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Cloning and sequencing of a phenol hydroxylase gene of Pseudomonas pseudoalcaligenes strain MH1: a bacterium able to mineralize various aromatic compounds.

The phenol-degrading strain Pseudomonas pseudoalcaligenes MH1, identified by the rRNA approach, was isolated from wastewater enrichment culture. It utilized phenol up to 1.5 g/L as the sole source of carbon and energy. In addition, cresols (o-, m-, p-), 4-hydroxybenzoic acid, syringic acid, and vanillic acid were metabolized as sole substrates by phenol-grown cells of strain MH1. Using primers, designed on the basis of the sequence of the dmp operon of P. putida strain CF600, a gene encoding phenol hydroxylase, which catalyzes the hydroxylation of phenol to catechol, was detected in strain MH1. The whole phenol hydroxylase operon of strain MH1 was amplified in a polymerase chain reaction fragment of 5.207 kb that was cloned and sequenced. The total sequence revealed a cluster of six ATG starting open reading frames (ORFs). Analysis of the regulatory signals showed a putative promoter region, 40 bp upstream from the transcriptional start of ORF1, which have a strong homology to a set of positively controlled promoters. Comparison of the MH1 phenol hydroxylase gene sequence with those of other Pseudomonas strains revealed higher homology except in the 5' region. Thus, the deduced amino acid sequence of the first subunit of phenol hydroxylase of P. pseudoalcaligenes strain MH1 exhibited a difference at the N-terminal region (the first 10 amino acids) compared with that of known phenol hydroxylase of Pseudomonas strains.

Amino Acid Sequence↗
PubMed1982

Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis.

We have determined the nucleotide sequence of two Bacillus subtilis promoters (veg and tms) that are utilized by the principal form of B. subtilis RNA polymerase found in vegetative cells (sigma 55-RNA polymerase) and have compared our sequences to those of several previously reported Bacillus promoters. Hexanucleotide sequences centered approximately 35 (the "--35" region) and 10 (the "--10" region) base pairs upstream from the veg and tms transcription starting points (and separated by 17 base pairs) corresponded closely to the consensus hexanucleotides (TTGACA and TATAAT) attributed to Escherichia coli promoters. Conformity to the preferred --35 and --10 sequences may not be sufficient to promote efficient utilization by B. subtilis RNA polymerase, however, since three promoters (veg, tms and E. coli tac) that conform to these sequences and that are utilized efficiently by E. coli RNA polymerase were used with highly varied efficiencies by B. subtilis RNA polymerase. We have also analyzed mRNA sequences in DNA located downstream from eight B. subtilis chromosomal and phage promoters for nucleotide sequences that might signal the initiation of translation. In accordance with the rules of McLaughlin, Murray and Rabinowitz (1981), we observe mRNA nucleotide sequences with extensive complementarity to the 3' terminal region of B. subtilis 16S rRNA, followed by an initiation codon and an open reading frame.

Bacillus subtilis↗
PubMed1999

Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism.

To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half-life of total pulse-labelled RNA along with decreased half-lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Long-term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3'-terminus of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an almost 20-fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript-dependent manner.

Blotting, Southern↗
PubMed1993

Linear chromosomal physical and genetic map of Borrelia burgdorferi, the Lyme disease agent.

A physical map of the 952 kbp chromosome of Borrelia burgdorferi Sh-2-82 has been constructed. Eighty-three intervals on the chromosome, defined by the cleavage sites of 15 restriction enzymes, are delineated. The intervals vary in size from 96 kbp to a few hundred bp, with an average size of 11.5 kbp. A striking feature of the map is its linearity; no other bacterial groups are known to have linear chromosomes. The two ends of the chromosome do not hybridize with one another, indicating that there are no large common terminal regions. The chromosome of this strain was found to be stable in culture; passage 6, 165 and 320 cultures have identical chromosomal restriction maps. We have positioned all previously known Borrelia burgdorferi chromosomal genes and several newly identified ones on this map. These include the gyrA/gyrB/dnaA/dnaN gene cluster, the rRNA gene cluster, fla, flgE, groEL (hsp60), recA, the rho/hip cluster, the dnaK (hsp70)/dnaJ/grpE cluster, the pheT/pheS cluster, and the genes which encode the potent immunogen proteins p22A, p39 and p83. Our electrophoretic analysis detects five linear and at least two circular plasmids in B. burgdorferi Sh-2-82. We have constructed a physical map of the 53 kbp linear plasmid and located the operon that encodes the two major outer surface proteins ospA and ospB on this plasmid. Because of the absence of functional genetic tools for this organism, these maps will serve as a basis for future mapping, cloning and sequencing studies of B. burgdorferi.

Blotting, Southern↗
PubMed1982

Gene for Escherichia coli glycyl-tRNA synthetase has tandem subunit coding regions in the same reading frame.

Glycyl-tRNA synthetase is one of two Escherichia coli aminoacyl tRNA synthetases which has two different subunits. A 5.1-kilobase pair HindIII chromosomal DNA fragment was isolated, cloned into pBR322 (to give plasmid pTK201), and shown to direct synthesis in maxicells of both subunits (Mr = 35,000 (alpha) and Mr = 65,000 (beta) of glycyl-tRNA synthetase. Locations of alpha- and beta-subunit coding regions were established by introduction of Tn5 insertions into various positions within the 5.1-kilobase pair HindIII segment of pTK201 and by determining the effect of each Tn5 insertion on synthesis of alpha- and beta-subunits and on enzymatic activity. From the Tn5 insertion analysis, regions encoding the NH2 terminus of the alpha-subunit and of the beta-subunit were approximately defined and these regions were sequenced. To locate rigorously the respective NH2-terminal encoding sections in the DNA sequence, NH2-terminal amino acid sequences of alpha- and beta-subunits were established by standard Edman degradations and these sequences were aligned with the DNA sequence. This analysis established the following: 1) coding regions for the subunits are in tandem; 2) a single promoter is used for transcription of both coding sections and the order of transcription is from alpha to beta; 3) in the 500 nucleotides 5' to the start of the alpha-subunit coding section, there is no sequence arrangement like that found for regulatory regions of bacterial amino acid biosynthetic operons; 4) nine nucleotides serve as the spacer between the TAA stop of the alpha- and the ATG start of the beta-subunit coding regions, thus making both coding regions in the same reading frame; and 5) the TAA stop of the alpha-subunit and the next for nucleotides associated with the intersubunit region are complementary to the 3'-end of 16 S rRNA; this arrangement suggests ribosome re-initiation in the spacer region gives balanced synthesis of both subunits.

Amino Acid Sequence↗
PubMed1981

Pausing and attenuation of in vitro transcription in the rrnB operon of E. coli.

We have determined the effects of the nusA gene protein and the regulatory nucleotide guanosine tetraphosphate (ppGpp) on pausing and termination of transcription in the leader region of the rrnB operon in vitro. The leader region of rrnB contains several types of potential regulatory sequences that act at the level of RNA chain elongation and may be involved in control of bacterial growth. We have mapped a termination site, tL, located 260 bases from rrnB promoter P1. Termination at tL is dependent on the nusA protein and is enhanced by ppGpp. The DNA sequence at tL shows striking homologies with trp t', a terminator also strongly affected in vitro by the nusA protein. These in vitro results suggest that rRNA transcription in vivo may be regulated in part through an attenuation mechanism that leads to termination of rrnB chains in the leader region. In addition to tL, elongation of transcription in the leader region is affected by several pause sites that are sensitive to the concentration of ppGpp and the presence of the nusA gene protein. The location and properties of these pause sites suggest that they may also play a role in regulation of rrnB transcription through a mechanism we have termed "turnstile" attenuation. One pause site, located 90 and 91 bases from P1, is unique in that it is not normally a site for transcriptional pausing, but is dependent on simultaneous binding of polymerase at rrnB promoter P2. This leads to blockage of P1 transcripts at high RNA polymerase densities and may provide an additional locus for regulation of rrn transcription.

Bacterial Proteins↗
PubMed1983

Nested control regions promote Xenopus ribosomal RNA synthesis by RNA polymerase I.

The DNA regions required for accurate initiation by RNA polymerase 1 on the ribosomal RNA genes of Xenopus laevis consist of at least two domains within a 150 bp segment adjoining the initiation site. A region of only 13 nucleotides (from -7 to +6) appears sufficient to specify accurate and efficient initiation when various 5' and 3' deletion mutants are injected into oocyte nuclei. In contrast, a 10-fold larger region (from -142 to +6) is required for maximal synthesis when deletion mutants are assayed in oocyte nuclear homogenates; only a low level of synthesis is specified by the 13 bp promoter domain in this in vitro system. Site-specific mutants demonstrate that a sequence adjoining nucleotide -75 is also essential for efficient in vitro initiation. Under other conditions, transcription can be strongly affected by sequences that extend several kb upstream in the rDNA spacer and contain duplications of sequences found within the 150 bp promoter. We propose a model in which transcription of the X. laevis rRNA genes is regulated by interaction of at least three sequence domains that extend over a several kb region.

Animals↗
PubMed1983

Expression of an mRNA coding gene under the control of an RNA polymerase I promoter.

We have placed a 225-bp fragment from the 5' end of the mouse rDNA transcription unit (from -169 to +56) in front of the SV40 tumor antigen coding sequence. After microinjection of this chimeric plasmid into nuclei of mouse L-cells expression of SV40 large T antigen has been observed. The expression of T antigen was dependent on the correct orientation of the rDNA fragment relative to the T antigen-coding region and was seen only in mouse cells. This indicates that the 225-bp rDNA fragment contains the sequence information required for pre-rRNA transcription and demonstrates for the first time that a protein-coding gene can be transcribed and expressed under the control of an RNA polymerase I promoter.

Animals↗
PubMed1982

Analysis of transcriptional initiation of yeast mitochondrial DNA in a homologous in vitro transcription system.

We have developed an in vitro transcription system for yeast mitochondrial rRNA genes. Using highly purified yeast mitochondrial RNA polymerase and bacterial plasmids carrying DNA segments containing the mitochondrial rRNA sites of transcriptional initiation, we have been able to demonstrate correct initiation of transcription in vitro. By directly sequencing the transcription products, we show that transcription in vitro of both the 14S and 21S rRNAs is initiated at precisely the same site as it is in vivo. Transcription of the rRNA genes is highly sensitive to ionic strength and RNA polymerase concentration. Additional factors or modified conditions may be necessary to permit accurate transcription of mitochondrial protein genes.

Base Sequence↗
PubMed1991

Complete cloning of the chloroplast genome of safflower in lambda EMBL3 and mapping of 23S and 16S rRNA genes.

A recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of lambda EMBL3. Seventeen lambda recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.

Bacteriophage lambda↗
PubMed1993

Analysis of the self-defense gene (fmrO) of a fortimicin A (astromicin) producer, Micromonospora olivasterospora: comparison with other aminoglycoside-resistance-encoding genes.

The nucleotide (nt) sequence of a 3409-bp PvuII fragment carrying the self-defense gene (fmrO) of a fortimicin A (FmA; astromicin) producer, Micromonospora olivasterospora, was determined; four open reading frames (ORFs) exist in this region. Expression analysis of fmrO using the lac promoter in Escherichia coli revealed that ORF-3 encodes fmrO. ORF-1 was considered to encode a FmKH epimerase (fms11). ORF-2, ORF-3 and ORF-4 seemed to form an operon. No homology was detected between fmrO and the resistance-encoding gene fmrT of Streptomyces tenjimariensis, a producer of the Fm-group antibiotic, istamycin. Gene organization around the fmr genes differed considerably between M. olivasterospora and S. tenjimariensis. The deduced amino acid sequence of fmrO showed an identity of 30.8% to grmA of M. purpurea and 35.8% to grmB of M. rosea, respectively, suggesting that fmrO encodes a 16S rRNA methyltransferase. We found in M. purpurea a novel resistance gene distinct from grmA and highly similar to fmrO which conferred a resistance phenotype similar to that of fmrO. These results suggest that fmrO of M. olivasterospora and the two resistance-encoding genes of M. purpurea were derived from a common ancestral gene.

Amino Acid Sequence↗
PubMed2004

Comparison of ATPase-encoding type III secretion system hrcN genes in biocontrol fluorescent Pseudomonads and in phytopathogenic proteobacteria.

Type III protein secretion systems play a key role in the virulence of many pathogenic proteobacteria, but they also occur in nonpathogenic, plant-associated bacteria. Certain type III protein secretion genes (e.g., hrcC) have been found in Pseudomonas sp. strain SBW25 (and other biocontrol pseudomonads), but other type III protein secretion genes, such as the ATPase-encoding gene hrcN, have not been found. Using both colony hybridization and a PCR approach, we show here that hrcN is nevertheless present in many biocontrol fluorescent pseudomonads. The phylogeny of biocontrol Pseudomonas strains based on partial hrcN sequences was largely congruent with the phylogenies derived from analyses of rrs (encoding 16S rRNA) and, to a lesser extent, biocontrol genes, such as phlD (for 2,4-diacetylphloroglucinol production) and hcnBC (for HCN production). Most biocontrol pseudomonads clustered separately from phytopathogenic proteobacteria, including pathogenic pseudomonads, in the hrcN tree. The exception was strain KD, which clustered with phytopathogenic pseudomonads, such as Pseudomonas syringae, suggesting that hrcN was acquired from the latter species. Indeed, strain KD (unlike strain SBW25) displayed the same organization of the hrpJ operon, which contains hrcN, as P. syringae. These results indicate that the occurrence of hrcN in most biocontrol pseudomonads is not the result of recent horizontal gene transfer from phytopathogenic bacteria, although such transfer might have occurred for a minority of biocontrol strains.

Adenosine Triphosphatases↗
PubMed1990

Organization and nucleotide sequence of ribosomal RNA genes on a circular 73 kbp DNA from the colourless flagellate Astasia longa.

Three tandemly arranged repeats (A, B, C) of 16S and 23S rDNA, and one supplementary (S) 16S rDNA adjacent to the 16S rDNA of repeat A, are present within an 18 kbp segment of a circular 73 kbp DNA from the colourless flagellate Astasia longa. The repeat units are separated by a short region containing a 5S rRNA gene and a gene for tRNA-Val (UAC). Sequence comparisons reveal 78%, 81%, and 67% identical nucleotides of the 23S rDNA (A), the 16S rDNA (B), and the 5S rDNA (A), respectively, with the corresponding genes of the Euglena gracilis chloroplast genome. As in Euglena chloroplasts, the 3'-terminal portion of the 23S rDNA is homologous to the 4.5S rRNA gene of higher plant chloroplast genomes. These results are supportive of a common evolutionary origin for the Astasia 73 kbp DNA and the Euglena 145 kbp chloroplast DNA.

Animals↗
PubMed2002

Hyaloraphidium curvatum: a linear mitochondrial genome, tRNA editing, and an evolutionary link to lower fungi.

We have sequenced the mitochondrial DNA (mtDNA) of Hyaloraphidium curvatum, an organism previously classified as a colorless green alga but now recognized as a lower fungus based on molecular data. The 29.97-kbp mitochondrial chromosome is maintained as a monomeric, linear molecule with identical, inverted repeats (1.43 kbp) at both ends, a rare genome architecture in mitochondria. The genome encodes only 14 known mitochondrial proteins, 7 tRNAs, the large subunit rRNA and small subunit rRNA (SSU rRNA), and 3 ORFs. The SSU rRNA is encoded in two gene pieces that are located 8 kbp apart on the mtDNA. Scrambled and fragmented mitochondrial rRNAs are well known from green algae and alveolate protists but are unprecedented in fungi. Protein genes code for apocytochrome b; cytochrome oxidase 1, 2, and 3, NADH dehydrogenase 1, 2, 3, 4, 4L, 5, and 6, and ATP synthase 6, 8, and 9 subunits, and several of these genes are organized in operon-like clusters. The set of seven mitochondrially encoded tRNAs is insufficient to recognize all codons that occur in the mitochondrial protein genes. When taking into account the pronounced codon bias, at least 16 nuclear-encoded tRNAs are assumed to be imported into the mitochondria. Three of the seven predicted mitochondria-encoded tRNA sequences carry mispairings in the first three positions of the acceptor stem. This strongly suggests that these tRNAs are edited by a mechanism similar to the one seen in the fungus Spizellomyces punctatus and the rhizopod amoeba Acanthamoeba castellanii. Our phylogenetic analysis confirms with overwhelming support that H. curvatum is a member of the chytridiomycete fungi, specifically related to the Monoblepharidales.

Animals↗
PubMed1993

Hybridization patterns of Actinobacillus actinomycetemcomitans serotypes a-e detected with an rRNA gene probe.

This study reports a genetic characterization of Actinobacillus actinomycetemcomitans strains in relation to serotypes by using rRNA gene restriction patterns. Eighty-eight clinical strains were isolated from 20 unrelated subjects at one or several occasions. The strains were serotyped by using serotype-specific rabbit antisera against serotypes a, b, c, d or e. Three subjects harbored 2 A. actinomycetemcomitans serotypes, 15 subjects 1 serotype and 2 subjects untypable strains. Chromosomal DNA was digested with restriction endonuclease ClaI, BamHI, BglI or HindIII and hybridized to the rrnB ribosomal RNA operon of the Escherichia coli chromosome. Isolates belonging to the same serotype were genetically identical in the same individual but nonidentical if they belonged to different serotypes. Isolates of the same or different serotypes were genetically nonidentical in different individuals. The banding patterns of A. actinomycetemcomitans isolates recovered from the same individuals during several years always remained identical. The hybridization method using pKK3535 as a probe seemed suitable as an epidemiological tool for comparing the clonal identity of A. actinomycetemcomitans strains.

Adolescent↗
PubMed1992

Gene phylogenies and the endosymbiotic origin of plastids.

The endosymbiotic origin of chloroplasts from cyanobacteria has long been suspected and has been confirmed in recent years by many lines of evidence. Debate now is centered on whether plastids are derived from a single endosymbiotic event or from multiple events involving several photosynthetic prokaryotes and/or eukaryotes. Phylogenetic analysis was undertaken using the inferred amino acid sequences from the genes psbA, rbcL, rbcS, tufA and atpB and a published analysis (Douglas and Turner, 1991) of nucleotide sequences of small subunit (SSU) rRNA to examine the relationships among purple bacteria, cyanobacteria and the plastids of non-green algae (including rhodophytes, chromophytes, a cryptophyte and a glaucophyte), green algae, euglenoids and land plants. Relationships within and among groups are generally consistent among all the trees; for example, prochlorophytes cluster with cyanobacteria (and not with green plastids) in each of the trees and rhodophytes are ancestral to or the sister group of the chromophyte algae. One notable exception is that Euglenophytes are associated with the green plastid lineage in psbA, rbcL, rbcS and tufA trees and with the non-green plastid lineage in SSU rRNA trees. Analysis of psbA, tufA, atpB and SSU rRNA sequences suggests that only a single bacterial endosympbiotic event occurred leading to plastids in the various algal and plant lineages. In contrast, analysis of rbcL and rbcS sequences strongly suggests that plastids are polyphyletic in origin, with plastids being derived independently from both purple bacteria and cyanobacteria. A hypothesis consistent with these discordant trees is that a single bacterial endosymbiotic event occurred leading to all plastids, followed by the lateral transfer of the rbcLS operon from a purple bacterium to a rhodophyte.

Amino Acid Sequence↗
PubMed2002

Initiation of tRNA maturation by RNase E is essential for cell viability in E. coli.

RNase E, an essential endoribonuclease in Escherichia coli, is involved in 9S rRNA processing, the degradation of many mRNAs, and the processing of the M1 RNA subunit of RNase P. However, the reason that RNase E is required for cell viability is still not fully understood. In fact, recent experiments have suggested that defects in 9S rRNA processing and mRNA decay are not responsible for the lack of cell growth in RNase E mutants. By using several new rne alleles, we have confirmed these observations and have also ruled out that M1 processing by RNase E is required for cell viability. Rather, our data suggest that the critical in vivo role of RNase E is the initiation of tRNA maturation. Specifically, RNase E catalytic activity starts the processing of both polycistronic operons, such as glyW cysT leuZ, argX hisR leuT proM, and lysT valT lysW valZ lysY lysZ lysQ, as well as monocistronic transcripts like pheU, pheV, asnT, asnU, asnV, and asnW. Cleavage by RNase E within a few nucleotides of the mature 3' CCA terminus is required before RNase P and the various 3' --> 5' exonucleases can complete tRNA maturation. All 59 tRNAs tested involved RNase E processing, although some were cleaved more efficiently than others.

Cell Division↗
PubMed1998

Differentiation of Actinobacillus pleuropneumoniae strains by sequence analysis of 16S rDNA and ribosomal intergenic regions, and development of a species specific oligonucleotide for in situ detection.

The aims of this study were to characterize and determine intraspecies and interspecies relatedness of Actinobacillus pleuropneumoniae to Actinobacillus lignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in situ detection of A. pleuropneumoniae. Amplification and sequence analysis of the 16S-23S rDNA ribosomal intergenic sequence (RIS) from the three species showed the existence of two RIS's, differing by about 100 bp. Both sequences contained a region resembling the ribonuclease III cleavage site found in Escherichia coli. The smaller RIS contained a Glu-tRNA gene, and the larger one contained genes encoding Ile-tRNA and Ala-tRNA. These tRNA's showed a high sequence homology to the respective tRNA genes found in E. coli. Sequence analysis of the RIS's showed a high degree of genetic similarity of 24 strains of A. pleuropneumoniae. The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A. pleuropneumoniae. These sequences showed only minor base differences, indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the target species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA probe labeled with fluorescein was used for in situ hybridization experiments to detect A. pleuropneumoniae in biopsies of diseased porcine lungs.

Actinobacillus Infections↗