Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes

The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.     

Nuclease S1 mapping of 16S ribosomal RNA in ribosomes

Escherichia coli 16S rRNA and 16S-like rRNAs from other species have several universally conserved sequences which are believed to be single-stranded in ribosomes. The quantitative disposition of these sequences within ribosomes is not known. Here we describe experiments designed to explore the availability of universal 16S rRNA sequences for hybridization with DNA probes in 30S particles and 70S ribosomes. Unlike previous investigations, quantitative data on the accessibility of DNA probes to the conserved portions of 16S rRNA within ribosomes was acquired. Uniquely, the experimental design also permitted investigation of cooperative interactions involving portions of conserved 16S rRNA. The basic strategy employed ribosomes, 30S subunits, and 16S rRNAs, which were quantitatively analyzed for hybridization efficiency with synthetic DNA in combination with nuclease S1. In deproteinated E. coli 16S rRNA and 30S subunits, the regions 520-530, 1396-1404, 1493-1504, and 1533-1542 are all single-stranded and unrestricted for hybridization to short synthetic DNAs. However, the quantitative disposition of the sequences in 70S ribosomes varies with each position. In 30S subunits there appear to be no cooperative interactions between the 16S rRNA universal sequences investigated.     

菌种1137116S rRNA序列分析及鉴定

通过PCR方法扩增菌种11371的16S rRNA基因并测序,将序列提交GenBank(登录号:DQ531606),并与其他链霉菌属种进行比较,通过DNAStar软件得到菌种16S rRNA基因序列进化树。同时采用插片法、显微镜观察等方法对株菌11371进行形态特征、培养特征、生理生化特征鉴定。结果表明,11371的16S rRNA序列与其他链霉菌具有一定的同源性,结合生理、生化指标鉴定结果,进一步确定菌种为不吸水链霉菌一株新亚种(Streptomyces ahygroscopicus subsp.wuzhouensis n.sub-sp.),菌株11371 16S rRNA序列为GenBank中首例Streptomyces ahygroscopicus的16S rRNA序列。     

Cotranscription and processing of 23S, 4.5S and 5S rRNA in chloroplasts from Zea mays

The termini of rRNA processing intermediates and of mature rRNA species encoded by the 3' terminal region of 23S rDNA, by 4.5S rDNA, by the 5' terminal region of 5S rDNA and by the 23S/4.5S/5S intergenic regions from Zea mays chloroplast DNA were determined by using total RNA isolated from maize chloroplasts and 32P-labelled rDNA restriction fragments of these regions for nuclease S1 and primer extension mapping. Several processing sites detectable by both 3' and 5' terminally labelled probes could be identified and correlated to the secondary structure for the 23S/4.5S intergenic region. The complete 4.5S/5S intergenic region can be reverse transcribed and a common processing site for maturation of 4.5S and 5S rRNA close to the 3' end of 4.5S rRNA was detected. It is therefore concluded that 23S, 4.5S and 5S rRNA are cotranscribed.     

16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences analysis of the genus Myxococcus.

Phylogenetic relationships of the species belonging to the genus Myxococcus were elucidated based on the sequences of 16S rRNA genes and 16S-23S rRNA gene internal transcribed spacer (ITS) regions. The Myxococcus species were consequently classified into four distinct groups. The type strain of Myxococcus coralloides occupied an independent position (Group 1); it has been recently reclassified as Corallococcus coralloides. Group 2 comprised the type strains of both Myxococcus virescens and Myxococcus xanthus, and some strains assigned to Myxococcus flavescens. The type strain of M. flavescens was contained in Group 3 along with the strains of Myxococcus fulvus. Group 4 included the strains belonging to C. coralloides, M. fulvus, and M. stipitatus. The type strain of M. fulvus that was allocated outside Group 4 in the 16S rRNA gene tree belonged to Group 3 in the ITS tree. These results strongly suggest that the morphological characteristics of Myxococcus species are not consistent with the phylogenetic relationships. The Myxococcus species must therefore be redefined according to the phylogenetic relationships revealed in this study.     

Ribosomal ribonucleic acid synthesis in Bacillus subtilis

The mode of biosynthesis of the 16S and 23S ribosomal ribonucleic acids (rRNA) was studied in Bacillus subtilis 168thy(-). Three criteria were used to define the characteristics of the rRNA species: (i) the time required at 37 degrees C to synthesize 16S and 23S rRNA chains de novo in growing cultures; (ii) the degree of reactivity of the 3'-terminal groups of the rRNA molecules with periodate and [carbonyl-(14)C]isonicotinic acid hydrazide; and (iii) the reactivity of the 5'-terminal regions of the rRNA molecules with the bacterial exonuclease purified by Riley (1969). The 16S and 23S chains of B. subtilis were synthesized at rates of 22+/-2 and 21+/-2 nucleotides added/s. The periodate-[(14)C]isonicotinic acid hydrazide and the exonuclease techniques for estimating apparent chain lengths of RNA indicated that the chain length of the 23S rRNA was 1.8 times that of the 16S fraction. The apparent chain lengths of each rRNA species were: 16S rRNA, 1650+/-50 nucleotide residues; 23S rRNA, 3050+/-90 nucleotide residues. It appears that, the 16S and 23S rRNA molecules in B. subtilis are synthesized in the expected manner, by simple polymerization of the final products on independent cistrons.     

Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome

DNAs from Mycobacterium tuberculosis, M. intracellulare, M. phlei and M. smegmatis were digested by restriction enzymes and hybridized with three probes consisting of the 5' (16S rRNA), the middle (16S and 23S rRNA), and the 3' (23S and 5S rRNA) portions of the Escherichia coli rrnB operon. The resulting hybridization patterns indicate that slow-growing Mycobacteria species (i.e., M. tuberculosis and M. intracellulare), with genome size 3.13 - 4.29 X 10(9) daltons, appear to possess only one rRNA operon, whereas fast-growing species (i.e., M. phlei and M. smegmatis), with genome size 4.30 - 5.20 X 10(9) daltons, appear to possess two rRNA operons.     

Genetic relationships among mycoplasmas based on the 16S-23S rRNA spacer sequence

The nucleotide sequences of the spacer regions between the 16S and 23S rRNA genes of 20 Mycoplasma species were determined following amplification by PCR. Although the spacer regions lacked spacer tRNA genes, they contained the box B and box A sequences in this order from the 5' terminus. The sequence alignment indicated that the 20 species were divided into four clusters, the M. pneumoniae, M. hominis, M. hyorhinis and M. fermentans clusters, and a single floating species, M. hyopneumoniae.     

Prediction of the recognition sites on 16S and 23S rRNAs from E. coli for the formation of 16S-23S rRNA complex

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E. coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891-899 and 1195-1203 on 16S rRNA along with the corresponding complementary sites 1904-1912 and 760-768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630-638 on 16S rRNA with its complementary site 2031-2039 on 23S rRNA cannot be ruled out.     

Identification of Enterococcus spp. based on specific hybridisation with 16S rDNA probes

The conventional methods for routine enterococci species identification are usually based on phenotypic characteristics. However, in recent years, some studies have defined specific probes based on both 16S and 23S rRNA genes for the identification of some Enterococcus spp. A set of probes based on the 16S rRNA gene has been developed in order to evaluate the usefulness of a six-step biochemical key for species level identification of enterococci. Probe specificity has been evaluated with type collection and environmental strains by dot blot hybridisation. A high correlation was obtained between biochemical key and hybridisation identifications. This set of probes provides a confirmative method for phenotypic species identification.     

Ribosomal RNA and rDNA sequence analyses.

The potential of ribosomal (r) RNA and the encoding genes (rDNA) to elucidate natural relationships has been dramatically extended by improved sequencing approaches and the application of polymerase chain reaction. Sequence information on 16S and 23S rRNA/DNA from 69 strains of 53 Streptomyces species allows determination of regions that can be used as target sites for diagnostic probes, and for amplification and sequencing primers. To generate phylogenetic trees, sequence similarities are converted into distance values. The topologies of the trees based on different parts of the molecule are compared among each other and to the numerical phenotypic clustering of the strains.     

Prediction of the Recognition Sites on 16S and 23S rRNAs from E.coli for the Formation of 16S-23S rRNA Complex

Abstract

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E.coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891–899 and 1195–1203 on 16S rRNA along with the corresponding complementary sites 1904–1912 and 760–768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630–638 on 16S rRNA with its complementary site 2031–2039 on 23S rRNA cannot be ruled out.     

Characterization of Streptomyces venezuelae ATCC 10595 rRNA gene clusters and cloning of rrnA.

Streptomyces venezuelae ATCC 10595 harbors seven rRNA gene clusters which can be distinguished by BglII digestion. The three rRNA genes present in each set are closely linked with the general structure 16S-23S-5S. We cloned rrnA and sequenced the 16S-23S spacer region and the region downstream of the 5S rRNA gene. No tRNA gene was found in these regions.     

Molecular cloning and comparative sequence analyses of rRNA operons in Streptomyces nodosus ATCC 14899.

The genome of Streptomyces nodosus contains six ribosomal RNA (rRNA) operons. Four of the rRNA operons; rrnB, rrnD, rrnE and rrnF were cloned. We have completely sequenced all four operons, including a region 750 base pairs (bp) upstream of the 16S rRNA gene. The three rRNA genes present in each operon were closely linked in the order 16S-23S-5S. A sequence comparison of the four operons showed more than 99% sequence similarity between the corresponding 16S and 23S rRNA genes, and more than 97% similarity between 5S rRNA genes. The sequence differences observed between 23S rRNA genes appeared to be localized in two specific regions. Substantial sequence differences were found in the region upstream of the 16S rRNA gene as well as in the internal transcribed spacers. No tRNA gene was found in the 16S-23S spacer regions.     

Species-specific PCR for identification of common contaminant mollicutes in cell culture.

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5' end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5' end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.     

Identification of Staphylococcus carnosus and Staphylococcus warneri isolated from meat by fluorescent in situ hybridization with 16S rRNA-targeted oligonucleotide probes.

16S rRNA targeted oligonucleotide probes were designed by sequence analysis of an rRNA database to discriminate S. carnosus, S. warneri, and S. saprophyticus species. After establishing hybridization conditions by RNA dot blot hybridization with reference species, our probes were shown to be specific. By in situ hybridization only S-S-S.carno-0440-a-A-23 and S-S-S.war-0180-a-A-23 can specifically detect S. carnosus and S. warneri, respectively. The detection of old cells of S. carnosus 833 was more limited by the permeabilisation than by the low rRNA content. One day old cells could be permeabilized with lysostaphin, whereas young cells were permeabilized with lysozyme.     

The 16S rRNA nucleotide sequence of Mycobacterium leprae: phylogenetic position and development of DNA probes

The almost complete 16S rRNA sequence from Mycobacterium leprae was determined by direct sequencing of the chromosomal gene amplified by the polymerase chain reaction. The primary sequence revealed an insertion of 12 nucleotides at the 5' end of the 16S rRNA gene, which consists of an A-T stretch and appears to be unique for M. leprae. Within the mycobacteria M. leprae branches off with a group of slow-growing species comprising M. scrofulaceum, M. kansasii, M. szulgai, M. malmoense, M. intracellulare and M. avium. A systematic comparison of the nucleotide sequence resulted in the characterization of oligonucleotide probes which are highly specific for M. leprae. The probes hybridized exclusively to 16S rRNA nucleic acids from M. leprae, but not to nucleic acids from 20 cultivable fast- and slow-growing mycobacteria.     

Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA

We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.     

Evidence for indigenous Streptomyces populations in a marine environment determined with a 16S rRNA probe.

A 16S rRNA genus-specific probe was used to determine whether Streptomyces populations are an indigenous component of marine sediment bacterial communities. Previous debates have suggested that marine Streptomyces isolates are derived not from resident populations but from spores of terrestrial species which have been physically transported to marine ecosystems but remain dormant until isolation. Rigorously controlled hybridization of rRNA extracted from coastal marsh sediments with the genus-specific probe indicated that Streptomyces rRNA accounted for 2 to 5% of the sediment community rRNA and that spores are not the source of the hybridization signal. Streptomyces populations must therefore be at least the 26th most abundant genus-level source of bacterial rRNA. the relative amounts of rRNAs from Streptomyces spp. and members of the Bacteria (69 to 79%) and Archaea (4 to 7%) domains were highly consistent in these marine sediments throughout an annual cycle, indicating that the species composition of sediment bacterial communities may be more stable than recent studies suggest for marine planktonic bacterial communities. Laboratory studies designed to investigate the possible functional roles of Streptomyces populations in coastal sediments demonstrated that population levels of this genus changed relatively rapidly (within a time frame of 6 weeks) in response to manipulation of substrate availability. Amendments of intact sediment cores with two compounds (vanillic acid and succinic acid) consistently resulted in Streptomyces populations contributing an increased percentage of rRNA (6 to 15%) to the total bacterial rRNA pool.