Many bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) contain the nahAc gene that encodes a component of multimeric naphthalene dioxygenases. Because the nahAc gene is highly conserved, this gene serves as a potential biomarker for PAH degradation activity. The aim of this research was to examine the relationship between the rate of naphthalene degradation and the copy number of the nahAc gene in soils using conventional and real-time PCR. Four sets of degenerate primers for real-time PCR were designed based on the nahAc DNA sequences of 33 bacterial species. Before addition of naphthalene, copy numbers of the nahAc gene were below the detection limits of the assay at 5 x 10(3) copy numbers per gram of soil, but increased by over a thousand fold to 10(7) copies after 6 days of exposure to naphthalene vapors (approximately 30 ppm soil water concentration). Two unreported naphthalene dioxygenase homologs were found in the naphthalene-spiked soil by cloning and sequencing of the PCR products from the nahAc primers. Results of these experiments demonstrate the highly dynamic changes that occur in soil microbial communities after exposure to naphthalene and suggest that there is a direct relationship between gene copy numbers and degradation rates for naphthalene in PAH-contaminated soils.

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The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.

A novel halophilic bacterium of the genus Kangiella was isolated from a marine sponge collected from the Florida Keys, USA. Strain A79T , an aerobic, Gram-negative, non-motile, rod-shaped bacterium, grew in 2-15 % (w/v) NaCl, at a temperature of 10-49 6C and at pH 4.5-10. Phylogenetic analysis placed strain A79 T in the family Alcanivoraceae in the class Gammaproteobacteria. Strain A79 T showed 98.5 % 16S rRNA gene sequence similarity toKangiella japonica KMM 3899 T , 96.6 % similarity to Kangiella koreensis DSM 16069 T and 95.6 % similarity to Kangiella aquimarina DSM 16071 T . The major cellular fatty acids were iso-C 11 : 0 , iso-C 11 : 0 3-OH, iso-C 15 : 0 , iso-C 17 : 0 and iso-C 17 : 1 v9c and the G+C content of the genomic DNA was 44.9 mol%. On the basis of physiological, chemotaxonomic and phylogenetic comparisons, strain A79 T represents a novel species in the genus Kangiella, for which the name Kangiella spongicola sp. nov. is proposed. The type strain is A79 T (5ATCC BAA-2076Marine sponges are filter-feeders, and, although microorganisms are a major component of the sponge diet, several studies have shown that sponges also harbour a diverse array of bacterial species, many of which have yet to be cultivated (Hill, 2004;Hentschel et al., 2006;Taylor et al., 2007). Sponges are highly diverse and are found in nearly every aquatic habitat and play important ecological roles (Wörheide & Erpenbeck, 2007). Hentschel et al. (2006) found great differences in the density and diversity of sponge microbial communities compared with those of the immediate surrounding seawater, indicating selection for particular bacterial species by the sponge animal. Studies of the symbiotic relationship between bacteria and marine sponges suggest that many associated micro-organisms aid in the homeostasis of the sponge (Hentschel et al., 2006;Hill, 2004;Lee et al., 2001). The precise roles of the sponge microbiota, although intimately linked to the host animal, have not been clearly determined.In this study, a novel bacterium, strain A79 T , was isolated from a Chondrilla nucula sponge, collected from the Florida Keys, USA (Carolina Biological Supply Company), and delivered to the laboratory in plastic bags in seawater within a few days of collection. Sponge tissue was aseptically cut and ground with a mortar and pestle. The cell extract was inoculated into marine broth 2216 (37.4 g l 21 ; BD Difco; MB) and incubated at 28 u C for 7 days. A 100 ml aliquot of a 10 7 dilution of this enrichment culture was spread-plated on marine agar, pH 7.6 (MA; BD Difco). Individual colonies were isolated and purified through sequential streak plating. One of the isolates, designated strain A79 T , was selected (based on preliminary identification) for further analysis. Bacterial stocks were stored at 280 u C in MB containing 40 % glycerol.Colony and cell morphology of strain A79T were observed after growth at room temperature and at 28 u C on MA. Gram staining was determined following the protocols of Murray et al. (1994). Cell morphology was ...

Alluvial aquifers are one of the mainwater resources in many countries. Iron reduction in alluvial aquifers is often a major anaerobic process involved in bioremediation or causing problems, including the release of As trapped in Fe(III) oxide. We investigated the distribution of potential iron-reducing bacteria (IRB) in riverine alluvial aquifers (B1, B3, and B6 sites) at the Mankyeong River, Republic of Korea. Inactive iron reduction zones, the diversity and abundance of IRB can be examined using a clone library and quantitative PCR analysis of 16S rRNA genes. Geobacter spp. are potential IRB in the iron-reducing zone at the B6 (9 m) site, where high Fe(II) and arsenic (As) concentrations were observed. At the B3 (16 m) site, where low iron reduction activity was predicted, a dominant clone (10.6%) was 99% identical in 16S rRNA gene sequence with Rhodoferax ferrireducens. Although a major clone belonging to Clostridium spp. was found, possible IRB candidates could not be unambiguously determined at the B1 (18 m) site. Acanonical correspondence analysis demonstrated that, among potential IRB, only the Geobacteraceae were well correlated with Fe(II) and As concentrations. Our results indicate high environmental heterogeneity, and thus high spatial variability, in thedistribution of potential IRB in the riverine alluvial aquifersnear the Mankyeong River.