Small Subunit Ribosomal Gene Research Articles

Metagenomic signatures of the Peru Margin subseafloor biosphere show a genetically distinct environment

The subseafloor marine biosphere may be one of the largest reservoirs of microbial biomass on Earth and has recently been the subject of debate in terms of the composition of its microbial inhabitants, particularly on sediments from the Peru Margin. A metagenomic analysis was made by using whole-genome amplification and pyrosequencing of sediments from Ocean Drilling Program Site 1229 on the Peru Margin to further explore the microbial diversity and overall community composition within this environment. A total of 61.9 Mb of genetic material was sequenced from sediments at horizons 1, 16, 32, and 50 m below the seafloor. These depths include sediments from both primarily sulfate-reducing methane-generating regions of the sediment column. Many genes of the annotated genes, including those encoding ribosomal proteins, corresponded to those from the Chloroflexi and Euryarchaeota. However, analysis of the 16S small-subunit ribosomal genes suggests that Crenarchaeota are the abundant microbial member. Quantitative PCR confirms that uncultivated Crenarchaeota are indeed a major microbial group in these subsurface samples. These findings show that the marine subsurface is a distinct microbial habitat and is different from environments studied by metagenomics, especially because of the predominance of uncultivated archaeal groups.

Exophiala dermatitidis and Sarcinomyces phaeomuriformis: ITS1-sequencing and nutritional physiology

The internal transcribed spacer 1 (ITS1) sequences of the nuclear rRNA gene (approximately 200 bp) of 33 strains of the Exophiala dermatitidis complex were determined; two similar species were added for comparison. A core group (I), including the type strain CBS 207·35, contained 20 identical strains which had previously been found to have introns in their small sub-unit (SSU) rDNA. Eleven remaining strains identified as E. dermatitidis (groups II–V) differed from the core group in 1–4 nucleotide positions (plus a deletion in one strain); most of them lacked introns in their SSU ribosomal genes. The type strain of the meristematic species Sarcinomyces phaeomuriformis CBS 131·88 was found to differ significantly from E. dermatitidis. One strain had the annellidic morphology of E. dermatitidis, but the ITS1 sequence of S. phaeomuriformis. Strain CBS 709·95, an E. dermatitidis reported to have a meristematic synanamorph, was found to have ITS1 identity to the type strain of E. dermatitidis, although SSU sequences established previously suggested a close relationship with S. phaeomuriformis. Slight physiological differences were found between E. dermatitidis and S. phaeomuriformis. An oligonucleotide probe specific for E. dermatitidis was designed, thus able to discriminate this species from closely related black yeasts.

First record of Eimeria furonis infection in a ferret, Japan, with notes on the usefulness of partial small subunit ribosomal RNA gene sequencing analysis for discriminating among Eimeria species

Eimeria is a genus of apicomplexan parasites found in a variety of vertebrates including the weasel. At present, three species have been reported in members of the weasel family, but the presence of weasel Eimeria in Japan have been quite unclear. The identification of Eimeria species has been performed based on oocyst morphology, host species, and habitat in the host, but sometimes discriminating among morphologically similar species under light microscopy is impossible. The present study detected for the first time the weasel coccidium, E. furonis, in a ferret in Japan. Since molecular information for E. furonis has been quite unclear, this species was compared molecularly with other Eimeria species, and also the usefulness of sequencing analysis of the small subunit ribosomal ribonucleic acid gene (SSUrDNA) for discriminating among Eimeria species was examined. About the 350-bp sequence of SSUrDNA of all species including E. furonis compared differed from each other, and its differences found in Eimeria species were also reflected in the phylogram constructed using the partial nucleotide sequences. The sequencing analysis of partial SSUrDNA examined in the present study thus appears useful for discriminating among morphologically similar Eimeria species.

Using DNA Technology To Explore Marine Bacterial Diversity in a Coastal Georgia Salt Marsh

An important aspect of teaching biology is to expose students to the concept of biodiversity. For this purpose, bacteria are excellent examples. Prokaryotes were the first inhabitants on Earth, surviving and even thriving under very harsh conditions as new species continuously evolved. In fact, it is believed that there are more than 5 x 1030 prokaryotes living on Earth today (Whitman et al., 1998). Our current knowledge of these tiny organisms is very limited, and less than 1% of all bacterial species have been described (Horner-Devine et al., 2004). However, the prominent roles bacteria play in nature are not easy to overlook: Their functions range from providing essential nutrients to plants through nitrogen-fixation (such as for Rhizobium leguminosarum) to enhancement of nutrient absorption in animal intestines (such as for Escherichia coli). As a result, identifying unknown species of bacteria and extending our understanding of known ones are important tasks for 21st Century scientists. Individual bacteria are too small to be seen or distinguished by the naked eye, or even by a microscope, making identification difficult. The traditional ways to identify bacterial species are based on their morphological, developmental, and nutritional characteristics, but these may be both inefficient and inaccurate. Microbiologists now take advantage of the rapid development in DNA technology, using the sequence of the small subunit ribosomal gene, or 16S rRNA gene, as a type of molecular fingerprint to classify bacteria (Johnson, 1984). The advanced placement (AP) biology class at Cedar Shoals High School in Athens, Georgia, learned how to explore bacterial biodiversity using molecular fingerprinting. We collected marine water samples, isolated bacterial colonies, extracted DNA, amplified and sequenced the 16S rRNA genes, and then compared the sequences to an Internet database to reveal the identity of the isolates. The project began with a field trip to the salt marshes on Sapelo Island, a barrier island in coastal Georgia, and was completed at our high school and in a laboratory at the University of Georgia. Here we describe how the bacterial biodiversity exercise was carried out, and discuss options for source material for bacterial isolation and flexibility in scheduling the laboratory exercise modules. This laboratory has both educational and practical value for the students; it is appropriate for both AP biology and introductory college biology classes. Methods The laboratory is divided into three modules. In the first, basic ecology and microbiology techniques are used to collect a water sample from the environment and isolate bacterial colonies on solid growth medium in Petri dishes. In the second, molecular biology methods are used to extract genomic DNA from selected colonies, and amplify and sequence the 16S rRNA gene. In the third, bioinformatic tools are used to compare the sequences to those available in a national DNA database to obtain information about the identity and ecology of the isolates. Our class of 20 students was divided into groups of four. Each student isolated his/her own bacterial colony so that each group worked with four isolates (although not all were successfully taken through to sequencing). Class size for this exercise is flexible, but 24 or fewer students is recommended. The entire exercise takes about one month to complete, but time can elapse between the scheduling of each module. Phase One: Isolating Bacterial Colonies In our class, a seawater sample from a salt marsh on Sapelo Island, Georgia, was used for bacterial isolation. However, water, sediment, or soil samples can be obtained from virtually any natural environment as starting material for this exercise. Collecting Water & Isolating Bacteria Materials * sterile collection bottle * thermometer * salinity meter (refractometer) * 10 test tubes filled with 9 ml of sterile seawater for serial dilution * 10 plastic Petri dishes filled with YTSS agar or other appropriate medium (To make YTSS agar, mix 4 g yeast extract, 2. …

In vitro cultivation of a newly recognized Babesia sp. in dogs in North Carolina

A novel large Babesia sp. from an infected dog was cultivated in vitro by microaerophilous stationary phase culture methodology. A primary culture initiated in enriched RPMI-1640 medium supplemented with 40% canine serum and incubated in a 2% oxygen environment supported parasite growth in vitro. Subsequent subcultures into enriched HL-1 medium with 20% fetal bovine serum also supported parasite propagation. Cultures were successfully introduced to 5% carbon dioxide in air atmosphere at passage 4. To date, the parasites have been continuously cultured through 35 passages, although the parasitemias are low, ranging from 0.2 to 0.3%. Parasites cultured in RPMI with canine serum were cryopreserved and successfully recovered from liquid nitrogen storage. The small subunit ribosomal rRNA gene sequence was identical in blood-derived and culture-derived parasites, differing in a single base position from the previously reported sequence for this Babesia sp. The ultrastructure of the parasite was consistent with that of other large Babesia spp., except that the spherical body contained numerous round particles unlike the inclusions previously described in Babesia spp.

Higher-level phylogeny of Foraminifera inferred from the RNA polymerase II (RPB1) gene

Macroevolutionary relations among main lineages of Foraminifera have traditionally been inferred from the small subunit ribosomal genes (SSU rDNA). However, important discrepancies in the rates of SSU rDNA evolution between major lineages led to difficulties in accurate interpretation of SSU-based phylogenetic reconstructions. Recently, actin and β-tubulin sequences have been used as alternative markers of foraminiferal phylogeny and their analyses globally confirm results obtained with SSU rDNA. In order to test new protein markers, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1), a nuclear encoded single copy gene, for 8 foraminiferal species representing major orders of Foraminifera. Analyses of our data robustly confirm previous SSU rDNA and actin phylogenies and show (i) the paraphyly and ancestral position of monothalamid Foraminifera; (ii) the independent origin of miliolids; (iii) the monophyly of rotaliids, including buliminids and globigerinids; and (iv) the polyphyly of planktonic families Globigerinidae and Candeinidae. Additionally, the RPB1 phylogeny suggests Allogromiidae as the most ancestral foraminiferal lineage. In the light of our study, RPB1 appears as a valuable phylogenetic marker, particularly useful for groups of protists showing extreme variations of evolutionary rates in ribosomal genes.

High rate of detection of mixed infections of Plasmodium vivax and Plasmodium falciparum in South-East of Iran, using nested PCR

Sistan and Baluchestan province, South-East of Iran, has been reported as an endemic area of malaria [Sadrizadeh B. Malaria in the world, in the eastern Mediterranean region and in Iran: Review article. WHO/EMRO Report 2001: 1–13.]. The main objective of this research was to perform rapid and correct diagnoses of malaria infection. Blood specimens were collected from 140 suspected volunteers. The Giemsa-stained slides examination and nested PCR for amplification of the Plasmodium small subunit ribosomal genes (ssrRNA) were utilized. The results demonstrated 118 out of 140 cases (84.3%) positive for malaria parasites, including 60.7%, 20.7% and 2.9% as having Plasmodium vivax ( P. v), Plasmodium falciparum ( P. f) and mixed infections ( P. v + P. f), respectively by microscopy. The nested PCR detected malaria parasites in 134 samples (94.3%), consisting of 51.4% P.v, 12.6% P.f and 29.3% mixed infections. The PCR analysis detected 37 cases of mixed infections more than that of the routine microscopy. These results suggested that there are a considerable number of cases with mixed infections in the study area that mainly remain undiagnosed by microscopy. It is also concluded that the nested PCR is a suitable complement to microscopy for accurate specific diagnosis of malaria species in field.

Kleptoplasty in an Antarctic dinoflagellate: caught in evolutionary transition?

Photosynthetic dinoflagellates contain a diverse collection of plastid types, a situation believed to have arisen from multiple endosymbiotic events. In addition, a number of heterotrophic (phagotrophic) dinoflagellates possess the ability to acquire chloroplasts temporarily by engulfing algae and retaining their chloroplasts in a functional state. These latter relationships typically last from a few days to weeks, at which point the chloroplasts lose function, are digested and replaced with newly acquired plastids. A novel and abundant dinoflagellate related to the icthyotoxic genera Karenia and Karlodinium was recently discovered by us in the Ross Sea, Antarctica. Sequencing of its plastid small subunit ribosomal gene indicated that it did not share evolutionary history with the plastids of Karenia or Karlodinium, but was closely related to the free-living haptophyte Phaeocystis antarctica, a species that often dominates phytoplankton blooms in the Ross Sea. Chloroplast uptake was observed to occur rapidly (within 2 days), with retention in cultures being long-lived (several months) but not permanent. The dinoflagellate was also incapable of growing indefinitely in continuous darkness with algae as prey. Our findings may indicate an emerging endosymbiotic event yielding a dinoflagellate that is presently neither purely phototrophic nor purely heterotrophic, but occupies a niche juxtaposed between these contrasting nutritional modes.

Classification and Identification of Xanthomonas translucens Isolates, Including Those Pathogenic to Ornamental Asparagus

ABSTRACT In order to confirm and refine the current classification scheme of Xanthomonas translucens and to identify novel strains from ornamental asparagus, a collection of field and reference strains was analyzed. Rep-polymerase chain reaction (PCR) genomic fingerprint profiles were generated from 33 isolates pathogenic to asparagus as well as 61 X. trans-lucens reference strains pathogenic to cereals and grasses. Amplified ribo-somal gene restriction analysis profiles were obtained from most of these and 29 additional Xanthomonas reference strains. Rep-PCR genomic fingerprint profiles of all strains were compared with those in a large Xanthomonas database using computer-assisted analysis. Rep-PCR ge-nomic fingerprinting facilitated the characterization and discrimination of X. translucens, including the pathovars arrhenatheri, graminis, phlei, phleipratensis, and poae, as well as a number of strains received as X. translucens pv. cerealis. Strains received as pathovars hordei, secalis, translucens, undulosa, and other cerealis strains were grouped in two subclusters that correspond to the recently redefined pathovars X. trans-lucens pvs. undulosa and translucens. All 33 novel isolates from ornamental asparagus (tree fern; Asparagus virgatus) were identified as X. translucens pv. undulosa. Moreover, a unique amplified small subunit ribosomal gene MspI/AluI restriction profile specific for all X. translucens strains tested, including those pathogenic to asparagus, allowed discrimination from all other Xanthomonas spp. Although phage tests were inconclusive, the classification of the asparagus strains within the X. translucens complex was supported by pathogenicity assays in which all the isolates from ornamental asparagus induced watersoaking on wheat. Surprisingly, several X. translucens reference strains affected asparagus tree fern as well. That the novel asparagus isolates belong to X. translucens pv. undulosa is extraordinary because all hosts of X. translucens pathovars described to date belong only to the families Gramineae and Poaceae, whereas asparagus belongs to the phylogenetically distant family Liliaceae.

Phylogenetic Position of the Green FlagellateMesostigma virideBased on α‐Tubulin and β‐Tubulin Gene Sequences

The green alga Mesostigma viride has attracted considerable recent attention as a model system useful for understanding the evolutionary origin of the Viridiplantae (all green algae plus embryophytes) or the Streptophyta (charophycean algae plus embryophytes). Mesostigma’s utility for such studies depends on its phylogenetic position, which has been controversial. Phylogenetic analyses based on organellar genes have given conflicting results; some support a hypothesis that Mesostigma represents the first branch of the Viridiplantae, whereas others support an alternative hypothesis that it occupies a basal position within Streptophyta. Nuclear‐encoded actin and small subunit ribosomal gene sequences also mark Mesostigma as a basal lineage in Streptophyta. We tested these two alternative hypotheses by analyses of nuclear‐encoded α‐ and β‐tubulin sequences. We obtained new α‐ and β‐tubulin gene sequences from M. viride as well as from the charophycean Coleochaete scutata and the green flagellates Pterosperma cristatum, Nephroselmis rotunda, and Mantoniella squamata, each representing a major clade within Prasinophyceae, in order to increase the taxon richness of the databases. We employed Bayesian and maximum likelihood procedures and paid particular attention to codon position effects on the phylogenetic analysis. On balance, our results support the hypothesis that M. viride is a basal streptophyte.

A redescription of Eucyclops serrulatus (Fischer, 1851) (Crustacea: Copepoda: Cyclopoida) and some related taxa, with a phylogeny of the E. serrulatus‐group

Eucyclops serrulatus (Fischer, 1851), the type species of the genus Eucyclops, is redescribed from specimens found in the St. Petersburg area, Russia (type locality) and compared with specimens from Siberia, western Europe and North Africa. A neotype is selected. Cultures were set up, and interpopulation hybridization as well as hybridization with related species was attempted. The classical description of external body morphology was combined with pore signature mapping and with DNA nuclear small subunit (18S) ribosomal gene sequence analysis. Comparisons with E. dumonti Alekseev, 2000, E. hadjebensis Kiefer, E. speratus Lilljeborg, E. turcomanus Lindberg, and E. pectinifer (Cragin, 1883) were carried out. A phylogenetic tree based on molecular information shows that E. serrulatus and E. speratus should be regarded as separate species. E. dumonti also deserves species status, but not E. hadjebensis. A cladistic tree based on the pore pattern of the cephalosome agrees well with a tree based on the sequence of the 18S rDNA gene. Cephalosome (and probably metasome) pore patterns seem useful to elucidate relationships within genera, while urosomal pore patterns better reflect the relationship between genera. E. serrulatus occurs in three morphological forms over most of its range; one of these (C) might be a rare (‘recessive’) morphotype, while forms A and B differ in microhabitat choice, but hybridize when living together. The same polymorphism also occurs in an American species (E. prionophorus), and therefore two hypotheses regarding its origin are advanced: either forms A and B evolved during the glacial episode (Pleistocene origin), separately on both sides of the Atlantic, or the polymorphism was already present in the ancestor of the serrulatus‐group, and was later lost in some but not in all species (Pliocene origin.)

ABUNDANCE OF A NOVEL DINOFLAGELLATE PHYLOTYPE IN THE ROSS SEA, ANTARCTICA1

The biodiversity of protistan assemblages present in microhabitats of the Ross Sea, Antarctica, was examined using molecular biological methods to obtain a greater understanding of the genetic diversity present. Sequencing of 18S clone libraries indicated genetically diverse collections of organisms in the water column, ice, and meltwater layer (slush), but a single small subunit ribosomal DNA (srDNA) sequence type dominated clone libraries (>30%) from seawater and slush samples taken within the ice pack of this ecosystem. The BLAST searches indicated that this dominant clone was derived from a dinoflagellate, and that it shared sequence similarity (97.6%–98.3%) with both Karenia and Karlodinium species. Phylogenetic analyses based on small subunit ribosomal gene sequences supported its placement as a sister group to these taxa, and suggested that it represented a novel genus. The dinoflagellate was successfully recovered in culture, and morphological analyses have shown that it contains chloroplasts, is gymnodinoid, appears not to have thecal plates, and has an apical groove and sulcal structure that confirm its placement as a relative of the Karenia/Karlodinium group. The abundance of this phylotype in natural samples was confirmed by quantitative PCR analyses of water and slush communities, and suggests that this dinoflagellate can be a major constituent of the protistan assemblages of some Antarctic microhabitats of the Ross Sea.

Assessment of microbial population dynamics during yoghurt and hard cheese fermentation and ripening by DNA population fingerprinting

The application of Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis of the small subunit ribosomal gene to examine the bacterial dynamics of relatively fast dairy fermentation processes is illustrated. All individual starter strains yielded characteristic terminal restriction fragments (TRFs). Growth independent T-RFLP analysis was used to identify and follow the dynamics of streptococci, lactobacilli, lactococci and propionibacteria in yoghurt and hard cheese production while samples had been stored frozen until analysis. Starter cultures could be differentiated and followed during production and ripening of hard cheese (Gouda type and Maasdam) as well as production of yoghurt. T-RFLP analysis enabled the characterization of bacterial population structure and dynamics between short time spans, i.e., within hours. Eight samples taken at different times within 5.5 h yoghurt fermentation revealed shifting relative signal intensities in TRF peaks between streptococci and lactobacilli, indicating a (semi) quantitative relation of the TRFLP signal with the actual numbers of bacteria.

Comparative morphology and molecular phylogeny of aplanochytrids (Labyrinthulomycota)

Aplanochytrids comprise one of three major subgroups within the Labyrinthulomycota. We have surveyed the diversity of aplanochytrids and have discovered that most isolates are difficult to identify to species because of character plasticity and ambiguity. Ten isolates were studied using molecular phylogenies based on small subunit ribosomal gene sequences (SSU rDNA) and morphological characters derived from light microscopy, SEM and TEM (e.g., colony size, colony shape, colony pattern, agar penetration, cell shape, cell surface patterns, cell inclusion characteristics and ectoplasmic net morphology). Of these isolates, we could positively identify two of them to species, namely Aplanochytrium yorkensis (Perkins, 1973) Leander and Porter, 2000 and A. minuta (Watson and Raper, 1957) Leander and Porter, 2000. We used standardized conditions for growing aplanochytrid isolates in order to minimize environmentally induced phenotypic plasticity in our comparative studies of morphology. By mapping the morphological characters listed above onto a conservative phylogenetic topology derived from SSU rDNA sequences, we were able to identify several synapomorphies (e.g., gross colony characteristics and cell surface patterns) that serve as valuable taxonomic characters for the identification of species and specific clades of aplanochytrids.

DESCRIPTION OF THECADINIUM MUCOSUM SP. NOV. (DINOPHYCEAE), A NEW SAND‐DWELLING MARINE DINOFLAGELLATE, AND AN EMENDED DESCRIPTION OF THECADINIUM INCLINATUM BALECH1

A new thecate, phototrophic, marine, sand‐dwelling dinoflagellate, Thecadinium mucosum Hoppenrath et Taylor sp. nov., is described from a culture isolated from Boundary Bay, British Columbia, Canada. It was illustrated with LM as well as SEM and TEM, and its position in the phylogenetic tree of dinoflagellates was investigated using molecular methods. Cells are asymmetrical, oval, laterally flattened, and strongly pigmented, with the plate formula P 3′ 1a 6′′ 7/8c 5 s 6′′′ 2′′′′. Thecal plates are smooth with scattered pores, and there is a distinctive anterior intercalary plate that could be involved in mucus secretion. Thecadinium inclinatum Balech (=Sabulodinium inclinatum (Balech) Saunders et Dodge), a thecate, marine, sand‐dwelling species that has been previously confused with what we now call T. mucosum, was also examined and illustrated through LM and SEM. New information on T. inclinatum is provided, including its plate formula P 3′ 6′′ 7c ?s 5′′′ 1p 1′′′′; we consider T. inclinatum to be related to most other Thecadinium species and not to Sabulodinium. Molecular phylogenetic analyses based on the small subunit ribosomal gene of T. mucosum, T. kofoidii (the type species of the genus), and T. dragescoi weakly support earlier suspicions based on morphology that T. dragescoi is not a member of Thecadinium. Tabulational patterns of the species suggest a relationship to the genus Amphidiniopsis.

Speciation in the large-spored Metschnikowia clade and establishment of a new species, Metschnikowia borealis comb. nov.

The reproductive boundaries among species in the large-spored Metschnikowia clade were studied by prototrophic recombinant selection, electrophoretic karyotyping, mitochondrial DNA restriction analysis, and DNA sequence analysis. Inviable ascospores arose from crosses between the two varieties of Metschnikowia continentalis, indicating that they should be recognized as separate species. Prototrophic recombinants were recovered from crosses between auxotrophic mutants of Metschnikowia borealis, M. continentalis, Metschnikowia lochheadii, Metschnikowia sp. UWO(PS)00-154.1, and Candida ipomoeae, showing that some genetic exchange is possible in spite of the sterility of the asci formed in interspecific crosses. Metschnikowia hawaiiensis, although capable of ascus formation when its h(-) mating type is crossed with the h(+) mating type of the other species, did not give rise to recombinants. In the other species, some recombinants acquired the ability to form asci directly from single cells. These often contained the chromosomes of both parents, suggesting formation of allodiploid hybrids. Other recombinants behaved as haploids and were similar to one parent except for having inherited the selectable wild-type allele from the other parent. In most, but not all cases, inheritance of the mitochondrial genome was uniparental and correlated with the inheritance of the nuclear chromosome complement. In some cases, what appeared to be a recombinant mitochondrial genome was observed. Phylogenies derived from the sequences of various DNA regions were not congruent, indicating that hybridization may have taken place in nature as the large-spored species diverged from their common ancestor. Further evidence that C. ipomoeae arose from a natural recombination event was obtained, but a pair of Metschnikowia species that might represent derived forms of the parents could not be identified conclusively. C. ipomoeae and most of its closely related Metschnikowia species contained a group-II intron in the mitochondrial small-subunit ribosomal gene. The intron was absent in M. borealis, M. hawaiiensis, and other species in the genus Metschnikowia.

A group I intron-like sequence in the nuclear small ribosomal subunit gene of the ophiostomatoid fungus Gondwanamyces proteae

During a phylogenetic study of ophiostomatoid fungi, a group I intron-like sequence was noted in the SSU rDNA gene of Gondwanamyces proteae. Secondary structure and sequence characteristics assigned the intron to the I E class. We then examined 27 related Group I-like sequences deposited in GenBank, and as a result 15 additional and previously uncategorized I E rDNA introns were identified. This study, with other recent publications, suggests that the I E class might represent a major family of group I introns that are located within the nuclear SSU and, to some extent LSU, genes in fungi.

Molecular profiling of microbial communities associated with seeds of Beta vulgaris subsp. Vulgaris (sugar beet)

The composition of microbial communities on and within seeds may effect their storage and field performance, whether they are indigenous or applied as biocontrol agents. In this study, we have explored the usefulness of profiling small subunit ribosomal (SSR) gene fragments for studying the microflora associated with seeds. DNA was amplified by the polymerase chain reaction (PCR) and the amplicons separated using denaturing gradient gel electrophoresis (DGGE). Primers targeting eukaryotic SSRs were used to investigate fungal communities, and primers targeting bacterial SSRs were employed to study the eubacterial microflora. As a case study, we attempted to profile the fungi and bacteria associated with seeds of Beta vulgaris (sugar beet) to permit an insight into the varying field performance of several well-characterised commercial seed lots. Serious interference with the microbial signals was observed from the plant's own nuclear 18S rRNA genes and chloroplast 16S rRNA genes using standard PCR conditions and DNA extracted from whole seeds as template. Hot-start and touchdown PCR made no appreciable improvement to these signals. Seed imbibition and dissection into operculum and fruit wall and true seed prior to DNA extraction improved signal recovery in the fruit fraction. With primer modification, bacteria and fungi were detected in an excess of plant DNA of 100:1 and 10:1, respectively. With this method, microbial communities on seeds could be profiled, however, it is likely that targeted depletion of plant rDNA targets will be a necessary extra step before this approach can be used to screen seeds routinely.

Plasmodium Juxtanucleare associated with mortality in black-footed penguins (Spheniscus demersus) admitted to a rehabilitation center.

Five black-footed penguins (Spheniscus demersus) admitted to the Southern African Foundation for the Conservation of Coastal Birds, in Cape Town, South Africa, died from malaria infection. Evidence for malaria as the cause of death included antemortem clinical signs, parasitemia, splenomegaly, pulmonary edema, and the presence of histologically visible schizonts in the reticuloendothelial system. A portion of the malarial small subunit ribosomal ribonucleic acid gene was detected by polymerase chain reaction from postmortem blood samples from all the birds. A species-specific variable region of this gene was compared with the same region on genes from other known avian malarial organisms, establishing that Plasmodium juxtanucleare was involved.

Cr(VI) reduction by sulfidogenic and nonsulfidogenic microbial consortia.

In time course experiments, bacterial community compositions were compared between a sulfidogenic and two nonsulfidogenic Cr(VI)-reducing consortia enriched from metal-contaminated sediments. The consortia were subjected to 0 and 0.85 mM or 1.35 mM Cr(VI), and Cr(VI) reduction, growth, and denaturing gradient gel electrophoresis profiles of PCR products of small-subunit (16S) ribosomal genes were compared. Results showed that although Cr(VI) was completely reduced by the three consortia, Cr(VI) inhibited cell growth, with sulfate-reducing bacteria being particularly sensitive to Cr(VI) toxicity relative to other bacteria in the consortia.