Partial 16S Ribosomal DNA Sequencing Research Articles

New species of the genus Spio (Annelida, Spionidae) from the southern and western coasts of Korea.

A new spionid polychaete, Spiopigmentatasp. nov., is described from the southern and western coasts of Korea. This new species differs from its congeners by the combination of the following morphological characteristics: the presence of orange-brown pigmentation on the anterior part of the prostomium, black pigmentation on the peristomium and along the body, U-shaped nuchal organs, a comparatively long extension of metameric dorsal ciliated organs, three pairs of white dots per chaetiger, two to three posterior abranchiate chaetigers, and the presence of tridentate neuropodial hooded hooks. The partial 16S ribosomal DNA (rDNA) and nuclear 18S rDNA sequences of the new species and Spio sp. 2 reported by Abe and Sato-Okoshi (2021) from Japan showed high similarity, indicating that these two specimens belong to the same species. A detailed description and illustrations of the new species, together with molecular information, are provided.

Inoculation with Metal-Mobilizing Plant-Growth-Promoting Rhizobacterium Bacillus sp. SC2b and Its Role in Rhizoremediation

A plant growth-promoting bacterial (PGPB) strain SC2b was isolated from the rhizosphere of Sedum plumbizincicola grown in lead (Pb)/zinc (Zn) mine soils and characterized as Bacillus sp. based on (1) morphological and biochemical characteristics and (2) partial 16S ribosomal DNA sequencing analysis. Strain SC2b exhibited high levels of resistance to cadmium (Cd) (300 mg/L), Zn (730 mg/L), and Pb (1400 mg/L). This strain also showed various plant growth-promoting (PGP) features such as utilization of 1-aminocyclopropane-1-carboxylate, solubilization of phosphate, and production of indole-3-acetic acid and siderophore. The strain mobilized high concentration of heavy metals from soils and exhibited different biosorption capacity toward the tested metal ions. Strain SC2b was further assessed for PGP activity by phytagar assay with a model plant Brassica napus. Inoculation of SC2b increased the biomass and vigor index of B. napus. Considering such potential, a pot experiment was conducted to assess the effects of inoculating the metal-resistant PGPB SC2b on growth and uptake of Cd, Zn and Pb by S. plumbizincicola in metal-contaminated agricultural soils. Inoculation with SC2b elevated the shoot and root biomass and leaf chlorophyll content of S. plumbizincicola. Similarly, plants inoculated with SC2b demonstrated markedly higher Cd and Zn accumulation in the root and shoot system, indicating that SC2b enhanced Cd and Zn uptake by S. plumbizincicola through metal mobilization or plant-microbial mediated changes in chemical or biological soil properties. Data demonstrated that the PGPB Bacillus sp. SC2b might serve as a future biofertilizer and an effective metal mobilizing bioinoculant for rhizoremediation of metal polluted soils.

Plant genotype and nitrogen fertilization effects on abundance and diversity of diazotrophic bacteria associated with maize (Zea mays L.)

This research has investigated the effect of two genotypes of maize (Zea mays L.) on the number and diversity of diazotrophic bacteria under different N-fertilization rates. Cultivars NK940 and PAU871 were grown in soil:sand with 0, 40, and 80 kg N ha−1 added as NH4NO3 under controlled conditions for 60 days. The number of diazotrophic bacteria of rhizosphere (S), disinfected roots (R), and stems (T) was determined by most probable number (MPN) method. Diazotrophic bacteria were isolated by N-free media, confirmed by amplification of nifH gene fragments, characterized by amplified 16S rDNA restriction analysis (ARDRA) and (GTG)5 fingerprinting, and identified by partial 16S ribosomal DNA (rDNA) sequence analysis. The diversity of the diazotrophic community from S and R was determined by Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis considering the nifH gene. Maize genotype had a marked effect on the number and diversity of endophytic communities, the NK940 community being more abundant and diverse than that of PAU871. Additionally, N-fertilization increased the number and diversity of diazotrophs in endophytic communities but not in rhizosphere samples. One hundred and six diazotrophs were isolated from S, R, and T samples. Pseudomonas and Enterobacter were the dominant and ubiquitous genera isolated and detected by culture-independent method. T-RFLP showed that the N-fixing populations of the rhizosphere of both cultivars were more diverse than those of inside roots. Principal component analysis (PCA) separated the samples by cultivar and demonstrated a more marked effect of N-fertilization on the NK940 diazotrophic community than on PAU871. These results support the hypothesis that plant genotype and fertilization conditions should be taken into account when searching for N-fixer inocula.

Diversity of Dickeya Strains Collected from Pineapple Plants and Irrigation Water in Hawaii.

Bacterial heart rot caused by a yet undetermined species of Dickeya was first observed in December 2003 in Oahu, HI, on a pineapple cultivar (Ananas comosus 'PRI 73-114') recently imported from Central America. Identical symptoms were later seen in the same plantation in fields that had been planted with propagules from the Philippines. Dickeya strains isolated from symptomatic plants and irrigation water collected over subsequent years were identified using bacteriological tests and partial 16S ribosomal DNA sequencing and characterized using repetitive sequence-based polymerase chain reaction (rep-PCR) with the BOXA1R primer (BOX-PCR), pathogenicity on pineapple leaves, and reactivity with two monoclonal antibodies (MAbs). Strains exhibited variability in pathogenicity and in reactivity with MAbs. BOX-PCR separated the plant-isolated Dickeya strains into seven haplotypes that were placed into four fingerprint groups (A to D). Strains from the A and B groups were isolated from the Central American stocks, whereas strains in the D group were identified from Philippine material. Strains from the C group were isolated from both planting materials. Most strains from water sources were placed into three haplotypes that loosely formed group E. BOX-PCR polymorphisms between the Dickeya strains isolated from foreign pineapple plants, a local collection of Dickeya strains, and strains from the plantation's water sources support the possibility that at least two separate introductions of genetically distinct strains occurred via imported planting stocks.

Characterization of metal‐resistant plant‐growth promoting Bacillus weihenstephanensis isolated from serpentine soil in Portugal

A metal-resistant bacterial strain SM3 isolated from a serpentine soil in the north-east of Portugal was characterized as Bacillus weihenstephanensis based on the morphological and biochemical characteristics and on the comparative analysis of the partial 16S ribosomal DNA sequence. Bacillus weihenstephanensis SM3 showed a high degree of resistance to nickel (1500 mg l(-1)), copper (500 mg l(-1)) and zinc (700 mg l(-1)) and also to antibiotics (ampicillin, penicillin, kanamycin and streptomycin). Strain SM3 has also exhibited the capability of solubilizing phosphate and producing indole-3-acetic acid (IAA) both in the absence and in the presence of metals (Ni, Cu and Zn). A pot experiment was conducted to elucidate the effects of strain SM3 on plant growth and uptake of Ni, Cu or Zn by Helianthus annuus. Inoculation with strain SM3 increased the shoot and root biomass of H. annuus grown in both non-contaminated and contaminated soil. Furthermore, strain SM3 increased the accumulation of Cu and Zn in the root and shoot systems. A batch experiment was also conducted to assess the metal mobilization potential of strain SM3 in soil. Inoculation with this strain increased the concentrations of water soluble Ni, Cu and Zn in soil. Metal solubilization by this bacterial strain may be an important process to promote the uptake of heavy metals by plants. This study elucidates the multifarious role of strain SM3 in plant growth promotion and its metal mobilizing potential.

Pseudooutbreak of Pantoea species bacteremia associated with contaminated cotton pledgets

A total of 22 isolates of Pantoea strains, unusual causative agents of clinical infection, was isolated from blood cultures from 9 patients and 1 ear swab from 1 of the patients within a period of 1 month in a tertiary-care hospital. Pseudooutbreak was suspected because specimens were collected from a limited number of places and the patients did not show consistent signs or symptoms of bacterial sepsis. Enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR) and partial 16S ribosomal DNA sequencing were performed to determine the clonal relationship among the isolates. Screening environmental cultures revealed that cotton pledgets were contaminated with Pantoea species. Molecular typing suggested that 2 different clones of Pantoea strains were responsible for the pseudooutbreak. Cotton materials may be a possible source of Pantoea pseudooutbreak. Molecular typing is useful for investigating epidemics and identifying unusual clinical isolates.

A survey of ammonia-assimilating micro-organisms in cattle manure composting

To evaluate the ammonia-assimilating abilities of micro-organisms isolated from cattle manure composting processes and to determine the distribution of cultivable species of ammonia-assimilating micro-organisms in microbial communities during the composting processes. Compost samples were collected from four stages of treatment. Trypto soya agar was used for the isolation of ammonia-assimilating aerobes. Many of the isolates showed high ammonia-assimilating ability in a medium containing basal components and a compost extract. Partial 16S ribosomal DNA sequencing showed that the cultivable species of highly efficient ammonia-assimilating isolates changed during the composting process. The community structure of micro-organisms and actinomycetes was analysed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Two species of actinomycetes identified by PCR-DGGE coincided with those found among the cultured isolates. Ammonia-assimilating micro-organisms obtained by the cultivation method were not predominant in the microbial community during the composting process: however certain cultured actinomycetes were members of predominant species in the actinomycetes community. Ammonia assimilation by micro-organisms is one of the important mechanisms for ammonia retention in the composting process. Cultivable actinomycetes are a means for preventing ammonia emission from the composting process.

Mining the microbiota of the neonatal gastrointestinal tract for conjugated linoleic acid-producing bifidobacteria.

This study was designed to isolate different strains of the genus Bifidobacterium from the fecal material of neonates and to assess their ability to produce the cis-9, trans-11 conjugated linoleic acid (CLA) isomer from free linoleic acid. Fecal material was collected from 24 neonates aged between 3 days and 2 months in a neonatal unit (Erinville Hospital, Cork, Ireland). A total of 46 isolates from six neonates were confirmed to be Bifidobacterium species based on a combination of the fructose-6-phosphate phosphoketolase assay, RAPD [random(ly) amplified polymorphic DNA] PCR, pulsed-field gel electrophoresis (PFGE), and partial 16S ribosomal DNA sequencing. Interestingly, only 1 of the 11 neonates that had received antibiotic treatment produced bifidobacteria. PFGE after genomic digestion with the restriction enzyme XbaI demonstrated that the bifidobacteria population displayed considerable genomic diversity among the neonates, with each containing between one and five dominant strains, whereas 11 different macro restriction patterns were obtained. In only one case did a single strain appear in two neonates. All genetically distinct strains were then screened for CLA production after 72 h of incubation with 0.5 mg of free linoleic acid ml(-1) by using gas-liquid chromatography. The most efficient producers belonged to the species Bifidobacterium breve, of which two different strains converted 29 and 27% of the free linoleic acid to the cis-9, trans-11 isomer per microgram of dry cells, respectively. In addition, a strain of Bifidobacterium bifidum showed a conversion rate of 18%/microg dry cells. The ability of some Bifidobacterium strains to produce CLA could be another human health-promoting property linked to members of the genus, given that this metabolite has demonstrated anticarcinogenic activity in vitro and in vivo.

Morphological, chemical, and genetic diversity of tropical marine cyanobacteria Lyngbya spp. and Symploca spp. (Oscillatoriales).

Although diverse natural products have been isolated from the benthic, filamentous cyanobacterium Lyngbya majuscula, it is unclear whether this chemical variation can be used to establish taxonomic relationships among disparate collections. We compared morphological characteristics, secondary-metabolite compositions, and partial 16S ribosomal DNA (rDNA) sequences among several collections of L. majuscula Gomont, Lyngbya spp., and Symploca spp. from Guam and the Republic of Palau. The morphological characteristics examined were cell length, cell width, and the presence or absence of a calyptra. Secondary metabolites were analyzed by two-dimensional thin-layer chromatography. Each collection possessed a distinct cellular morphology that readily distinguished Lyngbya spp. from Symploca spp. Each collection yielded a unique chemotype, but common chemical characteristics were shared among four collections of L. majuscula. A phylogeny based on secondary-metabolite composition supported the reciprocal monophyly of Lyngbya and Symploca but yielded a basal polytomy for Lyngbya. Pairwise sequence divergence among species ranged from 10 to 14% across 605 bp of 16S rDNA, while collections of L. majuscula showed 0 to 1.3% divergence. Although the phylogeny of 16S rDNA sequences strongly supported the reciprocal monophyly of Lyngbya and Symploca as well as the monophyly of Lyngbya bouillonii and L. majuscula, genetic divergence was not correlated with chemical and morphological differences. These data suggest that 16S rDNA sequence analyses do not predict chemical variability among Lyngbya species. Other mechanisms, including higher rates of evolution for biosynthetic genes, horizontal gene transfer, and interactions between different genotypes and environmental conditions, may play important roles in generating qualitative and quantitative chemical variation within and among Lyngbya species.

Evaluation of partial 16S ribosomal DNA sequencing for identification of nocardia species by using the MicroSeq 500 system with an expanded database.

Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate Nocardia species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5' 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microorganisms). The expanded MicroSeq 500 system was compared to (i). conventional identification obtained from a combination of growth characteristics with biochemical and drug susceptibility tests; (ii). molecular techniques involving restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes; and (iii). when necessary, sequencing of a 999-bp fragment of the 16S rRNA gene. An unknown isolate was identified as a particular species if the sequence obtained by partial 16S rDNA sequencing by the expanded MicroSeq 500 system was 99.0% similar to that of the reference strain. Ninety-four nocardiae representing 10 separate species were isolated from patient specimens and examined by using the three different methods. Sequencing of partial 16S rDNA by the expanded MicroSeq 500 system resulted in only 72% agreement with conventional methods for species identification and 90% agreement with the alternative molecular methods. Molecular methods for identification of Nocardia species provide more accurate and rapid results than the conventional methods using biochemical and susceptibility testing. With an expanded database, the MicroSeq 500 system for partial 16S rDNA was able to correctly identify the human pathogens N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. otitidiscaviarum, and N. veterana.

Seasonal shifts in population structure of Vibrio vulnificus in an estuarine environment as revealed by partial 16S ribosomal DNA sequencing

The partial sequence (600 bp) containing the most variable region of Vibrio vulnificus 16S ribosomal DNA (rDNA) was determined for 208 randomly selected V. vulnificus strains isolated from Galveston Bay, TX, USA between June 2000 and June 2001. A comparative analysis of the determined partial 16S rDNA sequences revealed the existence of two different partial 16S rDNA sequences (type A and type B, 1.3% base substitutions) among the 208 V. vulnificus isolates. A higher proportion of 16S rDNA type A strains was isolated in June and July while a considerably higher proportion of type B strains was isolated in September. In addition, after no V. vulnificus strains were detected during the winter months (December–February), only type A strains were isolated during the following months (March–May). The results suggest that the relative abundance of type A and type B V. vulnificus strains in Galveston Bay varies with the season and that the differences between the two 16S rDNA types may affect the viability of these organisms in the natural environment.

The phylogeny of Paralabrax (Perciformes: Serranidae) and allied taxa inferred from partial 16S and 12S mitochondrial ribosomal DNA sequences

Partial sequences of 16S and 12S mitochondrial ribosomal DNA were used to examine the phylogenetic relationships of the primarily eastern Pacific genus Paralabrax (Perciformes: Serranidae) and allied taxa. Paralabrax is considered a basal serranine, which is itself considered the basal subfamily in the Serranidae. Multiple serranines reported closely related to Paralabrax from the genera Serranus, Hypoplectrus, Cratinus, and Centropristis were used as outgroups. Species from the remaining two subfamilies, Epinephilinae and Anthiinae, of the Serranidae were also used in the analyses. The tree of the Serranidae was rooted with the families Polyprionidae and Priacanthidae. Paralabrax, the Serranidae, and the Serraninae were monophyletic in this study. Serranus was found to be paraphyletic. Centropristis, formerly considered the sister taxon to Paralabrax, was not closely related in these analyses. Cratinus agassizii, a monotypic genus from the eastern Pacific, was found to be the sister taxon to Paralabrax. There is greater resolution for intergeneric and subfamily relations than interspecific relationships. A single most parsimonious tree for the interspecific relationships of Paralabrax and allied taxa is proposed. This proposed molecular phylogeny is consistent with known biogeographic processes in the eastern Pacific.

Microheterogeneity in 16S ribosomal DNA-defined bacterial populations from a stratified planktonic environment is related to temporal changes and to ecological adaptations.

Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.

Cyanophycin production in a phycoerythrin-containing marine synechococcus strain of unusual phylogenetic affinity.

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.

Biodegradation of 2,6-Naphthalenedisulfonic Acid by a Floc-Forming Bacterium, Pigmentiphaga sp. NDS-1.

Two 2, 6-naphthalenedisulfonate (2, 6NDS) degrading bacteria were isolated from the activated sludge of a wastewater treatment plant and designated NDS-1 and NDS-2. Only strain NDS-1 showed a floc-forming ability in such media as synthetic wastewater. Sequence analysis of the partial 16S ribosomal DNA (rDNA) sequence of strain NDS-1 revealed that it is a strain closely related to Pigmentiphaga kullae, which was recently established in the family Alcaligenaceae, and that the sequence was in perfect agreement with that of strain NDS-2. Strain NDS-1 could mineralize 2, 6NDS and 1, 6-naphthalenedisulfonate (1, 6NDS) with sulfate ion release, but showed no assimilating or desulfonating activity on 8 other sulfonated naphthalenes, indicating that it has a very narrow substrate specificity. 2, 6NDS-induced NDS-1 cells exhibited a high oxygen uptake for 2, 6NDS, while non-induced cells showed a very low oxygen uptake, demonstrating that 2, 6NDS oxidation is inducible in strain NDS-1.

Mitochondrial Phylogeny of Extant Hawaiian Tree Snails (Achatinellinae)

Hawaiian tree snails in the endemic subfamily Achatinellinae display a staggering variety of shell colors and banding patterns. Despite numerous attempts to classify this morphological variation, a conclusive phylogeny has not been proposed. To improve conservation efforts, we sought to better understand the species identities and phylogenetic relationships among the extant species of Achatinella and Partulina using partial mitochondrial 16S ribosomal DNA sequences. The reconstructed phylogeny showed a high degree of support for more recent branches, but gave little support to deeper nodes. The most confident branches challenge previous systematic arrangements of these snails, grouping species that previously had been placed into different subgenera. High levels of sequence divergence within some species may reflect the long-term isolation of subpopulations. Rapid rates of sequence divergence may have saturated base substitutions and contributed to the lack of resolution of higher-order relationships. We did not find support for the monophyly of the Achatinella species, nor thus for a single colonization of Oahu from Maui Nui.

Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-1, isolated from chitin-containing compost.

A thermophilic and actinic bacterium strain, MH-1, which produced three different endochitinases in its culture fluid was isolated from chitin-containing compost. The microorganism did not grow in any of the usual media for actinomyces but only in colloidal chitin supplemented with yeast extract and (2, 6-O-dimethyl)-beta-cyclodextrin. Compost extract enhanced its growth. In spite of the formation of branched mycelia, other properties of the strain, such as the formation of endospores, the presence of meso-diaminopimelic acid in the cell wall, the percent G+C of DNA (55%), and the partial 16S ribosomal DNA sequence, indicated that strain MH-1 should belong to the genus Bacillus. Three isoforms of endochitinase (L, M, and S) were purified to homogeneity and characterized from Bacillus sp. strain MH-1. They had different molecular masses (71, 62, and 53 kDa), pIs (5.3, 4.8, and 4.7), and N-terminal amino acid sequences. Chitinases L, M, and S showed relatively high temperature optima (75, 65, and 75 degreesC) and stabilities and showed pH optima in an acidic range (pH 6.5, 5.5, and 5.5, respectively). When reacted with acetylchitohexaose [(GlcNAc)6], chitinases L and S produced (GlcNAc)2 at the highest rate while chitinase M produced (GlcNAc)3 at the highest rate. None of the three chitinases hydrolyzed (GlcNAc)2. Chitinase L produced (GlcNAc)2 and (GlcNAc)3 in most abundance from 66 and 11% partially acetylated chitosan. The p-nitrophenol (pNP)-releasing activity of chitinase L was highest toward pNP-(GlcNAc)2, and those of chitinases M and S were highest toward pNP-(GlcNAc)3. All three enzymes were inert to pNP-GlcNAc. AgCl, HgCl2, and (GlcNAc)2 inhibited the activities of all three enzymes, while MnCl2 and CaCl2 slightly activated all of the enzymes.

Characterization of Root-Associated Methanotrophs from Three Freshwater Macrophytes: Pontederia cordata , Sparganium eurycarpum , and Sagittaria latifolia

Root-associated methanotrophic bacteria were enriched from three common aquatic macrophytes: Pontederia cordata, Sparganium eurycarpum, and Sagittaria latifolia. At least seven distinct taxa belonging to groups I and II were identified and presumptively assigned to the genera Methylosinus, Methylocystis, Methylomonas, and Methylococcus. Four of these strains appeared to be novel on the basis of partial 16S ribosomal DNA sequence analysis. The root-methanotroph association did not appear to be highly specific, since multiple methanotrophs were isolated from each of the three plant species. Group II methanotrophs were isolated most frequently; though less common, group I isolates accounted for three of the seven distinct methanotrophs. Apparent K(m) values for methane uptake by representative cultures ranged from 3 to >17 muM; for five of the eight cultures examined, apparent K(m) values agreed well with apparent K(m) estimates for plant roots, suggesting that these strains may be representative of those active in situ.

Sporogenous Midgut Bacteria of the Leafroller, Pandemis pyrusana (Lepidoptera: Tortricidae)

The composition of the microbial flora within the midgut of Pandemis pyrusana Kearfott was analyzed by using the polymerase chain reaction (PCR) and culturing techniques. Universal 16S ribosomal RNA gene-specific eubacterial primers were used in PCR, and the resulting products cloned and sequenced. High homology (86-95%) was obtained between the cloned products and Clostridium ramosum and Clostridium spiroforme when compared with computer database sequence information. Initial culturing steps aimed at endospore-forming bacteria yielded reproducible growth from a bacterium that matches most closely with Bacillus licheniformis when analyzed by partial 16S ribosomal DNA sequencing and biochemicals cultural profiling. This isolate was transformed with an Escherichia coli-Bacillus subtilis shuttle vector and reintroduced into the insect, where it remained stable under nonselective conditions for at least 9 d after initial ingestion. This confirmed the ability of the genetically altered bacterium to survive within the leafroller midgut in the midst of competing microorganisms and perhaps to serve as a suitable vehicle for biological control.

Discrimination of Streptomyces albidoflavus strains based on the size and number of 16S-23S ribosomal DNA intergenic spacers.

In an attempt to develop a rapid and accurate method for discrimination of streptomycetes at the strain level, 21 strains identified by fatty acid analysis as Streptomyces albidoflavus and the type strains of nine subjective synonyms of S. albidoflavus were selected for a full or partial 16S ribosomal DNA (rDNA) sequence analysis and an investigation of the 16S-23S rDNA intergenic spacer region. 16S rDNA sequence analysis showed that 27 of the strains exhibited 100% sequence similarity; these strains included the type strain of S. albidoflavus and the type strains of the subjective synonyms Streptomyces canescens, Streptomyces coelicolor, Streptomyces felleus, Streptomyces limosus, Streptomyces odorifer, and Streptomyces sampsonii. The type strains of the other subjective synonyms of S. albidoflavus (i.e., Streptomyces gougerotii, Streptomyces intermedius, and Streptomyces rutgersensis) were found to have levels of 16S rDNA sequence difference of 1.0 to 1.1% when they were compared to the type strain of S. albidoflavus. In order to discriminate between the strains which had identical 16S rDNA sequences, the 16S-23S rDNA intergenic spacer regions were amplified and cloned, and the sequences of the spacer regions were determined for four S. albidoflavus strains, including the type strain. The 16S-23S rDNA intergenic spacer region was found to vary in length and sequence composition among the strains and within each strain. The sizes and numbers of 16S-23S rDNA intergenic spacer regions for the 27 strains with identical 16S rDNA sequences were determined by high-resolution electrophoresis of FAM-labeled PCR products and a subsequent size analysis with GeneScan 672 software. This was shown to be a useful method for discrimination of S. albidoflavus strains. Strains with the same 16S-23S rDNA intergenic spacer band pattern, as determined by high-resolution electrophoresis of FAM-labeled PCR products, could be further discriminated on the basis of the sequence composition of the spacer region.