Microbiology Reader
Equipment to run microbiology work automatically

Growth Curves of any strain.
Microbiological calculations.

Microbiology Home
Microbioloy Reader
Growth Curves
Photo Album
Microorganisms
Software
Download
Purchasing
Contact Us

Journal of Bacteriology, March 2002, p . 1481-1487, Vol . 184, No . 5

Novel Group I Intron in the tRNALeu(UAA) Gene of a {gamma}-Proteobacterium Isolated from a Deep Subsurface Environment

Alexey A . Vepritskiy,* Inna A . Vitol, and Sandra A . Nierzwicki-Bauer*

Department of Biology and New York Center for Studies on the Origins of Life (NSCORT), Rensselaer Polytechnic Institute, Troy, New York 12180

Received 3 July 2001/ Accepted 2 November 2001


   ABSTRACT

 
A group I intron has been found to interrupt the anticodon loop of the tRNALeu(UAA) gene in a bacterium belonging to the {gamma}-subdivision of Proteobacteria and isolated from a deep subsurface environment . The subsurface isolate SMCC D0715 was identified as belonging to the genus Pseudomonas . The group I intron from this isolate is the first to be reported for {gamma}-proteobacteria, and the first instance of a tRNALeu(UAA) group I intron to be found in a group of bacteria other than cyanobacteria . The 231-nucleotide (nt) intron's sequence has group I conserved elements and folds into a bona fide group I secondary structure with canonical base-paired segments P1 to P9 and a paired region, P10 . The D0715 intron possesses the 11-nt motif CCUACG .. . UAUGG in its P8 region, a feature not common in bacterial introns . To date, phylogenetic analysis has shown that bacterial introns form two distinct families, and their complex distribution suggests that both lateral transfer and common ancestry have taken part in the evolutionary history of these elements .


   TEXT

 
Introns that belong to a class called group I are often capable of autocatalytic activity and are characterized by sharing a common secondary structure and a conserved core region essential for autocatalysis . Group I introns have been found to interrupt a number of RNA- and protein-coding genes in diverse eukaryotic organisms, viruses, and bacteria (5, 10, 19, 21) . The discovery of group I introns in divergent bacteria has extended the phylogenetic diversity of small catalytic RNAs . However, their origin and distribution are not well understood . In the domain Bacteria, the majority of group I introns have been discovered in different filamentous and unicellular cyanobacteria, and there are only two other bacterial phyla, {alpha}- and ß-proteobacteria, that have been shown to possess group I introns (6, 18, 23, 24, 26, 28, 35) . All bacterial group I introns found to date reside in the anticodon loop regions of tRNA genes . While cyanobacteria have been demonstrated to possess a group I intron in either the tRNALeu(UAA) or tRNAfMet gene, or both (6, 24), introns from three divergent species of {alpha}-proteobacteria (23, 26) and one species of a ß-proteobacterium (26) interrupt the tRNAArg(CCU) and tRNAIle(CAU) genes, respectively .

It appears that tRNAfMet and tRNAArg(CCU) group I introns are sporadically distributed among cyanobacteria and {alpha}-proteobacteria, respectively, and they are thought to have arisen recently by horizontal transfer (23, 24) . The data available on the ß-proteobacterial tRNAIle(CAU) intron are insufficient to draw any conclusions regarding its distribution and/or origin (26, 32) . To date, the bacterial tRNALeu(UAA) group I intron has only been found in cyanobacteria . It has been suggested that there are at least two subfamilies of this intron originating by different evolutionary pathways and independently of each other . The phylogenetic distribution of this intron in one subfamily has been demonstrated to be consistent with an ancient origin and an inheritance through common ancestry (24) . Conversely, the tRNALeu(UAA) introns in a second subfamily seem to share a more recent common ancestor with the {alpha}-proteobacterial tRNAArg(CCU) introns and originated from a tRNAArg(CCU)-like intron through horizontal transfer (27, 28) .

Inasmuch as it has become evident from previous studies (5, 24) that determination of the phylogenetic distribution of group I introns is a valuable tool to assess their evolutionary history, we were interested in undertaking a systematic investigation of the abundance, phylogenetic distribution, and diversity of group I introns among {alpha}-, ß-, and {gamma}-proteobacteria obtained from deep subsurface environments . The earth's deep subsurface (depth of 100 to 1,000 m), once considered a lifeless place, has now firmly been shown to be home to a host of bacteria (1, 3, 13, 31) . In such environments, bacteria often obtain only limited nutrients and energy and hence are capable of maintaining only very slow growth . The available geological data indicate that in certain subsurface environments, there has been no or minimal transport of microorganisms between different types of sediments (14, 16, 30) . Bacteria in such environments are likely to be descendants from the original bacteria present at the time of deposition, being isolated from modern surface populations for hundreds of millions of years (14) . Taken together with the possibility of reduced selective pressure for rapid multiplication and hence for genome streamlining in subsurface environments, such bacteria might have a higher probability of retaining genetic features of original ancestor bacteria and, as such, may provide a unique source of intron sequences mostly lost from surface populations .

The present study explores the presence and distribution of group I introns in proteobacteria isolated from subsurface environments . These isolates are part of the Deparment of Energy (DOE) Subsurface Microbial Culture Collection (SMCC) housed at the Florida State University in Tallahassee (1, 4) and obtained from sediments of different depths (up to 526 m) from the DOE Savannah River site in Aiken, S.C . (30) . We report results of our survey of a limited number of subsurface {gamma}-proteobacterial isolates and the discovery of a novel group I intron, which resides in the anticodon loop of the tRNALeu(UAA) gene of a {gamma}-proteobacterium belonging to Pseudomonas strain D0715 .

Bacterial strains and growth conditions. Subsurface bacterial strains were obtained from the SMCC as living cultures recovered from glycerol stocks prior to shipping . All cultures originated from samples collected in 1986 to 1988 at the DOE Savannah River site in Aiken, S.C . (30) . The cultures were grown in PTYG medium (2) at 30°C on a shaker . In our laboratory, the cultures were stored as glycerol stocks (at -80°C) and recovered only for DNA extraction as needed . Fifty subsurface isolates were screened for the presence of group I introns by dot blot hybridization with the group I intron-specific oligonucleotide probe Q20 (M . E . Frischer and S . A . Nierzwicki-Bauer, unpublished data) . Fifteen of the 18 isolates, which were identified as potentially harboring group I introns, were shown to belong to the {gamma}-proteobacteria and were selected for the present study (Fig . 1) .


FIG . 1 . Phylogenetic affiliations of 15 subsurface bacterial isolates with other members of the {gamma}-subdivision of Proteobacteria as inferred from SSU 16S rRNA gene sequences . The SSU 16S ribosomal DNA (rDNA) from subsurface isolates was PCR amplified from purified DNA with universal oligonucleotide primers (4, 34) . Bacterial SSU 16S rRNA gene sequences were retrieved from GenBank . The tree was constructed by the neighbor-joining method (29) and drawn by using TREECON 1.3b software (33) . A distance matrix was calculated from the Galtier and Gouy two-parameter equation (15) . Bootstrap percentage values, based on 100 resamplings, are shown at the internal nodes . Only values higher than 50% are shown . The scale bar corresponds to 10 nt substitutions per 100 sequence positions . The SSU rRNA gene sequence of the {alpha}-proteobacterium Agrobacterium tumefaciens was used as an outgroup to position the root (not shown) . The sediment depth from which the isolates were obtained is indicated in brackets next to the SMCC number.

 
PCR amplification of tRNA genes from subsurface {gamma}-proteobacteria. The initial approach used was to PCR amplify genes coding for tRNAfMet, tRNAArg(CCU), and tRNALeu(UAA), because all group I introns discovered in bacteria to date reside only in the anticodon loop regions of tRNA genes . Amplification of an uninterrupted tRNA gene should result in a product of approximately 75 bp, whereas an amplification product of a gene with a residing intron should be 300 to 400 bp in size or greater, provided the intron contains an open reading frame . To exclude possible PCR-induced sequence errors, two to three different clones from independent PCRs were analyzed .

PCR amplification of tRNAfMet and tRNAArg genes. Genes coding for tRNAfMet and tRNAArg(CCU) were amplified by PCR using previously published primers (6, 23) . The results suggested that tRNAfMet and tRNAArg(CCU) genes in the subsurface {gamma}-proteobacteria were not interrupted by introns . However, with respect to the tRNAfMet genes from subsurface {gamma}-proteobacteria, in 14 of 15 isolates, amplification of tRNAfMet gene produced products of one of three sizes, namely 236, 263, or 376 bp, in addition to the tRNA-size (76 bp) product (data not shown) . In all instances, the larger products were clusters of two uninterrupted tRNAfMet genes . A similar observation was reported by Biniszkiewicz et al . (6) . Twelve isolates possessed identical tRNAfMet genes, with 2 other tRNAfMet gene sequences found in isolates B0138 and B0208 (Fig . 2) . All of the gene sequences had hallmark diagnostic features for bacterial tRNAfMet, including 3 consecutive G-C bp closing the anticodon stem (25), and they shared identical sequences up to the T stem region . When pairwise comparisons were made, these sequences revealed 5.2 and 6.5% mismatches, most of which were in the T stem (see the legend to Fig . 2) .


FIG . 2 . Alignment of three reconstructed tRNAfMet gene sequences amplified by PCR from subsurface {gamma}-proteobacteria . The sequence of each tRNA was derived from the cloned PCR products of clusters of two uninterrupted tRNAfMet genes . In tandems, the 5" and 3" ends consisted primarily of the primer sequences . Presuming that tRNAs in tandems were identical, chimeric tRNA genes were reconstructed . The reconstructed genes consisted of the 5" end of the second tRNA gene (in the tandem) and the 3" end of the first, and the anticodon stem-loop region was a consensus of both . Base pairings (underline) in the acceptor stem (A), anticodon stem (Ac), D stem (D), and T stem (T), as well as the anticodon (double underline), are indicated . Sequences: 1, tRNAfMet from isolate B0138; 2, tRNAfMet from isolate B0208; 3, tRNAfMet from isolates B0205, B0251, B0252, B0259, B0280, B0307, B0310, B0361, B0608, B0628, B0631, and D0715 . Pairwise comparison of sequences 1 and 2 and of 1 and 3 shows 5.2% mismatches; pairwise comparison of sequences 2 and 3 shows 6.5% mismatches.

 
PCR amplification of tRNALeu genes. Genes coding for tRNALeu(UAA) were amplified by using both previously published primers (6, 26) and primers designed in this study . Two forward and three reverse primers were designed to amplify the entire anticodon loop, the 3" part of the anticodon stem, and almost the entire extra arm of the tRNALeu(UAA) gene (see Fig . 5B) . The forward primers were V5 (TGG TGG AAT TGG TAG ACG CAG KGG ACT) and W5 (TGG TGG AAT TGG TAG ACG CWG KGG ATT) . The reverse primers were P3 (TGG GGG CGG GGG GAC TTG AAC CCW CAC G), V3 (TGG TSC CSA TGR CCG GAC TTG AAC CCG CAC), and W3 (TGG TSC CSG WGG CSG GAC TTG AAC CCW CAC) . These primers were used in six pairwise combinations . The reverse primers V3 and W3 were also used in combination with the forward tRNALeu(UAA) primer from reference 18 (denoted here as primer M5) . The reaction and cycling conditions were as follows: sixty nanograms of template DNA, 1x TaqExtender PCR buffer (Stratagene), 1.5 mM MgCl2, 200 µM each deoxynucleoside triphosphate (dNTP) (Amersham Pharmacia Biotech), 1 µM each primer, 1.2 µg of T4 gene 32 protein (Ambion, Inc.), 1.5 U of TaqExtender (Stratagene), and 1.5 U of Taq DNA polymerase (Sigma) were combined in a total volume of 50 µl; there was 1 cycle of 4 min at 94°C, followed by 35 cycles of 20 s at 94°C, 30 s at 41°C, and 40 s at 72°C . Amplification with either primer pair invariably yielded a tRNA-size product from each of the templates, and for most of them, a larger product was detected . However, only three templates produced amplification products of approximately 310 to 320 bp in size: B0208 (primers W5-V3, V5-P3, and W5-P3), B0333 (primers W5-V3), and D0715 (primers V5-V3, V5-W3, W5-W3, and W5-V3, as well as M5-V3 and M5-W3) (Fig . 3) . Template D0715 also yielded a strong amplification product of 250 bp with primers V5-P3 (not shown) . Amplification products from templates B0208 and D0715 (V5-P3) turned out to be clusters of two uninterrupted genes of the tRNALeu(NAG) family: UAG and CAG, respectively . A 310-bp product from template B0333 appeared to be a sequence with some similarities to a tRNA interrupted by 236 bp of a potential group I intron . However, the sequence did not provide enough evidence to infer the type of the tRNA as well as the exact location of the intron insertion site . The 236-nucleotide (nt) intron-like sequence was reproducibly amplifiable in independent experiments and with independently obtained DNA preparations by using the W5-V3 primer pair (Fig . 3), as well as primers from reference 26 . The sequence can be folded into a secondary structure that contains elements common to group I introns: conserved residues, primary sequence elements, common double-stranded segments P1 to P9, and region P10 (not shown) . The structure, however, has several ambiguities and gross disruptions of canonical features and, from a canonical point of view, is not likely to represent a "true" group I intron . For these reasons, the sequence was not analyzed further in the present study . A 310-bp amplification product from template D0715 (V5-V3, V5-W3, W5-W3, W5-V3, M5-V3, and M5-W3) was found to be tRNALeu(UAA) interrupted by a group I intron (Fig . 4) . The amplified portion of the exon sequence contained almost the entire anticodon loop, the entire 3" portion of the anticodon stem, and almost the entire extra arm with conserved features characteristic of tRNALeu(UAA) (Fig . 5B) . The intron (231 bp) was inserted between the U and A of the anticodon, which was the same position at which all known tRNALeu(UAA) introns from cyanobacteria had been previously localized (18, 24, 35) .


FIG . 5 . (A) Secondary structure of the tRNALeu(UAA) intron from the subsurface {gamma}-proteobacterium isolate D0715 . The representation format is that of Cech et al . (8) . Exon sequences are in lowercase, and intron sequences are in uppercase letters . P1 to P9 refer to conserved double-stranded segments of group I introns . Boxed sequences in P1 and the 3" exon show the P10 pairing . Putative splice sites are marked by arrows . The circled G-C pair in P7 locates the G-binding site (20) . The 13-nt motif in P6 and the putative tertiary interaction between P2 and P8 are boxed . (B) Proposed secondary structure of the tRNALeu(UAA) from the subsurface isolate D0715 . PCR-amplified portions of the anticodon loop and stem and the extra arm are shown in black . The sequences of the PCR primers are shown in light gray lowercase italics . Missing nucleotides in the 5" portion of the acceptor stem are shown as a dotted arrow . The D0715 intron insertion site is indicated by a solid arrow.

 

FIG . 3 . PCR amplification of tRNALeu genes from subsurface {gamma}-proteobacteria . Shown is migration in a 2.5% agarose gel of the amplification products obtained by PCR with some tRNALeu primer pairs and various {gamma}-proteobacterial DNAs . (A) tRNALeu PCR primers W5-W3 . (B) tRNALeu PCR primers W5-V3 . (C) tRNALeu PCR primers W5-P3 . Lanes: 1, B0138; 2, B0205; 3, D0715; 4, B0208; 5, B0251; and 6, B0333 . -, no DNA (negative control); M, 1-kb DNA marker (Gibco BRL) . Sizes in base pairs are indicated on the left side of the gel.

 

FIG . 4 . Partial alignment of group I intron sequences from proteobacteria . Shown are (i) tRNAArg(CCU) introns from three {alpha}-proteobacteria, Agrobacterium tumefaciens A136 (Ag . tumef) (26), Azospirillum halopraeferens Au5 (Az . halop) (23), and Anaplasma marginale (A . marg) (23); (ii) the tRNAIle(CAU) intron from the ß-proteobacterium Azoarcus sp . strain BH72 (Azoarcus) (26); and (iii) the tRNALeu(UAA) intron from the subsurface {gamma}-proteobacterium isolate D0715 . Group I intron secondary structure elements (P1 to P9) are indicated above the sequences . Putative base pairings are underlined . Numbers in brackets indicate the number of bases in the omitted sequence . Dashes represent gaps used to improve the alignment.

 
Structural features of the tRNALeu(UAA) group I intron. All group I introns possess similar structural features, which include four conserved primary sequence elements at defined locations and a common secondary structure with nine paired regions and a core centered on paired regions P3, P4, P6, and P7 . There is also the P10 region, which is potentially present in most, but not all, group I introns . The 5" splice site resides within the paired region P1 (11, 19, 36) . Sequence and structural features of the group I intron from the subsurface isolate D0715 place it in the subgroup IC3 according to the classification proposed by Michel and Westhof (19) . It has well-defined conserved sequence elements P, Q, R, and S, and its 231-nt sequence folds into a bona fide group I secondary structure (Fig . 4 and 5A) . Like other bacterial group I introns, the intron from isolate D0715 has an exceptionally short P1 helix, with only 3 bp preceding the proposed 5" splice site; its P2 stem consists of 9 bp, and it has a T-shaped P9 region with a 4-bp P9.1 stem . Unlike other bacterial group I introns, the D0715 intron possesses a well-defined P10 pairing in the P1 and the 3" exon regions, which consists of 4 bp (boxed in Fig . 5A) . Four out of six hairpin terminal loops in the D0715 intron are tetraloops . Three tetraloops are of the GNRA type: two are GAAA (L2 and L9.1) and one is GUAA (L8) . These loops are common in structured RNAs and are known to participate frequently in RNA tertiary interactions (19) . One feature, which seems to be conserved in introns interrupting tRNALeu(UAA), as well as tRNAfMet, is an AA bulge in the P8 stem . In contrast, the D0715 tRNALeu(UAA) intron contains an 11-nt motif (CCUACG .. . UAUGG) in its P8 stem (boxed in Fig . 5A) . The 11-nt motif CCUAAG .. . UAUGG, described by Costa and Michel (9), is quite abundant in eukaryotic group I and group II self-splicing introns at sites known to interact with hairpin terminal GNRA loops and has been shown to have high affinity to GAAA loops . However, the 11-nt motif is not that common in bacterial group I introns . To date, it has only been described in the tRNAIle(CAU) intron from Azoarcus sp . strain BH72 (26, 32), where there are two perfect copies of it in P5 and P8 . The Azoarcus 11-nt motif in P8 has been demonstrated to bind to the L2 GAAA tetraloop and provide exceptional stability for the intron's tertiary structure (32) . The 11-nt motif in the D0715 intron has a C5 instead of the usual A5 . The possibility of a PCR-induced error was excluded because the sequence has been reproduced in independent experiments with different primer pairs . In the D0715 intron, the 11-nt motif occupies the same position in P8 that it does in the Azoarcus intron and likewise could bind to the P2 GAAA tetraloop and contribute to the stability of the intron's tertiary structure . In the P6 region, the D0715 intron possesses a 13-nt motif (GCCAAGC .. . GAAGGU) that seems to be universally conserved in bacterial introns . To our best knowledge, the exact copy of the 13-nt motif is present in all known bacterial group I introns (subgroup IC3) regardless of the gene they interrupt, with the exception of four tRNAfMet introns from cyanobacteria . These four introns have A1 instead of the usual G1 . Our analysis of 50 chloroplast tRNALeu(UAA) introns from the database, which also belong to the subgroup IC3, has demonstrated that approximately 40% of them possessed a structurally similar 13-nt feature in the same position, but the sequence was not conserved, varying among species with a consensus of GCCAARD .. . WDWGGU . The subgroup IA1 is the only other one, which has introns with a similar 13-nt structural feature . This subgroup consists of chloroplast and mitochondrial introns, which interrupt various genes and are characterized by the presence of a P7.1 region and P11 pairing . Out of 30 IA1 introns analyzed, 95% possessed the 13-nt feature . However, its sequence was more variable than in the case of chloroplast IC3 introns, varying among different species of the same genus and having a consensus of GCSAADY .. . RVASGU . It is likely that the 13-nt motif GCCAAGC.. . GAAGGU, which is always present in bacterial introns, represents a hallmark diagnostic feature . This structural feature could plausibly be dated back to the common ancestor of the domain Bacteria . The presence of the 13-nt structural feature in introns from two different subgroups, IC3 and IA1, might suggest close evolutionary relatedness . In addition, apparent structural similarity between the 13-nt motif GCCAAGC .. . GAAGGU and the 11-nt motif CCUAAG ...UAUGG might imply that they are functionally similar, and perhaps originally they were both present in bacterial group I introns . For reasons yet to be discovered, the 13-nt motif appears to have remained associated exclusively with bacterial group I introns, whereas the 11-nt motif is found distributed sporadically in introns from eukaryotes .

Phylogenetic relationship between the D0715 group I intron and related bacterial introns. To analyze the phylogenetic relationship between the D0715 group I intron and other bacterial group I introns, a representative sample of 20 sequences of known bacterial introns was aligned based on the potential secondary structure, rather than similarities in the primary sequence . There were 289 positions that could be unambiguously aligned . Five introns in this alignment represented proteobacteria: three were {alpha}-proteobacteria [tRNAArg(CCU) introns from Agrobacterium tumefaciens, Azospirillum halopraeferens, and Anaplasma marginale], one was a ß-proteobacterium [tRNAIle(CAU) intron from Azoarcus sp.], and one was a {gamma}-proteobacterium [tRNALeu(UAA) intron from subsurface isolate D0715] . All of the rest were tRNALeu(UAA) and tRNAfMet introns from various cyanobacteria . The phylogenetic analysis demonstrated that bacterial group I introns dichotomized, forming distinct families (Fig . 6) . The branches separating two intron families were reasonably supported by bootstrap values between 53 and 100% . This separation was reproducible in trees inferred by the parsimony method (not shown) . Within the families, however, not all branches were well supported, with bootstrap values below 50% . Additionally, the topology varied slightly, depending on the method used and the data set . This presumably reflects the small number of positions that could be used in this analysis and the high degree of divergence of these sequences . One intron family was homogeneous and consisted exclusively of group I tRNALeu(UAA) introns from various cyanobacteria, whereas in a second family, introns, interrupting different tRNAs, seemed to be scattered across bacterial phylogenetic boundaries, comprising tRNAArg(CCU) introns from {alpha}-proteobacteria, the tRNAIle(CAU) intron from a ß-proteobacterium, tRNAfMet introns from cyanobacteria, and tRNALeu(UAA) introns from a {gamma}-proteobacterium and cyanobacteria . In the second family, the tRNAfMet introns formed a very well supported tight cluster with bootstrap value of 98%, the topology remaining unchanged regardless of the data set and method used . The cyanobacterial tRNALeu(UAA) intron subtree within this family formed a stable cluster of two filamentous and one unicellular species with relatively good bootstrap values and a stable topology, whereas the {alpha}-proteobacterial tRNAArg(CCU) intron subtree was in general poorly supported . However, the tRNAArg(CCU) intron subtree was reasonably well supported (bootstrap values between 50 and 67%) in maximum parsimony trees (not shown) . The D0715 tRNALeu(UAA) intron branched deeply and appeared to be more related to either tRNAArg(CCU) introns from {alpha}-proteobacteria or the tRNAIle(CAU) intron from Azoarcus sp . than to any tRNALeu(UAA) or tRNAfMet introns from cyanobacteria . In some distance trees, the D0715 intron clustered with the tRNAIle(CAU) intron from Azoarcus sp . (not shown), albeit with relatively low bootstrap values . Nevertheless, this clustering was well supported by bootstrap values of up to 85% in maximum parsimony trees (not shown) . This intron never clustered with cyanobacterial tRNALeu(UAA) introns from either family or tRNAfMet introns .


FIG . 6 . Phylogenetic analysis of bacterial tRNA group I introns . The tree was inferred from an alignment of the intron core sequences (289 positions) by using the neighbor-joining algorithm (29) and drawn with the TREECON 1.3b software (33) . A distance matrix was calculated from the Kimura two-parameter equation (17) . Bootstrap percentage values, based on 100 resamplings, are shown at the internal nodes . Only values higher than 50% are shown . The scale bar corresponds to 10 nt substitutions per 100 sequence positions . The tRNALeu(UAA) intron sequence from the chloroplast of Marchantia polymorpha (CP) (22) was used as an outgroup to position the root . Branches separating two intron families are shown in boldface . [tArg], tRNAArg(CCU) intron; [tfMet], tRNAfMet intron; [tLeu], tRNALeu(UAA) intron . The intron sequences were obtained from the following sources: for {alpha}- and ß-proteobacteria, see the legend to Fig . 4; for Anabaena cylindrica, Anabaena sp . strain PCC7120, Chlorogloeopsis fritschii, Cylindrospermum sp . strain PCC7417, Dermocarpa sp . strain PCC7437, Gloeobacter violaceus PCC7421, Oscillatoria sp . strain PCC6304, and Pseudoanabaena sp . strain PCC7403, see reference 24; and for Aphanizomenon flos-aquae NIVA-CYA 142(1) and 142(2), Microcystis aeruginosa NIVA-CYA 57, Nostoc sp . strain NIVA-CYA 246, Nostoc sp . strain NIVA-CYA 308(1) and 308(2), see reference 28 . The clustering of Aphanizomenon flos-aquae NIVA-CYA 142(1) and 142(2) and of Nostoc sp . NIVA-CYA 308(1) and 308(2) is different from that in the original report (28) . This discrepancy was caused by an error in the database (K . Rudi, personal communication).

 
The phylogenetic analysis has revealed the complex distribution of group I introns within bacteria, which suggests that both lateral transfer and common ancestry are part of the evolutionary history of these elements . Our data support the view that the group I intron in the first family is relatively stable and has a monophyletic origin . A cluster of related bacteria possessing this intron inherited it from the common ancestor of the group (24) . The intron in the second family seems to be less stable and is likely to have originated by horizontal transfer . It provides an interesting instance of intron transposition and horizontal transfer between widely divergent bacterial species . The fact that there is an intronic open reading frame with endonuclease activity in at least one strain (6, 7, 24) suggests that this intron is capable of mobility . On the other hand, recent evidence (12) suggests that lightning-mediated gene transfer could take place in soil and might be a mechanism involved in bacterial evolution . The introns from this family possibly share a more recent common ancestor with each other than they do with introns from the first family . However, the apparent clustering of introns that interrupt different tRNAs within this family might suggest that there were multiple invasive elements, which were independently inserted into different bacterial tRNAs . It also seems plausible that after transfer some of these elements might have been vertically inherited within specific tRNA genes or lost .

Nucleotide sequence accession number. The nucleotide sequences generated in this study have been deposited in GenBank under the following accession numbers: AF336311, isolate D0715 SSU 16S rRNA gene; AF336313, isolate D0715 tRNALeu(UAA) group I intron; AY029759, isolate B0333 SSU 16S rRNA gene; and AY029760, isolate B0333 tRNALeu(UAA) group I intron-like sequence .

 


   ACKNOWLEDGMENTS

 
This work was supported by the New York Center for Studies on the Origins of Life, a NASA Specialized Center of Research and Training (NSCORT) (funded under agreement NAG5-7598 to S.A.N.-B) .

We thank David Balkwill for providing us with the bacterial cultures; Jennifer C . Reineke for technical assistance; Ellen Braun-Howland and Marc Frischer for sharing unpublished data; and David Shub for helpful discussions, suggestions, and critical reading of the manuscript .


   FOOTNOTES

 
* Corresponding author . Mailing address for S . A . Nierzwicki-Bauer: Department of Biology, Rensselaer Polytechnic Institute, MRC 306, 110 8th Street, Troy, NY 12180 . Phone: (518) 276-2696 . Fax: (518) 276-2162 . E-mail: nierzs{at}rpi.edu . Mailing address for A . Vepritskiy: Department of Biology, Rensselaer Polytechnic Institute, MRC 307, 110 8th St., Troy, NY 12180 . Phone: (518) 276-2359 . Fax: (518) 276-2162 . E-mail: vepria{at}rpi.edu .


   REFERENCES

 

  1. Balkwill, D . L. 1989 . Numbers, diversity, and morphological characteristics of aerobic, chemoheterotrophic bacteria in deep subsurface sediments from a site in South Carolina . Geomicrobiol . J . 7:33-52.
  2. Balkwill, D . L., and W . C . Ghiorse. 1985 . Characterization of subsurface bacteria associated with two shallow aquifers in Oklahoma . Appl . Environ . Microbiol . 50:580-588.
  3. Balkwill, D . L., J . K . Fredrickson, and J . M . Thomas. 1989 . Vertical and horizontal variations in the physiological diversity of the aerobic chemoheterotrophic bacterial microflora in deep southeast coastal plain subsurface sediments . Appl . Environ . Microbiol . 55:1058-1065.
  4. Balkwill, D . L., R . H . Reeves, G . R . Drake, J . Y . Reeves, F . H . Crocker, M . B . King, and D . R . Boone. 1997 . Phylogenetic characterization of bacteria in the Subsurface Microbial Culture Collection . FEMS Microbiol . Rev . 20:201-216.
  5. Bhattacharya, D., B . Surek, M . Rüsing, S . Damberger, and M . Melkonian. 1994 . Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae) . Proc . Natl . Acad . Sci . USA 91:9916-9920.
  6. Biniszkiewicz, D., E . Cesnaviciene, and D . Shub. 1994 . Self-splicing group I intron in cyanobacterial initiator methionine tRNA: evidence for lateral transfer of introns in bacteria . EMBO J . 13:4629-4635.
  7. Bonocora, R . P., and D . A . Shub. 2001 . A novel group I intron-encoded endonuclease specific for the anticodon region of tRNAfMet genes . Mol . Microbiol . 39:1299-1306.
  8. Cech, T . R., S . H . Damberger, and R . R . Gutell. 1994 . Representation of the secondary structure of group I introns . Nat . Struct . Biol . 1:273-280.
  9. Costa, M., and F . Michel. 1995 . Frequent use of the same tertiary motif by self-folding RNAs . EMBO J . 14:1276-1285.
  10. Damberger, S . H., and R . R . Gutell. 1994 . A comparative database of group I intron structures . Nucleic Acids Res . 22:3508-3510.
  11. Davies, R . W., R . B . Waring, J . A . Ray, T . A . Brown, and C . Scazzocchio. 1982 . Making ends meet: a model for RNA splicing in fungal mitochondria . Nature 300:719-724.
  12. Demaneche, S., F . Bertolla, F . Buret, R . Nalin, A . Sailland, P . Auriol, T . M . Vogel, and P . Simonet. 2001 . Laboratory-scale evidence for lightning-mediated gene transfer in soil . Appl . Environ . Microbiol . 67:3440-3444.
  13. Fredrickson, J . K., T . R . Garland, R . J . Hicks, J . M . Thomas, S . W . Li, and K . M . McFadden. 1989 . Lithotrophic and heterotrophic bacteria in deep subsurface sediments and their relation to sediment properties . Geomicrobiol . J . 7:53-66.
  14. Fredrickson, J . K., J . P . McKinley, S . A . Nierzwicki-Bauer, D . C . White, D . B . Ringelberg, S . A . Rawson, S.-M . Li, F . J . Brockman, and B . N . Bjornstad. 1995 . Microbial community structure and biogeochemistry of Miocene subsurface sediments: implications for long-term microbial survival . Mol . Ecol . 4:619-626.
  15. Galtier, N., and M . Gouy. 1995 . Inferring phylogenies from DNA sequences of unequal base compositions . Proc . Natl . Acad . Sci . USA 92:11317-11321.
  16. Kieft, T . L., J . K . Fredrickson, J . P . McKinley, B . N . Bjornstad, S . A . Rawson, T . J . Phelps, F . J . Brockman, and S . M . Pfiffner. 1995 . Microbiological comparisons within and across contiguous lacustrine, paleosol, and fluvial subsurface sediments . Appl . Environ . Microbiol . 61:749-757.
  17. Kimura, M. 1980 . A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences . J . Mol . E vol . 16:111-120.
  18. Kuhsel, M . G., R . Strickland, and J . D . Palmer. 1990 . An ancient group I intron shared by eubacteria and chloroplasts . Science 250:1570-1573.
  19. Michel, F., and E . Westhof. 1990 . Modeling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis . J . Mol . Biol . 216:585-610.
  20. Michel, F., M . Hanna, R . Green, D . P . Bartel, and J . W . Szostak. 1989 . The guanosine binding site of the Tetrahymena ribozyme . Nature 342:391-395.
  21. Nishida, K., S . Suziki, Y . Kimura, N . Nomura, M . Fujie, and T . Yamada. 1998 . Group I introns found in Chlorella viruses: biological implications . Virology 242:319-326.
  22. Oda, K., K . Yamato, E . Ohta, Y . Nakamura, M . Takemura, N . Nozato, K . Akashi, T . Kanegae, Y . Ogura, T . Kohchi, and K . Ohyama. 1992 . Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA . A primitive form of mitochondrial genome . J . Mol . Biol . 223:1-7.
  23. Paquin, B., A . Heinfling, and D . A . Shub. 1999 . Sporadic distribution of tRNACCUArg introns among {alpha}-purple bacteria: evidence for horizontal transmission and transposition of a group I intron . J . Bacteriol . 181:1049-1053.
  24. Paquin, B., S . D . Kathe, S . A . Nierzwicki-Bauer, and D . Shub. 1997 . Origin and evolution of group I introns in cyanobacterial tRNA genes . J . Bacteriol . 179:6798-6806.
  25. RajBhandary, U . L. 1994 . Initiator transfer RNAs . J . Bacteriol . 176:547-552.
  26. Reinhold-Hurek, B., and D . A . Shub. 1992 . Self-splicing introns in tRNA genes of widely divergent bacteria . Nature (London) 357:173-176.
  27. Rudi, K., and K . S . Jakobsen. 1997 . Cyanobacterial tRNALeu(UAA) group I introns have polyphyletic origin . FEMS Microbiol . Lett . 156:293-298.
  28. Rudi, K., and K . S . Jakobsen. 1999 . Complex evolutionary patterns of tRNAUAALeu group I introns in cyanobacterial radiation . J . Bacteriol . 181:3445-3451.
  29. Saitou, N., and M . Nei. 1987 . The neighbor-joining method: a new method for reconstructing phylogenetic trees . Mol . Biol . E vol . 4:406-425.
  30. Sargent, K . A., and C . B . Fliermans. 1989 . Geology and hydrology of the deep subsurface microbiology sampling site at the Savannah River plant, South Carolina . Geomicrobiol . J . 7:3-13.
  31. Sinclair, J . L., and W . C . Ghiorse. 1989 . Distribution of aerobic bacteria, protozoa, algae, and fungi in deep subsurface sediments . Geomicrobiol . J . 7:15-31.
  32. Tanner, M . A., and T . R . Cech. 1996 . Activity and thermostability of the small self-splicing group I intron in the pre-tRNAIle of the purple bacterium Azoarcus . RNA 2:74-83.
  33. Van de Peer, Y., and R . De Wachter. 1994 . TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment . Comput . Appl . Biosci . 10:569-570.
  34. Weisburg, W . G., S . M . Barns, D . A . Pelletier, and D . J . Lane. 1991 . 16S ribosomal DNA amplification for phylogenetic study . J . Bacteriol . 173:697-703.
  35. Xu, M.-Q., S . D . Kathe, H . Goodrich-Blair, S . A . Nierzwicki-Bauer, and D . A . Shub. 1990 . Bacterial origin of a chloroplast intron: conserved self-splicing group I introns in cyanobacteria . Science 250:1566-1570.
  36. Zaug, A . J., M . M . McEvoy, and T . R . Cech. 1993 . Self-splicing of the group I intron from Anabaena pre-tRNA: requirement for base pairing of the exons in the anticodon stem . Biochemistry 32:7946-7953.

 

 

 

Free Online Full-text Article

 

 

 

 

What Is Water Purification?, What Is Protein?, What Is Growth Medium?, What Is Staphylococcus Aureus?, What Is Molecular Microbiology?, c, Microbes, n, Microbiology, e, Bacteria, a, Microbe, c, Microorganism, n, Microorganism, c, Gram negative, c, Cell cultures, a, Acinetobacter, o, Salmonella, e, Escherichia coli, o, Rhodotorula, s, Listeriosis, r, Antibiotics, o, Photobacteria, a, Prokaryotes, n, Nitrifying, r, Prokaryotes, c, Yeasts, r, Bacteriological, n, Cell cultures, n, Yeasts, r, Streptococcal, e, Microorganisms, r, Escherichia coli, s, Penicillin




 

   Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology
Anaerobic Microbiology
Antimicrobial Susceptibility
Artificial Atmosphere
Bioassay of Antibiotics
Biofilm Microbiology
Bioreactor Technology
Biotechnology
Cell Biology
Clinical Microbiology
Environmental Microbiology
Experiments with Yeast		 Fermentation
Food Microbiology
Functional Genomics
Gene Technology
Growth Media Development
Growth Rate and Lag Time
Industrial Microbiology
Medical/Pharmaceutical Field
Microbiological Assay
Microbiological Research
Microbiology of Cosmetics

go to a specific theme...

 Military Microbiology
Molecular Microbiology
Mutagenicity and Genotoxicity
Oral Microbiology
Patents
Postantibiotic Studies
Soil Microbiology
Spore Microbiology
Veterinary Microbiology
Waste/Wastewater Treatment
Water Microbiology
Wine Microbiology

 


 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

 

 

Last modified: May 25, 2005