Detection of representatives of the Planctomycetes in Sphagnum peat bogs by molecular and cultivation approaches

By means of fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH), it has been shown that members of the phylum Planctomycetes represent a numerically significant bacterial group in boreal Sphagnum peat bogs. The population size of planctomycetes in oxic layers of the peat bog profile was in the range of 0.4–2.0 × 10⁷ cells per g of wet peat, comprising 4 to 13% of the total bacterial cell number. A novel effective approach that combined a traditional cultivation technique with FISH-mediated monitoring of the target organism during the isolation procedure has been developed for the isolation of planctomycetes. Using this approach, we succeeded in isolating several peat-inhabiting planctomycetes in a pure culture. Sequencing of the 16S rRNA genes from two of these isolates, strains A10 and MPL7, showed that they belonged to the planctomycete lineages defined by the genera Gemmata and Planctomyces, respectively. The 16S rRNA gene sequence similarity between strains A10 and MPL7 and the phylogenetically closest organisms, namely, Gemmata obscuriglobus and Planctomyces limnophilus, was only 90%. These results suggest that the indigenous planctomycetes inhabiting Sphagnum peat bogs are so far unknown organisms.     

Assembly of a complete genome sequence for <Emphasis Type="Italic">Gemmata obscuriglobus</Emphasis> reveals a novel prokaryotic rRNA operon gene architecture

Gemmata obscuriglobus is a Gram-negative bacterium with several intriguing biological features. Here, we present a complete, de novo whole genome assembly for G. obscuriglobus which consists of a single, circular 9 Mb chromosome, with no plasmids detected. The genome was annotated using the NCBI Prokaryotic Genome Annotation pipeline to generate common gene annotations. Analysis of the rRNA genes revealed three interesting features for a bacterium. First, linked G. obscuriglobus rrn operons have a unique gene order, 23S–5S–16S, compared to typical prokaryotic rrn operons (16S–23S–5S). Second, G. obscuriglobus rrn operons can either be linked or unlinked (a 16S gene is in a separate genomic location from a 23S and 5S gene pair). Third, all of the 23S genes (5 in total) have unique polymorphisms. Genome analysis of a different Gemmata species (SH-PL17), revealed a similar 23S–5S–16S gene order in all of its linked rrn operons and the presence of an unlinked operon. Together, our findings show that unique and rare features in Gemmata rrn operons among prokaryotes provide a means to better define the evolutionary relatedness of Gemmata species and the divergence time for different Gemmata species. Additionally, these rrn operon differences provide important insights into the rrn operon architecture of common ancestors of the planctomycetes.     

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes

Summary In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors EMBL3 and EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.     

16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria: implications for phylogeny and in situ detection

Recently, anaerobic ammonium-oxidizing bacteria (AAOB) were identified by comparative 16S rDNA sequence analysis as a novel, deep-branching lineage within the Planctomycetales . This lineage consists currently of only two, not yet culturable bacteria which have been provisionally described as Candidatus 'Brocadia anammoxidans' and Candidatus 'Kuenenia stuttgartiensis'. In this study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2 000 bases of the 23S rDNA, was polymerase chain reaction (PCR) amplified, cloned and sequenced from both AAOB. The retrieved 16S rDNA sequences of both species contain an insertion at helix 9 with a previously overlooked pronounced secondary structure (new subhelices 9a and 9b). This insertion, which is absent in all other known prokaryotes, is detectable by fluorescence in situ hybridization (FISH) and thus present in the mature 16S rRNA. In contrast with the genera Pirellula , Planctomyces and Gemmata that possess unlinked 16S and 23S rRNA genes, both AAOB have the respective genes linked together by an ISR of approximately 450 bp in length. Phylogenetic analysis of the obtained 23S rRNA-genes confirmed the deep branching of the AAOB within the Planctomycetales and allowed the design of additional specific FISH probes. Remarkably, the ISR of the AAOB also could be successfully detected by FISH via simultaneous application of four monolabelled oligonucleotide probes. Quantitative FISH experiments with cells of Candidatus 'Brocadia anammoxidans' that were inhibited by exposure to oxygen for different time periods demonstrated that the concentration of transcribed ISR reflected the activity of the cells more accurately than the 16S or 23S rRNA concentration. Thus the developed ISR probes might become useful tools for in situ monitoring of the activity of AAOB in their natural environment.     

Comparative and functional analysis of the rRNA-operons and their tRNA gene complement in different lactic acid bacteria

The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.     

Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis

Summary The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S–16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.     

Diversity and evolution of 5S rRNA gene family organization in Pythium

The 5S rRNA gene family organization among 87 species and varieties of Pythium was investigated to assess evolutionary stability of the two patterns detected and to determine which pattern is likely the ancestral state in the genus. Species with filamentous sporangia (Groups A-C according to the ITS phylogenetic tree for Pythium) had 5S genes linked to the rDNA repeat that were predominantly coded for on the DNA strand opposite to the one with the other rRNA genes (‘inverted’ orientation). A small group of species with contiguous sporangia (Group D) is related to Groups A-C but had unlinked 5S genes. The main group of species with spherical zoosporangia (Groups E-J) generally had unlinked 5S genes in tandem arrays. The six species in Group K, although they also have spherical sporangia, had linked genes on the same strand as the other rRNA genes ‘non-inverted’ and most of them had pairs of tandem 5S genes. The evolutionary stability of 5S sequence organization was compared with the stability of morphological characters as interpreted from a phylogeny based on ITS sequence analysis. Features of 5S sequence organization were found to be just as consistent within groups as were the morphological characters. To determine the ancestral type of 5S family organization, a survey of Phytophthora strains was conducted to supply an outgroup reference. The most parsimonious interpretation of the data in this survey yielded the tentative conclusion that the linked condition of the 5S sequences was ancestral.     

rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi.

Lyme disease is the most common vector-borne disease in the United States. The causative agent is the spirochete Borrelia burgdorferi. The copy number and organization of the genes encoding the rRNAs of this organism were determined. There is a single gene for 16S rRNA and two copies each of the 23S rRNA and 5S rRNA genes. All of the genes are located within a chromosomal fragment of approximately 9.5 to 10.0 kb. The 23S and 5S rRNA genes are tandemly duplicated in the order 23S-5S-23S-5S and are apparently not linked to the 16S rRNA gene, which is situated over 2 kb upstream from the 23S-5S duplication. The individual copies of the 23S-5S duplication are separated by a 182-bp spacer. Within each 23S-5S unit, an identical 22-bp spacer separates the 23S and 5S rRNA sequences from each other. The genome organization of the 23S-5S gene cluster in a number of different B. burgdorferi isolates obtained at a number of different geographical locations, as well as in several other species of Borrelia, was investigated. All isolates of B. burgdorferi tested displayed the tandem duplication, whereas the closely related species B. hermsii, B. anserina, and B. turicatae all contained a single copy of each of the genes. In addition, different geographical isolates of B. burgdorferi can be differentiated on the basis of a restriction fragment length polymorphism associated with the 23S-5S gene cluster. This polymorphism can be a useful tool for the determination of genetic relatedness between different isolates of B. burgdorferi.     

Corresponding 16S rRNA gene segments in Rhizobiaceae and Aeromonas yield discordant phylogenies

Previous evidence has indicated that the 16S rRNA genes in certain species of Aeromonas may have a history of lateral transfer and recombination. A comparative analysis of patterns of 16S nucleotide sequence polymorphism among species of Rhizobium and Agrobacterium was conducted to determine if there is similar evidence for chimeric 16S genes in members of the Rhizobiaceae. Results from phylogenetic analyses and comparison of patterns of nucleotide sequence polymorphism in portions of rhizobial 16S genes revealed the same type of segment-dependent polymorphic site partitioning that was previously reported for Aeromonas. These results support the hypothesis that certain 16S segments in rhizobia may have a history of lateral transfer and recombination.Abbreviations 16S rRNA 16S ribosomal ribonucleic acid - 16S the 16S rRNA gene     

Sequence Heterogeneity of the Ten rRNA Operons in Clostridium perfringens

We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis. The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon. The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle. Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction. Two of the counter clockwise operons contained tRNA^Ile genes between the 16S and 23S rDNAs, and the other had a tRNA^Ile genes between the 16S and 23S rDNAs and a tRNA^Asn gene in the place of the 5S rDNA. Microheterogeneity was found within the rRNA structural genes and spacer regions. The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome. Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.     

基于16S rRNA和rpoB基因的分子系统发育分析在铜绿假单胞菌鉴定中的应用

为对比16S rRNA和rpo B基因分子系统发育分析与传统表型分类法对铜绿假单胞菌的鉴定,评估16S rRNA和rpo B基因序列分析在铜绿假单胞菌鉴定中的应用,用表型分类方法对临床自动微生物鉴定系统鉴定为铜绿假单胞菌的23株分离株进行再鉴定,PCR扩增23株分离株16S rRNA和rpo B基因片段,并测序进行系统发育分析。结果表明,表型再鉴定结果与自动微生物鉴定系统鉴定结果一致。基于两个基因的系统发育分析均显示分离株p22与不动杆菌属序列聚为一枝,其余22株分离株与铜绿假单胞菌序列聚为一枝。因此p22应鉴定为不动杆菌,16S rRNA和rpo B基因序列分析均能准确鉴定铜绿假单胞菌并能较好建立假单胞菌属内种间关系。     

Isolation, Crystallization, and Investigation of Ribosomal Protein S8 Complexed with Specific Fragments of rRNA of Bacterial or Archaeal Origin

The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8–RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Unusual characteristics of Codium fragile chloroplast DNA revealed by physical and gene mapping

Summary A complete physical map of the Codium fragile chloroplast genome was constructed and the locations of a number of chloroplast genes were determined. Several features of this circular genome are unusual. At 89 kb in size, it is the smallest chloroplast genome known. Unlike most chloroplast genomes it lacks any large repeat elements. The 8 kb spacer region between the 16 S and 23 S rRNA genes is the largest such spacer characterized to date in chloroplast DNA. This spacer region is also unusual in that it contains the rps12 gene or at least a portion thereof. Three regions polymorphic for size are present in the Codium chloroplast genome. The psbA and psbC genes map closely to one of these regions, another region is in the spacer between the 16 S and 23 S rRNA genes and the third is very close to or possibly within the 16 S rRNA gene. The gene order in the Codium genome bears no marked resemblance to either the consensus vascular plant order or to that of any green algal or bryophyte genome. Present address: Department of Biology, Texas A&M University, College Station, TX 77843; USA     

Evolution of the plastid ribosomal RNA operon in a nongreen parasitic plant: Accelerated sequence evolution,altered promoter structure,and tRNA pseudogenes

The nucleotide sequence of a 7.4 kb region containing the entire plastid ribosomal RNA operon of the nongreen parasitic plant Epifagus virginiana has been determined. Analysis of the sequence indicates that all four rRNA genes are intact and almost certainly functional. In contrast, the split genes for tRNA^Ile and tRNA^Ala present in the 16S-23S rRNA spacer region have become pseudogenes, and deletion upstream of the 16S rRNA gene has removed a tRNA^Val gene and most of the promoter region for the rRNA operon. The rate of nucleotide substitution in 16S and 23S rRNAs is several times higher in Epifagus than in tobacco, a related photosynthetic plant. Possible reasons for this, including relaxed translational constraints, are discussed.     

Species-level identification of <Emphasis Type="Italic">Bacillus</Emphasis> strains isolates from marine sediments by conventional biochemical, 16S rRNA gene sequencing and inter-tRNA gene sequence lengths analysis

The aim of this study was to compare the ability of commonly used conventional biochemical tests, sequencing analysis of 16S rRNA genes and tDNA-intergenic spacer length polymorphism (tDNA-PCR) to identify species of the genus Bacillus recovered from marine sediments. While biochemical tests were not sufficiently sensitive to distinguish between the 23 marine strains analyzed, partial 16S rRNA gene sequences allowed a correct identification, clustering them into four species belonging to Bacillus licheniformis (n = 6), Bacillus cereus (n = 9), Bacillus subtilis (n = 7) and Bacillus pumilus (n = 1). The identification results obtained with 16S rRNA sequencing were validated by tDNA-PCR analysis of 23 marine isolates that were identified by the similarities of their fingerprints to those of reference strains. tDNA-PCR fingerprinting was as discriminatory as 16S rRNA sequencing analysis. Although it was not able to distinguish among the species of the B. cereus and B. subtilis groups, it should be considered a rapid and easy approach for the reliable identification of unknown Bacillus isolates or at least for the primary differentiation of Bacillus groups.     

Variation of 16S-23S rRNA intergenic spacer regions (ISRs) in Acinetobacter baylyi (strain B2) isolated from activated sludge

To determine the variability of the 16S-23S rRNA intergenic spacer region (ISR) of the newly described Acinetobacter baylyi, 88 clones containing ISR amplicons were screened and 14 chosen for further analysis. Two different sized 16S-23S rRNA ISRs were distinguished comprising five variable and four conserved nucleotide blocks. The major regions of heterogeneity between the different sized ISRs were due to blocks of substitutions with unique secondary structures interspersed with nucleotide substitutions, rather than differences caused by presence or absence of tRNA genes, which is often the case. Recombination events causing shuffling of nucleotide blocks are considered the most likely explanation for the mosaic structure observed between the different copies of the ISR. Single base differences present in the long ISR (LISR) were then exploited in attempts to detect possible heterogeneity between rrn copies in Acinetobacter baylyi but variability was not detected by RFLP analysis of LISR-specific PCR products. These primers were shown to be highly specific for 3 Acinetobacter baylyi strains based on LISR sequence homogeneity.