Degradation of Fucoidan by the Marine Proteobacterium Pseudoalteromonas citrea

It was found that Pseudoalteromonas citrea strains KMM 3296 and KMM 3298 isolated from the brown algae Fucus evanescens and Chorda filum, respectively, and strain 3297 isolated from the sea cucumber Apostichopus japonicus are able to degrade fucoidans. The fucoidanases of these strains efficiently degraded the fucoidan of brown algae at pH 6.5–7.0 and remained active at 40–50°C. The endo-type hydrolysis of fucoidan resulted in the formation of sulfated -L-fucooligosaccharides. The other nine strains of P. citrea studied (including the type strain of this species), which were isolated from other habitats, were not able to degrade fucoidan.     

Particulate methane monooxygenase from Methylosinus trichosporium is a copper-containing enzyme

Particulate methane monooxygenase (pMMO) has been exfoliated and isolated from membranes of the Methylosinus trichosporium IMV 3011. It appears that the stability of pMMO in the exfoliation process is increased with increasing copper concentration in the growth medium, but extensive intracytoplasmic membrane formed under higher copper concentration may inhibit the exfoliation of active pMMO from membrane. The highest total activity of purified pMMO is obtained with an initial concentration of 6 microM Cu in the growth medium. The purified MMO contains only copper and does not utilize NADH as electron donor. Treatment of purified pMMO with EDTA resulted in little change in copper level, suggesting that the copper in the pMMO is tightly bound with pMMO.     

Isolation and Characterization of a Fucoidan-Degrading Marine Bacterium

Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from the algal bodies of Cladosiphon okamuranus (class Phaeophyceae, order Chordariales, family Chordariaceae) with a yield of 2.0% of the wet weight of the alga. To obtain enzymes that digest the fucoidan, we screened bacteria in the gut contents of the sea cucumber Stichopus japonicus for their ability to decrease the fucoidan in their culture media, and successfully isolated one bacterial strain that could decrease it. The bacterial strain was gram-negative and possessed menaquinone 7 as the predominant respiratory quinone, and the GC content of its genomic DNA was 52%. The results of the phylogenetic analysis of its 16S ribosomal DNA sequence indicated that the bacterial strain was a member of the division Verrucomicrobia. However, as the bacterial strain is phylogenetically and phenotypically distinct from verrucomicrobial species described previously, the strain was assumed to be a new member of the division Verrucomicrobia. When the bacterial strain was cultivated in an algal fucoidan-containing medium, the strain decreased fucoidan from C. okamuranus (44%), Nemacystus decipiens (19%), Laminaria japonica (31%), Kjellmaniella crassifolia (23%), sporophyl of Undaria pinnatifida (22%), Fucus vesiculosus (42%), and Ascophyllum nodosum (61%).     

Intraspecies variability of Desulfovibrio desulfuricans strains determined by the genetic profiles

Fifteen (soil and intestinal) strains of Desulfovibrio desulfuricans species were typed by PCR method with the use of primers specific for repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) sequences. As a result, characteristic DNA fingerprints for the strains were obtained. Moreover, the genetic profiles were found to be useful for typing and distinguishing the strains of D. desulfuricans. According to cluster analysis, PCR with primers complementary to the sequences REP appeared to be slightly more discriminatory than PCR with ERIC primers for the investigated strains. Distinct fingerprint patterns of two isolates derived from the same patient pointed to the different origin of both strains.     

Development of specific fluorescent oligonucleotide probes for in situ identification of wine lactic acid bacteria

A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising technique for the rapid identification of LAB, allowing positive identification in a few hours (4-16 h).     

Broad and complex antifungal activity among environmental isolates of lactic acid bacteria

More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dual-culture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined. Several of the strains showed strong inhibitory activity against the moulds A. fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa. Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited. Several isolates showed reduced antifungal activity after storage and handling. The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis. Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates. The degree of fungal inhibition was not only related to production of lactic or acetic acid. In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.     

Chlorobium Tepidum: Insights into the Structure,Physiology, and Metabolism of a Green Sulfur Bacterium Derived from the Complete Genome Sequence

Green sulfur bacteria are obligate, anaerobic photolithoautotrophs that synthesize unique bacteriochlorophylls (BChls) and a unique light-harvesting antenna structure, the chlorosome. One organism, Chlorobium tepidum, has emerged as a model for this group of bacteria primarily due to its relative ease of cultivation and natural transformability. This review focuses on insights into the physiology and biochemistry of the green sulfur bacteria that have been derived from the recently completed analysis of the 2.15-Mb genome of Chl. tepidum. About 40 mutants of Chl. tepidum have been generated within the last 3 years, most of which have been made based on analyses of the genome. This has allowed a nearly complete elucidation of the biosynthetic pathways for the carotenoids and BChls in Chl. tepidum, which include several novel enzymes specific for BChl c biosynthesis. Facilitating these analyses, both BChl c and carotenoid biosynthesis can be completely eliminated in Chl. tepidum. Based particularly on analyses of mutants lacking chlorosome proteins and BChl c, progress has also been made in understanding the structure and biogenesis of chlorosomes. In silico analyses of the presence and absence of genes encoding components involved in electron transfer reactions and carbon assimilation have additionally revealed some of the potential physiological capabilities, limitations, and peculiarities of Chl. tepidum. Surprisingly, some structural components and biosynthetic pathways associated with photosynthesis and energy metabolism in Chl. tepidum are more similar to those in cyanobacteria and plants than to those in other groups of photosynthetic bacteria.     

Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.     

Relative abundance of denitrifying and dinitrogen-fixing bacteria in layers of a forest soil

The populations of N(2)-fixing and denitrifying bacteria in an acid forest soil near Cologne were characterized by gene probing. The DNA isolated from the soil for this purpose was suitable for DNA-DNA hybridization using 0.4-0.7-kb probes targeting denitrification enzymes, dinitrogenase reductase (nifH) and eubacterial 16S rRNA. The densitometrical comparison of band intensities obtained in these Southern hybridizations indicated that the highest number of total bacteria, of denitrifying and N(2)-fixing microorganisms always occurred in the upper ( approximately 5 cm) soil layer. The concentration of all these organisms decreased in parallel with the soil depth. The soil investigated was rich in nitrate in all layers, and the availability of nitrate apparently did not govern the distribution of denitrifying and N(2)-fixing bacteria in this soil. Soil cores investigated in the laboratory formed N(2)O on addition of nitrate irrespective of the presence of C(2)H(2). Hybridization intensities, with a gene probe for the 16S rRNA, and MPN numbers were generally higher in soil samples taken from the roots of plants than in the bulk soil. There was no selective enrichment of denitrifying or N(2)-fixing bacteria at the roots. The data obtained by hybridizing isolated soil DNA generally matched previous results obtained with culturable bacteria.     

Microbes involved in dissimilatory nitrate reduction in the human large intestine

Nitrate-limited batch cultures, incorporating 20 different fermentation substrates and inoculated with human faeces, mainly selected for the growth of enterobacteria. The microbial diversity involved was determined by a combination of phenotypic and genotypic procedures. Continuous culture with lactate as the sole electron donor selected for similar micro-organisms, but when antibiotics were incorporated to inhibit Escherichia coli and lactate was replaced with choline, there was a wider microbial diversity recovered. Clostridium ramosum and Bacteroides vulgatus were then isolated as well as enterobacteriaceae.