Cryptoendolithic microorganisms from stratified communities in Antarctic sandstone were studied for physiological diversity and possible interactions. Cultures of 25 bacteria, five fungi, and two green algae from one boulder grew with a wide variety of organic carbon or nitrogen sources, they exhibited varied exoenzymatic activities and were psychrophilic or psychrotrophic. Many isolates excreted vitamins into the medium and were stimulated by other vitamins. Organic acid excretion and siderophore formation were common, but antibiotic activity was rare. Plasmids were found in 24% of the bacteria, and many of these strains showed resistance to antibiotics and heavy metals. A small plasmid (2.9 kb) from strain AA-341 was electrotransferred into sensitive isolates, thereby rendering these resistant to ampicillin and Cr 3÷. Bacterial cultures in spent algal medium and coculture with algae demonstrated beneficial (rarely inhibitory) interactions. A search for free organic compounds in zones of the sandstone community revealed sugars, sugar alcohols, organic acids and amino acids -in many cases the same compounds that were excreted into the laboratory medium. Data presented here indicate low taxonomic but high physiological diversity among these heterotrophic cryptoendoliths. This physiological diversity, as well as the spatial separation in layers with distinct activities, allows coexistence within the community and contributes to the stability of this ecosystem.
I nst it ut fur Al lg emei ne Mi kro biologie, U n iversitat Kiel, Am Botanischen Garten 1-9, 0-241 18 Kiel, Germany Almost complete 16s rDNA sequences from the type strains of seven species of the genus Hyphomicrobium and of Filomicrobium fusiforme have been determined. The Hyphomicrobium species form two phylogenetic clusters that are only moderately related to each other. While cluster I contains the type species Hyphomicrobium vulgare, Hyphomicrobium aestuarii, Hyphomicrobium hollandicum and Hyphomicrobium zavarzinii, cluster II comprises Hyphomicrobium facilis, Hyphomicrobium denitrificans and Hyphomicmbium methylovomm. Within the two species clusters, the species are highly related. Phylogenetically, Filomicmbium fusiforme clusters moderately with Hyphomicrobium species. The lack of distinguishing phenotypical properties presently excludes the possibility of describing cluster II as a new genus.
Abstract. The aim of this study was to compare structural differences in the nirS-type denitrifying microbial communities along the environmental gradients observed in the water column and coastal sediments of the Baltic Sea. To link community structure and environmental gradients, denitrifier communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) based on nirS as a functional marker gene for denitrification. nirS-type denitrifier community composition was further evaluated by phylogenetic analysis of nirS sequences from clone libraries. T-RFLP analysis indicated some overlap but also major differences between communities from the water column and the sediment. Shifts in community composition along the biogeochemical gradients were observed only in the water column while denitrifier communities were rather uniform within the upper 30 mm of the sediment. Specific terminal restriction fragments (T-RFs) indicative of the sulfidic zone suggest the presence of nitrate-reducing and sulfide-oxidizing microorganisms that were previously shown to be important at the suboxic-sulfidic interface in the water column of the Baltic Sea. Phylogenetic analysis of nirS genes from the Baltic Sea and of sequences from marine habitats all over the world indicated distinct denitrifier communities that grouped mostly according to their habitats. We suggest that these subgroups of denitrifiers had developed after selection through several factors, i.e. their habitats (water column or sediment), impact by prevalent environmental conditions and isolation by large geographic distances between habitats.
Budding methylotrophic bacteria resembling Hyphomicrobium spp. were counted for 12 months in a German sewage treatment plant by most-probable-number (MPN) methods. Influent samples contained up to 2 ؋ 10 4 cells ml ؊1 , activated sludge consistently contained 1 ؋ 10 5 to 5 ؋ 10 5 cells ml ؊1 , and the effluent contained 1 ؋ 10 3 to 4 ؋ 10 3 cells ml ؊1 . The receiving lake had only 2 to 12 cells ml ؊1 . Six morphological groups with different growth requirements could be observed among 1,199 pure cultures that had been isolated from MPN dilutions. With dot blot DNA hybridizations, 671 isolates were assigned to 30 hybridization groups (HGs) and 84 could not be classified. Only HG 22 hybridized with a known species, Hyphomicrobium facilis IFAM B-522. Fourteen HGs (HGs 8 to 20 and HG 22) were specific for the lake; most others occurred only in the treatment plant. HGs 1, 3, and 26 were found in the activated sludge tank throughout the year, and HGs 27 and 28 were found for most of the year. In summary, it was demonstrated that bacteria with nearly identical and specific morphologies and nutritional types showed a high level of genetic diversity, although they were isolated under the same conditions and from the same treatment plant or its receiving lake. A directional exchange of these genetically different populations was possible but less significant, as was shown by the establishment of distinct populations in specific stations.
Genomic DNA of Hyphomicrobium spp., Hirschia baltica ATCC 49814T, Hyphomonas oceanitis ATCC 33897T, and Pedomicrobium ferrugineum S-1290Twas investigated with gene probes specific for nitrate reductase (narG), cytochrome cd1containing nitrite reductase (nirS), Cu-containing nitrite reductase (nirK), nitrous oxide reductase (nosZ), ammonia monooxygenase (amoA), and nitrogenase reductase (nifH) by Southern or dot blot hybridization. The presence of denitrification genes could be demonstrated for Hyphomicrobium denitrificans 1869T, Hyphomicrobium aestuarii IFAM NQ-521GrT, Hyphomicrobium zavarzinii IFAM ZV-580, Hyphomicrobium zavarzinii subsp. chengduense, in the Hyphomicrobium DNA-DNA hybridization groups 3, 12, 13, 18, 26a1, 26a2, 26c, 26d, 26e, 26f, 26g2, and 29, and in three isolates from a denitrifying sand filter in a municipal wastewater treatment plant. The Cu-containing nitrite reductase appeared to be more frequent than the cytochrome cd1containing nitrite reductase in hyphomicrobia. Resulting positive DNA-DNA hybridization signals correlated with physiological activity measurements of intact cells in all cases determined. The nifH-like gene fragment was found in Hyphomicrobium aestuarii, Hyphomicrobium zavarzinii, Hyphomicrobium zavarzinii subsp. chengduense, Hyphomicrobium facilis and eight additional DNA-DNA hybridization groups. Ammonia monooxygenase was not genetically detected in any of the strains investigated. The results significantly extended the previous findings that genetically different hyphomicrobia of a sewage treatment plant and its adjacent receiving lake could occupy different ecological niches. Denitrification genes or the nifH-like gene fragment were not found in the other budding bacteria investigated.Key words: Hyphomicrobium, denitrification, nitrification, nitrogen fixation, activated sludge, biofilm.
Microbial respiration-based microbiosensors used for quantification of available dissolved organic carbon (ADOC) instantaneously respired by microorganisms are described. The sensing membranes contained aerobic seawater microorganisms immobilized in a polyurethane hydrogel. Molecular investigations revealed that the bacterial strain used was most closely related to Staphylococcus warneri. This strain was characterized by low substrate selectivity, which was reflected in the response to various mono-and disaccharides, short-chain fatty acids, and amino acids, as determined using Biolog microplates. Specific emphasis was placed on critically assessing biosensor functioning that was affected by preconditioning of the selected bacterial strain, chemical and geometric properties of the sensing membrane (e.g., composition, permeability, and thickness), and the distribution, biomass, and physiological state of immobilized cells, as well as the exposure conditions (e.g., temperature and nutrient supply). The sensors revealed that there was a linear response up to a glucose concentration of 500 M depending on the type, characteristics, and recent history of the sensors. The detection limit of the sensors was equivalent to about 6 to 10 M glucose. The 90% response time ranged from 1 to 5 min. Generally, the response of the biosensors became weaker with time. The shelf lives of individual sensors were up to 2 weeks. Measurements based on optical ADOC microbiosensors revealed that in photoautotrophically dominated sandy coastal sediments, the pool sizes and turnover of ADOC were regulated by the photosynthetic activity of benthic microalgae and microbial aerobic respiration. A large increase in ADOC production was observed shortly after the microphytobenthic primary production reached the maximum value at midday, whereas ADOC was consumed by microbial respiration during the night.A relatively new and challenging technique in microbial ecology is the use of whole-cell microbial biosensors (12, 14, 48) for measurement of various organic and inorganic compounds in aquatic environments. Such biosensors consist of a transducer (e.g., an electrochemical electrode or optode) (for reviews, see references 2, 4, 9) in conjunction with prokaryotic or eukaryotic microorganisms that either are immobilized in a polymeric matrix or are suspended in a reaction chamber in front of the sensor tip. Metabolic functions, such as respiration or the luminescence of cells, are used to monitor the concentration of analytes that are either substrates or inhibitors of these processes. Depending on their selectivity, biosensors enable workers to measure either a specific microbial response to a single compound (specific biosensors) or a common response to groups of environmental compounds (nonspecific biosensors). The rapid and sensitive responses of biosensors have been utilized to determine the presence of a broad spectrum of substances, especially for environmental control (e.g., biosensors for environmental contaminants [47]) and toxic substances ...
An efficient system for electroporation of the methylotrophic bacteria Hyphomicrobium facilis, Hyphomicrobium denitrificans, Methylobacillus glycogenes, Methylobacterium extorquens, and Methylophilus methylotrophus is described. It could be demonstrated that vectors based on the broad-host-range plasmid pBBR1 could be transferred into these strains. Plasmid pBBR1KAN (3.9 kb), a kanamycin-resistant derivative of pBBR1, was suitable for transformation experiments in these methylotrophic bacteria. Transformation efficiencies up to 10(4) transformants/microgram plasmid pBBR1KAN were obtained. The broad-host-range plasmid pLA2917 was transferred into Hyphomicrobium species by a triparental mating. However, this plasmid was integrated into the genome of Hyphomicrobium spp. Plasmids pLA2917, pKT231, pSUP2021, pRZ705, and phage DNA could not be transferred in Hyphomicrobium spp. by electroporation under the conditions applied.
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