Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

Streptomonospora litoralis sp. nov., a halophilic thiopeptides producer isolated from sand collected at Cuxhaven beach

Antonie van Leeuwenhoek - Strain M2T was isolated from the beach of Cuxhaven, Wadden Sea, Germany, in course of a program to attain new producers of bioactive natural products. Strain M2T produces...     

Striptease on glass: validation of an improved stripping procedure for in situ microarrays

Microarrays have rapidly become an indispensable tool for gene analysis. Microarray experiments can be cost prohibitive, however, largely due to the price of the arrays themselves. Whilst different methods for stripping filter arrays on membranes have been established, only very few protocols are published for thermal and chemical stripping of microarrays on glass. Most of these protocols for stripping microarrays on glass were developed in combination with specific surface chemistry and different coatings for covalently immobilizing presynthesized DNA in a deposition process. We have developed a method for stripping commercial in situ microarrays using a multi-step procedure. We present a method that uses mild chemical degradation complemented by enzymatic treatment. We took advantage of the differences in biochemical properties of covalently linked DNA oligonucleotides on in situ synthesized microarrays and the antisense cRNA hybridization probes. The success of stripping protocols for microarrays on glass was critically dependent on the type of arrays, the nature of sample used for hybridization, as well as hybridization and washing conditions. The protocol employs alkali hydrolysis of the cRNA, several enzymatic degradation steps using RNAses and Proteinase K, combined with appropriate washing steps. Stripped arrays were rehybridized using the same protocols as for new microarrays. The stripping method was validated with microarrays from different suppliers and rehybridization of stripped in situ arrays yielded comparable results to hybridizations done on unused, new arrays with no significant loss in precision or accuracy. We show that stripping of commercial in situ arrays is feasible and that reuse of stripped arrays gave similar results compared to unused ones. This was true even for biological samples that show only slight differences in their expression profiles. Our analyses indicate that the stripping procedure does not significantly influence data quality derived from post-primary hybridizations. The method is robust, easy to perform, inexpensive, and results after reuse are of comparable accuracy to new arrays.     

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.     

Phylogenetic diversity of Flavobacteria isolated from the North Sea on solid media

Flavobacteria are abundant in the North Sea, an epeiric sea on the continental shelf of Europe. However, this abundance has so far not been reflected by the number of strains in culture collections. In this study, Flavobacteria were isolated from pelagic and benthic samples, such as seawater, phytoplankton, sediment and its porewater, and from surfaces of animals and seaweeds on agar plates with a variety of carbon sources. Dilution cultivation with a new medium, incubation at low temperatures and with long incubation times, and colony screening by a Flavobacteria-Cytophagia-specific PCR detecting 16S rRNA gene sequences led to a collection of phylogenetically diverse strains. Two strains affiliated with Flammeovirgaceae and seven strains affiliated with Cyclobacteriaceae, whereas within the Flavobacteriaceae 20 isolated strains presumably represented seven novel candidate genera and 355 strains affiliated with 26 of 80 validly described marine Flavobacteriaceae genera, based on a genus boundary of 95.0% 16S rRNA gene sequence identity. The majority of strains (276) affiliated with 37 known species in 16 genera (based on a boundary of 98.7% 16S rRNA gene sequence identity), whereas 79 strains likely represented 42 novel species in 22 established Flavobacteriaceae genera. Pigmentation, iridescence, gliding motility, agar lysis, and flexirubin as a chemical marker supported the taxonomy at the species level. This study demonstrated the culturability on solid medium of phylogenetically diverse Flavobacteria originating from the North Sea.     

Carbon source — A strong determinant of microbial community structure and performance of an anaerobic reactor

Industrial effluents differ in their organic composition thereby providing different carbon sources to the microbial communities involved in its treatment. This study aimed to investigate the correlation of microbial community structure with wastewater composition and reactor's performance. Self-immobilized granules were developed in simulated wastewater based on different carbon sources (glucose, sugarcane molasses, and milk) in three hybrid anaerobic reactors operated at 37 °C. To study archaeal community structure, a polyphasic approach was used with both qualitative and quantitative analysis. While PCR-denaturing gradient gel electrophoresis of 16S rRNA gene did not reveal major shifts in diversity of archaea with change in substrate, quantification of different groups of methanogens and total bacteria by real-time PCR showed variations in relative abundances with the dominance of Methanosaetaceae and Methanobacteriales. These data were supported by differences in the ratio of total counts of archaea and bacteria analyzed by catalyzed reporter deposition – fluorescence in situ hybridization. During hydraulic and organic shocks, the molasses-based reactor showed the best performance followed by the milk- and the glucose-based reactor. The study indicates that carbon source shapes the microbial community structure more in terms of relative abundance with distinct metabolic capacities rather than its diversity itself.     

Physiological diversity of Roseobacter clade bacteria co-occurring during a phytoplankton bloom in the North Sea

Organisms of the Roseobacter clade are an important component in marine ecosystems, partially due to their metabolic variety. Not much is known, however, about the physiological diversity of different roseobacters present within one habitat. By using serial dilution cultures with low-nutrient media seven roseobacter strains, co-occurring during a phytoplankton bloom in the southern North Sea, were obtained in this study. Physiological characterization exhibited distinct substrate spectra of the isolates. Although no isolate showed growth on algal osmolyte dimethylsulfoniopropionate (DMSP), feeding experiments revealed that all new strains converted [²H₆]DMSP into a variety of volatile compounds. Six strains mainly decomposed DMSP via the demethylation pathway, but four strains were also capable of cleaving DMSP to DMS and acrylate. It is hypothesized that the great physiological diversity of the roseobacters reflects their ability to inhabit different ecological niches and enables the organisms to cope differently with changing substrate supplies during phytoplankton blooms. Denaturing gradient gel electrophoresis and sequencing of excised bands resulted in detection of five additional roseobacters. Three of these sequences showed affiliation with three of the four major clusters of the Roseobacter clade, consisting predominantly of uncultured organisms (i.e. the Roseobacter clade-affiliated (RCA)), the NAC11-7 and the CHAB-I-5 clusters.     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.