, there was evidence of manganese oxide precipitation at high Mn concentrations (≥1 mM) as a dark brown coloration on the sides of culture tubes. Highest Mn oxidation rates were observed at 10 mM Mn(II) concentration with CR35 oxidizing 27 and 25 μM Mn day⁻¹ in unamended and amended condition, respectively. CR48 oxidized Mn at the rate of 26 μM Mn day⁻¹ in unamended medium and 35 μM Mn day⁻¹ in amended medium. Scanning electron microscope (SEM) observations of both isolates revealed free-living cells in clustered matrices ≈2 μm diameter. Energy dispersive spectrum of the cell matrix of CR35 cultured in 1 mM Mn detected 30% Mn, while the cell aggregates of CR48 harbored 7–10% Mn. The relatively high specific activity of these mixotrophic bacteria under relatively oligotrophic conditions suggests that they may be responsible for scavenging dissolved Mn from the Carlsberg Ridge waters and could potentially participate in oxidation.     

Prokaryotic abundance and 16S rRNA gene sequences detected in marine aerosols on the East Sea (Korea)

Modern studies on marine airborne prokaryotes over open seas have been rare. Here, to understand their distribution and composition, aerosol as well as surface seawater samples were collected at three sites in the East Sea. The abundance of airborne prokaryotes was 0.7-1.2 × 10(5) cells m(-3) . Partial sequences of the 16S rRNA gene from the aerosols were dominated by Gammaproteobacteria and Bacteroidetes. Interestingly, the numbers of phylotypes of airborne bacteria were comparable to those of seawater bacteria in coastal and remote offshore sites. Over half of the bacterial operational taxonomic units (OTUs) detected in these aerosols (12 out of 20) were of marine origin. Eight of the 19 bacterial OTUs found in the surface waters occurred in the aerosols. Thus, surface seawater seems to be a main source of marine airborne bacteria. However, in the intermediate offshore site, airborne bacterial OTUs (n=4) were all terrestrial, indicating that variations of dominant airborne bacterial phylotypes occurred over the sites (each ～160 km apart) sampled at 1-4-day intervals. Euryarchaeota sequences belonging to the MSP8 clade reported from geographically distant regions were detected in marine aerosols. Long-distance aerial transport of the marine prokaryotes seems to be quite possible.     

Mercury and methylmercury detoxification potential by sponge-associated bacteria

Ionic and organic forms of mercury (Hg) are powerful cytotoxic and neurotoxic agents in both humans and wild life. The aim of this study was to analyze the resistance profile and potential detoxification of inorganic and organic forms of Hg of bacteria isolated from marine sponges on the coast of Rio de Janeiro, Brazil. Out of the 1,236 colony forming units associated with eleven species of marine sponges, 100 morphologically different bacterial strains were analyzed in this study. Of these, 21 strains were resistant to Hg, 14 of which were classified as highly resistant because they grew despite exposure to 100 µM HgCl₂. Fifteen resistant strains reduced Hg and presented merA in their genomes. The remaining six strains produced biosurfactants, suggesting that they may tolerate Hg by sequestration. Eleven strains grew in the presence of methylmercury. Our results suggest a potential for mercury detoxification by marine sponge-associated resistant bacteria, either through reduction or sequestration, as well as the possibility of bioremediation of toxic waste containing mercury.     

Isolation of Marine Bacteria by In Situ Culture on Media-Supplemented Polyurethane Foam

Polyurethane foam (PUF) supplemented with various agar media was used in situ to trap marine bacteria and it consequently provided a substrate on which they could be cultivated while exposed to natural seawater in the coral reef area. The bacterial population on the PUF blocks was analyzed by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. Changing the composition of the cultivation medium in the PUF blocks and selecting different sampling sites resulted in different bacteria being detected on the PUF blocks. For example, iron-utilizing (IU) bacteria, siderophore-producing (SP) bacteria, and petroleum-degrading (PD) bacteria were isolated from PUF blocks and it was discovered that IU and SP contained iron and PD contained hydrocarbon. This method opens up the possibility for isolating novel and useful marine bacteria.     

Growth characteristics of low-nutrient bacteria from the north-east and central Pacific Ocean

Abstract Low-nutrient, or oligotrophic, marine bacteria, defined operationally as those which grow well in the dilute nutrient concentrations typical of natural seawaters, were found in waters of the eastern and central Pacific Ocean. All isolates tested grew in sterile unsuplemented seawater, ultraviolet-irradiated seawater, artificial seawater and charcoal-treated artificial seawater. Under optimal conditions, doubling times for low-nutrient bacteria were generally 2–5 h. Low-nutrient bacteria are heterotrophs responding to added organic substrates including glucose, proline, acetate, peptone, algal extract and yeast extract. Organic substrate concentrations ( K _s values) necessary to yield half the maximum growth rates of low-nutrient bacteria ranged from 0.8–12 μg · 1⁻¹ (0.2–4.9 μg C · 1⁻¹). Bacterial inoculum size had little effect on doubling times, whereas low oxygen tensions and low temperatures reduced growth rates. Surfaces (glass beads) increased growth rates and maximum cell densities. The results of these laboratory studies indicate that low-nutrient bacteria play a major role in marine heterotrophic transformations of dissolved organic matter.     

A-Factor as a selective agent for isolation of the soil Gram-negative bacterium strain--the producent of an antibacterial antibiotic]

It was shown the stimulating role of A-factor on soil prokaryotes growth. Soil sample culturing on agar medium, containing A-factor, resulted in the colony forming units (CFU) increasing in comparison with culturing on the medium without this regulator. Gram-negative bacteria were the reason of CFU increasing; previously the effect of A-factor on bacteria of this group was not shown. Gram-negative rod bacterial strain No. 35 was isolated and shown that CFU number was approximately twice increased at A-factor concentration in agar medium 2 and 7 mcg/ml; high A-factor concentration up to 28 mcg/ml was not effective. Isolated strain No. 35 is the producer of antibiotic, effective against gram-positive bacteria.     

南海冷泉区深海沉积物中细菌的分离培养及多样性分析

为了认识南海深海冷泉区沉积物中可培养微生物的多样性,本文以冷泉区与非冷泉区两个站点的深海沉积物为样品,通过两种培养基(R2A海水培养基和2216E培养基)直接涂布或富集后平板分离纯化,从9个样品中共得到395株菌株,并通过16SrRNA基因鉴定,分属10个属。发现产芽胞细菌分布最广、丰度最大,包括3个属、15个种。其中芽胞杆菌(Bacillus)无论是在数量还是在种类上都分布最多。并且,随着水深和沉积物深度的增加,分离到的可培养微生物丰富度降低。本研究表明,即使在冷泉区,南海深海沉积物中产芽胞细菌也比较丰富。     

Characterization of marine bacteria highly resistant to mercury exhibiting multiple resistances to toxic chemicals

Several strains of bacteria unusually highly resistant to mercury were isolated from seawater and marine sediment samples and identified by 16S rDNA sequencing and were also characterized by a battery of biochemical and morphological tests. The bacterial isolates were identified to belong to the genera Pseudomonas, Alcaligenes, Brevibacterium and Bacillus. Many of the chosen isolates were tested for growth in the presence of different heavy metals and a variety of xenobiotics. Growth curves of all six bacteria highly resistant to mercury examined for growth at different concentrations of Hg exhibited prolonged lag phase, during which time necessary physiological adaptations to toxic milieu were undergone. All the strains tested for antibiotic resistance showed little to no effect of antibiotics on their normal growth. Results of this study demonstrate the occurrence of diverse groups of marine prokaryotes capable of high tolerance to mercury with a potential to degrade a variety of toxic heavy metals and xenobiotics.     

"Bactericidal" property of seawater: death or debilitation?

Coliform colony-forming units in sewage-contaminated seawater were observed to decrease rapidly with time in water that was collected from St. John's Harbour, Newfoundland, and isolated in dialysis bags; this confirms observations made in warmer climates. Adenosine 5'-triphosphate biomass, however, did not decline, nor did the particle size distribution of radioactively labeled coliforms change. It was observed that the coliforms were not killed by seawater but were debilitated to the extent that they would not form colonies on selective media. However, they recovered and grew on nutrient agar made with seawater. The adenosine 5'-triphosphate content per cell apparently did not decline during debilitation.