'Aeromonas intestinalis' and 'Aeromonas enterica' isolated from human faeces, 'Aeromonas crassostreae' from oyster and 'Aeromonas aquatilis' isolated from lake water represent novel species.

Four Aeromonas strains from clinical and environmental samples differed from known species on the basis of rpoD gene sequence. Multilocus phylogenetic analysis and in silico DNA-DNA hybridization confirmed them as four new species even though their 16S rRNA gene sequence similarity with their closest relatives was >98.7%, as occurred for other Aeromonas spp.

Identification of novel toluene monooxygenase genes in a hydrocarbon-polluted sediment using sequence- and function-based screening of metagenomic libraries.

The microbial potential for toluene degradation within sediments from a tar oil-contaminated site in Flingern, Germany, was assessed using a metagenomic approach. High molecular weight environmental DNA from contaminated sediments was extracted, purified, and cloned into fosmid and BAC vectors and transformed into Escherichia coli. The fosmid library was screened by hybridization with a PCR amplicon of the α-subunit of the toluene 4-monooxygenase gene to identify genes and pathways encoding toluene degradation. Fourteen clones were recovered from the fosmid library, among which 13 were highly divergent from known tmoA genes and several had the closest relatives among Acinetobacter species. The BAC library was transferred to the heterologous hosts Cupriavidus metallidurans (phylum Proteobacteria) and Edaphobacter aggregans (phylum Acidobacteria). The resulting libraries were screened for expression of toluene degradation in the non-degradative hosts. From expression in C. metallidurans, three novel toluene monooxygenase-encoding operons were identified that were located on IncP1 plasmids. The E. aggregans-hosted BAC library led to the isolation of a cloned genetic locus putatively derived from an Acidobacteria taxon that contained genes involved in aerobic and anaerobic toluene degradation. These data suggest the important role of plasmids in the spread of toluene degradative capacity and indicate putative novel tmoA genes present in this hydrocarbon-polluted environment.

Aeromonas aquatica sp. nov., Aeromonas finlandiensis sp. nov. and Aeromonas lacus sp. nov. isolated from Finnish waters associated with cyanobacterial blooms.

Three groups of Aeromonas strains isolated from Finland lakes experiencing cyanobacterial blooms could not be assigned to any known species of this genus on the basis of 16S rRNA and rpoD gene sequences. The Multilocus Phylogenetic Analysis (MLPA) of the concatenated sequence of seven genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD; 4093bp) showed that the three groups of strains did not cluster with any known Aeromonas spp. and formed three independent lineages. This was confirmed by performing the analysis with their closest relatives using 15 genes (the latter 7 and cpn60, dnaK, gltA, mdh, radA, rpoB, tsf, zipA; 8751bp). Furthermore, ANI results between the genomes of the type strains of the three potential new species and those of their close relatives were all <96% which is the previously proposed cutoff value for differentiating species within this genus. The in silico DDH values of the three type strains of the new species also showed a similarity<70% with the most closely related species indicating they belong to different taxa. The three groups of strains could be differentiated from each other and from other known Aeromonas species on the basis of several phenotypic characters. This polyphasic study revealed that the 3 groups of strains represent 3 novel Aeromonas species for which the names Aeromonas aquatica sp. nov. (type strain AE235T=CECT 8025T=LMG 26712T), Aeromonas finlandiensis sp. nov. (type strain 4287DT=CECT 8028T=LMG 26709T) and Aeromonas lacus sp. nov. (type strain AE122T=CECT 8024T=LMG 26710T) are proposed.

Comparison of soil fungal community structure in different peanut rotation sequences using ribosomal intergenic spacer analysis in relation to aflatoxin-producing fungi.

The present study focuses on determining soil fungal community structure in different peanut-cropping sequences by using a high-resolution DNA fingerprinting technique: ribosomal intergenic spacer analysis (RISA). This study was initiated to determine fungal community profiles in four peanut-cropping sequences (continuous peanut, 4 years of continuous bahiagrass followed by peanut, peanut-corn-cotton, and peanut-cotton rotations), with a special focus to evaluate whether the profiles under investigation may have also indicated microbial differences that could affect Aspergillus flavus populations. Results indicated 75% similarities among fungal communities from the same cropping sequences as well as with similar times of sampling. Polymerase chain reaction (PCR)-based detection of A. flavus directly from these soils was carried out using A. flavus-specific primers (FLA1 and FLA2) and also through quantitative estimation on A. flavus and A. parasiticus agar medium. Population levels of A. flavus in soil samples ranged from zero to 1.2 × 10(3) CFU g(-1) of soil (based on culturable methods); however, the fungus was not detected with A. flavus-specific primers. The minimum threshold limit at which these aflatoxin-producing fungi could be detected from the total soil genomic DNA was determined through artificial inoculation of samples with 10-fold increases in concentrations. The results indicated that a minimum population density of 2.6 × 10(6) CFU g(-1) of soil is required for PCR detection in our conditions. These results are useful in further determining the relative population levels of these fungi in peanut soils with other soil fungi. This is a new approach to understanding soil fungal communities and how they might change over time and under different rotation systems.

Identification and characterization of bacteriophages specific to the catfish pathogen, Edwardsiella ictaluri.

AIMS: To identify and characterize bacteriophages specific for Edwardsiella ictaluri, the causative agent for enteric septicemia of catfish (ESC). METHODS AND RESULTS: Two bacteriophages were isolated that infect Edw. ictaluri. They both produce clear plaques, have icosahedral heads with a non-rigid tail, and are tentatively classified as Siphoviridae. Phages PhieiDWF and PhieiAU are dsDNA viruses with approximate genome sizes of 40 and 45 kb, respectively. The addition of 500 micromol l(-1) CaCl(2) enhanced phage titres. Both phages have a latent period of 40 min and an estimated burst size of 270. Every Edw. ictaluri strain tested was susceptible to phage infection with variable plaquing efficiencies and with no evidence of lysogeny, with no plaques detected on other bacterial species. CONCLUSIONS: Two unique bacteriophages were isolated that show host-specificity for Edw. ictaluri, have temperature and metal cation-dependent infectivity, and are tentatively placed within the family Siphoviridae. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of bacteriophages specific to Edw. ictaluri, an important fish pathogen affecting farm-raised channel catfish. Initial characterization of these bacteriophages has demonstrated their potential use as biotherapeutic and diagnostic agents associated with ESC.

Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays.

Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species. In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array. For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1,500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (10(12) molecules/cm2) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity. Fresnel calculations are used to show that changes in percent reflectivity as measured by SPR imaging are linear with respect to surface coverage of adsorbed DNA oligonucleotides. Data from SPR imaging is used to construct a quantitative adsorption isotherm of the hybridization adsorption on a surface. DNA and RNA 18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir isotherm with an adsorption coefficient of 1.8 x 10(7) M(-1).

Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms.

Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.

Discovery of a nonclassical siderophore, legiobactin, produced by strains of Legionella pneumophila.

The mechanisms by which Legionella pneumophila, a facultative intracellular parasite and the agent of Legionnaires' disease, acquires iron are largely unexplained. Several earlier studies indicated that L. pneumophila does not elaborate siderophores. However, we now present evidence that supernatants from L. pneumophila cultures can contain a nonproteinaceous, high-affinity iron chelator. More specifically, when aerobically grown in a low-iron, chemically defined medium (CDM), L. pneumophila secretes a substance that is reactive in the chrome azurol S (CAS) assay. Importantly, the siderophore-like activity was only observed when the CDM cultures were inoculated to relatively high density with bacteria that had been grown overnight to log or early stationary phase in CDM or buffered yeast extract. Inocula derived from late-stationary-phase cultures, despite ultimately growing, consistently failed to result in the elaboration of siderophore-like activity. The Legionella CAS reactivity was detected in the culture supernatants of the serogroup 1 strains 130b and Philadelphia-1, as well as those from representatives of other serogroups and other Legionella species. The CAS-reactive substance was resistant to boiling and protease treatment and was associated with the <1-kDa supernatant fraction. As would also be expected for a siderophore, the addition of 0.5 or 2.0 microM iron to the cultures repressed the expression of the CAS-reactive substance. Interestingly, the supernatants were negative in the Arnow, Csáky, and Rioux assays, indicating that the Legionella siderophore was not a classic catecholate or hydroxamate and, hence, might have a novel structure. We have designated the L. pneumophila siderophore legiobactin.

The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila.

Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage-like U937 cells. As reintroduction of pilD into the mutant restored inefectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100-fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.

Identification and temperature regulation of Legionella pneumophila genes involved in type IV pilus biogenesis and type II protein secretion.

Previously, we had isolated by transposon mutagenesis a Legionella pneumophila mutant that appeared defective for intracellular iron acquisition. While sequencing in the proximity of the mini-Tn10 insertion, we found a locus that had a predicted protein product with strong similarity to PilB from Pseudomonas aeruginosa. PilB is a component of the type II secretory pathway, which is required for the assembly of type IV pili. Consequently, the locus was cloned and sequenced. Within this 4-kb region were three genes that appeared to be organized in an operon and encoded homologs of P. aeruginosa PilB, PilC, and PilD, proteins essential for pilus production and type II protein secretion. Northern blot analysis identified a transcript large enough to include all three genes and showed a substantial increase in expression of this operon when L. pneumophila was grown at 30 degrees C as opposed to 37 degrees C. The latter observation was then correlated with an increase in piliation when bacteria were grown at the lower temperature. Southern hybridization analysis indicated that the pilB locus was conserved within L. pneumophila serogroups and other Legionella species. These data represent the first isolation of type II secretory genes from an intracellular parasite and indicate that the legionellae express temperature-regulated type IV pili.

Absence of siderophore-like activity in Legionella pneumophila supernatants.

Conflicting reports have been given as to the existence of a Legionella pneumophila siderophore. Hence, we rigorously examined the reported siderophore-like activity using the chrome azurol S indicator. Although chrome azurol S reactivity was detected in supernatants, control experiments indicate that it was due to cysteine in the media. When bacteria were cultured in media without cysteine, no siderophores were detected.

Evaluation of oxidative processes in human pigment epithelial cells associated with retinal outer segment phagocytosis.

To investigate the nature of the oxidative event that occurs during phagocytosis of retinal outer segments (ROS) by cultured human retinal pigment epithelial (RPE) cells, cells were incubated with isolated bovine ROS labeled with either the fluorescence probe carboxy-SNAFL-2 or the nonfluorescent, oxidizable probe 2',7'-dichlorodihydrofluorescein (H2DCF). The increase in fluorescence following phagocytosis was measured by a flow cytometer. Other measurements included: oxygen consumption using a Clark-type oxygen electrode, extracellular superoxide release by superoxide dismutase inhibitable lucigenin chemiluminescence, intracellular hydrogen peroxide (H2O2) production, and the effect of catalase inhibition on cellular thiobarbituric acid-reactive substances (TBARS) caused by phagocytosis. The activities of the enzymes NADPH oxidase and palmitoyl-CoA oxidase were also measured. H2DCF attached to bovine ROS was oxidized during phagocytosis with a time course suggesting oxidation subsequent to ROS uptake. Measurements of oxygen consumption showed a time-dependent increase of 10%, 4 h after ROS feeding, attributable to a doubling of the cyanide-resistant oxygen consumption. Intracellular H2O2 production also doubled 4 h after ROS phagocytosis. ROS uptake by RPE cells produced no significant extracellular superoxide, while extracellular superoxide production was readily demonstrated in a control macrophage cell line. Enzyme activity measurements showed that incubation of RPE cells with ROS doubled catalase activity without affecting superoxide dismutase or glutathione peroxidase activities. Inhibition of catalase during ROS uptake increased TBARS by 66%. Other enzyme activity measurements showed that human RPE cells possess both NADPH oxidase and palmitoyl-CoA oxidase activities. We conclude that ROS phagocytosis subjects RPE cells to an oxidative event on the same order of magnitude as measured in a macrophage. The event is not an extracellular macrophage-type respiratory burst and may be due to intracellular H2O2 resulting from an NADPH oxidase in the phagosome or from beta-oxidation of ROS lipids in peroxisomes. Irrespective of case, the enzyme catalase appears to be essential in protecting the RPE cell against reactive oxygen species produced during phagocytosis.

Antioxidant enzymes in the aging human retinal pigment epithelium.

The antioxidant enzymes catalase and superoxide dismutase have integral roles in controlling reactive oxygen radicals that can harm cells. In the present study, we quantitated catalase activity in retinal pigment epithelium, retina, iris, and vitreous from human donors. To our knowledge, our results represent the first quantitation of catalase activity in human retinal pigment epithelium and show six-fold greater catalase activity in retinal pigment epithelium than in other ocular tissues analyzed (P less than .0001). To investigate whether aging or macular degeneration affects retinal pigment epithelium catalase or superoxide dismutase activities, we measured enzyme levels in retinal pigment epithelium from donors 50 to 90 years of age with and without evidence of macular degeneration. Superoxide dismutase activity showed no significant correlations with aging or macular degeneration, while catalase activity decreased with age (P less than .02) and macular degeneration (P less than .05) in both macular and peripheral retinal pigment epithelium.

Human retinal pigment epithelium contains two distinct species of superoxide dismutase.

Superoxide dismutase (SOD) molecules occur in all cells exposed to an oxygen-containing environment, including retinal pigment epithelial (RPE) cells. Previous studies of nonhuman RPE have either probed specifically for copper-zinc-containing SOD (CuZn-SOD) or have not distinguished between CuZn-SOD and the SOD molecule that contains manganese (Mn-SOD). The authors used specific enzymatic assays and immunologic probes, both in vivo and in vitro, to show that human RPE cells contain both CuZn-SOD and Mn-SOD. The CuZn-SOD had a diffuse cytosolic distribution, whereas the Mn form was located primarily in the mitochondria. The role of SODs in protecting the chorioretinal complex against oxidative damage and with regard to aging processes is not well understood and warrants further investigation, and the two cellular forms of SOD should be considered in future studies.